calcium signalling in cancer-sh erbet

CalciumSignallingin Cancer

0942/fm/frame Page 2 Wednesday, October 25, 2000 5:14 PM

Gajanan V. SherbetCancer Research UnitThe Medical School

University of Newcastle upon Tyne,UKand

The Institute for Molecular MedicineHuntington Beach, CA USA

CalciumSignallingin Cancer

Boca Raton London New York Washington, D.C.CRC Press

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No claim to original U.S. Government worksInternational Standard Book Number 0-8493-0982-4

Library of Congress Card Number 00-062115Printed in the United States of America 1 2 3 4 5 6 7 8 9 0

Printed on acid-free paper

Library of Congress Cataloging-in-Publication Data

Sherbet, G. V. (Gajanan V.)Calcium signalling in cancer : the role of calcium binding proteins in signaltransduction, cell proliferation, invasion, and metastasis / Gajanan V. Sherbet.

p. cm.Includes bibliographical references and index.ISBN 0-8493-0982-4 (alk. paper)

1. Carcinogenesis. 2. Calcium-binding proteins. 3. Metastasis. 4. Cancer invasiveness.I. Title.[DNLM: 1. Neoplasm Proteins—physiology. 2. Calcium Signaling—physiology. 3.Calcium-Binding Proteins—physiology. 4. Cell Differentiation—physiology. 5. NeoplasmInvasiveness. 6. Neoplasm Metastasis. QZ 200 S551c 2000]RC268.5 .S53 2000616.99

′

407—dc21 00-062115


Dedication

toShri Sai Baba


Preface

The perfect messenger commands respect, delivers the message uncorrupted, at theright time, at the right place, with integrity.

Thiru Valluvar(Tamil poet, second century, India)

Thirukkural

, Chapter 69, verse 688

Calcium signalling occupies a preeminent position in the signal transduction systemof the cell by virtue of its participation in a wide range of physiological functionsand in the biological events associated with genetic expression, cell proliferationand apoptosis, and cell differentiation and morphogenesis. Calcium signalling is animportant feature of cell adhesion and motility, and of cancer invasion and metastasis.The calcium ion virtually unifies several pathways of signal transduction. It hasrightly been described as a second messenger. Calcium-binding proteins are animportant link in the calcium-signalling pathway. These proteins not only maintainthe integrity of the calcium signal, but they are also responsible for transmitting themessage in a temporally and spatially coordinated manner. It follows therefore thatthe integrity of the calcium binding proteins themselves is a basic requirement ofnormal biological function. This concept is encapsulated most eloquently in theTamil quotation given above. If their integrity is compromised or lost from abnormalor inappropriate expression, or by genetic changes, that could lead to a profoundderegulation of signal transduction with dramatic and wide-ranging effects on thelife of the cell and its biological behaviour. That is the simple and singular justifi-cation for focusing on this protein species in this book.

This volume is a natural sequel to my previous works. They are:

The Biochemicaland Biophysical Characterisation of the Cell Surface

(Academic Press, 1978)

, TheBiology of Tumour Malignancy

(Academic Press, 1982)

, The Metastatic Spread ofCancer

(Macmillan, 1987),

and

The Genetics of Cancer

(Academic Press, 1997).In these one can discern the evolution of thought relating to the biological behaviourof cells and the pathogenesis of cancer, from inception of the tumour to its progres-sion to the overt metastatic state. This book has not been written with the specialistonly in mind. The discussion of topics is self-contained, as far as it was practicable,and I believe, therefore, it would be useful to students and research scientists alike.


Also, the presentation and discussions cater to the needs of scientists in related butperipheral fields of discipline.

I had considerable help from my colleagues in the preparation of this book.Professor P.A. Riley of the University College School of Medicine and Dr. M.S.Lakshmi very kindly read the entire manuscript. Their comments and criticisms havebeen taken and consequently the manuscript has greatly improved in presentationand coverage. Dr. Lakshmi also kindly provided the Tamil quotation and its trans-lation. I wish to thank them most sincerely for their help. It is needless to say,however, that the responsibility for errors and omissions rests solely with me. I thankPaula Rutter of the Audio-visual Centre for preparing the figures and for the patienceand skill that she has displayed. CRC Press LLC received this book most enthusi-astically and with great fervour. It has been a considerable pleasure working withthem, especially with Fequiere Vilsaint, on this project. Finally, the North of EnglandCancer Research Campaign largely supported the research work in my laboratoryand I wish to record my gratitude to them.

Gajanan V. SherbetCancer Research Unit

The Medical SchoolUniversity of Newcastle upon Tyne, UK

andThe Institute for Molecular Medicine

Huntington Beach, CA, USA


Abbreviations

β

APP

β

-amyloid precursor proteinABP Amyloid-

β

proteinACTH Adrenocorticotropic hormoneACV AcycloviraFGF Acidic fibroblast growth factorALS Amyotrophic lateral sclerosisANN Artificial neural networkAPC Adenomatous polyposis coli (protein)AR Androgen receptorBA-1p Bone-specific alkaline phosphataseBAE Bovine aortic endothelial (cell)BDNF Brain-derived neurotropic factorbFGF Basic fibroblast growth factorBMD Becker muscular dystrophybp Base pair(s)BPH Benign prostatic hyperplasiaBrDU Bromo-deoxyuridineCAI Carboxyamido-triazoleCaM CalmodulincAMP Cyclic 3

′

,4

′

-adenosine monophosphateCAMPK Calcium/calmodulin-dependent protein kinaseCapn CalpainCAR Cancer-associated retinopathyCBD Calbindin D-28KCBD9K Calbindin D-9KCBP Calcium-binding proteincdk Cyclin-dependent protein kinasecGMP Cyclic guanosine monophosphateCH Calponin homology (domain)CHO Chinese hamster ovary (cell line)CK Casein kinaseCNS Central nervous systemCP (Actin) capping proteinCRE Cyclic AMP response elementCREB Cyclic AMP response element binding proteinCS Cowden’s syndromeDAG 1,2-DiacylglycerolDAGK Diacylglycerol kinasedb-cAMP Dibutyryl cyclic AMP


DD Darier’s diseaseDMD Duchenne muscular dystrophyDMSO Dimethyl sulphoxideEAE Experimental allergic encephalomyelitisEC Endothelial cellECM Extracellular matrixEDC Epidermal differentiation complexEFABP Epidermal-type fatty acid-binding proteinEGF Epidermal growth factorEGFr Epidermal growth factor receptoreNOS Endothelial nitric oxide synthaseEp-ICAM Epithelial intercellular adhesion moleculeER Endoplasmic reticulumER Oestrogen receptorERK Extracellular signal-regulated receptor kinase (or MAPK)ES Embryonic stem (cells)FC Follistatin-like (domain)FN FibronectinFGF Fibroblast growth factorFLG ProfilaggrinFSP1 Fibroblast-specific protein 1 (S100A4)GABA Gamma-amino butyric acidGAP GTPase-activating proteinGC Guanylate cyclaseGCAP Guanylate cyclase-activating proteinGDNF Glial cell-derived growth factorGDP Guanosine diphosphateGFAP Glial fibrillary acidic proteinGRP Gastrin-releasing peptideGRPr Gastrin-releasing peptide receptorGTP Guanosine triphosphateGTPase Guanosine triphosphatase4-HNE 4-HydroxynonenalHO-1 Heme oxygenase-1HSP Heat shock proteinHUVEC Human umbilical vein endothelial cellICAM Intercellular adhesion moleculeICE Interleukin-1

β

-converting enzymeICTP Pyridinoline cross-linked telopeptide of type I procollagenIF Intermediate filamentsIFN InterferonIGF Insulin-like growth factorIL InterleukinIP3 Inositol 1,4,5-trisphosphateIP3R Inositol 1,4,5-trisphosphate receptorIP3R-P Phosphorylated form of inositol 1,4,5-trisphosphate receptor


JAK Janus tyrosine kinaseLAK Lymphokine-activated killer (cells)LGMD Limb girdle muscular dystrophyL-NAME NG-Nitro-l-arginine methyl esterLOH Loss of heterozygosityLPS LipopolysaccharideMAP Microtubule associated proteinMAPK Mitogen-activated protein kinase (or ERK)MB-40 Basement membrane-40 protein (SPARC, osteonectin)MBP Myelin basic proteinMBPK Myelin basic protein kinase5-MC 5-MethylcytosineMDBK Madin-Darby bovine kidney (cell line)MHC Major histocompatibility complexMHC Myosin heavy chainMLC Myosin light chainMLCK Myosin light chain kinaseMMP Matrix metalloproteinasesMMTV Murine mammary tumour virusMS Multiple sclerosisMSH Melanocyte-stimulating hormoneMTase MethyltransferaseNAD Nicotinamide adenine dinucleotideNCAM Neural cell adhesion moleculeNCBP Neural calcium binding proteinNDP Nucleoside diphosphateNF2 Neurofibromatosis type 2NF-AT Nuclear factor of activated T cellsNFT Neurofibrillary tanglesNGF Nerve growth factorNMDA

N

-Methyl-d-aspartateNOS Nitric oxide synthaseNSCLC Non-small cell lung carcinomaNT3 Neurotropin-3NMU

N

-methyl-

N

-nitrosoureaOA OsteoarthritisOM OncomodulinORF Open reading frameOSE Osteonectin silencer elementOSE-bp Osteonectin silencer element-binding proteinOT OxytocinPA Plasminogen activatorPAI Plasminogen activator inhibitorPARP Poly (ADP-ribose) polymerasePCNA Proliferating cell nuclear antigenPCR Polymerase chain reaction


PDE PhosphodiesterasePDGF Platelet-derived growth factorPDI Protein disulphide isomerasePEP1 Profilaggrin endopeptidase 1PEST Pro, Asp/Glu, Ser, Thr sequence (in proteins)PGE Prostaglandin EPgR Progesterone receptorPH Pleckstrin homology [domain]PHF Paired helical filamentsPI3K Phosphoinositide-3 kinasePICP C-Terminal peptide of type I procollagenPIIINP N-Terminal peptide of type III procollagenPIP Phosphatidyl inositol-3 phosphatePIP2 Phosphatidyl inositol 4,5-bisphosphatePIP3 Phosphatidyl inositol 3,4,5-trisphosphatePIP4 Phosphatidyl inositol 1,3,4,5-tetrakisphosphatePKA Protein kinase APKC Protein kinase CPLA2 Phospholipase A2PLC Phospholipase CPMA Phorbol 12-myristate 13-acetatePMCA Plasma membrane Ca

2+

-ATPasePMN Polymorphonuclear leukocytePP-1A/PP-1B Protein phosphatases 1A and 1BPS Presenilin (genes 1 and 2 in Alzheimer’s disease)PSA Prostate-specific antigenPSA-NCAM Polysialylated NCAMPT Demyelinating paralytic tremor rabbit mutantPTTH Prothoracicotropic hormonePV ParvalbuminRA Retinoic acidRA Rheumatoid arthritisrb Retinoblastoma susceptibility (gene product)RBP Retinol-binding proteinRCN RecoverinRGS Regulator of G-protein signalling (proteins)RP Retinitis pigmentosarptn RepetinRSV Rous sarcoma virusRT Reverse transcriptaseRTK Receptor tyrosine kinaseRT-PCR Reverse transcriptase polymerase chain reactionRV Rubella virusRXR Retinoid X receptorRyR Ryanodine receptorSCCA Squamous cell lung carcinoma antigen


SCLC Small cell lung carcinomaSDK Sphingosine-dependent kinaseSERCA Sarcoplasmic–endoplasmic reticulum Ca

2+

-ATPaseSLE Systemic lupus erythematosusSMS Smith–Magenis syndromeSPARC Secreted protein, acidic, rich in cysteine (osteonectin; BM-40)SR Sarcoplasmic reticulumSTAT Signal transducer and activator of transcription factorsSVZ Subventricular zoneT

β

Beta thymosinsTCR T-cell antigen receptorTGF Transforming growth factorTHH TrichohyalinTIMP Tissue inhibitor of metalloproteinaseTK Thymidine kinaseTn TroponinTNF Tumour necrosis factorTPA 12-

O

-Tetradecanoyl phorbol 13-acetatetPA Tissue plasminogen activatoruPA Urokinase-type plasminogen activatorVASP Vasodilator-stimulated phosphoproteinVCAM Vascular cell adhesion moleculeVD3 Vitamin D3VDR Vitamin D3 receptorVDRE Vitamin D response elementVEGF Vascular endothelial growth factorVGCC Voltage-gated calcium channelVILIP Visinin-like proteinVP VasopressinWAS Wiskott–Aldrich syndromeWASP Wiskott–Aldrich syndrome protein


Author

Dr. Gajanan Sherbet

is a professor at the Institute for Molecular Medicine, Hun-tington Beach, CA. He received his D.Sc. and M.Sc. degrees from the Universityof London, and Ph.D., M.Sc., and B.Sc. degrees from the University of Poona. Dr.Sherbet was reader in Experimental Oncology and deputy director of the CancerResearch Unit in the Medical School of the University of Newcastle upon Tyne,England. Previous to this Dr. Sherbet was a staff member of the Chester BeattyResearch Institute, Institute of Cancer Research, and University College HospitalMedical School in London. He has held prestigious fellowships such as the BeitMemorial and Williams Fellowship of the University of London. He held a careerfellowship awarded by the North of England Cancer Research Campaign. For a briefperiod, he was a fellow of Harvard University, Cambridge, MA. Dr. Sherbet is afellow of the Royal College of Pathologists (FRCPath), Royal Society of Chemistry(FRSC), and The Institute of Biology (FIBiol) of U.K. He served as editor of

Oncology

and

Experimental Cell Biology

and was senior editor of

Pathobiology

.Currently, he is a member of the editorial boards of

Pathobiology

and

AnticancerResearch

.Dr. Sherbet’s major interest is in cancer metastasis. In recent years he has been

investigating the role of the calcium binding protein S100A4 in cell proliferation,cancer invasion, and metastasis, focusing mainly on melanomas, neuroectodermaltumors, and breast cancer. He recently demonstrated the potential value of S100A4as a marker for assessing the progression of breast cancer. He is also studying thepotential of artificial neural networks in the management of breast cancer, especiallythe analysis of expression of cancer markers and image cytometric data of breastcancer by using artificial neural networks.

Dr. Sherbet has published numerous scientific papers in international journals.He has written and edited several books on cancer. Notable among them are

TheMetastatic Spread of Cancer

(Macmillan, 1987),

The Biology of Tumour Malignancy

(Academic Press, 1982), and

The Biochemical and Biophysical Characterisation ofthe Cell Surface

(Academic Press, 1978). His latest book,

The Genetics of Cancer

(Academic Press, 1997), was co-authored with Dr. M.S. Lakshmi. He was guesteditor of

Retinoids: Their Physiological Function and Therapeutic Potential

(1997).Dr. Sherbet is co-editor of

Artificial Neural Networks in Cancer Diagnosis

,

Prog-nosis, and Patient Management

to be published soon by CRC Press LLC.


Contents

PrefaceAbbreviations

Chapter 1

Introduction................................................................................................................1

Chapter 2

The Calcium Signalling Pathway..............................................................................5Homeostasis of Cell Calcium...........................................................................5

The Plasma Membrane Ca

2+

-ATPase Pump ........................................5The Sarcoplasmic-Endoplasmic Reticulum Ca

2+

-ATPase Pump .....................................................................................................7Voltage-Gated Calcium Channels.........................................................8The Deregulation of Calcium Homeostasis as a Primary Event in Carcinogenesis .......................................................................9

Phospholipid Signalling..................................................................................10PTEN Phosphatase in the Regulation of Lipid Signalling ............................11The Protein Kinase C Pathway ......................................................................13Protein Kinase C and Its Isoforms in Signal Transduction ...........................14Inositol Phosphates in Calcium Signal Transduction ....................................16Deregulation of Inositol 1,4,5-Trisphosphate Pathway and Its Consequences ............................................................................................18Ryanodine and Related Receptors in Calcium Mobilisation.........................20Cyclic AMP in Calcium Signalling ...............................................................21Architectural Aspects of the Signal Transduction Machinery.......................25

The Role of Caveolae in Signal Transduction...................................25Caveolin Expression in Cancer ..........................................................28

Chapter 3

Calcium Binding Proteins and Their Natural Classification ..................................29

Chapter 4

Non-EF-Hand Calcium Binding Proteins ...............................................................35Annexins .........................................................................................................35

Structure..............................................................................................35Biologic Functions..............................................................................36Annexins in Cancer Growth and Progression....................................38Annexins in Morphogenesis and Differentiation ...............................39


The Gelsolin Family of Calcium Binding Proteins .......................................40Gelsolin in Severing and Capping of Actin Filaments ......................40Gelsolin in Embryonic Development and Morphogenesis ................41Gelsolin Expression in Amyloidosis ..................................................42Gelsolin in Cancer ..............................................................................42Severin and Cytoskeletal Reorganisation...........................................44Villin in Differentiation and Neoplasia..............................................44

Calreticulin and Its Functional Diversity .......................................................46Structure and Molecular Features of Calreticulin..............................46Regulation of Calreticulin Expression ...............................................46Phosphorylation of Calreticulin..........................................................47Intracellular Distribution of Calreticulin............................................48Calreticulin in Intracellular Calcium Storage ....................................48Calreticulin and Calnexin as Molecular Chaperones.........................49Calreticulin in Cell Proliferation and Differentiation........................50Calreticulin in Cell Adhesion .............................................................50Calreticulin in Neoplasia ....................................................................51Immunological Implications of Calreticulin Function.......................52

Calsequestrin and Intracellular Calcium Storage...........................................53Osteocalcin in Bone Metabolism and Osteotropism of Cancer ....................54

The Biology of Osteocalcin ...............................................................54Calcium-Binding Properties of Osteocalcin.......................................55Osteocalcin Gene Structure and Function..........................................55Regulation of Osteocalcin by Vitamin D3 .........................................56Osteocalcin in Cell Proliferation and Differentiation........................57Osteotropism of Metastatic Dissemination ........................................60

Chapter 5

The EF-Hand Calcium-Binding Proteins ................................................................63Molecular Organisation of Calcium Binding EF-Hand Proteins ..................63Calcium Binding and the Molecular Configuration of Calcium-Binding Proteins ...........................................................................................................65The Structure and Organisation of S100 Family Genes................................68Alternatively Spliced Variants of S100A4 .....................................................68Functional Significance of Alternatively Spliced Isoforms ...........................69Regulation of Expression of S100 Family Genes..........................................71

Transcriptional Regulation of S100 Genes ........................................71Regulation of Gene Expression by DNA Methylation......................72DNA Methylation in Cancer ..............................................................72Regulation of S100 Gene Transcription by Methylation...................74

Chapter 6

The Calmodulin Family of Calcium Binding Proteins...........................................75Calmodulin and Its Physiological Function...................................................75

Structure and Mode of Action of Calmodulin ...................................75


Calmodulin-Mediated Signal Transduction........................................76Calmodulin and Cell Proliferation .....................................................77Calmodulin in Neoplasia ....................................................................78

Recoverin Subfamily of Neural Calcium-Binding Proteins and Their Function ..........................................................................................................79

The G-Protein Signalling Pathway.....................................................79Recoverin and Its Function ................................................................80Mode of Action of Recoverin.............................................................82Post-translational Modification of Recoverin ....................................82Recoverin and Cancer-Associated Retinopathy .................................83

Recoverin and Cancer-Associated Retinopathy inSmall Cell Lung Cancer ......................................................83Retinopathy Associated with Other Forms of Human Cancer .....................................................................84Is Recoverin Involved in Retinitis Pigmentosa? .................85

Guanylate Cyclase-Activating Proteins..........................................................85

Chapter 7

The Structure of Contractile Proteins .....................................................................87The Actin Component of Contractile Machinery of the Cell........................87

Actin Isoforms ....................................................................................87Regulation of Actin Dynamics ...........................................................88Cofilin in the Regulation of Actin Dynamics ....................................88Profilin in the Regulation of Actin Dynamics ...................................90Rho GTPases in Actin Dynamics and Signal Transduction ..............90Interaction of Formin with Profilin and Rho GTPases......................92

The Role of Thymosin Family Actin-Binding Proteins in Actin Dynamics ..............................................................................................93

Sequestration of Actin by Thymosins ................................................93Effects of Thymosins on Cell Proliferation .......................................93Thymosins and Cell Motility and Differentiation .............................94Expression of Thymosins in Embryonic Development .....................94Potential Role of Thymosins in Cancer Progression.........................95

The Fimbrin Family of Actin-Binding Proteins.............................................96Molecular Features of Fimbrin...........................................................96Function of Fimbrin in Cytoskeletal Organisation ............................97Regulation of Fimbrin Expression .....................................................99Is Fimbrin Involved in Cancer?........................................................100Modulation of Actin Dynamics and Cancer Cell Dissemination ...................................................................................101

α

-Actinin.......................................................................................................102Molecular Structure of

α

-Actinin ....................................................102

α

-Actinin Isoforms ...........................................................................103Function of

α

-Actinin.......................................................................104


Actinins in Cell Adhesion, Motility, and Signal Transduction........104The Cadherin–Catenin Complex in Signal Transduction and Cell Adhesion ...................................................................................104

Myosin Filaments .........................................................................................110Myosin Heavy Chain (MHC) Isoforms ...........................................111Actomyosin Assembly......................................................................112Myosin Light Chain (MLC) Phosphorylation and Function ...........115

Troponins and Tropomyosins in the Regulation of Muscle Cell Contraction....................................................................................................118

The Regulatory Role of Troponins and Tropomyosins in Muscle Contraction........................................................................................118Tropomyosin Isoforms in Benign and Malignant Cells ..................119The Regulatory Role of Caldesmon.................................................121Calponin: Its Function and Regulation ............................................122Caltropin-Mediated Reversal of Myosin-ATPase Inhibition by Caldesmon and Calponin..................................................................124

Chapter 8

Structure and Biology of Calbindin ......................................................................125Calbindin in Neuronal Populations ..............................................................125Neural Cell Lineage and the Regulation of Calbindin Expression .............125Calbindin Expression in Embryonic Development and Ageing..................126Physiological Function of Calbindin............................................................127Neuroprotective Function of Calbindin........................................................129Calbindin Expression and the Metastatic Phenotype ..................................129

Chapter 9

Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker......................................................................................................131

Calretinin and Its Alternatively Spliced Isoforms........................................131Regulation of Calretinin Expression ............................................................132Calretinin Expression in Cell Proliferation and Differentiation..................133Calretinin and Its Possible Neuroprotective Property..................................133Calretinin as a Potential Tumour Marker.....................................................133

Chapter 10

Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading ................135Molecular Features of Calcineurin...............................................................135Calcineurin in Cell Proliferation and Adhesion-Related Phenomena .........136

Putative Role of Calcineurin in Cell Cycle Progression .................136The Effects of Calcineurin on Cell Adhesion and Motility ............138

Calcineurin in Alzheimer’s Disease .............................................................140Calcineurin in Immunosuppression..............................................................141


Chapter 11

Centrins (Caltractins) and Their Biological Functions .........................................145

Chapter 12

Reticulocalbin Family of EF-Hand Proteins.........................................................149Molecular Features of Reticulocalbin Homologues ....................................150Putative Functions of Reticulocalbin and Its Homologues .........................150

Chapter 13

Calpains in Normal and Aberrant Cell Physiology ..............................................153The Calpain Family of Calcium-Binding Proteins ......................................153Molecular Organisation of Calpains ............................................................154Regulation of Physiological Events by Proteolytic Function......................155Involvement of Calpains in Development and Differentiation....................157Calpains in Cell Proliferation and Apoptosis ..............................................158Calpains in Cell Spreading and Migration ..................................................160Calpains in Integrin-Mediated Cell Adhesion and Signal Transduction..................................................................................................161Calpains in Cancer Growth and Progression ...............................................162Calpains in Myelodegenerative Diseases .....................................................163Calpains in Muscular Dystrophy..................................................................165

Association of Calpains with Duchenne Muscular Dystrophy .......165Calpains and Limb Girdle Muscular Dystrophy..............................166

Chapter 14

Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia .................169Caspases in Apoptotic Cell Death................................................................169Poly (ADP-Ribose) Polymerase as a Marker of Apoptosis ........................172Caspase-Mediated Apoptosis and Cell Growth Inhibition in Tumour Expansion........................................................................................173Caspase-Mediated Proteolysis of Fodrin: Implications for Apoptosis, Cell Adhesion, Cell Migration, and Neoplastic Transformation .................176Caspases and Neuronal Loss in Alzheimer’s Disease .................................177

Chapter 15

Parvalbumins in Neuronal Development, Differentiation, and Proliferation .......181

Chapter 16

Osteonectin in Cell Function and Behaviour........................................................183Molecular Structure of Osteonectin .............................................................183Functions and Functional Domains of Osteonectin.....................................184Regulation of Osteonectin Expression .........................................................184Osteonectin in the Remodelling of the Extracellular Matrix ......................186


Osteonectin in Embryonic Development and Differentiation .....................187Modulation of Cellular Adhesion, Cell Shape, and Motility by Osteonectin ..............................................................................................188Modulation of Cell Proliferation by Osteonectin ........................................190Effects of Osteonectin on Angiogenesis ......................................................191Osteonectin Expression in Cancer Development and Progression..............193Osteonectin Involvement in Other Disease States .......................................196Osteonectin Homologues and Their Putative Tumour Suppressor Properties ......................................................................................................197

Chapter 17

S100 Proteins: Their Biological Function and Role in Pathogenesis ..................199S100 Proteins in Cell Differentiation, Motility, and Cancer Invasion ........202

Profilaggrin (FLG) in Keratinocyte Differentiation.........................202The Molecular Characteristics of Profilaggrin..................202

Trichohyalin (THH)..........................................................................205Effects of S100 Proteins on Cell Deformability and Cellular Morphology ..................................................................................................205

Cell Adhesion and Invasive Potential of Cancer Cells ....................210S100 Proteins in Remodelling of the Extracellular Matrix.............213S100 Proteins in Cell Proliferation ..................................................214Cell Cycle-Related Expression of S100 Proteins ............................217Postulated Mechanism of Cell Cycle Control by S100A4..............219

S100A Isoforms............................................................................................222S100A2 as a Putative Tumour Suppressor...................................................223S100A3 Expression in Cell Differentiation and Neoplasia.........................224

Molecular Features of S100A3 ........................................................224S100A3 Expression in Cell Differentiation and Human Gliomas .............................................................................................225

S100A4 in Cancer Development and Progression.......................................225S100A4 Expression and Metastatic Potential of Cancers ...............225Clinical Potential of S100A4 as a Marker for Cancer Prognosis...........................................................................................230S100A4 in Human Breast Cancer ....................................................230S100A4 in Other Forms of Human Cancer .....................................234

S100A6 (Calcyclin) in Cancer .....................................................................235The Biological Properties of S100A7 (Psoriasin) .......................................236Structure and Molecular Properties of S100A7...........................................236

S100A7 in Skin Pathology ...............................................................237S100A7 in Neoplastic Disease .........................................................238

S100A8 and S100A9 Proteins in Inflammatory Diseases ...........................239S100A11 (S100C) and Possible Modes of its Function..............................239S100P in Cancer Progression .......................................................................241

S100P and Its Putative Functions.....................................................241


Potential Value of S100 Proteins as Markers of Cancer Progression and Prognosis.......................................................................................................243

Epilogue

................................................................................................................245

R

eferences

.............................................................................................................249

Index

......................................................................................................................349


1

1

Introduction

Calcium-binding proteins (CBPs) are a family of unique importance in normal andaberrant cell biology, by virtue of their participation in a wide spectrum of physio-logical processes. These proteins appear to function from the inception of life,participating in sperm maturation and motility and implantation of the fertilisedovum, to cell differentiation and morphogenesis. Among other important physiolog-ical functions of CBPs are their involvement in Ca

2+

transport and the maintenanceof intracellular Ca

2+

levels, as well as metabolic processes such as nucleotide metab-olism. Signal transduction is another area of cellular physiology in which CBPs areheavily involved. Defective signal transduction is often associated with aberrant cellcycle regulation, which in turn can lead to the development and progression ofcancer. Besides, several CBPs, especially those belonging to the S100 protein family,have recently been demonstrated to be actively engaged in the regulation of the cellcycle in normal as well as in aberrant cell proliferation and in apoptotic cell death.It follows, therefore, that CBPs may be associated with the pathogenesis of severaldiseases, including neoplastic disease. Directly relevant in the context of carcino-genesis is the accumulation of a substantial body of evidence demonstrating thatCBPs participate in the formation and emergence of neoplastic foci of cells and inthe invasive and metastatic spread of these cells constituting the progressive phaseof the disease. A major pathway by which CBPs affect neoplastic progression is byaltering cell proliferation via the modulation of the process of transduction ofproliferative signals imparted by extracellular growth factors and hormones, andthus the regulation of the cell cycle traverse and apoptotic cell death. Influencingcell adhesion and motility by modulating the function of enzymes implicated in theremodelling of the extracellular matrix, leading to metastatic dissemination of cancercells, are also important features of CBP function in cancer. CBPs are associatedwith other pathological conditions of the central nervous system (CNS) and skin,such as amyloidosis, Alzheimer’s disease, and Smith–Magenis syndrome (SMS),among others. The purview of the present volume is to review, analyse, and assimilatethese apparently diverse functions of CBPs into a coherent picture.

The conceptual basis of this book, summarised in Figure 1, is to recognise,redefine, and establish CBPs as second messengers themselves. Intracellular calciumlevels modulate in response to extracellular signals and calcium transport across thecell membrane. CBPs play a crucial role in calcium homeostasis, by calcium buff-ering, calcium transport, and release of calcium from intracellular stores. They carrythe information downstream to activate the phosphorylation of target proteins, whichleads to enzyme function and metabolism, muscle contraction, and a host of otherphysiological functions. They also influence cytoskeletal dynamics, actively partic-ipate in the remodelling of the extracellular matrix, and consequentially affect cellmorphology, motility, and intercellular interactions. A recent development is the

0942/C01/frame Page 1 Tuesday, October 24, 2000 7:30 PM

2

Calcium Signalling in Cancer

demonstration that certain CBPs may actively control the progression of the cellcycle in consort with other proteins such as p53 and stathmin. Apoptosis or pro-grammed cell death is a calcium-dependent phenomenon, and CBPs can activateseveral pathways of molecular degradation leading to apoptosis. In light of thesemultifarious functions, the thesis developed here is that CBPs are an integral com-ponent of the mechanisms of cell population homeostasis and tumour growth.Genetic activation is the coup de grace

of signal transduction. The inappropriateexpression, temporally and spatially, of some CBPs induces invasive behaviour and

FIGURE 1

Summary of the conceptual basis of this book, providing an outline of thepostulate that forms the core of this work: calcium binding proteins (CBPs) are secondmessengers participating in the calcium signalling cascade. CBPs not only regulate intracel-lular calcium levels, but they also are involved in the regulation of normal cell physiology,as well as cell behaviour, proliferation, apoptosis, tumorigenesis, and the secondary spreadof cancer.


Introduction

3

secondary spread of immortalised cells. These genetic and phenotypic changesinduced by CBPs are integrated here to provide a postulate for the growth andprogression of cancers. The formulation of this postulate underpins the evolutionand development of new strategies for controlling the growth of the tumour and itsmetastatic spread.


5

2

The Calcium Signalling Pathway

HOMEOSTASIS OF CELL CALCIUM

Ion channels are involved in a large number of cell functions and in the inductionof cellular responses to extracellular stimuli. There are three types of voltage-sensitive ion channels: the sodium channel, the potassium channel, and the calciumchannel. These function in a coordinated fashion in determining cellular responsesto extraneous signals. The Na

+

channel is involved in the generation of actionpotentials and the K

+

channel is involved in the regulation of membrane potentialand is significantly involved in synaptic plasticity.

The regulation of intracellular levels of calcium is an important element in thesignalling process mediated by calcium as a second messenger. There are severalmechanisms by which calcium levels in the cell are exquisitely controlled andcalcium homeostasis is achieved. These are listed together with their characteristicfeatures and properties in Table 1. The Ca

2+

-ATPase pumps in the plasma membraneand the sarcoplasmic–endoplasmic reticulum play a highly significant part in theregulation of calcium homeostasis. Calcium homeostasis also involves the ligand-gated and voltage-gated channels. Calcium-binding proteins are of special interestin the present context because of their ability to regulate intracellular calcium levels.

T

HE

P

LASMA

M

EMBRANE

C

A

2+

-ATP

ASE

P

UMP

The plasma membrane Ca

2+

-ATPase extrusion pump (PMCA) and the sarcoplas-mic–endoplasmic reticulum Ca

2+

-ATPase pump (SERCA) are specifically targetedto the two membrane systems of the cell. The N-terminal region of the SERCAmolecule contains a domain that has an endoplasmic reticulum (ER)-retention signalsequence. Certain mutations of the PMCA can cause it to become localised to theER (Guerini

et al.

1998). There are four genes which code for four isoforms. Theseisoforms show differential localisation in different organs and the subcellular com-partment. PMCA-1 and PMCA-2 are expressed ubiquitously, but PMCA-3 andPMCA-4 occur specifically in neural cells (Carafoli

et al.

1996). The expression of PMCA genes seems to be developmentally regulated. Thus,

in mouse development, PMCA genes show an overlapping, but, nonetheless, adistinctive pattern of expression (Zacharias and Kapper, 1999). Furthermore, PMCAand SERCA genes are co-ordinately regulated. The differentiative effects of nervegrowth factor (NGF) on PC12 cells are an example of this. NGF not only up-regulatesPMCA but also down-regulates SERCA expression (Keller and Grover, 2000).


6


Membrane depolarisation can also differentially regulate PMCA and SERCA expres-sion (Guerini

et al.

1999). Kuo

et al.

(1997) had previously reported that theirexpression might indeed be interdependent.

The N-terminal region of PMCA molecule is highly variable and is essential forenzymatic activity. There seems to be an absolute requirement of a specific aminoacid sequence for its function (Talgham and Adamo, 1999).

The transcription of the PMCA isoforms is controlled by calcium itself. Calcium-binding proteins are intricately connected with this process. In developing neuronescalcineurin seems to regulate PMCA expression (Carafoli

et al.

1999). Besides,PMCA isoforms show a marked differential sensitivity to calcium, calmodulin(CaM), ATP, and kinases. The calcium pump activity appears to be regulated byphosphorylation, and phosphorylation might be the means by which different

TABLE 1Mechanisms of Homeostasis of Cell Calcium

Mode of Homeostasis Characteristics

Calcium Pumps Na

+

/Ca

2+

exchanger Plasma membrane–associated low affinity mechanism; pumps out large quantities of Ca

2+

PMCA Ca

2+

-ATPase pump Plasma membrane calcium-ATPase pump involved in calcium extrusion

SERCA Ca

2+

-ATPase pump High affinity ER-located mechanism; leads to luminal accumulation of Ca

2+

and reduces cytosolic levels Ligand-gated calcium channels Initiation of calcium influx upon binding of ligands to

respective receptors; direct influx of Ca

2+

or mediated by IP3Voltage-gated calcium channels (VGCCs)

Ca

2+

influx/release from intracellular stores

Subtypes: T Transient currents in response to membrane depolarisation L Prolonged currents in response to depolarisation N Inactivate in response to moderate, not severe depolarisation;

not sensitive to intracellular cations P/Q Nerve terminals; initiation of neurotransmitter release RCalcium binding proteins Regulation of VGCC, PMCA and SERCA (e.g., by CaM);

NMDA receptor channel by CaM and references cited in the text

Note

: CaM, calmodulin; ER, endoplasmic reticulum; IP3, inositol 1,4,5-triphosphate; NMDA,

N

-methyl-

D

-aspartate; PMCA, plasma membrane Ca

2+

-ATPase; SERCA, sarcoplasmic–endoplas-mic reticulum Ca

2+

-ATPase.

Source

: Collated from Tsien

et al.

(1988), Tsunoda (1993), Campbell

et al.

(1988), Marin

et al.

(1998); Pascale and Etcheberrigaray (1999); Tsien

et al.

(1991); Dunlap

et al.

(1995); Ehlers

etal.

(1996), and references cited in the text.



7

isoforms become functional in a temporal dimension (Monteith

et al

. 1998). PMCAis activated by Ca

2+

/CaM, and the isoforms also show marked differences in theirsensitivity to Ca

2+

/CaM (Elwess

et al.

1997, 1998). Human PMCA-4a is phospho-rylated at a serine residue that occurs within the CAM-binding domain (Verma

etal.

1999; Carafoli, 1997; Carafoli

et al.

1996). Phosphorylation appears to preventCaM binding and lead to the inactivation of the pump (Enyedi

et al.

1997; Pennistonand Enyedi, 1998).

T

HE

S

ARCOPLASMIC

–E

NDOPLASMIC

R

ETICULUM

C

A

2+

-ATP

ASE

P

UMP

SERCA is one of the modes that controls the homeostasis of intracellular calcium.SERCA2 transports Ca

2+

into the lumen of the reticulum by an ATP-dependentmechanism. By virtue of this ability, SERCA2 plays an important part in normalphysiological function such as the contraction and relaxation cycle of cardiac muscle,as well as in the pathogenesis of certain diseases. In addition, the correct localisationof the SERCA protein in the ER is of the utmost importance. As stated above, thelocalisation signal occurs in a stretch of 28 amino acids at the N-terminus of themolecule (Guerini

et al.

1998). Ankyrin, a cytoskeletal protein, also seems to beinvolved in the targeting of SERCA. This is indicated by its abnormal localisationin ankyrin –/– mice. In these mutant mice, the absence of ankyrin also affects thelocalisation of ryanodine and inositol, 1, 4, 5-trisphophate (IP3) receptors (IP3R),both involved in calcium mobilisation (Tuvia

et al.

1999). The ATP2A2 gene encodesthe sarcoplasmic–endoplasm reticulum ATPase isoform type 2. Periasamy

et al.

(1999) have demonstrated the loss of calcium sequestering activity and consequentimpairment of cardiac function in heterozygous ATP2A2 gene mutants.

Mutations of the ATP2A2 gene have been associated with the pathogenesis ofDarier’s disease (DD) (keratosis follicularis) (Sakuntabhai

et al.

1999a, 1999b). DDis an autosomal dominantly inherited skin disorder. It is characterised by the presenceof keratotic papules, and in histology is distinguished by acantholytic dyskeratosis.The keratotic papules occur mainly in the upper trunk, scalp, and palmar pits. Naildystrophy is also a prominent feature of DD (Soroush and Gurevitch, 1997).

Because ATP2A2 controls calcium homeostasis in the cell, it is conceivable thatits deregulation by mutation of the ATP2A2 gene might represent an early event incarcinogenesis. It was demonstrated many years ago that thapsigargin, an inhibitorof SERCA2, not only alters intracellular calcium levels, but also functions as atumour promoter (Hakii, 1986; Thastrup

et al.

1990). With the demonstration thatATP2A2 mutations are associated with the genesis of DD, there have also beenseveral attempts to investigate the incidence of neoplasia in association with DD.Soroush and Gurevitch (1997) have pointed out that basal cell carcinomas and otherskin neoplasms occur in patients with DD. Downs

et al.

(1997) described theoccurrence of a subungual squamous cell carcinoma in a DD patient. However, inthis case the human papilloma virus might have been the aetiological agent. Ingeneral, neoplasia may be associated only infrequently with DD.

There is an obvious common feature between DD and neoplasia that deservesa mention. This concerns the abnormal expression of the calcium-binding transmem-brane glycoprotein cadherin. Some isoforms of cadherin have been regarded as


8


invasion suppressors, because there is a significant body of evidence that associatesthe acquisition of invasive ability by tumour cells with the loss of cadherins. E-typecadherin is found in the cell membrane of epidermal cells. E-cadherin is distributedin the plasma membrane of keratinocytes, in the intercellular space between desmo-somes. Interestingly, this cadherin is expressed at greatly reduced levels in theacatholytic cells of DD and Hailey–Hailey disease (benign pemphigus) (Furukawa

et al

. 1997). Desmosomes provide the adhesion machinery in most epithelia, andabnormalities associated with them may be a key feature of inherited diseases suchas DD and Hailey–Hailey disease. Indeed, the loss of cadherin may be the deter-mining factor that causes the characteristic loss of adhesion between epidermal cellsencountered in DD. Furukawa

et al.

(1997) also point out that both E- and P-cadherins are absent in squamous carcinomas, malignant melanomas and in Paget’sdisease. But basal cell carcinomas, which are noninvasive, do not show a loss ofcadherin.

The above discussion underscores the importance of the SERCA2 calcium pumpand intracellular calcium homeostasis in the calcium-signalling events associatedwith cell differentiation and dedifferentiation, and in the pathogenesis of DD andneoplasia.

V

OLTAGE

–G

ATED

C

ALCIUM

C

HANNELS

The Voltage-gated calcium channels (VGCCs) are located in the plasma membrane.The high voltage-activated channel subtypes L,N, P, Q, and R occur as heterodimersof four subunits. The largest of the subunits is the

α

1 subunit, which spans theplasma membrane and the auxiliary subunits,

β

,

γ

, and

α

2

δ

(Isom

et al.

1994). Theproperties of the calcium channel appear to be determined by the differential expres-sion of

α

1, which is the pore-forming subunit. The

β

subunit seems to regulatechannel properties and the targeting of

α

1. The

β

subunit, of which four isoforms(

β

1–4) have been identified, is said to interact with

α

1. This interaction is mediatedby certain highly conserved domains (Pragnall

et al.

1994; De Waard

et al.

1994).The interaction between these subunits appears to regulate channel activity and thediversity of calcium currents (Varadi

et al.

1991; Isom

et al.

1994; Olcese

et al.

1994). The

β

subunits are believed to modulate the kinetics of channel activationand inactivation by means of phosphorylation.

Calcium-binding proteins feature prominently in calcium homeostasis as impor-tant regulators of calcium channel activity. The elevation of intracellular levels ofcalcium can provide a negative feedback and close the channel. On the other hand,the feedback can have a positive element or facilitation, which opens the calciumchannel. Zuhlke

et al.

(1999) have shown that CaM functions as a sensor for bothnegative and positive regulation of L-type channel activity. CaM appears to do thisby binding to the consensus sequence called the IQ motif that occurs in the C-terminal

α

1C

subunit of the channel protein. They substituted the isoleucine residuewith alanine and reduced Ca

2+

-dependent inactivation and enhanced facilitation ofthe channel. Both inactivation and facilitation were abrogated when the isoleucineresidue was converted to glutamate. CaM binds to the IQ motifs of other channelsubtypes N, P/Q, and R as well (Peterson

et al.

1999). Indeed, calcium-mediated



9

modulation of P/Q channel subtypes also seems to involve CaM function. Again thechannel activity is regulated CaM binding to the IQ motif of the

α

1A

channel subunit(A. Lee

et al.

1999).

T

HE

D

EREGULATION

OF

C

ALCIUM

H

OMEOSTASIS

AS

A

P

RIMARY

E

VENT

IN

C

ARCINOGENESIS

The operation of ligand-gated calcium channels and the role that calcium bindingproteins play in calcium homeostasis and as messengers of the calcium signal providethe major focus for this work. There is much justification for the view held by manyinvestigators that the deregulation of calcium homeostasis in the cell might be aprimary event in cell transformation and carcinogenesis. Indeed, several agents thatblock or retard the influx of calcium into cells appear to be able to inhibit the invasiveability of cancer cells, alter their adhesion properties, and inhibit tumour growth andstabilise the disease.

Carboxyamido-triazole (CAI) is one such compound. CAI has been reported tobe able to inhibit the invasive behaviour of breast carcinoma cell lines

in vitro.

Thematrix metalloproteinases associated with these cells also appear to be inhibited inparallel (Lambert

et al.

1997). Not surprising, therefore, is the finding that CAIinhibits the adhesion of glioma cells to collagen type IV coated substrata and alsoinhibits their invasion

in vitro

.

CAI also appears to be capable of inhibiting cellproliferation (Jacobs

et al.

1997). The antiproliferation and anti-invasion propertiesof CAI have also been described by Wasilenko

et al.

(1996) using human prostatecancer cell lines. These are clear indications of the variety of downstream pathwaysactivated by calcium influx that are inhibited by CAI. Finally, CAI has been reportedto stabilise the disease in patients with a variety of refractory solid tumours (Kohn

et al.

1996).Verapamil, a selective L-type calcium channel blocker, also has demonstrable

anti-proliferative and anti-invasive properties. These were recognised some yearsago. Thus verapamil inhibits tumour growth

in vitro

(Brocchieri

et al.

1996) andseems to arrest cells in the G

0

G

1

phase of the cell cycle (Zeitler

et al.

1997). Theantiproliferative activity has been confirmed by Hoffman

et al.

(1998) on retinalpigment cells. Verapamil is able to inhibit the migration of CD4+ and CD8+ Tlymphocytes across the endothelium (Blaheta

et al.

2000). Other calcium antagonistssuch as mibefradil can also inhibit the adhesion and diapedesis of lymphocytes acrossthe endothelium (Blaheta et al. 1998). Earlier Hailer et al. (1994) had noticed thatthe expression of endothelial adhesion molecules was unaltered by verapamil. Itmay be too simplistic to interpret these findings merely in terms of blocking ofcalcium channels. Nonetheless, some of these publications also indicate that the F-actin levels of the lymphocytes are reduced by the treatment. This could be due tochanges in cytoskeletal dynamics that occur after verapamil-mediated modulationof intracellular calcium levels. This in turn could alter cellular motility. Other factorswill have serious effects on the permeability of the endothelium. For instance, CD4+lymphocytes form two subsets known as Th1 and Th2 cells. Upon activation bothsubsets produce a wide range of cytokines. Th1 cells secrete interleukin (IL)-2 and(IFN)-γ, and Th2 cells secrete IL-4, 5, 10, and 13. Some of these lymphokines are


10 Calcium Signalling in Cancer

bound to affect endothelial permeability. An additional factor that must be reckonedwith is the generation of nitric oxide, which is known to inhibit the adhesion andspreading of endothelial cells. Nitric oxide also affects the formation of stress fibresin the cells, which is bound to influence cellular motility. Nitric oxide also activelyinfluences angiogenesis. IFN-γ is a powerful inducer of nitric oxide synthase (NOS),whereas IL-4 from Th2 cells inhibits NOS. It would be well to remember here thatendothelial NOS is a Ca2+/CaM-inducible enzyme. It is activated when it is bindsthe Ca2+/CaM complex.

More recently, the effects of verapamil have been tested on the local invasionand metastasis of a murine carcinoma cell line in BALB/C mice. Farias et al. (1998)found that it inhibited local invasion and also produced a 50% reduction in bothspontaneous and experimental metastasis. In common with CAI, the cytostatic andanti-invasive effects may be due to the inhibition of membrane-associated proteinasessuch as matrix metalloproteinases and urokinase-type plasminogen activator (Fariaset al. 1998). These events are virtually terminal events in the process of inhibitionof adhesion and invasiveness by verapamil. More upstream in the pathway, verapamilhas been shown to down-regulate the expression of S100A4 (Parker and Sherbet,1992), which, as discussed elsewhere in this book, is closely allied with p53 andstathmin genes in the control of cell cycle progression. Furthermore, S100A4 expres-sion is closely linked with the remodelling of the extracellular matrix.

PHOSPHOLIPID SIGNALLING

A host of regulatory molecules alter cell behaviour and physiological events. Thusextracellular signals imparted to the cells regulate their growth, proliferation, andapoptosis. They also regulate intercellular adhesion and cell adhesion to substrata,and consequently cell migration and invasive behaviour. A family of second mes-sengers, the inositol phospholipids, transduces the regulatory signals across themembrane. Phospholipids (phosphatides) are a major structural component of thecell membrane and also subserve important functions of transducing the extracellularsignals. The second messengers phosphatidylinositol 4,5-bisphosphate (PIP2), phos-phatidylinositol 3,4,5-trisphosphate (PIP3), and IP3 are generated from these phos-pholipids by the action of phospholipases A and C (PLC), which are activated bythe extracellular signals, and kinases. An intermediary in this process is the phos-phatidylinositol (PI) transfer protein, which presents the PI metabolites to lipidkinases. The phosphorylation of the inositol ring at the 3-position gives rise tophosphoinositides PIP (phosphatidylinositol), PIP2, and PIP3. From here the trans-duction of the signal occurs by the interaction of these second messengers withtarget proteins that possess specific phosphoinositide-binding domains, such as anFYVE domain or pleckstrin homology (PH) domain. PIP2 and PIP3 show specificbinding to PH domains (see Leevers et al. 1999).

The second pathway is the generation of IP3 from PIP2 by PLC. As discussedbelow, IP3 then mediates the mobilisation of calcium from intracellular stores. Thedepletion of the intracellular stores triggers the so-called capacitative entry of cal-cium into the cell. Naturally, a deregulation of these pathways will have seriousconsequences in terms of differentiation, intercellular relationships, adhesion, and


The Calcium Signalling Pathway 11

cellular morphology. Such profound changes might indeed lead to the developmentof cancers, their invasion, and the formation of metastases.

PTEN PHOSPHATASE IN THE REGULATION OF LIPID SIGNALLING

Phosphoinositide-3 kinase (PI3K) is responsible for the phosphorylation and gener-ation of PIP2 and PIP3. This phosphorylation is antagonised by a phosphatase thatis encoded by the gene called PTEN/MMAC1/TEP1 located on chromosome 10q23.The phosphatase, referred to here as PTEN, shows sequence homology to tensin(Steck et al. 1997). The phosphatase activity is essential for the normal functioningof PTEN (Tamura et al. 1999b). PTEN regulates the levels of the phosphoinositolphosphates by dephosphorylation of the phosphate group at the 3-position of theinositol ring (Maehama and Dixon, 1998). This seems to inhibit cell proliferationand migration (Furnari et al. 1997; J. Li et al. 1998; Ramaswamy et al. 1999; Tamuraet al. 1999c; Podsypanina et al. 1999). Ramaswamy et al. (1999) transfected cellsthat lack PTEN with wild-type PTEN cDNA and found that cells were arrested inthe G1 phase of the cell cycle. Similar transfection studies have been carried out onthe glioma cell line U373, which lacks the PTEN gene. In these cells, wild-typePTEN inhibits growth and reduces saturation density of U373 cultures. However,PTEN mutants lacking phosphatase activity have the opposite effect and they indeedenhance cell growth in soft agar (Morimoto et al. 1999), which merely confirms thepotential suppressor function of PTEN.

PTEN also seems to be able to regulate the apoptotic pathway. PIP3 is knownto activate the akt serine/threonine kinase, which has anti-apoptotic properties. Ithas been shown to inhibit akt kinase activity (Maehama and Dixon, 1999;Ramaswamy et al. 1999), which could push cells into the apoptotic pathway. Forinstance, extraneous PTEN induces apoptosis of breast cancer cells, which correlateswith the down-regulation of akt. Furthermore, akt can rescue cells from PTEN-induced apoptosis (J. Li et al. 1998). PTEN also increases the expression of p27kip1

cyclin-dependent kinase (cdk) inhibitor (Y.L. Lu et al. 1999). The cdk inhibitors,kip1 and kip2, inhibit the phosphorylation of rb protein and this prevents the entryof the cells into S-phase. This is consistent with the recent finding of Paramio et al.(1999) that PTEN cannot produce growth arrest in rb-deficient cells, but it inducesgrowth arrest when functional rb is reintroduced. This could provide an alternativemechanism by which PTEN induces cell arrest in the G1 phase. The overall effectsof growth arrest could be a combination of G1 arrest and apoptosis (Weng et al.1999), but Paramio et al. (1999) believe that apoptosis might not contribute to this.

It appears that PTEN might regulate cell adhesion and alterations in cell shapeand motility by influencing the downstream of the integrin-mediated signallingpathway (Tamura et al. 1999c). Tamura et al. (1999a) have reported that PTEN alsomay interact with focal adhesion kinase and regulate its phosphorylation and focaladhesions. A further suggestion is that PTEN function might take the nitrogen-activated protein kinase (MAPK) pathway in the regulation of motility (Gu et al.1999). It ought to be recognised, nonetheless, that these pathways may not be



mutually exclusive in facilitating the flow of information, and one or more pathwaysmay be involved in the process. Consistent with this, Morimoto et al. (2000) haveshown that PTEN not only affects PLC-mediated signalling, but also inhibits thekinases that regulate integrin function as well as the anti-apoptosis kinase akt.

PTEN is required at certain stages of embryonic development. Its loss appearsto be lethal in early embryogenesis and to lead to abnormalities of the skin, thegastrointestinal tract, and the pancreas. PTEN –/– cells have shown marked changesin their ability to differentiate into cell types characteristic of the three germ layers(i.e., the ectoderm, the endoderm, and the mesoderm) (Di Cristofano et al. 1998).Presumably, inactivation of the gene affects the state of “competence” of the cellsto respond to differentiation-inducing stimuli (see Sherbet, 1982). Thus the abnor-malities in the various organs seem to have an early developmental history. ThePTEN gene is mutated, or there is a consistent loss of heterozygosity (LOH) at thisgene locus, in many forms of human cancer. The mutations tend to occur mainly inthe phosphatase domain (Steck et al. 1997; J. Li et al. 1997; W.G. Liu et al. 1997;Guldberg et al. 1997; Cairns et al. 1997; Tashiro et al. 1997; Tang et al. 1997; Konget al. 1997; Okami et al. 1998; Ali et al. 1999). The seemingly consistent associationof inactivating mutations and LOH of PTEN with cancers suggests the possibilitythat the lipid-signalling pathway could be seriously deregulated in the pathogenesisof neoplasia, and the PTEN gene has been suggested as a putative tumour suppressorgene. This is suggested unambiguously by the ability of PTEN to inhibit ras-mediated cell transformation, with a concomitant dephosphorylation of PIP-1,3,4,5-tetrakisphosphate (Tolkacheva and Chan, 2000).

Germline mutations of PTEN also occur with the pathogenesis of Cowden’ssyndrome (CS) and the Bannayan-Zonana syndrome (Liaw et al. 1997; Nelen et al.1997; Tsou et al. 1998; Marsh et al. 1998a, 1988b). CS is an autosomal dominantinherited syndrome. Several benign and malignant neoplastic conditions are associ-ated with this syndrome. Women with CS are believed to be more prone to developbreast cancer, and most of them have been found to develop benign fibrocysticdisease. The CS mutations tend to be in exon 5, which contains the phosphatasecore motif (Marsh et al. 1998b; Nelen et al. 1997). Although PTEN germlinemutations are frequent in the Bannayan–Zonana syndrome, none of them have beenfound in exon 5 (Marsh et al. 1998a). An overview of the pattern of mutationssuggests that those occurring in tumours could differ in nature from germline muta-tions (see Ali et al. 1999). Perhaps it is premature to speculate whether the generationof these mutations in different tumours is related to their pathogenesis. On the otherhand, Guldberg et al. (1997) have found the same missense mutation in both primaryand metastatic melanomas. Although further confirmation of this is essential, thishas a considerable implication for metastatic dissemination. Nonetheless, whileconsidering the potential tumour suppressor function of PTEN, it should be bornein mind that in PTEN abnormalities do not occur as a generalised event of tumourdevelopment. Yeh et al. (2000) examined human hepatocellular carcinomas foraberrant expression of PTEN, but encountered none. Neither did they find anyabnormalities associated with the gene itself. Furthermore, the PTEN gene does notseem to be involved in the pathogenesis of acute myeloid leukaemia (T.C. Liu et al.2000). Therefore, it might be prudent to reserve judgment regarding this issue. On



the other hand, PTEN overexpression might be a safeguard against progression. Inlaryngeal papillomas, for example, PTEN is overexpressed and the anti-apoptoticakt kinase is inhibited (P. Zhang and Steinberg, 2000). This should have an overallcontrolling effect on cell population size, and as a consequence upon progression,because more often than not, tumour progression is related to the rate of tumourexpansion.

THE PROTEIN KINASE C PATHWAY

Growth factors and hormones are known to increase the intracellular Ca2+

(Ca2+[i]), either by releasing Ca2+ from intracellular stores or by promoting Ca2+

influx. Ca2+[i] controls several biological functions, mainly by mediating the phos-phorylation of cellular effector proteins. Calcium-binding proteins transduce thecalcium signals to specific subcellular compartments. The generation of secondmessengers such as IP3, 1,2-diacylglycerol (DAG), and cyclic 3′,4′-adenosine mono-phosphate (cAMP) is a major mechanism of activation of the Ca2+ signalling path-way. IP3 and DAG are formed through the agency of an inositide specific phospho-lipase, which is stimulated by biological response modifiers. This PLC hydrolysesPIP2 into DAG and IP3. IP3 activates the release of Ca2+ from intracellular stores.The activation of appropriate protein kinases is a downstream event, which occursin the transduction pathway. The major kinases involved are protein kinase C (PKC),the calcium/calmodulin-dependent protein kinases (CAMPKs) and protein kinase A(PKA). The CAMPKs can in turn activate the cAMP-mediated pathway by activatingadenylyl cyclase, resulting in an increase in cAMP levels (Figure 2).

FIGURE 2 The activation of signal transduction pathways upon binding of a ligand (L)to its appropriate receptor (R). AC, adenylyl cyclase; cAMP, cyclic 3′,4′-adenosine mono-phosphate; CAMPK, calcium/calmodulin-dependent protein kinase; DAG, 1,2-diacyglycerol;G, G-protein; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidyl 4,5-bis phosphate; PKA,protein kinase A; PKC, protein kinase C; PLC, phospholipase C.

R

LCa

GPL AC

cAMP

PK

Ca [i]

CAMP

DAG

PK

PIP IP

R

L

R

L

G

2+

C

C

2 3

K A

2+



Calcium ions and DAG successively activate PKC and induce its translocationto the plasma membrane or the nucleus. Both intracellular calcium and DAG bindingto PKC maintain it in a sustained activated state (Oancea and Meyer, 1998). DAGitself is regulated by phosphorylation by DAG kinase (DAGK). DAG phosphoryla-tion seems to reduce PKC activity (Sakane and Kanoh, 1997) (summarised in Figure3). It may be noted that as many as eight isoforms of DAGK have been identified.These have the characteristic zinc finger structures and PH domains. PH domainsare found in many proteins and consist of approximately 120 amino acid residues.They are electrostatically polarised and show binding to G-protein subunits, PKCs,and phospholipids, thus linking PH domains as a characteristic feature of signaltransduction proteins. The presence of these domains in DAGK isoforms suggeststhat these kinases are a component of the signal transduction network. Besides,DAGK-α, -β, and -γ also possess two consecutive EF-hand calcium-binding domains(Sakane et al. 1990; Yamada et al. 1997; Sakane and Kanoh, 1997). Therefore,DAGK isoforms show calcium-dependent activity. This effect is variable, which hasbeen attributed to differences in calcium affinities of and the conformational changesoccurring in the EF-hand domains as a response to calcium binding (Yamada et al.1997). Upon activation, DAG kinases also translocate to the nucleus and remaintightly associated with nuclear matrix (Wada et al. 1996). Some DAGK isoforms,such as DAGK -δ, -ε, -ζ, have no EF-hand domains (Sakane et al. 1996; W. Tanget al. 1996; Bunting et al. 1996). So calcium-mediated activation is not a universalphenomenon in the activation of these kinases.

PROTEIN KINASE C AND ITS ISOFORMS IN SIGNAL TRANSDUCTION

DAG, upon being activated by the calcium binding protein DAGK, now activatesPKC by enhancing its affinity for Ca2+ and altering its enzymatic activity. This leadsto the phosphorylation of substrate proteins, thus effectively transducing the calciumsignal into a physiological response. Some CBPs may inhibit PKC-mediated phos-phorylation of protein substrates. In other words, CBPs can actively regulate PKCfunction (see Sherbet, 1987; Sherbet and Lakshmi, 1997a, 1997b, for review). Theymay also be directly associated with the coupling of calcium signalling with generegulation. This role is discussed in detail elsewhere in this book. Several isoformsof PKC have been identified and it is believed that they may subserve specificfunctions in signal transduction. It is well known that PKCs are translocated fromcytoplasmic to nuclear location when cells are stimulated by growth factors. In Swiss3T3 cells, although a number of isoforms are detectable in the cytoplasm, onlyPKCα is found in nuclei isolated from cells that have been exposed to growth factors(Neri et al. 1994). In HL-60 cells that were induced by all-trans-retinoic acid todifferentiate, the translocation of PKCα and ζ to the nucleus has been observed(Zauli et al. 1996). This differential translocation has been attributed to possiblelinkage with the nuclear inositol lipid cycle (Neri et al. 1994). Further support forthis view comes from the observation by Mizukami et al. (1997) concerning the



translocation of PKCζ. They reported that translocation of this PKC isoform to thenucleus could be inhibited by inhibiting phosphatidylinositol-3 kinase.

With the plethora of PKC isoforms discovered over the years, their occurrenceis being rationalised by the postulate that these isoforms might be specificallyassociated with identifiable signal transduction pathways and signal transduction indifferent physiological functions in a tissue-specific manner. In PC12 cells, theepidermal growth factor (EGF) signal either is mitogenic or induces differentiation.The flow of information and the phenotypic effect appear to depend on the PKCisoform. EGF induces neurite formation and arrests cell proliferation if the cellsoverexpress PKC-ε, but not PKC-α or δ. Neurite differentiation is accompanied byphosphorylation of mitogen-activated protein kinase, and inhibition of this phospho-rylation event also inhibits neurite differentiation as well as arrest of proliferation.In sharp contrast, overexpression of PKC-α and -δ is strongly associated with themitogenic function of EGF (Brodie et al. 1999).

There are also examples of tissue-specific functioning of PKC isoforms. Forinstance, calcium-independent vascular smooth muscle contraction seems to involvePKC-ε rather than PKC-ζ, both calcium-independent isoforms of PKC (Walsh et al.1996). Such specificity of function might reflect the substrate specificity of thekinase. Walsh et al. (1996) suggested the possibility that calponin is the muscle-specific protein that might be a substrate of this PKC isoform. DAGKs that activate

FIGURE 3 The flow of information of ligand binding to its membrane receptor viaintracellular calcium increases and DAG-to-PKC activation and its translocation to specificintracellular sites are illustrated. DAG is in turn regulated by DAG kinases, which are EF-hand proteins.

Ligand binding

Receptor

Calcium spikes

DAG activation

PKC activation

DAG kinase



PKCs show considerable tissue-specific distribution themselves. It may be that thetissue-specific function of PKCs might flow from the distribution of the DAGKs.

INOSITOL PHOSPHATES IN CALCIUM SIGNAL TRANSDUCTION

The transduction of signals imparted to the cell by the binding of extracellular ligandsto their respective membrane receptors generates repetitive calcium spikes or oscil-lations, and these transient increases of intracellular calcium are a result of theproduction of IP3 by the mediation of PLC (see Figure 3). The repetitive nature ofthese oscillations is believed to be a consequence of a positive feedback loop inwhich calcium induces its own release, and a negative feedback effect which stopscalcium release. Such calcium oscillations occur in many physiological phenomena.An increase in intracellular calcium is a critical event in the activation of the egg(ovum) in a wide variety of species. Experimental enhancement of intracellularcalcium of mouse eggs using calcium ionophores or ethanol results in activation.Conversely, when intracellular calcium levels are reduced egg activation is prevented(Cuthbertson, 1983; Ducibella et al. 1988; Kline and Kline, 1992). That IP3 mediatesegg activation is indicated by the demonstration that microinjection of IP3 producescalcium release from intracellular stores and initiates some of the events associatedwith activation (Kurasawa et al. 1989; Ducibella et al. 1993). IP3 levels are increasedby fertilisation of the egg (Kline, 1996). An increase is also known to take place inthe number of IP3 receptors (Mehlmann et al.1996). IP3 receptors occur in theanterior acrosomal region of mammalian sperm, and by implication IP3 is involvedin the acrosome reaction of mammalian sperm (Walensky and Snyder, 1996).

IP3 regulates intracellular Ca2+ levels by mobilising calcium from intracellularER-associated stores and probably also by stimulating Ca2+ influx into cells. Onlya proportion (up to 50%) of the ER Ca2+ pool is IP3 sensitive, and the remaindercan be released by calcium ionophores. The IP3-sensitive calcium pool is clearlyassociated with IP3R function. IP3R is an intracellular calcium release channel onthe ER. IP3 stimulates IP3R, which results in calcium mobilisation from intracellularstores. Jayaraman et al. (1995) stably transfected antisense IP3R cDNA into JurkatT cells. The transfected cells, which then lacked functional IP3R, failed to elevateCa2+[i] in response to antigen-specific activation. However, the depletion of intra-cellular calcium stimulated calcium influx, which suggests that IP3R are requiredfor IP3-mediated mobilisation of calcium from intracellular stores, but not forcalcium influx.

The function of IP3R seems to be regulated by phosphorylation. Both cyclicguanosine monophosphate (cGMP) and cAMP are known to inhibit the mobilisationof intracellular calcium, and this has been found to be a consequence of the phos-phorylation of IP3R by cGMP-dependent kinase (Komalavilas and Lincoln, 1994,1996) (Figure 4). Komalavilas and Lincoln identified serine 1755 of IP3R as thephosphorylation site. Nonreceptor tyrosine kinases have also been shown to generateIP3, activate IP3R, and raise intracellular calcium levels. Jayaraman et al. (1996)demonstrated the association between nonreceptor tyrosine kinase Fyn and IP3R



following T-cell receptor stimulation. Furthermore, phosphorylation of tyrosine res-idues was found to be reduced in thymocytes derived from fyn (–/–) mice. Thesestudies showed that phosphorylation of tyrosine might also regulate IP3R function.It would appear that the phosphorylation of serine and tyrosine might have antago-nistic regulatory effects. This situation is somewhat analogous to the activation orinactivation of p34cdc2 kinase, which is involved with the phosphorylation of theretinoblastoma gene product in the regulation of cell cycle progression (see Sherbetand Lakshmi, 1997b). Although the molecular mechanisms by which IP3R raisesCa2+[i] are not fully understood to date, it is obvious that the process of phospho-rylation of IP3R is an important event in IP3-mediated calcium signalling.

The occurrence of IP3R was recognised many years ago (Supattapone et al.1988; Snyder and Supattapone, 1989). There are three types of IP3R. Three IP3Rgenes have been cloned. One of them, the IP3R1, is expressed predominantly inPurkinje neurones of the cerebellum. IP3R1 and IP3R2 genes have been found tobe expressed at low levels in several tissues studied to date. The distribution of themRNA of the three receptor genes appears to display a definable pattern, leading tothe suggestion that the different receptor types may have distinct functions (Fujinoet al. 1995; T.X. Yang et al. 1995). Indeed, multiple isoforms of IP3R genes maybe expressed in cells. Alternatively spliced isoforms could be fulfilling differentcellular requirements, that is, they may be functionally different (Iida and Bourgui-gnon, 1995, 1996; Katusic and Stelter, 1995). IP3R1 may be associated with intra-cellular calcium mobilisation, because IP3R1-deficient T-lymphocytes show a low-ered IP3-mediated calcium release by T-cell receptor stimulation (Jayaraman and

FIGURE 4 Possible mechanisms involved in IP3-mediated mobilisation of intracellularcalcium. cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate;cGMPK, cGMP kinase; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IP3R-P, phos-phorylated forms of IP3R.



Marks, 1997). The same study also implicated IP3R1 in apoptosis, by the observationthat IP3R1-deficient T lymphocytes are resistant to dexamethasone-induced apop-tosis. IP3R3 genes have been credited with the capacitative entry of calcium influx,when intracellular Ca2+ is depleted. In Xenopus oocytes IP3R3 was associated withthe plasma membrane, which facilitated the influx of calcium (Putney, 1997).

The transduction of signals imparted by a variety of biological response modi-fiers occurs via cytoskeletal structures. Compatible with this generalisation, thesignal transduction pathway of IP3 has been linked with cytoskeletal dynamics.Ribeiro et al. (1997) found that the disruption of cytoskeletal structures of NIH3T3cells by treatment with cytochalasin D resulted in the abolition of intracellularcalcium mobilisation normally induced by platelet-derived growth factor (PDGF).The depolymerisation of the tubulin network similarly abolished calcium mobilisa-tion. However, PDGF continued to stimulate the generation of IP3 even in thepresence of cytochalasin. This suggests that the cytoskeleton is closely involved inthe IP3-mediated transduction of the PDGF proliferative signal. It is of interest tonote that Bourguignon and Jin (1995) had previously identified a C-terminal aminoacid sequence of GGVGDVLRKPS in IP3R that possesses sequence homology tothe ankyrin-binding domain of the intercellular adhesion molecule CD44. Theyfurther had demonstrated that an 11 amino acid synthetic peptide with that sequenceshowed specific binding to ankyrin, which is a cytoskeletal protein.

DEREGULATION OF INOSITOL 1,4,5-TRISPHOSPHATE PATHWAY AND ITS CONSEQUENCES

It is hardly surprising that interference with signal transduction pathways and theirderegulation should have serious consequences for biological responses in develop-mental systems as well as in pathogenesis. Ecdysone, a steroid hormone, regulatesa variety of developmental processes and metamorphosis in insects. It also influencesneuronal responses, apoptosis, and histiolysis in a variety of larval tissues, binds tonuclear receptors, which show tissue- and stage-specific expression (Kamimura etal. 1997); and regulates ecdysone-responsive genes (No et al. 1996; Truman, 1996).The metamorphic changes involve “early” and “late” genes, with the early genesencoding proteins that regulate the expression of late genes (Baehrecke, 1996).Although our knowledge of the mechanics of ecdysone signal transduction is stillrudimentary, it is clear that the pathway involves calcium signalling. The prothorac-icotropic hormone (PTTH) stimulates the synthesis of ecdysone in the prothoracicgland of Galleria larvae (Birkenbeil, 1996). PTTH increased intracellular Ca2+ levelsof prothoracic gland in vitro. This increase could be abolished by removing extra-cellular calcium and by calcium channel inhibitors. The PTTH-stimulated increaseof intracellular calcium was not abolished by agents, which inhibit mobilisation ofcalcium from intracellular stores. Recently, Venkatesh and Hasan (1997) found thatdisruption of IP3 receptor gene function in Drosophila larvae grossly affected meta-morphosis and ecdysone synthesis and secretion. They generated IP3R gene (itpr)mutants. The mutations delayed moulting and were lethal at early larval stages.



Feeding ecdysone to mutant larvae partially counteracted the phenotypic effects ofthe itpr mutation, and down-regulated ecdysone-inducible genes.

General or specific abnormalities of signal transduction pathways (e.g., occur-rence of abnormalities in one or more components of the transduction pathway)may be associated with disease processes, including the invasive behaviour andmetastatic progression of cancers. Such disruptions of the signal transductioncascade in human cancers has been studied by measuring the levels of threeenzymes involved in the generation of IP3, namely, phosphatidylinositol 4-kinase,phosphatidyl 4-phosphate 5 kinase, and PLC. Weber et al. (1996) found that allthree enzymes were up-regulated with tumour progression. In rat hepatomas theenzyme levels rose by two- to eight-fold compared with normal liver. A three- tofour-fold increase in PIP kinase and PI kinase was found in ovarian epithelialcancers. Weber et al. (1996) found a dramatic 16- and 96-fold increase of theseenzymes in human breast carcinoma cells compared with normal breast parenchy-mal cells. These changes clearly correlated with enhanced IP3 expression andproliferative potential. Further evidence has also been reported by Singhal et al.(1997), who investigated the connection between elevated IP3 and DAG withproliferation of the breast cancer cell line MDA-MB-435 and the ovarian cancercell line OVCAR-5. They demonstrated that quercetin treatment of the cellsinhibited PI kinase and reduced IP3 before any inhibition in cell proliferation wasevident. The expression of these kinases seems to be up-regulated by differentia-tion-inducing agents. Thus Ai et al. (1995) found enhanced expression of PI kinaseas well as IP3 in murine erythroleukaemia cells induced toward erythroid differ-entiation by dimethyl sulphoxide (DMSO) treatment.

Another line of circumstantial evidence may be cited. For instance, monoterpe-nes such as limonene and perillyl alcohol have been reported not only to preventtumour initiation and promotion, but also to inhibit tumour progression. Thesemonoterpenes appear to inhibit the isoprenylation of G-proteins (Gould, 1997),which are a component of the signal transduction machinery (Figure 5). Similarly,abnormalities in the related pathway involving DAG and PKC may also be associatedwith cancers, as demonstrated by Hoelting et al. (1997) using the PKC agonist TPA(12-O-tetradecanoyl phorbol 13-acetate). They reported a 15% increase in the inva-sive ability of a follicular thyroid cancer cell line. In contrast, PKC inhibitors suchas staurosporine, chelerythrine and calphostin C reduced invasion by 62%. CAIwhich inhibits calcium influx into cells, has been reported to inhibit the proliferativeand invasive capacity of cell lines derived from human prostate cancer (Wasilenkoet al. 1996). The neuroendocrine cells of prostate cancer express bombesin andgastrin-releasing peptide (GRP). In vitro bombesin and GRP signals invoke cellproliferation via bombesin receptors of which there are three subtypes: GRP recep-tors (GRPr), neuromedin B receptor, and bombesin receptor subtype 3. Transcriptsof GRPr only, but not neuromedin B or bombesin receptor subtype 3, have beenfound in androgen-insensitive prostate cancer cell lines PC-3 and DU-145, suggest-ing the very important role GRPr play in signal transduction in these cells (Aprikianet al. 1996). In all lines derived from advanced prostate cancer, several agents suchas GRP, bombesin, and bradykinin, among others, raised intracellular calcium levels.



However, bombesin did not influence calcium levels of an immortalised prostate cellline (Wasilenko et al. 1997). The authors have, therefore, suggested not only thatthere might be multiple receptors that can mediate increases in intracellular calciumlevels of androgen-independent prostate cancer cell lines, but also that GRPr expres-sion may be associated with the progression of prostate cancer. Calcitonin-likepeptides may also function as neuropeptides in prostate cancer. The peptide bindsto high-affinity receptors in the plasma membrane and induces a rapid and markedincrease in intracellular calcium. Furthermore, the calcitonin signal is transducedinto a clear proliferative response. The pattern of calcium response is biphasic inthe form of a spike, and plateau is regarded as indicative of the phospholipid/calcium-signalling system (Shah et al. 1994).

RYANODINE AND RELATED RECEPTORS IN CALCIUM MOBILISATION

Besides IP3R, other receptor-mediated pathways are available to the cell for calciummobilisation, and the activation and availability of the pathways may be develop-mentally regulated. In embryonic development, the IP3-dependent calcium signallingis switched to an IP3-independent mode of signal transduction. The ryanodinereceptor (RyR) is also capable of regulating intercellular calcium. In the early stagesof development, IP3R are ubiquitously expressed. But in subsequent developmentRyR begin to be expressed. Eventually, although most cell types do continue to

FIGURE 5 The cAMP/Ca2+-signalling pathway. The binding of extracellular ligands tospecific receptors regulates adenylyl cyclase activity via the linking G-proteins. The cAMPgenerated activates protein kinases that phosphorylate substrate proteins. Another route startswith the raising of intracellular calcium levels, which in turn regulate adenylyl cyclase activity.



express IP3R, in excitable cells including muscle cells, RyR becomes the major Ca2+

release channel (Rosemblit et al. 1999). Ryanodine receptors, of which there are several isoforms, are activated by

calcium influx via voltage-gated Ca2+ channels of the plasma membrane. Recently,cyclic ADP-ribose has been identified as an activator of RyR (Petersen and Cancela,1999). The fatty acid metabolic products, the fatty acyl-CoA esters, can also activateRyR in skeletal muscle (Fulceri et al. 1994; El-Hayek et al. 1993; Dumonteil et al.1994). This seems to be mediated by the acyl-CoA binding protein (Fulceri et al.1997).

A reference to the multifunctional receptor called megalin would not be out ofplace here. Megalin is a member of the low-density lipoprotein receptor family,which function as endocytic receptors. The megalin glycoprotein of rat kidney isapproximately 330 kDa in size. The 550-kDa human homologue of megalin is foundin the luminal surface of cells of the renal proximal tubule and epididymis. It isexpressed in mammary epithelia, thyroid follicular cells, and the ciliary body of theeye (Lundgren et al. 1997). It is also found in parathyroid and trophoblast cells.Mackrill et al. (1999) raised antibodies against RyR from rabbit muscle. Theseantibodies recognised not only skeletal RyR but also another high-molecular-weightprotein (k-HMW) in kidney microsomes thought to be a rabbit homologue of mega-lin. Antibodies raised against k-HMW were unable to recognise RyR. Furthermore,this protein showed partial sequence homology to RyR.

Megalin functions as a receptor for thyroglobulin (Marino et al. 1999; G. Zhenget al. 1998), lipoprotein (a) (Niemeier et al. 1999), and insulin (Orlando et al. 1998).It is implicated in the transport of retinol. Megalin-deficient mice show greatlyincreased urinary excretion of retinol and retinol-binding protein (RBP). Further-more, the uptake of retinol and RBP is partially inhibited by antibodies againstmegalin (Christensen et al. 1999). Megalin also serves as a receptor for aminoglyo-coside antibiotics, such as gentamycin, and mediates the endocytosis and accumu-lation of the antibiotic in renal tubule cells (Decorti et al. 1999). There is a prelim-inary report that the nephrotoxicity of gentamycin is related to the loss of megalinreceptors (Vaamonde et al. 1996).

The megalin receptor has also been attributed with a putative role in calciumhomeostasis (Friedman, 1999; Christensen et al. 1998). This seems reasonable inlight of its similarity to RyR. The deregulation of this function might be responsiblefor its apparent involvement in pathogenesis. Megalin might function as an autoim-mune antigen in the pathogenesis of membranous glomerulonephritis (Mackrill etal. 1999). Its dysfunction might occur also in neoplasia. Apparently, the expressionof megalin is down-regulated in hyperplasia of the parathyroid.

CYCLIC AMP IN CALCIUM SIGNALLING

A majority of hormones, neurotransmitters, and growth factors generate cellularresponses by means of a signal transduction machinery that is composed of threeelements. These agents bind to specific receptors, which are linked to the effectorcomponent by means of G-proteins (Iyengar and Birnbaumer, 1990a, 1990b). Gilman(1989) identified these as components of the machinery regulating adenylyl cyclase



activity. A large number of hormones modulate cAMP levels by regulating adenylylcyclase activity. The G protein family encompasses a number of heterotrimerscomposed of subunits encoded by three G-protein-type genes, α, β, and γ genes.Several Gα, Gβ, and Gγ genes have been cloned (Iyengar and Birnbaumer, 1990b).The G-protein linker GS mediates stimulatory effects, whereas GI mediates inhibitoryeffects of hormones. Most G-protein intervention follows the activation of the αsubunit by binding to GTP, and this activation occurs upon the receptor being boundby the appropriate ligand. Numerous biological responses to extracellular stimuliare mediated by cAMP synthesised by adenylyl cyclase activity, and in turn cAMPactivates cAMP-dependent protein kinase, which phosphorylates a variety of cellularsubstrate proteins (Krebs, 1989). This pathway involves an elevation of intracellularcalcium levels either by increased uptake of extracellular calcium or release ofcalcium from intracellular stores. Calcium can modulate adenylyl cyclase activity.There is a considerable body of evidence that CBPs such as calmodulin may regulateadenylyl cyclase activity. It has been shown that Ca2+/CaM can stimulate adenylylcyclase activity (Minocherhomjee et al. 1988), but in some tissues the activity ofthis enzyme is insensitive to Ca2+/CaM. Furthermore, adenylyl cyclase activity maybe related to intracellular distribution, and this may or may not be affected byactivated GS. The cellular response generated by biological response modifiers maydepend on the G-protein and the appropriate effector component being expressedby cells (Iyengar and Birnbaumer, 1990b). The control of cellular response toexternal signalling ligands requires that the levels of the second messengers them-selves be regulated strictly. Enzymes such as phosphodiesterases that degrade thesemolecules regulate cAMP. Besides adenylyl cyclase, phosphodiesterases, proteinkinase II, and protein phosphatase are also CaM-dependent enzymes. Receptorproteins and kinases may amplify second messenger signals. Indeed, this mechanism,involving their amplification on the one hand and degradation on the other, maintainthe steady state levels of second messengers (see Figure 5).

Several biological responses have been identified in which the cAMP/calciumpathway of signal transduction is followed upon the binding of specific extracellularligands. The processes of aggregation and development and chemotactic responseof Dictyostelium discoideum involve the cyclic nucleotides cAMP and cGMP. Thechemotactic response appears to be a function of the elevation of intracellular Ca2+,which has been shown to correlate with the association of myosin with the cytosk-eleton (Menz et al. 1991). This suggests a close involvement of calcium uptake withthe process of cell motility. cAMP has been implicated in sperm motility (Garbersand Kopf, 1980). In fact, high extracellular levels of calcium have been known toincrease cAMP levels in sperm cells (Garbers et al. 1982), and possibly Ca2+ couldbe activating adenylyl cyclase activity.

Functionally it seems possible to differentiate between influxed calcium andCa2+ released from intracellular stores. This is illustrated by the modulation ofS100A4 expression in B16 murine melanoma cells by agents that modulate intra-cellular calcium levels. Melanocyte-stimulating hormone (MSH) increases calciuminflux and cAMP levels. Verapamil is a calcium channel blocker that reduces cAMPlevels (Atlas and Adler, 1981; Janis et al. 1987). Thapsigargin is a sesquiterpenelactone that raises intracellular calcium levels by releasing calcium from intracellular



stores, by inhibiting the ER-associated Ca2+-ATPase (Takemura et al. 1989; Thastrupet al. 1990). MSH has been reported to up-regulate S100A4, whereas verapamildoes the opposite. In contrast, thapsigargin down-regulates its expression despiteeffecting an increase in intracellular calcium levels (Parker and Sherbet, 1992)(Figure 6). This is compatible with the suggestion by Haverstick et al. (1991) thatCa2+ influx and release from intracellular stores have independent roles. It has alsobeen suggested that a low micromolar range of intracellular calcium levels inhibitsadenylyl cyclase, in contrast to calmodulin-stimulated adenylyl cyclase which maybe mediated by calcium influx (Cooper, 1991). The release of calcium from intra-cellular stores may therefore activate specific components of the calcium-signallingpathway (Figure 7). The transcription factor, the cAMP response element bindingprotein (CREB), is phosphorylated by Ca2+/CaM-dependent protein kinase PKII.The phosphatases PP-2A and -2B (calcineurin) dephosphorylate the phosphataseinhibitor (PI-1), which in turn inhibits the phosphatase PP-1. The inactivation of PI-1 by PP-2A or PP-2B may allow the phosphatase PP-1 to dephosphorylate CREB,thus rendering the latter inactive. Calcineurin is reported to have an affinity for CaMtwo to three magnitudes greater than that of PKII. Therefore, low levels of intra-cellular calcium, such as those resulting from its release from intracellular stores,might preferentially activate calcineurin.

FIGURE 6 The differential regulation of S100A4 expression by calcium influx into cellsas compared with the release of Ca2+ from intracellular stores. Calcium influx up-regulatesS100A4. Verapamil, which blocks calcium influx, down-regulates its expression. In sharpcontrast, thapsigargin, which raises intracellular calcium levels by releasing Ca2+ from intra-cellular stores, down-regulates S100A4 expression. This suggests that calcium released fromintracellular stores may be functionally differentiated from influxed calcium and may beactivating different components of the calcium signalling system. (Based on Parker andSherbet, 1992). cAMP cyclic AMP; MSH, melanocyte-stimulating hormone.



Several extracellular agents are known to activate the cAMP- and Ca2+-signallingpathway. There are also instances where the calcium-signalling pathway is indepen-dent of an upstream event of cAMP activation. cAMP and calcium signalling mediatethe biological responses of several hormones. Adrenocorticotropic hormone (ACTH)stimulates uptake of Ca2+ and elevates cAMP levels in lymphocytes. Splenic lym-phocytes that possess ACTH receptors show ACTH-dependent calcium uptake, butthymocytes, which lack these receptors, do not show enhanced calcium uptake(Clarke et al. 1994). In adrenocortical cells maintained in culture, ACTH exerts abiphasic effect, inhibiting cell proliferation in the initial phase followed by a stim-ulatory phase of cell differentiation, and synthesis and secretion of corticosteroneand 18-OH-deoxycorticosterone. cAMP and 8-bromo-cAMP can induce adrenocor-tical cells to differentiate and to secrete steroid hormones, but they do not inhibitcell proliferation. This suggests that although cAMP mediates the mitogenic, differ-entiative, and steroid stimulatory signals of ACTH, it is not involved in the processof inhibition of cell proliferation by ACTH (Arola et al. 1993). Similarly, althoughthe stimulation of bone resorption in foetal limb bud bones by tumour necrosis factor(TNF) appears to involve cAMP, the latter may not be directly associated with TNFsignalling (Shankar and Stern, 1993).

Calcitonin and calcitonin-like substances induce cell proliferation in prostatecells in culture and possibly also in the genesis of carcinoma of the prostate.Calcitonin induces a dose-dependent enhancement of cAMP and intracellular levels

FIGURE 7 Summary of cAMP and Ca2+/CaM-mediated regulation of “immediate early”gene expression. cAMP, cyclic AMP; CREBP, cyclic AMP response element binding protein;PI-1, phosphatase inhibitor l; PKA, protein kinase A; PKII protein kinase II; PP-1, proteinphosphatase 1; PP2B, calcineurin.



of Ca2+ (Shah et al. 1994). The alterations in intracellular calcium levels in Sertolicells induced by follicle-stimulating hormone (FSH) involve the generation of endog-enous cAMP in response to this hormone treatment (Gorczynska et al. 1994).Hypothalamic neurohormones that control a variety of normal biological processes,such as cell proliferation and differentiation as well as hyperplasia and neoplasticprocesses, transduce their signals by the cAMP/Ca2+ pathway (Spada et al. 1997).The expression of cytokines in T lymphocytes is a highly regulated feature, involvingcAMP. Benbernou et al. (1997) treated activated Jurkat cells with dibutyryl cAMPor pentoxifylline and found that IFN-γ mRNA expression was inhibited but that ofIL-10 was unaffected. Cholera toxin and prostaglandin E (PGE)-2 also inhibitedIFN-γ but greatly enhanced IL-10 mRNA expression. Using immunofluorescencetechniques to detect intracellular cytokines, these authors confirmed the inhibitionof IFN-γ by dibutyryl cAMP and the enhancement of expression of IL-10 by PGE-2. This clearly shows that a differential control is exerted by cAMP at both thetranscriptional and the protein levels. Downstream events such as the induction ofclass II major histocompatibility complex (MHC) genes by IFN-γ, on the other hand,can be inhibited by agents that trigger the cAMP signal transduction pathway(Ivashkiv et al. 1994).

The endpoint of the pathway is obviously the activation of responsive genes.There are certain regulatory regions called cAMP response elements (CREs) thatinteract with CREB. Besides CREB, the transcription factor known as ATF1 alsomediates the transcription of responsive genes upon transduction of the extracellularsignal via the cAMP/Ca2+-signalling pathway. Both these transcription factors arephosphorylated by the mediation of cAMP. CREB and ATF may integrate the signalsflowing from the cAMP/Ca2+ pathway. As stated before, cAMP activation can eitherinhibit or enhance biological responses. This has been suggested to be a consequenceof blocking or activation of CREB. P.Q. Sun et al. (1994) showed by transienttransfection studies with the Ca/CaM-dependent protein kinases CAMKIV andCAMKII that the former is a more potent activator of CREB than the latter. Thisthey found was due to the nature of CREB phosphorylation. CAMKIV phosphory-lated CREB at serine residue 133, but CAMKII phosphorylated serine 142 in additionto serine 133. The studies also showed that whilst phosphorylation of serine 133activated CREB, phosphorylation of serine 142 inhibited CREB activation. In ATF1,CAMKII phosphorylates the positive regulatory serine 63, corresponding to serine133 of CREB, but not serine 72, which corresponds to the negative regulatory serine144 of CREB (Shimomura et al. 1996). Thus, although CREB and ATF sharesubstantial sequence homology, it may be that these two transcription factors maydifferentially transactivate the responsive gene (Figure 8).

ARCHITECTURAL ASPECTS OF THE SIGNAL TRANSDUCTION MACHINERY

THE ROLE OF CAVEOLAE IN SIGNAL TRANSDUCTION

The signal transduction machinery contains several elements along which informa-tion flows. A wide variety of signals are imparted to the cell and these need to be



targeted and conveyed to specific subcellular compartments for eliciting cellularresponses. This inevitably means that there should be an optimal organisation of thevarious elements to achieve a coordinated functioning of signal transduction. Certaininvaginations involving specialised microdomains of the plasma membrane calledcaveolae were identified in epithelial and endothelial cells many years ago (Simio-nescu, 1993; Anderson, 1998). Caveolae do occur in many cell types. They havebeen identified with the endocytosis of macromolecules and in signal transductionand calcium homeostasis.

The status of caveolae as a dynamic structural component of signal transductionis underlined by the presence within these membrane microdomains of a variety ofsignal transduction molecules. They contain G-protein-coupled receptors, ras familyproteins, receptor tyrosine kinases (RTKs), and isoforms of PKC (Shaul and Ander-son, 1998). Also identified with them are adenylyl cyclase and CBPs such as CaMand annexin. Caveolin, a cholesterol and lipid-binding protein (Murata et al. 1995;Trigatti et al. 1999), of which a number of isoforms are now known, is an integralmembrane protein of caveolae. The caveolins are synthesised in the ER and aretransported to the plasma membrane. Specific domains of the molecule take part inthe movement of caveolins from the ER to the cell surface (Machleidt et al. 2000).The N-terminal cytoplasmic domains of caveolin-1 interact with G-proteins (S. Liet al. 1995), RTKs (Couet et al. 1997), and endothelial nitric oxide synthase (eNOS)(Okamoto et al. 1998).

FIGURE 8 Phosphorylation of the transcription factors CREB (cyclic AMP responseelement binding protein) and ATF in the negative and positive regulation of genetic activity.The Ca2+/CaM-activated protein kinases CAMKIV and CAMKII phosphorylate specific pos-itive and negative regulatory serine residues of the transcription factors, which thereforedifferentially transactivate the responsive genes.



eNOS is also a Ca2+/CaM-dependent enzyme. The increases in intracellularcalcium levels induced by extracellular signals and the binding of Ca2+/CaM complexwith eNOS seem to lead to its activation. eNOS also appears to be negativelyregulated by caveolin-1. The interaction between eNOS and caveolin seems to reducenitric oxide production (Shah et al. 1999). The vascular endothelial growth factor(VEGF) increases endothelial cell permeability as well as endothelial cell (EC)migration and proliferation. The VEGF signal appears to involve the production ofnitric oxide by eNOS (Yiyu et al. 1999), and this mediated by the Flt-1 receptor(Ahmed et al. 1997). The up-regulation could be attributed also to posttranscriptionalchanges leading to the stability of eNOS mRNA (Bouloumie et al. 1999). In anyevent, the serine/threonine kinase Akt phosphorylates the serine residue 1179 ofeNOS and activates the enzyme (Fulton et al. 1999). The nitric oxide synthaseinhibitor NG-nitro-l-arginine methyl ester (L-NAME) inhibits VEGF-mediated ECmigration and proliferation (Shizukuda et al. 1999). Nitric oxide is able to inhibitfocal adhesion and spreading of endothelial cells and affects the formation of stressfibres (Goligorsky et al. 1999). Human tumour cells have been transfected with theNOS gene. The transfectant cells have shown enhanced invasive ability, growth rate,and vascular density, with parallel constitutive expression of NOS (Thomsen andMiles, 1998). The mechanisms involved in the nitric oxide–mediated increase ininvasive and proliferative capacity have not yet been addressed, but there are sug-gestions that nitric oxide might up-regulate matrix metalloproteinases and blocktheir endogenous inhibitors (Lala and Orucevic, 1998). IFN-γ and lipopolysaccharide(LPS) induce nitric oxide synthesis in C3-L5 murine adenocarcinoma cells as wellas enhance their invasive ability in vitro. This is also accompanied by an up-regulation of matrix metalloproteinase (MMP)-2. Furthermore, endogenous nitricoxide seems to down-regulate TIMP-2 and TIMP-3 (tissue inhibitors of metallopro-teinases). This has led to the suggestion that nitric oxide alters the balance betweenMMPs and their inhibitors (Orucevi et al. 1999).

VEGF is a potent inducer of angiogenesis. This angiogenic signal also involvesnitric oxide production. L-NAME inhibits angiogenesis in vitro (Pipili-Synetos etal. 1995) as well as tumour-induced angiogenesis in vivo (Jadeski and Lala, 1999).Norrby (1998) supports the view that nitric oxide suppresses angiogenesis, but statesthat L-NAME did not suppress VEGF-induced angiogenesis. Nitric oxide synthaseis also inducible. The inducible form (INOS) is found in large quantities in malignantprostate epithelium and in bladder cancer epithelium, but normal epithelial cells areonly weakly positive. eNOS is expressed in the endothelial cells of tumour stroma,but not in stroma of normal bladder tissue (Klotz et al. 1998, 1999). In breast andgastric cancers also INOS occurs predominantly in the stromal component (Thomsenand Miles, 1998). This suggests a possible functional differentiation between INOSand eNOS. A marked up-regulation of NOS isoforms has also been reported innon–small cell lung cancers in relation to their progression (Ambs et al. 1998). Alsoof much interest is the finding by Gallo et al. (1998) that NOS activity in head andneck tumours with lymph node metastasis was higher than in tumours that had shownno metastatic spread. This has received some further support from Thomsen andMiles (1998) who state that NOS activity correlates with tumour grade in breastcancer.



It is possible that VEGF might follow two different pathways to increase endo-thelial permeability, endothelial cell migration, and proliferation, and induce angio-genesis. Whereas nitric oxide production is essential for effecting an increase inendothelial permeability, it appears to negatively regulate the angiogenic signal. Thisis partly borne out by the demonstration that VEGF reduces the activity of PKCisoforms, especially PKC-δ, and this could be blocked by pretreatment with L-NAME (Shizukuda et al. 1999). Overall, it seems that there is a switching mechanismoperating here, which apparently is distinct from the transduction pathway involvingeNOS activation. This is likely to be a very rewarding area of investigation in thecontext of cancer spread and metastasis.

The presence of IP3-like receptors (Patel et al. 1999; Fujimoto et al. 1995) andCa2+-ATPase (Fujimoto, 1993) amply testifies to the participation of caveolae incalcium homeostasis. Caveolae seem to sequester extracellular calcium into thevesicles and release it into intracellular locations, presumably by the mediation ofIP3 and Ca2+-ATPase. Furthermore, with possible lateral mobility of caveolae onecan envisage a regulation of entry and release of calcium to appropriate cellularcompartments.

CAVEOLIN EXPRESSION IN CANCER

On account of the highly significant participation of caveolae in signal transduction,the expression of caveolin has been the focus of some investigation in cancerprogression. Caveolin expression appears to decrease with tumour progression,which is compatible with the view that there is a serious deregulation of signaltransduction in neoplasms. Caveolin-1 and -2 genes, which occur at chromosome7q31, are frequently deleted in tumours (Fra et al. 1999; Hurlstone et al. 1999).Caveolin expression is lower in breast cancer cell lines as compared with normalepithelial cells of the breast. Furthermore, caveolin expression seems to be inhibitoryof growth (S.W. Lee et al. 1998). S.W. Lee et al. (1998) also transfected caveolincDNA into breast cancer cells that normally show no detectable caveolin and showedthat its overexpression in the transfectants resulted in a 50% reduction in growthrate and colony forming ability. Pflug et al. (1999) grew LNCaP prostate cancercells in androgen-depleted culture medium, and from these they derived tumorigenicandrogen-independent cell clones. These clones also showed greatly reduced levelsof caveolin expression. In contrast, benign prostate epithelial cells express caveolinat high levels. On the face of it, caveolin seems to function as a tumour suppressor.However, metastatic tumours also overexpress caveolin (Thompson, 1998). It wouldseem therefore that although its overexpression can lead to androgen resistance, itsassociation with metastatic progression could be a secondary event.

Caveolin-3, which is believed to be a muscle-specific isoform, is often mutatedin patients with limb girdle muscular dystrophy, and the mutations have been sug-gested to be associated with the pathogenesis of the disease (McNally et al. 1998a;Minetti et al. 1998).


29

3

Calcium Binding Proteins and their Natural Classification

Calcium-binding proteins comprise a large family of proteins that can be subdividedinto two subfamilies based on their molecular organisation. A large number of theseproteins have been identified and characterised, and their physiological functionshave been investigated (Tables 2 and 3). The subfamily of CBPs, referred to hereas non-EF-hand CBPs, binds to certain phospholipids in a calcium-dependent fashion(Heizmann and Hunziker, 1990). They do not possess the characteristic feature ofthe other subfamily, popularly described as the EF-hand CBPs. The EF-hand proteinsubfamily in turn can be divided into two groups (Da Silva and Reinach, 1991). Themembers of one group function as Ca

2+

sensor proteins. These are inactive at calciumconcentrations in the low range of 10

–7

to 10

–8

M

. They are activated into playingtheir regulatory role when the Ca

2+

concentration increases to around 10

–5

to 10

–6

M

. The second group comprises EF-hand proteins that are mainly concerned withcalcium buffering and transport. Calcium ions function as second messengers

parexcellence

in many pathways of cellular response. The translation of the calciumsignal into biological function is mediated by CBPs that occur ubiquitously inintracellular locations as well as in the extracellular matrix. In order to be able tomediate the transduction of calcium signalling into cellular responses, CBPs needto recognise and interact with downstream target proteins. The binding of Ca

2+

toCBPs produces conformational changes in these molecules and these changes appearto endow the CBPs with the ability to recognise and interact with their targetmolecules. This is discussed in greater detail in a later section.

Both EF-hand and non-EF-hand CBPs participate in a host of normal physio-logical functions and consequently they are also associated with an array of patho-logical conditions (Tables 2 and 3). Although many EF-hand proteins have beenstudied for their involvement in normal and aberrant physiology and in pathogenesis,only a small number of them have been studied extensively. Consequently, theinformation currently available about some EF-hand proteins has tended to besomewhat fragmentary. Nonetheless, there is an indisputable theme that underliesthe molecular features of CBPs as associated with their function in normal physi-ology and in pathogenesis. Their functions appear to be linked closely with themolecular organisation of the EF-hands, and furthermore, to the characteristic con-formational changes that they undergo and the new molecular configuration thatthey assume upon Ca

2+

binding.


30


TABLE 2Non-EF-Hand Calcium Binding Proteins

Non-EF-Hand CBP Function Ref.

Annexins (individual annexins also known as chromobindin, endonexin, lipocortin, etc.)

Membrane-related functions; adhesion; fusion; exocytosis; possible cell-cycle-related expression; morphogenesis; differentiation

Martin and Creutz (1987, 1990); Hamman

et al.

(1988); Martin

et al.

(1989); Creutz

et al.

(1992, 1994); further references cited in text

Gelsolin family proteins Gelsolin Cytoskeletal dynamics; severing and

capping of actin filaments; cell migration, cancer progression; amyloidosis

Cited in text

Severin Sequence homology with gelsolin in actin-interacting domains

Cited in text

Villin Regarded as a marker of differentiation; occurs in intestinal and brush border epithelia, and in Barrett’s metaplasia and adenocarcinoma

Cited in text

Cap G Mishra

et al.

(1994)Van Ginkel

et al.

(1998) Fragmin Constantin

et al.

(1998)Calreticulin ER-associated CBP; molecular

chaperone; intracellular calcium storage; cell adhesion; proliferation; autoimmune diseases

Khanna

et al.

(1987); Smith

et al.

(1989); Milner

et al.

(1991);Heilmann

et al.

(1993);N. Liu

et al.

(1993)Calsequestrin Muscle homologue of calretinin,

intracellular calcium storageCited in text

Osteocalcin Extracellular matrix (ECM) protein; bone metabolism; cell proliferation; differentiation; cancer invasion; osteotropism of cancer metastasis

Cited in text

Regucalcin [senescence marker protein-30 (SMP-30)]

a

Implicated putatively in cell proliferation; regulation activity of protein phosphatases and Ca

2+

/CaM-dependent kinases; adenosine triphosphate (ATP)-dependent transport of calcium across the plasma membrane

Fujita (1999); M. Yamaguchi (1998); M. Yamaguchi

et al.

(1996, 1997); T. Murata

et al.

(1997); Murata and Yamaguchi (1998); Kurota and Yamaguchi (1997); Hanahisa and Yamaguchi (1998); H. Takahashi and Yamaguchi (1997), M. Yamaguchi and Kanayama (1996)



31

TABLE 2 (CONTINUED)Non-EF-Hand Calcium Binding Proteins

Non-EF-Hand CBP Function Ref.

Calelectrin Morse and Moore (1988)Calcimedin Moore and Dedman (1984);

Moore

et al.

(1984); Morse and Moore (1988)

a

A large body of literature about regucalcin, mostly from the laboratory of M. Yamaguchi, has appearedin the past few years. The references cited indicate that it is a non-EF-hand CBP that shows tissue-specific expression mainly in the liver and to a lesser extent in the kidney. It is also found in hepatomacell lines. The mouse regucalcin gene is described as spanning 18 kbp of the genome and containingseven exons and six introns. The human regucalcin gene has been provisionally mapped to chromosomeXp11.3–q11.2.


32


TABLE 3Functional Grouping of Major EF-Hand Proteins and Their Involvement in Normal and Aberrant Physiology

EF-Hand Protein Function Ref.

Regulatory Proteins

S100 family members Modulation of enzyme function; cytoskeletal dynamics; binds to various cellular target proteins; control of cell cycle traverse and cell proliferation; apoptosis; intercellular communication, signal transduction modulation of cell shape; motility and invasion; metastasis; ageing; Alzheimer’s disease

Cited in text

Calmodulin Regulation of enzyme activity; inhibition of transcription; cell proliferation; invasion

James

et al.

(1995); Corneliussen

et al.

(1994)

Troponin C Muscle contraction J.D. Potter and Johnson (1982); Wnuk (1988)

Myosin light chains Muscle contractionRecoverin Activation of guanylate cyclase;

phototransductionStryer (1991); Gorczyca

et al.

(1995)Calbindins, 9 and 28 kDa

Calcium buffering and transport; neuronal function; motor coordination; neuroprotective action

Bronner

et al.

(1986); Chard

et al.

(1993); further references cited in text

Calretinin Calcium buffering and transport; phosphorylation; cell proliferation and differentiation; tumour marker

Rogers (1987); further references cited in text

Guanylate cyclase (GC)-activating protein

Photoreceptor-specific CBP Cited in text

Centrin (caltractin) Microtubule dynamics; cell polarity; cell motility; duplication of microtubule organising centre; cell proliferation; response to environmental signals

Cited in text

Calnuc (nucleobindin) Associated with luminal surface of Golgi membrane

P. Lin

et al.

(1998, 1999)

Reticulocalbin family (reticulocalbin, calumenin, ERC-55, Cab-45)

ER-associated proteins; membrane trafficking of macromolecules DNA supercoiling (?)

Cited in text

DAG kinases

α

,

β

, and

γ

Phosphorylation of DAG and activation of PKCs Cited in text; Sakane

et al.

(1990); Sakane and Kanoh (1997); Yamada

et al.

(1997)Calpains Keratin filament aggregation (proteolytic

processing of profilaggrin)Cited in text



33

TABLE 3 (CONTINUED)Functional Grouping of Major EF-Hand Proteins and Their Involvement in Normal and Aberrant Physiology

EF-Hand Protein Function Ref.

Osteonectin Embryonic development and differentiation; extracellular matrix (ECM) remodelling; modulation of cell adhesion; cell shape; angiogenesis; tumour development and progression

Cited in text

Calcineurin B Protein phosphatase B regulatory subunit Cited in textCalmyrin Calcineurin B- and CaM-related 191 amino

acids; two C-terminal EF-hands; binds to cytoplasmic domain of platelet integrin

α

II

b

β

3

; putative regulation of the integrin function

Naik

et al.

(1997)

Buffer Proteins

β

-Parvalbumin (oncomodulin)

Lymphocyte maturation Brewer

et al.

(1990)

α

-Parvalbumin Muscle contraction; electrophysiology of neurones

Heizmann (1984); Heizmann and Berchtold (1987); Heizmann and Braun (1990)

Calbindins, 9 and 28 kDa

Calcium buffering and transport Bronner

et al.

(1986); Chard

et al.

(1993)Calretinin Calcium buffering and transport;

phosphorylationRogers (1987)

Note

: The S100 protein family is separately listed in Table 17. Calretinin has been advisedly includedas both a regulatory and buffer protein, for although it was regarded preeminently as a buffer protein,its participation in cell proliferation and differentiation and utility as a tumour marker were consideredas providing a strong basis for classifying it with other calcium-binding regulatory proteins.

EF-hand proteins have also been identified in prokaryotes. An example of this is the

Escherichiacoli

lytic transglycosylase Slt 35. The EF-hand domain of this enzyme binds Ca

2+

as well as Na

+

, albeitin different molecular configurations. Ca

2+

ions are bound preferentially, and this appears to be con-ducive to the stability of the enzyme (Van Asselt and Dijkstra, 1999).

Source:

Based on B.W. Schafer and Heizmann (1996) and references cited in the text in the course ofdiscussions relating to their respective physiologic functions.


35

4

Non-EF-Hand Calcium Binding Proteins

Several non-EF-hand calcium-binding proteins have been studied in the past few yearswith regard to their participation in normal physiological processes and their apparentability to modulate biological behaviour of cells (see Table 2). Of these, annexins,gelsolin, calreticulin, and osteocalcin have been extensively investigated. Althoughthey are grouped together here as non-EF-hand CBPs, the pathways of their approachto the modulation of cell physiology and function are distinctively different. Annexinsare calcium-dependent membrane binding proteins that participate in membrane-related processes of fusion, adhesion, exocytosis, and the remodelling of the plasmamembrane. Calreticulin also subserves a membrane-associated function (e.g., the intra-cellular storage of calcium). Osteocalcin is an extracellular matrix protein and isinvolved in bone metabolism. It is apparently also significantly implicated in invasivebehaviour of cancers, their proliferation, and osteotropic metastasis. In contrast, gelso-lin activity is mainly targeted on the cytoskeletal dynamics.

ANNEXINS

S

TRUCTURE

Several non-EF-hand CBPs have been identified and sequenced. Of these, annexinshave been studied extensively. Annexins form a family of structurally related andhighly conserved proteins that bind phospholipids in a Ca

2+

-dependent manner.Several annexins have been identified and some of these have been well character-ised, e.g., annexins I, IV, V, and XII. A 40-kDa annexin, distinct from annexin XII,has been reported from

Hydra vulgaris

(Schlaepfer

et al.

1992a). Although basicallyannexins are intracellular proteins, it has been postulated that these proteins may beinserted into the phospholipid bilayer of the plasma membrane. Integration of themolecules into the plasma membrane is essential for their postulated functions ofmembrane trafficking, fusion, and the formation of ion channels.

The crystal structure of annexins is characterised by a common fold that consistsof four domains. Each domain has five helices with a short interconnecting loop.The loops between helices A and B (domain 3) and helices D and E (domain 2) areattributed with the ability to bind phosphatidyl serine in a Ca

2+

-dependent manner.It has been suggested that annexin XII forms a trimer on the surface of the membrane(Luecke

et al.

1995). Furthermore, it appears that an intramolecular Ca

2+

-bindingsite participates in the formation of the trimer, and this is quite distinct from theCa

2+

site involved in interaction with phospholipid (Mailliard

et al.

1997). Langen

et al.

(1998) have postulated that, at low pH, annexin is inserted into the membraneas a continuous transmembrane helix that is formed, in a reversible manner, from


36


the helix–loop–helix configuration. They regard this latter configuration as beingconducive to ion channel formation. Tyrosine phosphorylation of the tetramer mayoccur following the interaction of the tetramer with phospholipid. This is suggestedby the stimulation of pp60

src

RTK following the binding of the annexin II tetramerto plasma membrane or phosphatidyl serine vesicles (Bellagamba

et al.

1997).

B

IOLOGICAL

F

UNCTIONS

Annexins are regarded generally, although not exclusively, as intracellular proteinswith predominantly intracellular functions. Several biological functions have beenattributed to annexins, (e.g., membrane fusion, calcium-channel formation, etc.) thatinvolve cAMP as the second messenger in signal transduction. Thus membrane-bound annexins could be subserving a different class of function. Annexin II, forinstance, can be found in both soluble and membrane-bound states. It has beenidentified on the cell surface, and in this location it could be functioning as a receptorfor extracellular signalling ligands (Siever and Erickson, 1997). This argument isfurther reinforced by the apparent interaction of annexin V with the actin cytoskel-eton. Membrane preparations of activated platelets indicate the presence of an 85-kDa complex, which contains annexin V. Besides, this complex also contains actin(Tzima

et al.

1999). This suggests the presence of a link between the plasmamembrane and the actin cytoskeleton, which is potentially of considerable signifi-cance in defining the function of annexins as an early link in the transmembranesignal transduction machinery.

Annexins also show a major involvement down-stream in the flow of informationin PKC-mediated transduction of growth factor signals. It has been demonstratedthat annexin V is able to inhibit PKC and this in turn results in the inhibition ofphosphorylation of annexin I (Schlaepfer

et al.

1992b, 1992c). Annexin I has phos-phorylation sites in the

hinge

region, which is believed to be important for cellproliferation and differentiation. Annexin XII is also a high-affinity substrate forphosphorylation by PKC (Schlaepfer

et al.

1992b). This could be a reason why theyshow cell proliferation related expression (Schlaepfer and Haigler, 1990). Anotheraspect of PKC-mediated regulation of annexin function is the phosphorylation ofcertain amino acid residues that are involved in the interaction with EF-hand proteinsof the S100 family. Annexin II is known to interact with S100A10 (p11) protein.There is a serine residue in the N-terminal region of annexin. If this residue isphosphorylated by PKC, annexin II interaction with S100A10 appears to be inhib-ited. Other amino acid residues have been implicated in similar interactions betweenannexin I and S100A11 (S100C) (Seemann

et al.

1996). Annexin XI-A has beenshown to bind to S100A6 (calcyclin) by means of specific hydrophobic residues onthe surface of its

α

-helix (Sudo and Hidaka, 1999).It seems, however, that annexin V does not inhibit the phosphorylation of annexin

XII by the epidermal growth factor RTK (Schlaepfer

et al.

1992c). On the otherhand, annexin II, which is also involved with cell proliferation is a substrate forRTKs. Hubaishy

et al.

(1995) demonstrated that the phosphorylation of annexin IIis effected by RTKs. They reported that phosphorylation negatively regulates annexin



37

II function, because it inhibited the binding of annexin II to and the formation ofF-actin bundles.

The annexins show a definable pattern of intracellular distribution. Barwise andWalker (1996) studied the distribution of annexins I, II, IV, V, and VI in human foreskinfibroblasts. Annexins IV and V were predominantly located in the cytoplasm andannexin VI was partly associated with the endoplasmic reticulum, whereas annexinsI and II were associated with the plasma membrane. Furthermore, annexins I, IV, andV occurred in the nucleus at higher concentrations than in the cytoplasm. When thecells were treated with calcium ionophores and C

2+

[

i

] was raised, a marked relocationof the annexins occurred. Annexins II, IV, and V translocate from the nucleoplasm tothe nuclear membrane. In MG-63 cells a relocation of intranuclear annexins IV andV to the nuclear membrane occurs within 40 s after treatment (Mohiti

et al.

1995).Annexins located in the cytoplasm may be translocated to the plasma membrane.Human osteosarcoma MG-63 cells grown under normal serum conditions show highlevels of annexin V in the nucleus. Upon serum starvation, however, the cells lose 72%of the nuclear annexin. When serum levels are restored nuclear annexin levels are alsorestored (Mohiti

et al.

1997). These authors also observed that annexin phosphorylationis essential for the relocation of annexins. The translocation of annexin V appears tobe inhibited when tyrosine kinase activity is blocked. Whether PKC-mediated phos-phorylation plays any part in the translocation is unclear. PKC itself shows intracellulartranslocation upon being activated. It would have been of considerable interest also todetermine PKC translocation, especially in light of the role PKC plays in the regulationof annexin function. A different pattern of annexin II and V translocation has beendescribed in human neuroblastoma SH-SY5Y cells. Blanchard

et al.

(1996) found thatboth annexins were associated with membranes. Annexin II showed a uniform asso-ciation before elevation of intracellular calcium levels, but upon treatment with calciumionophores, it was relocated to discrete patches.

Annexins take part in the process of calcium-dependent aggregation of lipo-somes. Annexins I through IV, but not V or VI, seem to mediate liposome aggregation(L. Liu

et al.

1996). From this functional viewpoint, there is evidence of cAMPinvolvement in calcium-dependent aggregation of phosphatidyl serine liposomes andbovine chromaffin granules and in calcium channel activity (Cohen

et al.

1995).Blanchard

et al.

(1996) showed that membrane-associated annexin II is reorganisedinto discrete patches. It seems preferentially to partition into cholesterol-richdomains of the plasma membrane. Furthermore, the transmembrane adhesive gly-coprotein CD44 also partitions into the same type of domain, with the result thatCD44 clusters at the external membrane surface and annexin II at the cytoplasmicsurface (Oliferenko

et al.

1999). Whether this seemingly fortuitous partitioning ofannexin II has a functional provision in CD44-mediated cell adhesion is unclear atpresent. Nevertheless, with the demonstration by Tzima

et al.

(1999) that annexinII might be coupled with the actin cytoskeleton, this is highly suggestive of itsinvolvement in membrane-mediated functions of intercellular adhesion and cellfusion. Several mammalian annexins positively influence the process of exocytosis(Creutz

et al.

1992). Such a function seems to be strengthened by the observationby Oliferenko

et al.

(1999) that the redistribution of CD44 and annexin II wasaccompanied by changes in the cytoskeletal organisation. CD44 patching is induced


38


by overexpression of the S100A4 protein, and this appears to relate to the ability ofS100A4 to depolymerise cytoskeletal elements (Lakshmi

et al.

1993, 1997). There-fore, there is the distinct possibility that annexins and S100A4 might cooperate ingenerating the membrane-mediated behavioural changes of cells.

The intracellular translocation of annexins in the human osteosarcoma cell lineMG-63 has been found to be phosphorylation dependent. Serum depletion resultsin a reduction of nuclear annexin V levels. With serum stimulation these levelsincrease. Inhibition of tyrosine kinases has been found to reduce nuclear transloca-tion of the annexin (Mohiti

et al.

1997), which suggests that the relocation ofannexins requires tyrosine phosphorylation.

A

NNEXINS

IN

C

ANCER

G

ROWTH

AND

P

ROGRESSION

The influence of annexins on cell growth was recognised some years ago. Theexpression of annexin VII has been reported to inhibit the growth and viability ofsome secretory mutants of

Saccharomyces cerevisiae

at semipermissive tempera-tures. In contrast, annexin IV mitigated the growth defect of the sec2 secretorymutant (Creutz

et al.

1992). Recent studies have in fact elucidated in some detailthe significant part that annexins play in cell proliferation. Raynal

et al.

(1997)demonstrated convincingly that annexins exhibit a cell cycle-related expression.Apparently, annexins II, V, and VI are, on the whole, uniformly expressed at a lowlevel at all phases of the cell cycle. Annexin IV, on the other hand, showed a 50%increase in the S-phase of the cycle. Annexins I and V decreased by 40% in earlyG

2

M phase, although the corresponding gene expression did not vary. These obser-vations show that there are specific patterns of occurrence of annexins and theexpression may be regulated at the transcriptional level.

The apparent cell cycle-related expression of annexins and the deregulation ofPKC-mediated signal transduction with consequent deregulation of proliferation hasprompted the investigation of annexins in relation to the proliferative state andprogression of human cancers. Masaki

et al.

(1996) found that annexin I levels weregreatly increased, at both the transcriptional and the protein levels, in human hepa-tocellular carcinomas as compared with normal liver tissue. Annexin II was reportedto be overexpressed in human pancreatic carcinomas compared with normal pan-creatic tissue (Vishwanatha

et al.

1993). Furthermore, these authors found that theexpression of annexin II was associated with proliferating ductal adenocarcinomasand especially that its expression colocalised with that of proliferating cell nuclearantigen (PCNA). Roseman

et al.

(1994) also found some association between levelsof annexin II and the BrDU (bromo-deoxyuridine) proliferation index. Compatiblewith this is the reported histological grade-related expression of annexin II in gliomas(Roseman

et al.

1994). They found that annexin II levels were greater in glioblastomamultiforme than in anaplastic astrocytomas, which in turn showed higher levels thandid astrocytomas.

With variations of annexin levels seemingly in consonance with growth dereg-ulation in human cancers, there has been some effort directed at examining whethervariations in expression might relate to the stage of progression of cancers. As statedbefore, annexin levels have been found to correlate with histological grade of gliomas



39

(Roseman

et al.

1994). Annexin I does not occur in ductal luminal cells of normalbreast tissue, but it is expressed in a range of breast cancers including noninvasiveductal carcinomas as well as invasive and metastatic breast cancer (Ahn

et al.

1997).Annexin VI, in contrast, has been shown previously to inhibit cell proliferation.

Theobald

et al.

(1994) transfected A431 cells with annexin VI. The growth of thesecells, in normal serum concentration, was only moderately inhibited by the expres-sion of annexin VI. When grown in low serum, their doubling time seemed toincrease. However, Theobald

et al.

(1994) found that annexin VI-expressing cellsgrown in low serum conditions showed arrest at the G

1

phase of the cell cycle.Furthermore, annexin VI expression inhibited the growth of A431 cells injectedsubcutaneously into nude mice (Theobald

et al.

1995). It appears that the inhibitoryeffect is exerted only by the larger of the two splice variants of annexin VI. Fleet

etal.

(1999) found that the larger splice variant inhibited the Ca

2+

mobilisation andcell proliferation induced by EGF. The shorter splice variant of annexin VI was notcapable of such inhibition. Francia

et al.

(1996) identified several mRNAs that aredifferentially expressed in the murine melanoma cells B16F-10 and an immortalisedmelanocyte cell line called Melan A. Among these was an mRNA that was foundto be identical to the 3

′

region of murine annexin VI. The expression of this mRNAwas reduced in B16F-10 melanoma cells. Melan A may not be a proper control cellline with which to compare the mRNA profile of B16 melanomas, and the lowmetastasis variant B16F1 might have been a more appropriate control cell line.Nonetheless, Francia

et al.

(1996) followed this up with a study of the expressionof annexin VI in human melanomas. In these tumours also a reduced or loss ofannexin expression was encountered during progression. The expression of annexinV has been found to be markedly suppressed in carcinomas of the uterine cervixand the endometrium (Karube

et al.

1995).Thus, most of the annexins studied to date have related directly, however inde-

cisively, to cell proliferation and progression; however, annexin VI seems to differfrom the rest. If confirmed, the reported tumour suppressor function of annexin VIdoes add a new dimension to the biology of annexins. But clearly, more work needsto be done to examine whether the putative suppressor function is associated withthe larger splice variant and whether the second shorter variant of the annexin mightpossess some compensatory and antagonistic function.

Overall, in spite of the cell cycle-related expression of some of the annexins, noclear relationship has emerged between their expression and the neoplastic diseaseprocess. Annexin II, which seems to relate to histological grade and differentiationof gliomas, tends to be expressed uniformly in the cell cycle. The expression ofannexins I and V seems to change in opposite directions in the context of the cellcycle. Both are down-regulated in their expression at the G

2

M point in the cell cycle.Perhaps such comparisons are unhelpful because of the differences in the biologyand histogenesis of the tumours studied.

A

NNEXINS

IN

M

ORPHOGENESIS

AND

D

IFFERENTIATION

The expression patterns of annexins have been studied with respect to cell differen-tiation. Rahman

et al.

(1997) reported that annexins V and VI are not detectable in


40


undifferentiated mesenchymal cells of foetal rat limb buds. The progenitor cells ofskeletal muscle, which appear in the limb bud on day 14, express annexin VI on thecell surface upon differentiation from myogenic cells into myotubules. Annexins Vand VI also are associated with the differentiation of chondrocytes. King

et al.

(1997)have confirmed that annexin V is required for the formation of the pericellular matrixof chondrocytes.

THE GELSOLIN FAMILY OF CALCIUM-BINDING PROTEINS

Actin-binding proteins constitute a large family of proteins that actively participatein actin bundling, nucleation, actin capping, and severing. Gelsolin, severin, andvillin are three important proteins of the gelsolin family of actin-binding proteinsthat will be discussed here, for they possess the remarkable property of rapidlyreorganising the cytoskeleton and also rapidly modulating, in a Ca

2+

-dependentmanner, cell shape, substrate adhesion, and cell locomotion.

G

ELSOLIN

IN

S

EVERING

AND

C

APPING

OF

A

CTIN

F

ILAMENTS

Gelsolin was identified as an 82-kDa actin-binding protein that is involved in Ca

2+

-dependent severing of actin filament and capping of filaments. The kinetics ofpolymerisation of actin filaments, namely, the association and dissociation of mono-meric subunits, essentially involves F-actin elongation by the association of ATP-bound monomers with the barbed end of the filament and a slower loss of ADP-actin from the pointed end (Sheterline and Sparrow, 1994). Actin polymerisation isaltered by the sequestration of monomeric elements and by filament capping, whichprevents the addition of subunits. The exchange of subunits at the filament ends iscontrolled by gelsolin and gelsolin-related proteins. They bind to the filaments andinhibit the addition of actin subunits. Several proteins that participate in the regula-tion of cytoskeletal organisation have been identified (A. Schafer and Cooper, 1995).A large number (>60) of actin-binding proteins are known (Pollard

et al.

1994) andthe new ones are continually being identified and reported. A group of proteinscomposed of the gelsolin family and of tensin, and profilin bind to barbed ends.Others bind to pointed filament ends, whereas some proteins bind alongside thefilament and yet successfully influence filament assembly. Certain isoforms of tro-pomyosin have been found to protect actin filaments from gelsolin-mediated sever-ing. This protective function may be further enhanced by caldesmon (Ishikawa

etal.

1989a). Tropomyosins appear to be able to anneal the actin fragments and againcaldesmon is said to accentuate this process (Ishikawa

et al.

1989b).The

Drosophila

flightless-I gene and the homologous human FLII gene encodegelsolin-like actin-binding proteins (H.D. Campbell

et al.

1997). Constantin

et al.

(1998) have isolated a gelsolin-like protein from

Physarum polycephalum

that affectscytoskeletal integrity when introduced into mammalian cells. Tropomodulin andspectrin bind to pointed filament ends. Two isoforms of gelsolin had been identified:a cytoplasmic form and a secreted isoform called plasma gelsolin. A third isoformhas now been reported; all three isoforms arise by alternative splicing of the gelsolin



41

transcript (Vouyiouklis and Brophy, 1997). The FLII maps to chromosome 17p11.2(Chen

et al.

1995), in the region that is consistently deleted, and is associated withSmith-Magenis Syndrome (SMS).

Cytoplasmic-free calcium and inositol 4,5-bisphosphate regulate the functionof gelsolin family proteins. Gelsolin binding to PIP2 has been found to be modulatedby Ca

2+

(K.M. Lin

et al.

1997). Several functional domains that regulate actinfilament length have been identified in the gelsolin molecule. The N-terminaldomain (S1) is able to inhibit actin polymerisation. The inhibitory activity has beenattributed to a small peptide sequence. The interaction of the C-terminal domains(S4–6) with actin filaments occurring during the severing process has been localisedwith residues 112 to 120 of actin subdomain 1 (Feinberg

et al.

1997a, 1997b). Bydeletion mutagenesis, residues close to the S2 domain have also been implicatedin actin filament binding, capping and as well as interaction with phosphoinositides(H.Q. Sun

et al.

1994). Cofilin is another protein that binds actin and promotes itsdepolymerisation. There are important sequence homologies between cofilin andgelsolin. The actin-binding peptide of cofilin has been found to compete withgelsolin segment S2 for binding to actin, suggesting that they share a binding siteon the actin filament (Van Troys

et al.

1997). Interaction with gelsolin inducesconformational changes in actin, and nucleation of actin polymerisation may bepromoted by these conformational changes (Khaitlina and Hinssen, 1997).

G

ELSOLIN

IN

E

MBRYONIC

D

EVELOPMENT

AND

M

ORPHOGENESIS

Gelsolin and related proteins have been shown to be actively involved in normalembryonic development and morphogenesis as well as in pathogenesis of diseasessuch as amyloidosis and cancer. The gelsolin gene has been mapped to the chromo-some 17p11.2 region, which is a critical region deleted in SMS which encompassesshort stature, brachydactyly, dysmorphic features, retarded development, and behav-ioural problems. Gelsolin may also participate in the process of programmed celldeath or apoptosis.

Gelsolin shows a definable pattern of expression in the developing CNS. It isfound predominantly in oligodendrocytes and Schwann cells. It is also found in themyelin sheath. In oligodendrocytes, gelsolin is found in the soma and in culturedcells it is detectable in the branched cell processes (Lena

et al.

1994). The proteinis detectable in the brain of newborn rats. The expression of gelsolin graduallyincreases at 8 to 10 days after birth, reaching a maximum at 20 to 30 days whenmyelin formation is actively occurring. However, subsequently, its levels decreaseeven though myelin basic protein levels continue to rise until 6 months after birth.In Schwann cells gelsolin is found in the cytoplasm and in compact myelin (J. Tanakaand Sobue, 1994). It would appear therefore that gelsolin may be involved in thewrapping of myelin sheaths around the axons by promoting a process of motilitygenerated by means of its effects on the cytoskeleton (J. Tanaka and Sobue, 1994;Lena

et

al

. 1994). The gelsolin-like protein, fragmin, which has been isolated from

Physarum,

has been shown to interact with actin in a Ca

2+

-dependent fashion, whenintroduced into PtK2, CV1, and NIH3T3 cells. This has been reported to causecytoskeletal disruption and bring about changes in cellular morphology (Constantin



et al. 1998). Fibroblast migration has been found to be dependent on the actin-severing activity of gelsolin (P.D. Arora and McCulloch, 1996). Furthermore, gelso-lin has been implicated in cytoskeleton-mediated transduction of EGF signal andEGF-induced cell motility (P. Chen et al. 1996). The Drosophila melanogasterflightless-I gene product is a gelsolin-like protein that is also involved in embryonicdevelopment and in the structural organisation of the indirect flight muscle. However,it should be pointed out that gelsolin-null transgenic mice appear to develop nor-mally, albeit associated with certain abnormalities such as prolongation of bleedingtime caused by abnormalities of platelet shape, and also inhibition of neutrophilmigration. The transgenic mice also show increased stress fibre formation in dermalfibroblasts together with reduced motility and increased contractility (Witke et al.1995).

GELSOLIN EXPRESSION IN AMYLOIDOSIS

The strong association of gelsolin with CNS components has initiated studies ofpossible alterations in its expression in CNS-associated abnormalities. CNS abnor-malities in patients with familial amyloidosis may conceivably be related to gelsolinexpression (Kiura et al. 1995). Paunio et al. (1997) recently measured the mRNAlevels of both intracellular and secreted gelsolin isoforms in tissues of humansubjects. Intracellular gelsolin mRNA has been found to be a major component inall tissues. Most adult tissues can show mRNAs of both isoforms, with muscle andskin tissues showing especially high levels of expression. The high levels of skingelsolin may be related to skin amyloidosis. The levels of expression may differbetween adult and infant tissues. Patients with gelsolin-related amyloidosis hadhigher serum levels of gelsolin but did not show increased gene expression.

GELSOLIN IN CANCER

A major feature of cancer development is the perceived derangement of the cellsignalling system and the inappropriate acquisition of cell motility. The process ofactin filament severing and capping brings in its wake alterations in cellular motility.The involvement of gelsolin in the remodelling of the cytoskeleton has naturally ledto the investigation of the expression and possible involvement of gelsolin and relatedproteins in cancer development and progression. Neoplastic transformation alters cellbehaviour, morphology, and pattern of growth. These changes are indeed attributableto the loss of stress fibres and focal adhesion. Accompanying cell transformation,modulation of the expression of cytoskeleton-associated proteins is observed, andamong them is gelsolin. Van de Kerckhove et al. (1990) showed some time ago thatgelsolin expression is down-regulated in transformed human fibroblasts and epithelialcells. Transformed cells also show a down-regulation of tropomyosin. Upon restorationof tropomyosin status, cells return to the normal modes of spreading and organisationof stress fibres. These events are far from being coincidental, for the higher molecularweight forms of tropomyosin appear to protect actin filaments from the severingactivity of gelsolin (Matsumura et al. 1985; Ishikawa et al. 1989a).


Non-EF-Hand Calcium Binding Proteins 43

Although only a few studies have been carried out to date, gelsolin appears tobe consistently down-regulated in cancers. Moriya et al. (1994) found that gelsolinexpression was down-regulated in several tissue culture cell lines derived fromhuman gastric carcinomas. Gelsolin levels were found to be markedly reduced inhuman breast cancer cell lines as compared with normal breast epithelial cells andcell lines derived from benign breast diseases. Gelsolin loss was also found in 70%of 30 sporadic invasive breast carcinomas as well as in all chemically induced murineand rat mammary tumours (Asch et al. 1996). In human transitional cell bladdercarcinoma cell lines as well as tumour tissues (14 out of 18 samples) gelsolin wasnot detectable or detectable only at low levels compared with normal bladder epi-thelium (M. Tanaka et al. 1995). M. Tanaka et al. (1995) also transfected gelsolincDNA into the human bladder cancer cell line UMUC-2. The transfectants showeda greatly reduced colony forming ability as well as tumorigenicity.

One can see from these studies the emergence of the concept of tumour sup-pression by gelsolin. However, it is paradoxical that gelsolin on the one hand canenhance cellular motility and on the other suppress tumorigenicity. Tumorigenicityand cellular motility can be dissociated temporally, and therefore one can envisagea situation where the loss of gelsolin could lead to tumour development and beinvolved in a positive way at a later stage of tumour progression by conferringinvasive properties on tumour cells by altering cytoskeletal dynamics. There is littledoubt that this is a fruitful avenue to explore. One needs to investigate the patternof gelsolin alteration at various stages of tumour progression. The colon carcinomamodel immediately comes to mind. The progression of colon carcinoma has beendescribed in clearly identifiable phenotypic phases, and there is considerable evi-dence that different genetic changes are associated with the different phases ofprogression and that the development of carcinoma is a cumulative effect of thesegenetic alterations (Sherbet and Lakshmi, 1997b). It would be interesting to seewhether alterations of gelsolin or related gene alterations have a place in this picture.There is also the need to glean more information about the intracellular localisationof these proteins in the context of the alterations in cytoskeletal dynamics occurringin proliferating and transformed cells.

Other questions can be raised. For example, are gelsolin and related proteinsassociated with cell proliferation and apoptosis? We have noted earlier that EGF-induced cell motility involves gelsolin. The alteration of cytoskeletal dynamics isan essential ingredient of cell proliferation. There is compelling evidence that otherCa2+-binding proteins such as the S100A4 (18A2/mts1), which also causes pertur-bation of cytoskeletal dynamics (Lakshmi et al. 1993), can control cell cycle pro-gression by interfering with G1S and G2M checkpoint control exerted by the p53suppressor gene and stathmin (Parker et al. 1994a, 1994b; Cajone and Sherbet,2000). Therefore, if the gelsolin story is to be comprehended it is of the utmostimportance to determine whether gelsolin is associated with cell proliferation. Thereare indications that gelsolin may indeed influence cell population dynamics byinhibiting apoptotic death of cells (Ohtsu et al. 1997).



SEVERIN AND CYTOSKELETAL REORGANISATION

Severin is a 40-kDa Ca2+-activated actin-binding protein that was isolated from theamoeba Dictyostelium discoideum, and of this, mammalian homologues have alsobeen identified. Severin severs F-actin, nucleates actin assembly, and caps actin fila-ments. It is a gelsolin-related protein and shares sequence homology with gelsolin andprofilin in certain conserved functional domains that are involved with actin interaction(Schnuchel et al. 1995). Severin possesses two actin-binding sites located next to eachother, and these participate in both severing and nucleation functions. The third actin-binding site, situated near the N-terminus, suffices for the filament capping function(Eichinger et al. 1991). Eichinger et al. (1998) recovered a protein kinase fromcytosolic extracts of D. discoideum. This kinase phosphorylates severin, but phospho-rylation is reduced in the presence of calcium. Eichinger et al. (1998) have thereforesuggested that phosphorylation might be another regulatory mechanism.

Severin is known to alter rapidly the organisation of the cytoskeleton and asa consequence influence cell adhesion to the substratum, pseudopodial activity,and cell motility. Early work had suggested that mutants deficient in severin didnot affect the motility of Dictyostelium (Andre et al. 1989). But Schindl et al.(1995), who studied mutants that were deficient in severin, found the protrusionof pseudopodia was slower and the pseudopodia were shorter in the mutants. Asnoted in the previous section, the loss of gelsolin has been associated by somewith cell motility, especially in neoplastic transformation. Folger et al. (1999)have reported that severin replaces gelsolin in murine Lewis lung carcinoma (LL2)cells. No severin is detected in normal epithelial lung tissue, but gelsolin isexpressed at high levels. Folger et al. (1999) suggested tentatively that severintakes over from gelsolin the function of actin fragmentation in transformed cells.In light of the paradoxical effects of gelsolin of suppressing tumorigenicity on theone hand, and stimulating invasive behaviour on the other, further work on theexpression of gelsolin and severin in a temporal relationship to tumour progressionseems warranted.

VILLIN IN DIFFERENTIATION AND NEOPLASIA

Villin is another actin-binding protein that shows sequence homology with gelsolinwith respect to certain conserved sites involved in actin-binding. It occurs in intes-tinal and kidney brush border epithelia. Villin is known to take part in actin bundling,nucleation, filament capping, and severing in a Ca2+-dependent manner, in vitro.Villin cDNA has been transfected into cells that do not synthesise the protein, andit has been found that this induces the growth of microvilli and the reorganisationof the submembranous actin cytoskeleton of the transfectant cells. This process isinhibited by cytochalasin (Friederich et al. 1993). Friederich et al. (1999) haveprovided further experimental evidence for the involvement of villin in actin bun-dling. Villin not only induces the growth of microvilli, but also in parallel inducesactin bundling. This actin bundling ability seems to depend on the KKEK motif



occurring at the C-terminal end of the F-actin-binding site. However, some uncer-tainty about these events has arisen with the report by Ferrary et al. (1999) that invillin-null mice there is no disruption or disorganisation of microvilli, nor are thereany changes noticed in the localisation of actin-binding and membrane proteins ofthe intestinal brush border in these null animals.

Nevertheless, because the expression of villin appears to be a marker of differ-entiation, it has been investigated as a potential marker of tumour progression. Villinhas been employed as a means of differentiating between primary pulmonary ade-nocarcinomas and pulmonary metastases of neoplasms of the bronchial or digestivetract. Nambu et al. (1998) examined 57 primary pulmonary adenocarcinomas forvillin expression and found that 31% of the cancers expressed villin. Of the positives,10.5% showed a diffuse cytoplasmic distribution of villin and 21% showed cyto-plasmic distribution with minor brush border staining for villin. In contrast, meta-static lung tumours showed mainly primary brush border localisation that is char-acteristic of villin. The findings of J.Y. Tan et al. (1998) are similar. These authorsfound that villin, together with cytokeratin 7 and 20, is useful in differentiatingbetween primary pulmonary adenocarcinomas and pulmonary metastases of coloncarcinomas.

Villin is expressed in Barrett’s metaplasia as well as in Barrett’s adenocarcinoma.According to Regalado et al. (1998), all metaplasias and 93% (30) of adenocarci-nomas were villin positive. Furthermore, Northern analyses revealed the presenceof both 3.5- and 2.7-kb villin mRNAs. Thus, overall, villin seems to be expressedin most adenocarcinomas, metaplasias, as well as other oesophageal tumours. Villinalso has been investigated as a possible marker of neuroendocrine tumours of thegastrointestinal tract and also of hepatic tumours (P.J. Zhang et al. 1997; Velazquezet al. 1998), but definitive reports of these studies have not been published.

Another actin-regulatory protein called Cap G, which belongs to the gelsolinfamily, has been investigated somewhat superficially. Cap G is an actin-capping butnot -severing protein. The Cap G gene is located on the short arm of chromosome2 and consists of ten exons and nine introns. The open reading frame has nine exonsand eight introns (Mishra et al. 1994). Van Ginkel et al. (1998) found that this geneis differentially expressed between normal uveal melanocytes and uveal melanomasand cell lines derived from these tumours.

In summary, there seems to be ample evidence that villin gene function isinvolved in the organisation of the brush border cytoskeletal assembly and in theformation of microvilli. Therefore, the use of this protein as an indicator of brushborder differentiation, with an implicit relationship to neoplastic transformation,seems fully justified. However, as a marker, villin will need to perform more strin-gently and with greater specificity than merely providing a means of differentiatingbetween primary tumour of the lung and tumours metastatic to the lung. There ismuch scope for combining villin expression with other proteins such as ezrin andmoesin, which link the plasma membrane with the cytoskeleton. These proteins notonly localise with the actin cytoskeleton, but they are also specifically associatedwith the formation of microvilli and membrane ruffles.



CALRETICULIN AND ITS FUNCTIONAL DIVERSITY

STRUCTURE AND MOLECULAR FEATURES OF CALRETICULIN

Two types of CBPs are associated with the endoplasmic reticulum (ER), namelyreticulocalbin (an EF-hand CBP whose localisation and function are discussed inChapter 12 and calreticulin). Calreticulin is a non-EF-hand CBP that is ubiquitouslyassociated with the ER of a variety of tissues. It is regarded as the nonmuscleequivalent of calsequestrin, which is associated with the sarcoplasmic reticulum(SR). Calreticulin, which was identified as a CBP and isolated from SR (Ostwaldand MacLennan, 1974), is believed to be a homologue of calnexin, which is a 64-kDa transmembrane protein (Hammond and Helenius, 1995). It is a highly conservedprotein. Calreticulin obtained from rat liver has a molecular size of 60 kDa. In somecell-free systems a 62-kDa protein has been detected, which is probably processedinto 60-kDa calreticulin (Denning et al. 1997). Hershberger and Tuan (1998) havecloned a full-length cDNA for calreticulin from mouse trophoblast coding for a 57-kDa protein showing a high degree of sequence homology to calreticulin. Yamamotoand Nakamura (1996) isolated and sequenced the ER-associated calnexin from Ranarugosa, which shared 77% sequence homology with calreticulin. Two distinct iso-forms of calretinin have been isolated from spinach leaves and from the pollen ofLiriodendron tulipifera L. and Ginkgo biloba L. (Navazio et al. 1995, 1998; Nardiet al. 1998). These are reported to show very low sequence homology with animalcalreticulin. However, maize calreticulin is said to be a 48-kDa protein (as deducedfrom its cDNA sequence), highly acidic in nature, sharing 77 to 92% sequencehomology with other plant calreticulins, and have approximately 50% homologywith animal calreticulin (Dresselhaus et al. 1996).

Calreticulin consists of 400 amino acid residues. It has three distinct domains: anN-terminal domain containing 180 amino acid residues, a C-terminal domain withacidic residues and lysines, and a middle domain (P-domain) that is composed of threerepeats of a 17 amino acid motif. The C-domain binds calcium with high capacity butlow affinity. In contrast, the P-domain binds calcium with low capacity but high affinity.Overall, calreticulin may be seen as a high-capacity but low-affinity CBP.

The mouse calreticulin gene spans 4.2 kbp of genomic DNA and contains nineexons and eight introns (Waser et al. 1997). In the mouse the gene is located onchromosome 8 (Rooke et al. 1997). It should be pointed out here that P. Lin et al.(1998) state that nucleobindin, a mammalian protein showing a high degree ofhomology to calreticulin, is an EF-hand CBP. This protein is found in the cytosoland is associated with membranes, in the latter case mainly with the luminal surfaceof Golgi membranes.

REGULATION OF CALRETICULIN EXPRESSION

The expression of calreticulin appears to be regulated by intracellular calcium levels.The promoter region of the calreticulin gene contains elements responsive to the calciumionophore A23187 and to agents such as thapsigargin, which can raise intracellularcalcium levels by releasing calcium from intracellular stores. Both A23187 and thapsi-gargin have been shown to increase transcription of the calreticulin gene. It is not



affected by changes in extracellular or cytoplasmic levels of calcium. This has led tothe suggestion that loss of calcium levels in the intracellular stores could induce genetranscription (Waser et al. 1997). Calreticulin gene expression is also up-regulated byother stimuli such as heat shock and heavy metals like zinc and cadmium (Nguyen etal. 1996; Szewczenko-Pawlikowski et al. 1997). A number of heavy metal ions, e.g.,Ni2+, Zn2+, Cu2+, and La3+, stimulate the release of calcium from intracellular stores(McNulty and Taylor, 1999). Hyperthermia also raises intracellular calcium levels bycalcium mobilisation from intracellular stores as well as by simulating its influx intothe cell (Itagaki et al. 1998). Therefore, the up-regulation of calreticulin gene expressionmight, again, be mediated by the stress response of depletion of calcium held in theintracellular stores. This is in sharp contrast with the effects of hyperthermia andthapsigargin on the expression of S100A4. Both treatments have been shown to down-regulate S100A4 expression (Parker and Sherbet, 1992; Albertazzi et al. 1998a). Thismight suggest a different mode of regulation from that of calreticulin.

The thesis that the calreticulin gene is regulated by androgen has been advocatedvigorously by N. Zhu et al. (1998). This is based on the finding that androgenablation down-regulates and its restoration up-regulates both calreticulin mRNA andprotein expression in the prostate. Calreticulin is expressed at higher levels in theprostate than in the seminal vesicle and other organs and muscle. The regulation byandrogen occurs only in the prostate and seminal vesicles. In vitro, the induction ofcalreticulin by androgen is not inhibited by inhibitors of protein synthesis, hencethe suggestion that the calreticulin gene might be regulated by androgen. Further-more, in androgen-sensitive LNCaP prostate cancer cell lines, androgen is able toblock apoptosis induced by the calcium ionophore A23187. This effect of androgencan be negated by antisense calreticulin oligonucleotides (N. Zhu et al. 1999).

PHOSPHORYLATION OF CALRETICULIN

Calreticulin phosphorylation occurs under different physiological conditions. How-ever, it is unclear at present whether this is a physiological mechanism by whichcalreticulin activity is regulated. A phosphorylated form of calreticulin is detectablein cells infected with rubella virus (RV). It has been reported that the binding ofphosphorylated calreticulin to the 3′-end of RV genomic RNA is necessary forinitiating RNA replication (Singh et al. 1994). Phosphorylation is a requirement alsoin the binding of calreticulin to Leischmania donovani RNA (Joshi et al. 1996).Calreticulin from spinach leaves is phosphorylated by protein kinase (casein kinase)CK2, but those of animal origin are not substrates for this kinase. It appears thatthis is due to structural differences between the two types of calreticulin (Baldan etal. 1996). CK2 was identified as an ER-associated kinase involved in the phospho-rylation of calreticulin (Ou et al. 1992). CK2 might be localised in the lumen of theER (N.G. Chen et al. 1996). The in vitro phosphorylation of calreticulin and otherproteins in plasma membrane-enriched fractions has been attributed also to otherprotein kinases susceptible to inhibition by the PKC inhibitor staurosporine (Droil-lard et al. 1997). CK2 also phosphorylates calnexin in vitro as well as in vivo. Serineresidues that occur in the C-terminal half of the cytosolic domain of calnexin wereexclusively phosphorylated (Wong et al. 1998). Sphingosine-dependent kinases



(SDKs) are another kinase variety that have been shown recently to phosphorylatecalreticulin. SDK1 has been found specifically to phosphorylate calreticulin andprotein disulphide isomerase (PDI) (Megidish et al. 1999). Heat shock proteins arealso substrates of SDKs. Calreticulin, heat shock proteins, and PDI have a commonfunction as molecular chaperones. Furthermore, calreticulin and PDI both occur inthe ER. Therefore, these observations might be the closest researchers have cometo suggesting a relationship between calreticulin function and phosphorylation. Inspite of these various findings, how phosphorylation of these molecules regulatestheir participation in their apparently diverse physiological functions is yet to beelucidated. At present, no changes in the state of calreticulin phosphorylation havebeen found to correlate with a specific function. Furthermore, it has been suggestedthat calreticulin could occur in a constitutively phosphorylated form (Cala, 1999).This would deny the process of phosphorylation any regulatory control over calreti-culin function. Nevertheless, the induction of apoptosis of HL-60 cells by geranylge-raniol has been shown to be accompanied by a decrease in calreticulin as well as adecrease in the phosphorylation of another protein of 36 kDa molecular size. Thesereductions occur prior to DNA fragmentation (Nakajo et al. 1996). The occurrenceof these events in parallel suggests that calreticulin may influence the phosphoryla-tion of the 36-kDa protein.

INTRACELLULAR DISTRIBUTION OF CALRETICULIN

As stated earlier, calreticulin shows a predominant association with the intracellularmembrane system, the ER. The molecule contains KDEL/HDEL sequence at the C-terminal, which is the targeting signal for its localisation at the ER. Calreticulinoccurs also in the SR and membranes of the Golgi bodies. In protoplasts of plants,it is localised in the ER (Opas et al. 1996b). In animal as well as in some plantcells, calreticulin is found in the lumen of the ER, nucleus, and nuclear membrane,on the surface of the cells, and in the cytoplasm (Opas et al. 1991; Dedhar, 1994;White et al. 1995; Dresselhaus et al. 1996).

CALRETICULIN IN INTRACELLULAR CALCIUM STORAGE

Calreticulin is prominently associated with the endoplasmic reticulum, as its nomen-clature implies. Over the past few years, it has been found to participate in severalbiological phenomena.

With its occurrence in the ER, calreticulin has been linked empirically with thecellular faculty of intracellular storage of calcium. This role has been confirmed bya variety of experimental data. There are several lines of evidence on this score.Using reporter gene constructs carrying the promoter of calreticulin gene, Waser etal. (1997) showed that the promoter can be transactivated by agents such as brady-kinin and thapsigargin, which are known to release calcium held in intracellularstores. They have identified two regions of the promoter that are responsive tothapsigargin as well as the calcium ionophore A23187. Furthermore, both theseagents have been shown to be able to enhance the transcription of calreticulin genein NIH3T3 cells.



Mery et al. (1996) transfected a mouse fibroblast cell line with an expressionvector carrying a calreticulin cDNA insert. In the transfectant cells, a 1.6-foldoverexpression of calreticulin was found, and concurrently, intracellular calciumlevels rose to nearly two-fold. Furthermore, most of this calcium originated fromintracellular stores, as indicated by thapsigargin sensitivity. A study of the intracel-lular distribution of calreticulin in motor neurones of rat spinal cord has revealedthat it is localised not only in the ER but also in “coated” vesicles (Copray et al.1996). Copray et al. (1996) suggested that these vesicles may be counterparts ofcalciosomes, which are calcium storage vesicles found in liver cells and in cerebellarPurkinje cells. It ought to be pointed out, nonetheless, that Coppolino et al. (1996)found that the intracellular calcium stores were unaffected in cells in which thecalreticulin gene had been knocked out.

CALRETICULIN AND CALNEXIN AS MOLECULAR CHAPERONES

A second function attributed to calreticulin is that of a molecular chaperone. Theassembly of the T-cell antigen receptor (TCR) complex involves several genes,including calreticulin. Calnexin, which shares considerable sequence homology withcalreticulin, is known to be involved with the chaperoning of newly synthesisedTCR proteins. Similarly, calreticulin seems to be involved in the chaperoning ofnascent TCR-α and -β proteins (Van Leeuwen and Kearse, 1996). Indeed, on theone hand, both calnexin and calreticulin seem to bind to and promote correct foldingof proteins. They prevent aggregation, delay oligomerisation, and suppress degra-dation (Herbert et al. 1996). On the other hand, they possess the ability to bind toglycoproteins of the ER. Calnexin was shown to interact with and be involved inthe assembly of class I MHC (major histocompatibility complex) molecules (Degenand Williams, 1991). Calreticulin has also been shown to interact with class I MHCmolecules and probably with greater specificity than calnexin (Zhang and Salter,1998; Harris et al. 1998). Both calnexin and calreticulin are lectin-like proteins andare able to interact with newly synthesised glycoproteins that have been partiallytrimmed of the N-linked oligosaccharides and assist in their folding and assembly(Helenius et al. 1997; Zhang and Salter, 1998). Receptors for insulin and insulin-like growth factor (IGF)-1 are receptor tyrosine kinases that have to dimerise beforethey are exported to the ER. The receptor monomers form dimers through disulphidebonding, when the newly synthesised monomers are associated with calreticulin orcalnexin (Bass et al. 1998). Some of these studies implicate oligosaccharide-depen-dent binding to calnexin and calreticulin in a direct way in protein folding, assembly,and secretion. However, there are instances in which a lack of binding to theseproteins has been of little consequence.

It is possible that the chaperoning function of calreticulin might have implica-tions for gene function. Calreticulin-deficient cells show an inability to importtranscription factors belonging to the NF-AT (nuclear factor of activated T cells)family into the nucleus (Mesaeli et al. 1999). This could be a reflection of the partplayed by calreticulin-mediated chaperoning of the transcription factors. The NF-AT transcription factors are substrates for calcineurin, and thus calreticulin mightindirectly influence the expression of genes regulated by these factors. (See Figure



19.) This view is compatible with the intracellular distribution of calreticulin. Anoverall increase in the levels of calreticulin has often been associated with cancers.Yoon et al. (2000) have reported that the total calreticulin content of hepatocellularcarcinomas is similar to that of corresponding normal tissues, but carcinomas differfrom normal tissues in their intracellular distribution. Calreticulin occurs moreabundantly in the nuclear matrix of hepatocellular carcinomas as compared withcontrols. Whether this might reflect an enhanced transport of substances such astranscription factors into the nucleus of cancer cells is an interesting subject forspeculation. Such altered chaperoning of materials into the nucleus could greatlyalter the pattern of gene expression.

CALRETICULIN IN CELL PROLIFERATION AND DIFFERENTIATION

There is very little evidence at present that calreticulin is involved in cell proliferationand differentiation. A retinoblastoma susceptibility (rb) regulatory element has beenidentified in the promoter region of the calreticulin gene (Valente et al. 1996). Therb gene is a negative regulator of cell cycle progression (see Sherbet and Lakshmi,1997b). This observation provides some basis for implicating the calreticulin genein cell proliferation. Dresselhaus et al. (1996) found an enhancement in the expres-sion of calreticulin cDNA in the maize zygote during cell division. It has also beenreported that calreticulin reduces intimal hyperplasia following arterial injury (Daiet al. 1997).

There is considerably more information with regard to the involvement of cal-reticulin in cell adhesion and morphology, which, by implication, may be deemedto support the view that it might influence also cell proliferation. However, itsexpression has been related to differentiation in NG108-15 neuroblastoma/gliomahybrid cells (N. Liu et al. 1996; Johnson et al. 1998). Higher levels of calreticulinexpression were observed in these cells, when induced to differentiate by exposureto dibutyryl cAMP (db-cAMP). Although there could be a true effect on cell pro-liferation, calreticulin, in general, does not seem to affect the proliferative capacityof the cell. However, cell numbers might increase merely by the ability of calreticulinto protect cells from apoptosis (see below).

CALRETICULIN IN CELL ADHESION

Two other examples of the physiological involvement of calreticulin are in theprocess of cell adhesion and cell spreading and in disease states engendered byautoimmune conditions. The influence exerted by calreticulin on cell adhesion tosubstratum was appreciated from early experiments of White et al. (1995). Theydemonstrated that anticalreticulin antibodies inhibited the binding of murine B16melanoma cells to a substratum covered with laminin. They also showed the anti-bodies bound to the cell surface. The interaction of cells with the substratum alsoappeared to involve mannoside residues. Further, cell spreading could be inhibitedby adding purified calreticulin to the medium, which presumably competed withcell surface calreticulin and denied the cells interactive binding with the substratum.Incidentally, this also confirms the lectin-like properties of calreticulin.



An exciting avenue has opened in the continued search for potential functionsof calreticulin. It seems to possess the ability to influence cell adhesion mediatedby integrins. The walk along this avenue is exciting because integrins are a class oftransmembrane heterodimeric glycoproteins that function as receptors for adhesionmediating macromolecules. The integrin family of cell surface receptors has beenextensively studied and it has been established that their mediation of signal trans-duction as well as cell adhesion is accomplished by linking up with the cytoskeletalmachinery. Vinculin, α-actinin, and talin provide the linkage to the actin cytoskel-eton. This involves a specific interlinking of the three proteins as follows: inte-grin/talin/vinculin:α-actinin/actin. There is considerable evidence that adhesion-dependent phenomena such as invasion and metastatic deposition are modulated bymacromolecules that make up the extracellular matrix (Sherbet, 1982, 1987; Sherbetand Lakshmi, 1997b).

In the first place, there is much evidence that implicates integrins in the modu-lation of cell adhesion by calreticulin. Embryonic stem (ES) cells from calreticulinknock-out mice, which are deficient in calreticulin, and fibroblasts derived fromcalretinin mutant mice have been reported to show severe reductions in integrin-mediated cell adhesion. Transfecting ES cells with calreticulin cDNA restored thisfaculty (Coppolino et al. 1997). The cytoplasmic domain of the α subunit of integrinscontains the amino acid sequence KXGFFKR to which calreticulin is known to bind(Shago et al. 1997). Cell adhesion is modulated by a mechanism in which theexpression of the linking proteins is regulated. Overexpression of calreticulin in L-fibroblasts is accompanied by increased cell–substratum and cell–cell adhesion, andthis overexpression also enhances cell spreading and decreases motility. Fibroblastcells overexpressing calreticulin also overexpress vinculin protein as well as itsmRNA, and reduced levels of vinculin correlate with a down-regulation of calreti-culin expression (Opas et al. 1996a). It is intriguing to note that another link in thesystem, namely talin, is unaffected by calreticulin overexpression. Nevertheless,calreticulin does not seem to affect the expression of α-catenin. The latter reputedlylinks other CBPs such as cadherin to the cytoskeleton (Sherbet and Lakshmi, 1997a,1997b). Shago et al. (1997) have shown that calreticulin can interfere with signaltransduction mediated by retinoic acid receptors that contain an amino acid sequencesimilar to that found in the α subunit of integrins. Retinoids are known to be ableto inhibit invasive ability (Fazely and Ledinko, 1990). Furthermore, they can influ-ence the flow of information from ligand binding to integrins. Therefore, this possiblecomplication of the perceived events should be borne in mind when interpreting theeffects of calreticulin overexpression on cell adhesion.

CALRETICULIN IN NEOPLASIA

Although the above discussion indicates a significant involvement of calreticulin incell adhesion and possibly also in cell proliferation, only a few studies have dealtwith its occurrence in tumours. Of the multiple functions attributed to calreticulin,its perceived effects on cell proliferation are the most relevant in relation to cancer.Calreticulin is one of several peptides of human breast cancers studied by Franzenet al. (1997). They state that it is expressed at higher levels in carcinomas than in



nonmalignant conditions of the breast, confirming an earlier study in which calreti-culin expression in ductal carcinomas of the breast was compared with normal breasttissue (Bini et al. 1997). In the latter study, the enhancement of expression ofcalreticulin, and a number of other peptides, appeared to be specific for epithelialneoplasms.

It would be premature to speculate on the nature of these correlative observations,but it has been suggested that calreticulin might control tumour progression byinitiating a loss of apoptotic potential in tumours (Bruchovsky et al. 1996). Calreti-culin levels increased in NG 108-15 neuroblastoma/glioma hybrid cells wheninduced to differentiate by treatment with db-cAMP (Johnson et al. 1998). Interest-ingly, however, antisense oligonucleotides for calreticulin do induce cell death inundifferentiated cells but not in differentiated cells. When the apoptotic pathway isexperimentally induced in HL-69 cells by exposing them to geranylgeraniol, theexpression of calreticulin is inhibited (Nakajo et al. 1996). Similarly, antisensecalreticulin oligonucleotides seem to protect prostate cancer cell lines from calciumionophore A23187-induced apoptosis. In this experimental system both androgenand calreticulin seem to be involved in the control of apoptotic events. A23187induces apoptosis in both LNCaP and PC-3 cells. However, androgen is able toblock apoptosis only in the androgen-sensitive LNCaP cells and not in the androgen-insensitive PC-3 cells. Furthermore, the inhibition of apoptosis of LNCaP cells byandrogen is reversible by antisense calreticulin oligonucleotides (Zhu and Wang,1999). One should recall here that in experimentally induced apoptosis of HL-60cells, changes were also found in another protein species together with a down-regulation of calreticulin expression (Nakajo et al. 1996). There was a decrease inthe phosphorylation of a 36-kDa protein species. Thus it might be premature to linkloss of calreticulin expression exclusively with apoptosis. Nevertheless, these obser-vations suggest that calreticulin might protect cells against apoptosis. It is possiblethat deregulation of apoptosis is involved in both the growth of primary tumours aswell as their metastatic deposits. Deregulation of growth control as well as inhibitionof apoptosis can lead to cell population expansion in tumours. The phenomenon ofmetastatic dormancy may imply that cell mass is maintained by apoptosis-mediatedregulation. Protection of cells from apoptotic death may lead to the appearance ofovert metastasis. Most of these arguments are speculative. It would be interestingto see how the apoptosis regulating genes are affected by calreticulin expression.

IMMUNOLOGICAL IMPLICATIONS OF CALRETICULIN FUNCTION

Calreticulin has been implicated in the pathogenesis of some autoimmune diseaseconditions. In systemic lupus erythematosus (SLE), calreticulin might support theformation of the Ro/SS-A autoantigen complex (reviewed by Eggleton et al. 1997),and this could be related to its response to heat shock and stress factors.

An interesting aspect of calreticulin in autoimmune diseases that has recentlycome to light is the amino acid sequence identity between calreticulin and the C1qreceptor (Malhotra et al. 1993). C1q is a subunit of the first component of comple-ment C1. C1q recognises and binds to circulating immune complexes (N.R. Cooper,1985). A consequence of this binding is that C1q is released from the complex (Sim



et al. 1979). It can now interact with the surface receptors on the various types ofblood cells and endothelial cells (Tenner and Cooper, 1981; Malhotra and Sim, 1989;Peerschke and Ghebrehiwet, 1987; Peerschke et al. 1993). The binding of C1q tothe receptor elicits a variety of immunological responses. The binding site of C1qto the C1q receptor or calreticulin has been identified (Stuart et al. 1996). Calreticulinoccurs predominantly in association with immune complexes and C1q in sera ofpatients with SLE (Kishore et al. 1997). Kishore et al. (1997) suggest that inflam-matory episodes occurring in autoimmune conditions could be attributed to therelease of calreticulin from leukocytes, leading to an antigenic reaction, and furtherby virtue of its identity with C1q receptor, to an interference with C1q-mediatedinflammatory effects. It is unclear at present why calreticulin should function as anautoantigen, but one reason could be that some parasitic organisms contain proteinsthat possess partial sequence homology with calreticulin.

The inhibition of expression of steroid hormone-regulated genes is one of thepostulated functions of calreticulin. As Coppolino and Dedhar (1998) have stated,it is truly remarkable that a single protein can have such a diversity of function.Clearly more work needs to be done on the multiple functions attributed to this CBP.Calreticulin has been highly conserved in the evolutionary process. Its conservationin a structural sense is probably less stringent than its functional conservation insignal transduction involving Ca2+ as a second messenger. Also it should be pointedout that calreticulin may not be the only CBP with multiple functions. The S100family protein S100A4 has been described to participate in a wide spectrum offunctions (Sherbet and Lakshmi, 1997b, 1998).

CALSEQUESTRIN AND INTRACELLULAR CALCIUM STORAGE

Calsequestrin is a high-capacity, low-affinity CBP and is the muscle equivalent ofcalreticulin. Like calreticulin, calsequestrin is associated with specialised regions ofthe SR and participates in intracellular calcium homeostasis. Its major function seemsto be intraluminal calcium storage. When intracellular calcium channels are acti-vated, calcium is released from the intracellular pools leading to increases in cyto-solic calcium levels, and this is an important ingredient of sustained muscle con-traction. The release of calcium requires the activation of ryanodine receptor/Ca2+

channels of SR (Berridge, 1993). These ion channels are formed by tetramericcomplexes consisting of 565-kDa monomers of the ryanodine receptor (Sutko andAirey, 1996). Three isoforms of ryanodine receptor are known. The type I isoformis restricted to skeletal muscle and is involved in voltage-gated Ca2+ release. Thedistribution of types II and III is less restricted. Type II is the main mediator of Ca2+-induced Ca2+ release in cardiac cells (Lai et al. 1988; Gianini et al. 1992). Ryanodinereceptors resemble IP3 receptors, which are another mediator of Ca2+ release, bothin respect of their structure and function (Sutko and Airey, 1996).

The calsequestrin molecule binds to and releases 4 to 50 calcium ions. Calse-questrin must be linked with the ryanodine receptor/calcium release channel, andindeed a close physical relationship does exist between them (B.E. Murray and



Ohlendieck, 1998). The anchoring of calsequestrin to the SR has been found toinvolve a family of transmembrane proteins. One such transmembrane protein istriadin. Triadin seems to anchor calsequestrin to junctional regions of SR and couplesit with the ryanodine receptor/Ca2+ release channel (W. Guo and Campbell, 1995).A 26-kDa protein identified by Mitchell et al. (1988) as a calsequestrin bindingprotein was purified and characterised by L.R. Jones et al. (1995). This protein, nowcalled junctin, bears significant sequence homology to triadin and aspartyl β-hydrox-ylase. Junctin contains a transmembrane domain and a short N-terminal portionextending into the cytoplasm. But a major portion of this molecule extends into thelumen of the SR (L.R. Jones et al. 1995). Junctin is distributed with ryanodinereceptors and calsequestrin in the SR, which suggests that it may also be involvedin calcium release from intracellular stores. The binding interactions are attributedto specific amino acid motifs that occur in the luminal portion of both triadin andjunctin. L. Zhang et al. (1997) have suggested that calsequestrin, junctin, and triadinmight form a quaternary complex with the ryanodine receptor to achieve calciumrelease. It seems possible that calsequestrin might regulate the calcium releasemechanism. This is suggested by the finding that in transgenic mice overexpressingcalsequestrin, the expression of junctin, triadin, and ryanodine receptor is down-regulated (L.R. Jones et al. 1998).

Very little is known about the involvement of calsequestrin in pathogenesis. In arecent report the sera of some patients with ocular myesthenia gravis showed thepresence of antibodies that react against calsequestrin (Gunji et al. 1998). This suggeststhat an autoimmune mechanism might be operating in the pathogenesis of this disease.

OSTEOCALCIN IN BONE METABOLISM AND OSTEOTROPISM OF CANCER

THE BIOLOGY OF OSTEOCALCIN

Osteocalcin is a noncollagenous protein. It is the most abundant matrix protein ofbone and dentine (Price, 1992). It is regarded as a marker of bone turnover andmetabolism, where bone resorption and formation are coupled. Osteocalcin is syn-thesised exclusively by osteoblasts and secreted into the extracellular matrix (ECM)during bone mineralisation. It is released also during osteoclastic degradation. There-fore, it is considered to be a marker of bone formation when formation and resorptionof bone are uncoupled (J.P. Brown et al. 1984; Delmas et al. 1985, 1986; R.H.Christenson, 1997). Osteocalcin expression in developing chick and rat embryoscoincides with the onset of mineralisation of the bone and hence it is considered toplay a role in this process (Hauschka et al. 1989). Osteocalcin functions as achemoattractant for osteoblast progenitor cells (Mundy and Poser, 1983). Also, boneresorption is poor under conditions of osteocalcin deficiency (Lian et al. 1984).These observations have supported a role for osteocalcin in bone resorption. Meta-static spread to the bone might activate bone metabolism, and therefore osteocalcinhas been investigated as a potential marker of metastatic progression in certain formsof cancer, such as breast cancer and carcinoma of the prostate, which tend to showosteotropic secondary spread. Osteocalcin has turned out to be a good marker for



bone turnover in osteoporosis (Delmas et al. 1983; Eastell et al. 1993). Mutationsof the gla protein, a member of the osteocalcin family of proteins, have beensuggested as a causal factor in the autosomal recessive Keutel syndrome, which ischaracterised by abnormal calcification of the cartilage (Munroe et al. 1999).

CALCIUM-BINDING PROPERTIES OF OSTEOCALCIN

Osteocalcin is a non-EF-hand CBP and binds 10 Ca2+ per mole protein at pH 7.5.The binding is virtually halved when pH is lowered to 5.0 (Kobayashi et al. 1998).Kobayashi et al. (1998) produced, by proteolysis, peptides of less than 8 amino acidsfrom the N- and C-terminal ends of osteocalcin. The middle fragment bound 4 to 5Ca2+ per mole protein. A proline-rich segment and three γ-carboxyglutamic acidresidues are believed to participate in calcium binding (Heizmann, 1996).

OSTEOCALCIN GENE STRUCTURE AND FUNCTION

The osteocalcin gene has been mapped to human chromosome 1q25–q31 (Barille etal. 1996). The gene is regulated developmentally and in a tissue-specific manner. Thetissue-specific expression seems to be due to the fact that the osteocalcin promoterfunctions only in cells of the osteoblastic lineage. This is suggested by the work ofKo et al. (1996), who constructed a virus vector carrying the thymidine kinase (TK)gene under the control of osteocalcin promoter. When this vector was introduced intoosteoblastic cells TK gene was expressed, but no expression was detected in NIH3T3or in a cell line derived from a transitional cell carcinoma of the bladder. The regulationof the osteocalcin gene is mediated by several transcription factors. Sequences thatcan bind the general transcriptional promoter AP1 have been identified in the promoterregion of the osteocalcin gene (Goldberg et al. 1996; Lian et al. 1996). Lian et al.(1996) have also described two highly conserved regulatory motifs, which are, some-what surprisingly, reported to relate to the cell type-specific expression of osteocalcin,albeit being functional in osteoblastic as well as nonosteoblastic cell lines. The mod-ulation of expression of osteocalcin from the proliferative state to the differentiatedstate accompanying mineralisation of the ECM involves the function of a silencerelement. An osteonectin silencer element (OSE) has been identified in the humanosteocalcin gene, constituting a 7-bp (+29 to +35) sequence TGGCCT of the first exonof the osteocalcin gene (Y.P. Li et al. 1995). In proliferating cells, OSE is activatedby the binding of an OSE-binding protein (OSE-bp) and consequently osteocalcin iseither not expressed or is found in very low levels. When OSE-bp expression is down-regulated OSE is inactivated, which leads to enhanced osteocalcin expression and theonset of ECM mineralisation. A silencer element also occurs in the first intron of therat osteocalcin gene. Mutation of this suppressor element seems to inactivate thesuppressor function (Goto et al. 1996). Other genes such as Dix-5 and MsX-2 havebeen shown to regulate the expression of osteocalcin during osteoblast differentiation.Dix-5 is believed to repress osteocalcin gene transcription (Ryoo et al. 1997). Inter-estingly, there is an increase in the expression of Dix-5 with osteoblast differentiation,which suggests an important regulatory role for this gene in the control of ECMmineralisation.



Among other regulatory elements that have been identified is a short motif calledthe OSCARE-2. OSCARE-2 is reported to bind a number of proteins including AP1.Furthermore, this element appears to be able to bind also nuclear proteins that areinduced by vitamin D3 (VD3; 1α, 25-dihydroxyvitamin D3) (Goldberg et al. 1996).As described below, VD3 receptor response elements have also been identified inthe osteocalcin promoter region. This provides a molecular basis for the cooperationof VD3 and osteocalcin in the regulation of several processes such as calciumhomeostasis, osteoblast differentiation, and cell proliferation and apoptosis.

REGULATION OF OSTEOCALCIN BY VITAMIN D3

Vitamin D3 (VD3) is an important regulator of calcium homeostasis. It plays a majorrole in bone mineralisation of type I collagen matrix, the transport of calcium andphosphate. VD3 influences several important biological processes such as cell dif-ferentiation, apoptosis, inhibition of cell proliferation, and cell signalling. It is knownto suppress proliferation of T cells and their ability to produce cytokines.

The function of VD3 is mediated by the VD3 receptor (VDR). VDR is aphosphoprotein transcription factor that functions in the form of a heterodimer withthe retinoid X receptor (RXR). VD3 induces the formation of a heterodimericcomplex between VDR and RXR (Carlsberg and Polly, 1998). VDR has a numberof identifiable domains that participate in DNA binding, binding of the ligand,receptor dimerisation, and gene transactivation, as well as the C-terminal activationfunction (AF-2) domain required for co-factor interaction (reviewed by Issa et al.1998). RXR seems to enhance the DNA-binding affinity of VDR as well as itsspecificity. VDR/RXR transcription factor regulates transcription of target genes byzinc finger-mediated DNA binding and protein–protein interaction. However, theRXR ligand 9-cis retinoic acid (RA) prevents RXR from forming heterodimers withVDR and promotes the formation of RXR–RXR homodimers. This effectivelynegates the transcription of VDR responsive genes. On the other hand, onceVDR–RXR is generated, these seem to block the formation of RXR homodimers(Haussler et al. 1997).

The genes that encode the major bone matrix proteins, namely, osteocalcin,osteopontin, and β3-integrin, are among the target genes activated by VDR. A VDresponse element (VDRE), which consists of hexanucleotide repeats with inter-vening trinucleotide spacers, has been identified in the promoter regions of thesegenes. VD3 induces the synthesis of osteocalcin by osteoblast cells in both in vitroand in vivo conditions (Price and Baukol, 1980). VD3 not only increases thetranscription of the osteocalcin gene but also seems to increase the half-life ofosteocalcin mRNA (Mosavin and Mellon, 1996). However, species differencesmight exist in VD3-induced osteocalcin expression. Transgenic mice carryinghuman osteocalcin gene do respond to VD3 and show increased serum levels ofhuman osteocalcin, but there is no increase in the endogenous mouse osteocalcin.These species-specific differences are believed to be due to possible differencesin the regulatory sequences of the mouse and human osteocalcin genes (Clemenset al. 1997; Sims et al. 1997).



OSTEOCALCIN IN CELL PROLIFERATION AND DIFFERENTIATION

The osteocalcin gene is expressed in consonance with the inhibition of cell prolif-eration and the onset of cell differentiation and ECM mineralisation (Y.P. Li et al.1995). VD3, as discussed above, induces osteocalcin expression, but on the otherhand, it inhibits cell proliferation, apparently with the mediation of cdk inhibitors.M.J. Campbell et al. (1997) synthesised a number of VD3 analogues and demon-strated that these were capable of inhibiting proliferation of the prostate cancer celllines LNCaP, PC-3, and DU145. The inhibition of cell proliferation was accompaniedby an up-regulation of the expression of the cdk inhibitors p21waf1 and p27kip1. Anup-regulation of p21waf1 expression has also been encountered in VD3-induced dif-ferentiation of human MG-63 osteosarcoma cells, and this has been shown to beindependent of p53 function (Matsumoto et al. 1998). Therefore, these observationsseem to define a direct and novel pathway of inhibition of cell cycle progression byVD3.

Other hormones, such as thyroid hormones, that regulate the differentiation ofosteoblasts also seem to function through the mediation of osteocalcin. Triiodothy-ronine (T3) has been found to inhibit the proliferation of the osteoblast-type MC3T3-E1 cells and, in parallel, induce the expression of osteocalcin mRNA and proteinand alkaline phosphatase activity (Varga et al. 1997; Luegmayr et al. 1998). Oestro-gen has been reported to increase cell proliferation in the early stages of in vitroculture of osteoblasts derived from mouse bone marrow. The effects of oestrogenseem to involve the osteoblast-specific transcription factor osf2 (cbfa1) (Sasaki-Iwaoka et al. 1999). Oestrogen increases the expression of osteocalcin, alkalinephosphatase, osteopontin, and transforming growth factor (TGF)β-1 as well ascollagen type 1. Furthermore, exposure to oestrogen also increased the formationof bone nodules. Anti-oestrogens (Qu et al. 1998) blocked all these effects. Post-menopausal breast cancer patients treated with the anti-oestrogen tamoxifen havereduced (22%) levels of osteocalcin in serum (Marttunen et al. 1998).

Fibronectin (FN) is a component of the ECM that has been implicated in severalbiological activities. Thus FN influences cell adhesion to substratum, spreading, andmodulation of cell shape. It influences membrane ruffling and cell motility and alsois associated with cell differentiation (Sherbet, 1987). The pattern of FN expressionin osteoblast differentiation has attracted some attention. Although there is very littleinformation about its role in vivo, there seems to be some correlation between theexpression of FN and osteocalcin, in relation to the state of proliferation and differ-entiation in vitro. In the initial period of growth of osteoblasts, derived from foetalrat calvaria, on a collagen-coated substratum, a 50 to 70% reduction of FN occurs,but the expression of osteocalcin, osteonectin, and osteopontin is up-regulated sev-eralfold (Lynch et al. 1995). Similarly, in chicken osteoblast cell cultures at 6 to 18days, FN is associated with the cell membrane, but subsequently it remains associ-ated with the ECM in a fibrillary form. Overall, FN increases in the early periodsof cell culture and its levels are then maintained. In contrast, the major bone matrixECM markers show increased expression with the onset of differentiation (Winnardet al. 1995). There is no suggestion, however, that these events are necessarily related,



beyond the recognition that collagen type I could be involved in the signal trans-duction pathway. Possibly, in the initial stages where it is bound to the membrane,FN could be mediating extracellular signals through the occupancy of its specificintegrin receptors. The subsequent association with ECM in a fibrillary form isclearly a postsignalling event. This suggestion seems to be upheld by the experimentsdescribed by Moursi et al. (1996). These authors found that anti-FN antibodiesinhibited the formation of bone-like nodules and the expression of osteocalcin andalkaline phosphatase genes. Generally compatible with this is the ability of VD3 toregulate FN expression at the transcriptional level. A VDRE has been identified inthe FN gene (Polly et al. 1996). Interestingly, osteoblast differentiation requiresmore than the RGD domain of FN that is functional in cell adhesion. Certainly, theexperiments of Moursi et al. (1996) indicate that FN regulates the differentiation ofosteoblasts.

There is now general acceptance that integrin receptors form an important linkin the transduction of signals generated by ECM components (Sherbet and Lakshmi,1997a). Integrin receptors are actively involved in the recognition of and interactionwith ECM ligands occurring in the process of osteoblast differentiation (Uemura etal. 1997). The integrin α5β1 has been identified as the critical component in FNinteraction with osteoblasts (Moursi et al. 1997). The murine MC 3T3 cell lineresponds to ascorbic acid treatment by synthesising a collagen matrix, and collagentype 1 ligand seems to be essential for the subsequent expression of osteoblastmarkers as well as the activation of the osteocalcin promoter element, OSE2. OSE2is recognised by the osteoblast-specific transcription factor osf2 (also known ascbfa1, AML3, PEBP2, and alpha A). The latter is expressed only in osteoblastic celllines, such as MG63, ROS 17/2.8, and MC3T3-E1, but not in cell lines of nonos-teoblastic origin (Sasaki-Iwaoka et al. 1999). When the collagen type 1 receptor α2-subunit is blocked, the binding of osf2 to OSE2 is also blocked, with consequentinhibition of transcription of the osteocalcin gene (Xiao et al. 1998). These exper-iments indicate the importance of the interaction between collagen type 1 and itsintegrin receptor for transduction of the signal that can elicit osteocalcin geneactivation. Much effort needs to be directed toward a dissection of the pathway ofsignal transduction in order to provide the crucial evidence that can link these eventsin a coherent fashion.

Intracellular adhesion as well as cell–substratum adhesion is determined by thecomponents of the ECM. Their temporal and spatial expression is invariably asso-ciated with the alterations in the adhesive interactions, as well as changes in cellmotility or migration on a substratum or invasive behaviour of cancers. The facultyof migration could be an important feature in bone resorption. Osteoclast precursors,for example, need to target sites of bone resorption and they do possess the abilityof diapedesis across capillary endothelia. The modulation of certain ECM compo-nents such as FN, in conjunction with osteoblast differentiation and osteocalcinexpression, has inevitably led to investigations of a potential association of osteo-calcin with cell migration. Stringa et al. (1995) set up osteoblast cultures from 7-day-old rat tibia fragments. They found that when the cells were exposed to par-athyroid hormone they synthesised and secreted large amounts of osteocalcin



together with collagen type III. The conditioned medium from these cultures stim-ulated the migration of EA HY-926 endothelial cells in vitro. Osteoblast cellsobtained from rat calvaria adhere and show migratory behaviour upon plating onthree-dimensional matrices. These migrating cells expressed osteocalcin as indicatedby immunohistochemical staining (Attawia et al. 1995). TGFβ-1 mRNA expressionin regenerating tissue in distraction osteogenesis is said to coincide with osteoblastmigration and ECM mineralisation (Mehrara et al. 1999). However, there is alsomuch evidence that TGFβ-1 can inhibit osteonectin expression. Hence, it is con-ceivable that, in this experimental model, this promotion of migration by TGFβ-1could be an effect mediated by means other than involving osteocalcin. TGFβ-1 isa highly versatile cytokine that can elicit a wide-ranging cellular response. Not leastamong these is that it can induce the expression of S100A4 protein (Okada et al.1997), which has itself been implicated in the induction of cell motility.

The identity of the ECM component that might be instrumental in osteoblastmigration is uncertain at present. The two major adhesion mediating glycoproteinsFN and laminin both regulate adhesive interactions involving osteoblasts. As seenearlier, FN expression does vary with the state of cell proliferation and differentia-tion, but it has not been directly implicated in the motile behaviour of osteoblasts.Laminin, on the other hand, does seem to mediate the adhesion to substratum aswell as migration of osteoclasts. There is also a suggestion that there might be someform of cooperative functioning of laminin and FN in osteoblast migration. It hasbeen suggested, for instance, that FN might be secreted in response to laminin-2-mediated adhesion (Colucci et al. 1996). This postulate needs to be tested further.There are a number of possibilities that can be tested, e.g., whether there is de novosynthesis of FN, whether there is a deletion of FN into the medium, whether thereis a modulation in the expression of FN receptors, etc. But Colucci et al. (1996)have shown that osteocalcin induces the migration of osteoclasts on laminin-2- butnot collagen-coated substratum. One could concede easily that osteocalcin canpromote migration involving ECM components and their particular integrin recep-tors. However, it is unclear at present how one can envisage a physical mechanismthat transduces the osteocalcin signal to the cytoskeletal machinery to bring aboutcell locomotion. Also relevant in this context is the question of whether otherbiological macromolecules, such as the cadherins, might be involved too. Cadherinhas been studied extensively for its ability to suppress invasion by cancer cells and,indeed, it has often been described as an invasion suppressor gene. Now there isevidence that VD3 analogues up-regulate the expression of E-cadherin in prostatecancer cell lines (H.D. Campbell et al.1997). In osteoblastic cell lines, VD3 ana-logues up-regulate osteocalcin expression. It would be of much interest to examinethe status of cadherin expression in osteoblasts and determine what effects VD3exerts on cadherin.

Besides its effects on cell motility, TGFβ is also a powerful modulator of growthand differentiation. TGFβ peptides are known to inhibit proliferation of a numberof cell types. More than coincidental is that the mechanism by which TGFβ bringsabout growth inhibition involves cdk inhibitors, e.g., p27kip1 (see Sherbet and Lak-shmi, 1997b for references). TGFβ isoforms subserve many functions, includingbone turnover. Banerjee et al. (1996) found that TGFβ-1 down-regulated osteocalcin


60


expression in ROS 17/2.8 osteosarcoma cells. Similar effects have also beendescribed in foetal mouse long bone cultures (Staal

et al.

1998). Thus, although itwould appear that TGF

β

could be regulating bone metabolism by inhibiting osteo-calcin expression, its effects on cell proliferation are achieved by a different routeinvolving cyclin/cdks (Figure 9). This conclusion is supported by the suggestionmade by J.H. Liu

et al.

(1999) that TGF

β

might be inhibiting G

1

-S arrest partly byinactivating cyclin B/cdc2 kinase. TGF

β

treatment results in the phosphorylation ofthe cdc2 component in the TGF

β

receptor II–cyclin B2–cdc2 complex and down-regulates its kinase activity. Despite this, it should be recognised that there is muchobvious coordination in the functioning of osteocalcin, VD3, and TGF

β

in theinhibition of cell proliferation and induction of cell differentiation.

O

STEOTROPISM

OF

M

ETASTATIC

D

ISSEMINATION

Metastatic spread of cancer is a nonrandom process. The perceived organ specificityof metastasis can be attributed to a variety of factors intrinsic to the cancer cell aswell as to the target organs (see Sherbet, 1982 for review). Metastatic spread to thebone is a common occurrence in certain forms of cancer. It has been argued oftenthat bone metastases can activate the processes of bone metabolism. For that reason

FIGURE 9

A graphical illustration of how some of the functions of osteocalcin, VD3,and TGF

β

overlap, and of the putative mechanisms involved. AP1,

jun/fos

transcription factor;cdk, cyclin-dependent protein kinase; FN; fibronectin; RA, retinoic acid; RXR, retinoid Xreceptor; TGF

β

; transforming growth factor

β

; VD3; vitamin D3; VDR; VD3 receptor; VDRE;VD response element. (Based on references cited in the text.)

0942/C04/frame Page 60 Friday, October 27, 2000 9:13 AM


osteocalcin has been regarded as a potential surrogate method for detecting meta-static spread as well as for the purpose of monitoring the outcome of therapy onmetastatic bone lesions.

Several markers of bone metabolism have been employed in studies of this kind.Among those employed are osteocalcin, C-terminal peptide of type I procollagen(PICP), N-terminal peptide of type III procollagen (PIIINP), pyridinoline cross-linked C-terminal peptide of procollagen I (ICTP), and bone-specific alkalinephosphatase (BA-1p). Bloomqvist et al. (1996) found that ICTP and PICP levelscorrelated with that of osteocalcin, but not with urinary or serum calcium. All threemarkers correlated with the number of metastases detected by bone scans.

The aminobisphosphonate ibandronate has marked osteoclast inhibitory activityand has been investigated as a treatment modality for metastatic bone disease andcancer-induced hypercalcemia. In combination with taxol/taxotere, ibandronateappears to be able to inhibit invasion of the bone by the human breast cancer cellsMDA-MB231 (Magnetto et al. 1999) and the development of bone lesions in animalsinjected with myeloma cells (Dallas et al. 1999). Ibandronate markedly affects boneresorption in metastatic bone disease (Coleman et al. 1999). Osteocalcin, PICP, andBA-1p have been found to be reliable dose-dependent markers in a phase II clinicaltrial with ibandronate treatment of metastatic breast cancer. They were also foundto be suitable for monitoring the effects of treatment of osteoporosis (Schlosser andScigalla, 1997). However, Bombardieri et al. (1997) seem to disagree that any ofthese markers can replace bone scans.

So far as prostate cancer is concerned, ICTP has been reported to reflect bonemetastasis more accurately than other markers, including PSA (prostate-specificantigen). Osteocalcin showed no correlation with metastatic spread (Maeda et al.1997). In another study, PICP and BA-1p were found to increase with progressionas indicated by bone scans. A slight increase in osteocalcin was also reported inpatients with remission of metastatic bone lesions, but not related to progression(Koizumi et al. 1995). Obviously more clinical trials are needed to arrive at any firmconclusions. This need is underlined by laboratory work on the differentiation ofosteoblastic cells, using conditioned media of the human prostatic carcinoma PC-3cells and cell extracts. The effects of the conditioned medium have been studied ontwo osteoblastic cell lines, namely, a primary cell line derived from foetal rat calvariaand a rat osteosarcoma cell line ROS 17/2.8. The conditioned medium inhibitedbone nodule formation in both cell lines without affecting cell proliferation. Theconditioned medium also inhibited osteocalcin mRNA but not that of osteopontin(Kido et al. 1997). These data suggest there might be other factors involved in theformation of bone metastasis.

A development with much promise is the use of toxic gene therapy for cancer,with osteocalcin promoter for targeting the expression of the toxic gene in thetreatment of tumours of osteoblastic lineage and metastatic tumours. Ko (1996) madean adenovirus construct with the TK gene under the control of an osteocalcinpromoter (Ad-OC/TK). When this viral construct was introduced into cells of osteo-blastic lineage, e.g., murine ROS cells and human MG-63 cells, the TK gene wasexpressed. In contrast, no TK expression occurred in nonosteoblastic cell lines. Theaddition of acyclovir (ACV) caused cell death in vitro. Similar growth inhibition



and cytotoxicity were encountered in vivo when the adenovirus construct wasinjected into murine ROS osteosarcoma followed by intraperitoneal injection ofACV. (Cheon et al. 1997) has extended its findings and has reported that theadministration of the Ad-OC/TK construct coupled with methotrexate was highlyefficacious in the treatment of osteosarcoma. Shirakawa et al. (1998) adopted thisstrategy in another experimental tumour model. They introduced ROS rat osteosar-coma cells into nude mice by intravenous route. These cells formed tumour nodulesin the lung. They then injected the Ad-OC/TK construct into the tail vein withsubsequent intraperitoneal ACV treatment. This treatment markedly reduced thenumber of tumour nodules in the lungs and significantly enhanced survival. Further-more, the cell-type specificity of the functioning of this construct was also demon-strated by Shirakawa et al. (1998). They constructed the adenovirus vector with RSV(Rous sarcoma virus) promoter, rather than by osteocalcin promoter, and placed theE. coli β-galactosidase gene under its control. When this construct was used, therewas no osteoblast-specific expression of the β-galactosidase gene, but it wasexpressed nonspecifically in lung parenchyma.

This constitutes a rather elegant demonstration of cell-type-specific targeting ofgene therapy coupled with cytotoxic drugs and certainly deserves further testingusing experimental models of spontaneous metastasis.


63

5

The EF-Hand Calcium Binding Proteins

MOLECULAR ORGANISATION OF CALCIUM-BINDING EF-HAND PROTEINS

The members of the EF-hand CBPs share a general structural feature of possessingvariable numbers of domains that bind to Ca

2+

with high specificity and affinity.The EF-hand structural motif was first reported from the crystal structure of carpparvalbumin (Kretsinger and Kockolds, 1973; Kretsinger, 1980). Specific struc-tural features have been defined as being necessary for calcium binding. The EF-hand consists of a consensus sequence of 12 amino acid residues in a helix–loop–helix configuration that can ligate Ca

2+

(Linse and Forsen, 1995). The Kretsingerprinciple states that calcium binding is coordinated by five oxygen-containingamino acid residues and a conserved glycine residue, which causes the bendingof the loop. The calcium-binding affinities of these proteins range from K

d

10

–4

to 10

–9

M

, and this is dependent on the amino acid sequence of the EF-hand loop.EF-hand loops occur as a pair with antiparallel

β

-sheet interaction between thetwo loops. A high degree of cooperativity exists between calcium-binding loops(M. Zhang

et al.

1995; Ames

et al.

1995). The regulatory (sensor) and calciumbuffer functions of EF-hand CBPs are closely related to the conformational reor-ganisation that occurs as a consequence of calcium binding. Conformationalchanges are essential for the regulatory function of EF-hand proteins, whereas fortheir calcium buffer function only a small conformational change appears to suffice(Ikura, 1996). A pair of EF-hands may form a globular domain and the domainswithin a protein may be functionally different, i.e., they may be either a regulatorydomain or a buffer domain. Thus calmodulin has a regulatory domain at the N-and C-terminal regions of the protein. Troponin C and recoverin have an N-terminalregulatory domain and a C-terminal buffer or structural domain. In contrast, 9-kDa calbindin has only one buffer domain at the N-terminal end. The regulatorydomains undergo conformational changes upon binding to calcium and participatein the activation of target proteins (Ikura, 1996).

The remarkable similarity in the genetic organisation of the genes coding forthe EF-hand proteins has led to the formulation of an evolutionary profile of the EF-hand protein family. As shown in Table 4, three classes of EF-hand proteins can beidentified. Type I proteins include calmodulin, myosin light chains (MLCs), andspec I. The coding sequences of these are interrupted by five introns, of which fouroccur at identical sites. The type II proteins, the S100 and related proteins typicallypossess two EF-hands and the corresponding genes have two introns, one of them


64


in the 5

′

untranslated region and the second separating the two coding domains.Type III proteins, such as the 28-kDa calbindin and calretinin, have six EF-hands.The corresponding genes have ten introns that occur with the EF-hand coding domainand they occur at identical sites in the 28-kDa calbindin and calretinin genes (seeHeizmann and Hunziker, 1990 for a full review) (see Table 4).

Kawasaki

et al.

(1998) have proposed a more elaborate classification of EF-handCBPs based on the congruence of EF-hand domains. They have identified as manyas 43 subfamilies on this basis. Of these, a group composed of 13 subfamilies isthought to have evolved and diversified, by gene duplication and fusion, from aprogenitor domain. The remaining subfamilies do not show this probable evolution-ary relationship and, indeed, some EF-hand CBPs do not appear to lend themselvesto forming natural groups of subfamilies. The evolutionary relationship of EF-handCBPs is also highlighted by the similarity that odd and even numbered domains ofone protein show to the corresponding odd-and even-domains of another protein.Nonetheless, the framework for discussion adopted in the present work is based onthe functional, rather than structural, homology of the CBPs.

TABLE 4EF-Hand Calcium Binding Proteins and Their Genetic Characteristics

Protein Class Name

Number of EF

Domains Genetic Organisation

Type I Parvalbumin 3 5 introns interrupting EF-hand coding sequences

Calmodulin 4Myosin light chains 4Centrin (caltractin) 4Spec I (sea urchin) 4

Type II S100 proteins 2 2 introns: one in the 5

′

untranslated region and the other between the two EF-hand domains

CalcyclinS100A8 (MRP-8, myeloid-related protein)

In S100A8/A9, one EF-hand occurs at each terminal region

S100A9 (MRP-14)Type III Calbindin, 9 kDa 2 2.5 kbp; 3 exons and 2 introns; exons 2

and 3 coding for EF-hand domainsCalbindin, 28 kDa 6 10 introns; 7 within the EF-hand coding

domainsCalretinin 6 8 introns; distribution coinciding with

their distribution found in calbindin

Source

: Based on Heizmann and Hunziker (1990) and Ikura (1996) and other references cited in thetext.



65

CALCIUM BINDING AND THE MOLECULAR CONFIGURATION OF CALCIUM-BINDING PROTEINS

A characteristic feature of the binding of Ca

2+

to CBPs is the conformational changesthat these proteins undergo as a result. These changes in their molecular configurationhave a significant impact on their function. The calcium-induced switch betweenactive and inactive forms of target proteins is often a consequence of changes inmolecular conformation. Ca

2+

can induce conformational changes in both non-EF-hand and EF-hand CBPs. Calcium-mediated modulation of conformational changesin actin figures prominently in actin dynamics. For instance, following calciumbinding, actin seems to take a conformation that is conducive to strong binding withMLC (Avrova

et al.

1998). The gelsolin family proteins are regulated by calcium,interact with actin, and induce conformational changes. These conformationalchanges seem to be essential in actin dynamics. Cross-linking of actin is mediatedby actin-binding proteins such as fimbrin. The function of fimbrin itself is regulatedby conformational changes induced by the binding of calcium ions to fimbrin.

The EF-hand CBPs contain one to six EF-hand calcium-binding domains. TheEF domain consists of a loop of 12 amino acid residues flanked by

α

-helical domains.Calcium binding to these domains brings about changes in the conformation of thesedomains, the major changes being the relative orientation of EF-hands. The EF-hands open upon Ca

2+

binding and assume a closed conformation state in the absenceof Ca

2+

(M.R. Nelson and Chazin, 1998). The open configuration is conducive tointeraction with target proteins. However, CBPs might differ in the assumption ofthe closed position of EF-hands in the absence of Ca

2+

. Thus the C-terminal EF-hand of CaM shows a closed position, whereas the C-terminal EF-hand of MLCwould show a semi-open position. This would in effect mean a differential responseof these proteins to the presence of calcium. In other words, the interaction of someCBPs with their target molecules might not be calcium sensitive. Sastry

et al.

(1998)have studied the three-dimensional structure of calcyclin and other EF-hand proteinssuch as calbindin D-9K (CBD9K) and reported the occurrence of major differencesin the calcium-induced changes between these proteins. The flexibility and dynamicsof the EF-hand are very much dependent on the amino acid sequence of the entireEF-hand (Malmendal

et al.

1998). The degree as well as the nature of the variouschanges are probably characteristic of individual EF-hand proteins, as in the caseof S100 proteins. Furthermore, a combination of changes might endow individualS100 proteins with specific functional properties.

Conformational changes can affect protein function in many ways. For instance,the activation of calpains, which possess protease activity, seems to be a consequenceof conformational changes in the molecule that result in the re-orientation of theprotease domain to produce a functional active site of the enzyme (Hosfield

et al.

1999). A classical example of the effects of another facet of molecular configurationis provided by CaM. The changes undergone by CaM seem to enable the protein tobind to a wide spectrum of target proteins and bring about their activation. Guanylatecyclase-activating protein (GCAP), on the other hand, seems to be inactivated bycalcium binding to EF-hands 3 and 4. Mutations of EF-hand 3 suffice to inactivate


66


GCAP. Possibly, this suggests that calcium binding to EF-hand 4 is a secondary,albeit important, event. An intriguing example would be a CBP that at least putativelycontains domains that are involved in diametrically opposed functions. Osteonectinis a prime example of this. Two subdomains have been identified in the osteonectinmolecule, of which one is involved in the inhibition of endothelial cell proliferationand the other stimulates endothelial cell proliferation

in vitro

. In a similar fashion,osteonectin is able to exert differential effects on cell adhesion and spreading.Possibly, all these effects are regulated in a tissue-specific manner. The process ofregulation could involve a mechanism in which molecular conformation mightconceivably play a prominent part. One could postulate that changes in molecularconfiguration might change the relative orientation of one functional domain to theother. One can cite another example taken from the signal transduction system. Assuggested in relation to osteonectin, conformational changes occurring upon Ca

2+

binding might be linked closely with function. Only nonmuscle and cytoskeletalisoforms of

α

-actinin are capable of binding Ca

2+

, and only these isoforms arefunctional in the interaction with the cytoskeleton. This interaction is essential forsuccessful signal transduction.

Posttranslational changes of CBPs can influence calcium binding and in thisway also influence molecular configuration and function. Several S100 proteins aremodified at the posttranslational level. S100A8 and S100A9 are often found in thephosphorylated form. Phosphorylation can affect the functions of these proteins andcan be a positive or negative regulator. It can influence the binding of Ca

2+

and otherions and the configuration that molecules undergo as a consequence. These changesin molecular configuration are an essential feature of the process of target proteinrecognition. Phosphorylation can also target S100 proteins to specific subcompart-ments of the cell. For instance, S100A8 and S100A9 translocate to the cell mem-brane, following their phosphorylation (Guignard

et al.

1996). Recoverin is myris-toylated at its N-terminus. It has been suggested that this modification inducescooperative calcium binding. More precisely, calcium binding to EF-hand 3 of RCNis believed to produce reorientation of the molecule to enable EF-hand 2 to bindcalcium. The myristoylation of recoverin might be involved functionally in thetargeting of the molecule to the appropriate cellular compartment.

Some CBPs might influence molecular changes in other CBPs merely by alteringintracellular calcium homeostasis. It has been shown, for instance, that calretinindeficiency markedly alters intracellular calcium levels in calretinin-null Purkinjecells. As a result, calbindin D-28K (CBD) is saturated with calcium and undergoesconformational changes. Obviously, in this case calretinin functions as a calciumbuffer and thereby influences or controls the molecular configuration and functionof another CBP.

Calcium binding also alters the quaternary structure and consequent oligomer-isation of these molecules. Oligomerisation is essential for the binding of heat shockproteins to their targets and their oligomerisation is also known to be regulated bycalcium. Such changes could alter the pattern of target recognition and in this wayconfer on S100 proteins the ability to interact with diverse target molecules and thusdefine the specificity of their function. Besides calcium, the biological functions ofCBPs might be regulated by other divalent cations such as Zn

2+

and Cu

2+

(Heizmann



67

and Cox, 1998). EF-hand proteins can bind other divalent ions such as Mg

2+

andZn

2+

. Indeed, a subgroup of S100 proteins that binds Zn

2+

with high affinity hasbeen identified. These bind two to four Zn

2+

ions per protein monomer (Fohr

et al.

1995; Fritz

et al.

1998). As with Ca

2+

binding, Zn

2+

ion binding also occurs withmuch cooperativity. In S100A3, for instance, the binding of the first Zn

2+

ionfacilitates the binding of the second Zn

2+

ion. This cooperative binding appears tobe associated with and, indeed, requires structural reorganisation of the molecule(Fritz

et al.

1998). Whether these conformational changes also have functionalsignificance is still unclear.

S100 proteins are known to form dimers readily in both intracellular and extra-cellular environments. Both homo- and heterodimers are formed by means of dis-ulphide linkage, or the monomers may be held together by ionic bonds, dependingon the environment and conditions. The relevance of the formation of dimers ortetrameric complexes to their function is yet uncertain, except probably in the caseof S100A8 and S100A9. These are translocated from the cytosol to the plasmamembrane. They seem to be able to form tetrameric complexes (A8

2

/A9

2

).Some recent work has brought to light the possibility that, at least in some CBPs,

both Ca

2+

and Zn

2+

ions might be involved in the regulation of their function. Forinstance, the heterodimeric complexes of S100A8 and S100A9 bind arachidonicacid and transport it to its target site where it is metabolised. Kerkhoff

et al.

(1999c)have found that Ca

2+

induces the binding of arachidonic acid by the S100A8/A9complex, but this is reversed by Zn

2+

. This is an interesting joint functional involve-ment of these divalent cations, which contrasts with the demonstrations hitherto thatthey act on their own in bringing about changes in molecular configuration in thecontext of their function. Precise knowledge of the changes in conformation occur-ring here is still lacking. It would appear that S100A8 and S100A9 can formheterodimers in the absence of Ca

2+

. Possibly, Ca

2+

induces a primary change in themolecular configuration of the constituent monomer to expose Zn

2+

-binding sites.In the wake of binding of the zinc ions a secondary molecular reorganisation couldbe occurring, which is incompatible, by reason of spatial constraint, with the con-tinued association of arachidonic acid with the S100A8/A9 complex. It would beessential, therefore, to know the disposition of Zn

2+

-binding sites relative to theterminal calcium-binding EF-hand domains.

Fritz

et al.

(1998) pointed out that Zn

2+

-binding cysteine residues are clusteredin the C-terminal part of the S100A3 molecule that is said to be involved in therecognition of target proteins. Transcription factors recognise and bind to specificsequences in the promoter regions of genes, which initiates gene transcription. Thebinding of transcription factors to the DNA has been attributed to specific structuralmotifs that are present in the transcription factors. Many transcription factors possessZn

2+

-binding domains in the form of protrusions and these are described as zincfingers. The zinc finger motif binds to specific elements in the promoter. Zinc fingers,first identified in the transcription factor TFIIIA derived from

Xenopus

, are knownto be present in a large family of proteins that includes the thyroid hormone receptor,vitamin D3 receptor, and retinoic acid receptors, among others. Four types of zincfinger motifs have been identified. The TFIIIA zinc finger is regarded as the classicaltype or the prototype, which binds one Zn

2+

atom

via

two cysteine and two histidine


68


conserved residues and to DNA as a monomer with high affinity. The zinc atomsseem to stabilise the structure of the zinc finger (Miller

et al.

1985; Berg, 1990).The TFIIIA shows dual specificity in that it binds to DNA (5S RNA gene) as wellas to 5S RNA (Pelham and Brown, 1980). The second type of zinc finger motifoccurs in the steroid/nuclear receptor. Here the zinc finger binds two Zn

2+

atomsbinding to four cysteine residues each, and these receptors bind to the DNA as adimer (Hard

et al.

1990; Schwabe and Rhodes, 1991). The third type of motif isfound in retroviral proteins and is characterised by 2 cysteine, a histidine, and acysteine residues (Summers

et al.

1990). The GAL4-type zinc finger from yeastbinds two Zn

2+

atoms, with six cysteine ligands to each Zn

2+

(Keegan

et al.

1986;Tao and Coleman, 1990; Pan and Coleman, 1991). Fritz

et al.

(1998) found thatS100A3 binds two Zn

2+

atoms. Of these, one zinc atom bound four cysteines andthe second one bound four cysteines and one histidine residue. There is thereforesome similarity between S100A3 and the zinc finger organisation of GAL4 tran-scription factor. These observations underline the potential importance of Zn

2+

bind-ing and the related changes in the molecular configuration in the function of S100A3.

Gribenko and Makhatadze (1998) have proposed an attractive model in whichmolecular properties are postulated to change dependent upon relative the concen-trations of Ca

2+

and Mg

2+

. Calcium binding may not produce conformational changesin some proteins. S100A7 (psoriasin) has been reported to show very little changein its conformation upon calcium binding (Brodersen

et al.

1998), but there wereearlier reports of significant changes in conformation of S100A7 upon binding ofCa

2+

, Zn

2+

, and Mg

2+

(Vorum

et al.

1996). It is possible that in this particular caseCa

2+

-mediated changes may not be functionally relevant.

THE STRUCTURE AND ORGANISATION OF S100 FAMILY GENES

The members of the S100 gene family have a shared intron–exon organisation.S100

β

has three exons and two introns. The first exon codes for the 5

′

untranslatedregion and the second and third exons each code for an EF-hand (Allore

et al.

1990).Similarly, the S100A4 gene (

h-mts1

) has three exons interspersed with two introns.The first exon is a noncoding exon and exons 2 and 3 contain the coding sequencesfor the

mts1

protein (Ambartsumian

et al.

1995).

ALTERNATIVELY SPLICED VARIANTS OF S100A4

Ambartsumian

et al.

(1995) also reported the occurrence of a splice variant of the

h-mts1

cDNA. In the generation of this variant, alternative splicing seems to haveoccurred within the 5

′

untranslated region of the original cDNA transcript, and asecond noncoding exon is inserted in the variant form. This insertion, nonetheless,retains the main open reading frame (ORF), without creating a new longer ORF. Ina recent study of the expression of S100A4

(h-mts1

) in carcinomas of the breast,Albertazzi

et al.

(1998c) discovered the presence of another variant form, which hasbeen designated as

h-mts1v.

This variant is a shorter version of the original

h-mts1



69

message. They have deduced the molecular organisation of the

h-mts1v

by reversetranscriptase polymerase chain reaction (RT-PCR). These studies have suggestedthat a loss of exon 1 (1a as well as 1b) sequences corresponding to one or moreprimers used for PCR may have occurred in the variant cDNA. The organisation ofthe

h-mts1

gene and that of the variant cDNA is presented in Figure 10. The onlyand striking difference is the loss of exon 1 (1a as well as 1b) in the shorter splicevariant, which is present in the larger variant reported by Ambartsumian

et al.

(1995).

FUNCTIONAL SIGNIFICANCE OF ALTERNATIVELY SPLICED ISOFORMS

Alternative splicing of pre-mRNAs is a ubiquitous phenomenon in higher eukaryotes.However, the significance of the occurrence of splice variants is poorly understood.Alternative splicing has been regarded as a mechanism of regulation of gene function.Splice variants may have different functions and different intracellular locations, andmay indeed subserve opposing functions (Sherbet and Lakshmi, 1997b). Alternativesplicing of pre-mRNA can generate isoforms of biologically active molecules andof membrane receptors for extracellular ligands functioning as biological responsemodifiers (Gunthert

et al.

1991; Kim and Yamada, 1997; Shi

et al.

1994). This resultsin changes in the mode of function. Splicing out of the transmembrane domain ofthe IL-6 receptor appears to cause its deletion from the cell membrane (Horiuchi

et al.

1994).The generation of splice variants of the kinases and phosphatases might be a

common mechanism associated with the regulation of cell cycle progression. ThePITSLRE protein kinases are members of the p34 (cdc2) kinase family, and are sonamed on the basis of the amino acid sequence of an important regulatory region.Several splice variant isoforms of the PITSLRE protein kinases have been described

FIGURE 10

Molecular organisation of the

h-mts1

(S100A4) and that of the variant

h-mts1v

cDNA. Exon 1 (1a and 1b) of

h-mts1

is not translated. This is spliced out in

h-mts1v

. Exons2 and 3 encode the protein. (From Albertazzi

et al.

1998c.) Reprinted by permission of thepublisher Mary Ann Liebert Inc.


70


(Xiang

et al.

1994). They may be expressed differentially in different cell types andsubserve different functions (Lahti

et al.

1995). Similarly, the phosphatase cdc25B,which is involved with the function of the cyclin/cdk complex in cell cycle regulation,occurs in the form of several splice variants, which may be involved together withthe wild-type phosphatase in the regulation of the cell cycle (Forrest

et al.

1999).Another striking example is provided by the splice variants of annexin VI. AnnexinVI has marked effects on cell proliferation. In A431 cells, it inhibits the mobilisationof Ca

2+

induced by EGF. But this effect is produced only by the larger isoform. Theshorter isoform does not affect Ca

2+

mobilisation nor does it influence cell growth(Fleet

et al.

1999).Alternative splicing could also generate protein isoforms that may be unable to

interact with target proteins or, as in alternatively spliced p53, to bind to DNA (Bayle

et al.

1995). Possibly, the 5

′

region is most prone to splice modification, and thestability and translation efficiency could be affected in these splice variants (N. Roy

et al.

1992; Bingham et al. 1988). There is the further possibility that the 5′ untrans-lated region may have a role in the regulation of the mRNA translation, as demon-strated for the alternatively spliced variants of the muscle-specific enolase gene(Oliva et al. 1995). On similar lines, the noncoding exons 1 and 2 of the angiotensinII receptor gene have been shown to be able to regulate the translation of thetranscripts (Curnow et al. 1995). The 3′-untranslated region seems to inhibit theexpression of the cell membrane receptor of luteinising hormone. The inhibition hasbeen attributed to a decrease in the half-life of the receptor mRNA (Nair and Menon,2000).

As stated before, Ambartsumian et al. (1995) reported that the occurrence ofthe larger splice variant of h-mts1 that contained an additional noncoding exon. Ithas been argued that splice variants may subserve different, often opposing, func-tions. If this were the case, one would expect to find a differential expression ofsplice variants in tissues (Sherbet and Lakshmi, 1997b), which appears to be thecase with respect to the h-mts1 splice variants (Ambartsumian et al. 1995). Ambar-tsumian et al. (1995) have stated that this splice variant showed marked variationsin the level of expression in different human tissues. This large isoform was notfound in the breast cancers, which Albertazzi et al. (1998b) investigated. However,there are indications that the expression of the h-mts1v isoform described here mayhave some bearing upon nodal spread of the disease (Albertazzi et al. 1998c),although h-mts1v did not appear to influence nodal spread as decisively as h-mts. Itwould be premature to speculate on the significance or relevance of h-mts1v expres-sion to the state of the disease, because the deleted exon 1a/1b is not transcribedand exons 2 and 3 that encode the functional protein are intact. It may be arguedthat the loss of the 5′ untranslated exon could be affecting the secondary structureof the transcript and its stability and translatability as a consequence. Possibly, theintegrity of the S100A4 protein and its molecular properties might be affected bythe splicing process, and this might impair the ability of S100A4 to promote meta-static spread.


The EF-Hand Calcium Binding Proteins 71

REGULATION OF EXPRESSION OF S100 FAMILY GENES

The widespread involvement of S100 genes in diverse physiological function, andthe demonstration that abnormal expression of many of these genes is associatedwith aberrant physiological events and the pathogenesis of several diseases, hasinevitably led to the investigation of how the expression of S100 genes is regulated.Two modes of gene regulation have been investigated: transcriptional regulation andepigenetic regulation.

TRANSCRIPTIONAL REGULATION OF S100 GENES

The promoter regions of several S100 genes have now been characterised. Thefunctional promoter of S100β has been localised to a region –168 to +697 andcontains 168 bp upstream of the transcription start site. The activity of this promoterhas been identified in several cell types. Furthermore, there are both positive andnegative regulatory elements in the promoter region. There are positive regulatoryelements in –788 to –391 and –1012 to –788. Further upstream, in the region –4437to –1012 and in –1012 to –788, occur negative regulatory elements. The regulatoryelement in –4437 to –1012 has been found to suppress promoter activity in manycell types (Castets et al. 1997).

Several potential regulatory elements, such as the CRE and AP2, have beenidentified in the promoter region of S100β (Allore et al. 1990). AP1-like bindingsites are found also in the promoter region of the calcyclin gene, but the latter showsno binding by transcription factors such as AP-2, AP-3, or NF1 (Bottini et al. 1994).The murine homologue of S100A4 has been investigated by Tulchinsky et al. (1990,1992), who have found homology between the 5′ region of S100A4 and the promoterregions of rat fibrinogen and human prothrombin, and also with certain enhancerelements of SV40. Tulchinsky et al. (1992) found no cis-acting control elements inthe 5′ region of S100A4. Subsequently, a 16-nucleotide cis-acting transcriptionregulatory element has been identified in the first intron of the gene, and this elementhas been found to bear a high degree of sequence homology to the enhancer elementof the CD delta gene. The S100A4 promoter region also has been reported to containmotifs that bear a high degree of homology to p53 binding negative regulatoryelements and AP1-like enhancer elements in the 3′ region (Parker et al. 1994a).

Okada et al. (1998) have identified a cis-acting element stretching from –187to –88, upstream of the first intron, that they believe specifies the transcription ofthe S100A4 (FSP1) gene in fibroblasts. They have also reported the occurrence ofan enhancer element in the first intron. This does not show cell type specificity.Okada et al. (1998) have reported further that a 5-bp domain TTGAT at –177 to–173 interacts specifically with nuclear extracts derived from fibroblasts. This fibro-blast-specific expression is not a typical feature of S100A4, which is indeedexpressed to a variable degree in a variety of normal and neoplastic tissues. Thus,there are clear suggestions that the function of S100 genes may be controlled by avariety of transcription factors.



REGULATION OF GENE EXPRESSION BY DNA METHYLATION

DNA methylation is an epigenetic mechanism that regulates gene expressionand gene imprinting. The CpG dinucleotide sites or islands that occur within andaround genes are the targets of methylation (Ng and Bird, 1999). The process ofmethylation alters the conformational state of the DNA and it has been suggestedtherefore that methylation may play a role also in genomic stability. The associationof the state of DNA methylation with gene expression has been known for manyyears (Kass et al. 1997). Hypomethylation is associated with constitutive geneexpression. Demethylation of promoter regions of a gene, e.g., by treatment of cellswith 5-azacytidine, increases the level of its expression (S. Lu and Davies, 1997).The regulation of tissue-specific expression of a tyrosine hydroxylase gene is depen-dent on the methylation status of the cytosine bases occurring around the CRE ofthe promoter (Okuse et al. 1997). Similarly, the expression of the manganese super-oxide dismutase gene is suppressed when certain cytosines present in the 5′ flankingregion are methylated (Y.H. Huang et al. 1997). Methylation of regulatory elementsmay interfere with the binding of transcription factors, as shown by Ryhanen et al.(1997). These authors found that AP-1 transcription factor bound to unmethylatedresponse elements with far greater affinity than to methylated ones.

The pattern of DNA methylation has been studied extensively. Methylationpatterns may be somatically inherited, but probably not through the germ line.Epigenetic DNA methylation may be linked with genetic changes. However, incontrast with the generally held view that DNA methylation is a heritable and stablephenomenon, Ramchandani et al. (1999) have postulated that it is indeed a reversiblesignal. This postulate makes it easier to appreciate how DNA methylation might beinvolved in developmental processes as well as in neoplastic progression, bothinherently associated with changes in the patterns of gene expression. Wachsman(1997) has argued that DNA damage, e.g., by alkylation of bases, oxidative lesions,etc., can interfere with the methylation of CpG dinucleotides by DNA methyltrans-ferases (DNA-MTases) and alter the distribution of 5-methylcytosine (5-MC). Thepresence of 5-MC results in an increased risk of mutagenesis. CpG islands are hotspots for mutations with the presence of 5-MC and DNA-MTases. Furthermore, thelevels of DNA-MTases correlate with the state of DNA methylation. Quite clearly,epigenetic changes of DNA methylation and mutations are interrelated and also areassociated with carcinogenesis.

DNA METHYLATION IN CANCER

It has been known for many years that changes in methylation patterns are associatedwith normal developmental processes (E. Li et al. 1992; Neumann and Barlow, 1996)as well as human cancers (P.A. Jones, 1986; P.A. Jones and Chandler, 1986). Whetherabnormal methylation status is associated with neoplastic transformation has beenthe subject of many investigations. Tumorigenic and non-tumorigenic cell lines differmarkedly in their DNA-MTase levels (Kautiainen and Jones, 1986), and humantumours show a range of 5-MC levels (Gamasosa et al. 1983). The expression ofDNA-MTase is said to be deregulated in colonic carcinomas, when compared with


The EF-Hand Calcium Binding Proteins 73

normal colonic epithelia and adenomatous lesions. The carcinomas have also beenreported to be highly heterogeneous with regard to DNA-MTase expression (DeMarzo et al. 1999). However, because cancers tend to be intrinsically heterogeneous,the heterogeneity of DNA-MTase might not be sufficient grounds for postulating anassociation between possible deregulation of its expression and the process of car-cinogenesis.

The overexpression of certain oncogenes in animal tumour models has beenassociated with their demethylation (Wainfan and Poirier, 1992). Indeed, the onco-gene ras was reported many years ago to be hypomethylated in human cancers (A.P.Feinberg and Vogelstein, 1983). Using an experimental tumour model, Counts et al.(1997) have demonstrated that hypomethylation of Ha-ras and raf oncogenes isrelated to tumour promotion, and further that carcinomas were more hypomethylatedthan adenomas. In breast carcinomas, marked hypomethylation has been reportedin the vicinity of the promoter region of the calcitonin gene (Hakkarainen et al.1996). pS2 is a protein associated with oestrogen-induced breast cancers. The expres-sion of this protein has been found to be associated with hypomethylation of a CCGGsite within the promoter/enhancer region of the pS2 gene (V. Martin et al. 1997).

In contrast with oncogenes, tumour suppressor genes appear to be hypermeth-ylated and inactivated in association with tumorigenesis (Zingg and Jones, 1997;Denissenko et al. 1997). The suppressor gene p53 is mutated in a large number ofhuman tumours. Not only does a large number of somatic mutations occur atmethylated CpG dinucleotide sites, but also these might be preferred targets formutagenic agents (Denissenko et al. 1997; M.S. Tang et al. 1999). There is ageneralised DNA hypermethylation in chronic lymphocytic leukaemia. More spe-cifically, two tumour suppressor genes — cyclin-dependent kinase inhibitor genesCDKN2A and CDKN2B — have been reported to be methylated. Apparently theirexpression is suppressed thereby in B-cell chronic lymphocytic leukaemia (Martelet al. 1997), and also in many other forms of human cancer, such as carcinoma ofthe breast, prostate, and kidney (Herman et al. 1995). The candidate suppressor geneHIC1 is relatively unmethylated or hypomethylated in peripheral blood or in acutemyelogenous leukaemia as compared with recurrent acute lymphocytic leukaemiaand blast crisis chronic myelogenous leukaemia (Issa et al. 1998). The inactivationor silencing of other tumour suppressor genes has also been reported, e.g., the MUC2gene in colorectal cancer (Hanski et al. 1977) and the Wilms tumour gene (Kley-menova et al. 1998). The promoter region of the E-cadherin gene, which is regardedas a putative tumour and invasion suppressor, often has been found to be hyperme-thylated in gastric carcinomas. This has been associated with the reduced expressionof E-cadherin in these tumours, and it has been suggested that hypermethylation ofthe promoter might occur as an early event in gastric neoplasia (G. Tamura et al.2000). In prostate cancer, there is a decreased expression of endothelin receptor ascompared with normal prostate epithelium. This appears to be due to methylationof a 5′ CpG island covering the transcriptional regulatory region of the endothelinreceptor gene (J.B. Nelson et al. 1997).



REGULATION OF S100 GENE TRANSCRIPTION BY METHYLATION

The expression of S100 genes may also be regulated by methylation. This has beensuggested by Tulchinsky et al. (1995) for S100A4. They have implicated the meth-ylation of the first exon and the first intron in the repression of transcription ofS100A4. Subsequently, Tulchinsky et al. (1996) showed that the first intron of thisgene contains a methylation-dependent AP1 binding site. They further stated thatthe transcription of the gene is related to hypomethylation of the first intron in murineadenocarcinoma cells. In human colonic adenocarcinoma cell lines, the expressionof S100A4 closely correlates with hypomethylation of the second intron of the gene.Also, where S100A4 expression was low, this could be increased by treating thecells with 5-Aza-2′-deoxycytidine (N. Nakamura and Takenaga, 1998). Indeed, thepattern of methylation of the gene could be a reason why S100A4 often shows adifferential pattern of expression (D.S. Chen et al. 1999).

S100A2, on the other hand, may be regarded as a tumour suppressor because itis expressed in normal breast tissue but it is down-regulated in the progression ofbreast cancer. Wicki et al. (1997) demonstrated the presence of an enhancer element1.2 kb upstream of the transcription start site. Here they identified two AP1-likebinding sites. The same enhancer element regulates the expression of S100A2 innormal as well as neoplastic breast epithelia suggesting the involvement of anepigenetic mechanism of methylation. Although their observations are not conclu-sive, it would appear that an element proximal to the transcription start site showsdifferential states of methylation in normal cells, tumorigenic cells, and cells derivedfrom a breast tumour biopsy. Nonetheless, they have demonstrated that site-specificin vitro methylation of S100A4 gene in normal cells leads to a down-regulation ofits expression.

Perhaps it is unhelpful to overemphasise the significance of the regulation ofgene expression at the transcriptional level. Ambartsumian et al. (1999) have reportedthat, in S100A4 transgenic mice, S100A4 mRNA was expressed in all organs. ThemRNA was expressed at a higher level in some organs. However, they have madethe crucial observation that the S100A4 protein was down-regulated in the organsthat did not express the gene in the wild-type animal. It is obvious from this thatthere must exist a complex mechanism that regulates the expression of the proteinat the translational level or regulates the decay of the protein. This is not unique toS100A4, and the expression of other S100 proteins in common with a wide varietyof cellular proteins might also be controlled at the posttranscriptional level. Forinstance, S100α1 protein and its mRNA are differentially expressed in differentiatingneuronal and skeletal muscle cells (Zimmer and Landar, 1995).


75

6

The Calmodulin Family of Calcium Binding Proteins

A number of calcium regulatory proteins may be treated as belonging to the calm-odulin family of CBPs. In addition to calmodulin, troponins, calbindins, recoverin,and calretinin can be assigned to this family. The regulatory function of these CBPsin normal as well as aberrant physiology will be discussed in the following pages.

CALMODULIN AND ITS PHYSIOLOGICAL FUNCTION

S

TRUCTURE

AND

M

ODE

OF

A

CTION

OF

C

ALMODULIN

Calmodulin (CaM) is a 17-kDa CBP. Structurally it may be described as a dumbbell-shaped molecule with two lobes each containing two Ca

2+

-binding domains. Thetwo lobes are joined by an

α

-helix comprising 28 amino acid residues. A confor-mational change occurs in the CaM molecule in the presence of calcium and asresult CaM can bind to a large number of target molecules. The conformationalchange involves the exposure of two methionine-rich hydrophobic areas, one in eachglobular domain, and these take part in the interaction with target proteins and intheir activation. CaM can activate a large number of proteins, and this ability hasbeen attributed to the highly conserved methionine residues (Kincaid

et al.

1987;Perrino

et al.

1992; Milan

et al.

1994), the mutation of which might affect activationof target proteins. The

α

-helix linker also plays a crucial role in this process ofactivation. Tabernaro

et al.

(1997) studied a mutant form of CaM in which the aminoacid residues Thr 79 and Asp 80 are deleted from the

α

-helix. This deletion appearsto alter the relative orientation of the globular domains, which results in the hydro-phobic patches coming closer together and consequently becoming less accessibleto interaction with target proteins. The CaM binding domains of target proteins occurin short stretches of 15 to 25 amino acid residues (O’Neill and De Grado, 1990;Ikura

et al.

1992; Crivici and Ikura, 1995). CaM appears to recognise certainconserved structural features, which might differ from one target protein to anotherwith respect to their topographical arrangement. CaM orientates its globular domainsby virtue of the flexible nature of the linker.

An interaction with CaM is essential for the activation of several of the targetproteins (Kretsinger, 1992). Specificity of activation of the target enzymes seems tobe achieved by the phosphorylation of CaM (Williams

et al.

1994; Sacks

et al.

1995;Quadroni

et al.

1994; Saville and Houslay, 1994). Several kinases phosphorylate


76


CaM, among them the myosin light chain kinase (MLCK) (Davis

et al.

1996); caseinkinase II (Sacks

et al.

1992), and receptor tyrosine kinases (Graves

et al.

1986; SanJose

et al.

1992). Ubiquitination of calmodulin also has been suggested as a possibleregulatory mechanism (Laub and Jennissen, 1991). The occurrence of numeroustarget proteins indicates the broad spectrum of physiological function in which CaMparticipates. CaM mediates the function of enzymes that are involved in cyclicnucleotide metabolism, phosphorylation and dephosphorylation, and smooth musclecontraction (Means, 1988; Harrison

et al.

1988; MacNeil

et al.

1985; G. Li

et al.

1989).

C

ALMODULIN

-M

EDIATED

S

IGNAL

T

RANSDUCTION

There is currently a large body of evidence demonstrating the important role calm-odulin plays in the transduction of signals of cell proliferation and growth control,and in cell locomotion and invasion. The signalling pathways that are initiated bythe activation of cell membrane receptors, as alluded to earlier, involve the raisingof intracellular calcium levels and the subsequent phosphorylation of target proteins.Because Ca

2+

/CaM interacts with and activates target enzymes, CaM has beendeemed as an important component of the signalling pathway by mediating proteinphosphorylation via CAMKs. CaM kinases subserve a variety of functions. Severalexamples can be cited for the purposes of discussion and illustration. Thus T-cellactivation, cell migration, and proliferation will serve amply to underline the signif-icance of CaM as a signalling molecule.

The activation of the T-cell receptor not only increases intracellular calcium butalso activates CaM-dependent kinases, which results in the regulation of phos-phatases (Ostergard and Trowbridge, 1991). CAMKs are also involved in cell pro-liferation. CAMK II has been shown to be involved in the induction of S-phasedelay in fibroblasts exposed to

γ

-irradiation. The exposure to radiation also inducesCAMK II activity. This induction does not occur if the cells are pretreated withCAMK II antagonists (Famulski and Paterson, 1999).

An outstanding example of CaM-mediated signal transduction is the transductionof angiotensin signals. The hormone angiotensin II, derived from the decapeptideangiotensin I, generates many cellular responses. Angiotensin II possesses cytokine-like activity. It functions as a cardiovascular growth factor and is mitogenic to cardiacfibroblasts. It has also been attributed with the ability to induce angiogenesis. Notsurprisingly, therefore, angiotensin signal transduction has many similarities to thatof growth factors. Some of the effects of angiotensin are indeed mediated by growthfactors such as PDGF, EGF, basic fibroblast growth factor (bFGF), and TGF

β

.The transduction of the angiotensin signal involves two types of angiotensin

receptors: type 1 (AT-1) and type 2 (AT-2). The binding of angiotensin to AT-1triggers a cascade of signalling events and the activation of transduction pathways(Thomas, 1999). The C-terminal domain of the AT-1, close to the inner membrane,interacts with CaM in a Ca

2+

-dependent fashion. This interaction seems to be essen-tial for the transduction of angiotensin binding to AT-1A (Thomas

et al.

1999). Incardiac fibroblasts, AT-1-induced signals appear to transactivate epidermal growth



77

factor receptors (EGFr) and eventually result in DNA synthesis. This effect isinhibited by CAMK inhibitors (Murasawa

et al.

1998).Angiotensin is able to induce the synthesis of TGF

β

and the type II TGF

β

receptor (Wolf

et al.

1999). TGF

β

and its receptor activity have potentially profoundeffects on the composition and properties of the ECM. Furthermore, angiotensin andTGF

β

can both induce cytoskeletal changes, such as the formation of actin stressfibres and the phosphorylation of focal adhesion proteins (Riedy

et al.

1999). Therecan be little doubt that the cytoskeletal modulations and the remodelling of the ECMwill influence cell migration as well as cell proliferation and differentiation.

Cell migration is another cellular activity that demonstrably involves CaM insignal transduction. Cell migration is believed by some to involve activation of thepathway that includes members of a family of serine/threonine kinases called extra-cellular signal-regulated receptor kinases (ERK), which are also known as the MAPKpathway. In PC12 cell, calcium influx activates ERK. This activation is inhibited bythe CaM inhibitor W13, which indicates that CaM is involved in this pathway (Egea

et al.

1998). Egea

et al.

(1998) also showed that other receptors, e.g., trk A andEGFr, are not involved in the activation of the ERK pathway. In gastric epithelialcultures also CaM appears to be involved in the transduction of the cell migrationsignal (Ranta-Knuuttila

et al.

1998).Of late, much evidence has emerged that links CaM with G-protein-mediated

signal transduction. There is a marked parallelism between angiotensin signal trans-duction and the transduction of growth factor signals. It has been recognised, there-fore, that G-proteins might be involved in angiotensin signal transduction. Angio-tensin II has been known to bind to a high-affinity G-protein receptor (T.J. Murphy

et al.

1991; Sasaki

et al.

1991; Sandberg

et al.

1992). Recently, CaM has beenreported to enhance the Ca

2+

-dependent binding of guanosine triphosphate (GTP)to the

ras

-related protein Ral-A (Wang and Roufogalis, 1999). Fischer

et al.

(1998)found that the PI3K, an enzyme that functions downstream of G-proteins, possesseshigh-affinity binding sites for CaM. The association of CaM with G-proteins is notrestricted to animal cell systems. CaM appears to be closely involved with G-proteinsin pollen germination and the growth of pollen tubes (Ma

et al.

1999).

C

ALMODULIN

AND

C

ELL

P

ROLIFERATION

As stated earlier, among the targets of CaM are the cytoskeletal proteins MAP-2and the tau protein. The conclusion is inescapable, therefore, that CaM might beinvolved in cytoskeletal reorganisation. Both cell proliferation and cell motility havebeen regarded as the natural foci of CaM involvement. CaM seems to be activelyassociated with cell proliferation, as amply demonstrated by several investigators.Indeed, it is regarded as an essential ingredient of cell proliferation and the progres-sion of the cell cycle (Means, 1994). The exit of cells from the cell cycle has beenreported to be accompanied by a decrease in CaM levels (Christenson and Means,1993). CaM levels increase as cells progress into the mitotic phase, and there is anelegant demonstration that both Ca

2+

and CaM are essential for this process. Wheneither of these is reduced cells undergo G

2

arrest (K.P. Lu

et al.

1993).


78


Some investigators have examined the levels of CaM expression in relation togrowth responses. Exaggerated growth responses have been recorded in cardiomyo-cytes resulting from an overexpression of CaM (Gruver

et al.

1993). Recently, however,Prostko

et al.

(1997) found no effects on growth responses arising from an overex-pression of CaM in C6 glioma cells in culture, but reduction of CaM expression wasfound to inhibit their growth. The antiproliferative effects exerted by CaM inhibitorshave also provided a substantial body of evidence that suggests an association of CaMwith growth responses. Several CaM inhibitors have been tested to date. Schuller

etal.

(1991) found that B859-35, which is a dihydropyridine derivative, markedly inhib-ited proliferation of three human lung cancer cell lines. Hait

et al.

(1994) reported thatseveral phenothiazine antipsychotic drugs inhibit CaM and also the proliferation ofC6 glioma cells. They also found that the antiproliferative effects corresponded withthe inhibition of CaM-sensitive phosphodiesterases. Further work from the same lab-oratory has shown that KS-501 and KS-502 similarly affect cell proliferation, not bydirect action on the enzymes but by interfering with their function of activating CaM(Hait

et al.

1995). In other words, the failure to activate CaM appears to lead to aninhibition of cell proliferation. Glass-Marmor

et al.

(1996) and Glass-Marmor andBeitner (1997) have investigated the effects of another class of CaM inhibitors, whichreduce intracellular levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, andATP and also detach glycolytic enzymes bound to the cytoskeleton. Four antagoniststested — thioridazine, CGS 9343B, clotrimazole, and bifonazole — brought about amarked reduction in cell viability (Glass-Marmor

et al.

1996). The detachment ofglycolytic enzymes associated with the cytoskeleton can also affect the function of thelatter in cytokinesis.

CaM cDNA has been transfected in sense as well as in the antisense orientationinto C6 glioma cells. Transfection with sense-cDNA has been found to producemore clones than transfection with antisense constructs. The DNA content of cellshas been reported to correlate with CaM levels (Liu G.X.

et al.

1996). This iscompatible with the finding that cells experience a delay in DNA synthesis in thepresence of CaM inhibitors W7 and W13 and the CaM-dependent protein kinaseinhibitor KN-62 (Mirzayans

et al.

1995). CaM can influence the transduction ofgrowth factor signals via the receptor kinases by modulating their phosphorylationby means of CaM-dependent protein kinases. Thus one can visualise several waysin which CaM could regulate physiological processes.

C

ALMODULIN

IN

N

EOPLASIA

An enhancement of CaM has been recognised as a feature of cell transformationand of malignant cells. CaM levels of human lung cancer cells are higher than thatof benign tumours of the lung or normal lung tissue (Liu G.X.

et al.

1996). Theseauthors also describe a correlation between tumour grade and TNM stage and levelsof CaM. However, Edelman

et al.

(1994) have described a CaM-like protein that isapparently restricted to epithelial cells. This CaM-like protein was identical in sizeand largely homologous to CaM, but, unlike CaM, its expression seems to besignificantly lower in malignant cells.



79

The CaM antagonist J8 inhibits the invasive behaviour of the cutaneous mela-noma cell line A-375SM and uveal melanoma cells (Dewhurst

et al.

1997). Tamox-ifen, its metabolites

N

-desmethyltamoxifen, and 17

β

-oestradiol also inhibit invasionin the absence of oestrogen receptors, which suggests that the inhibition producedby these anti-oestrogens was mediated by mechanisms other than receptor binding,e.g., CaM inhibition. A deregulation of intracellular calcium resulting in cell deathis produced by tamoxifen at high concentrations where its effects are believed to benot mediated by oestrogen receptors (Jain and Trump, 1997).

CaM antagonists could inhibit invasion by starving the cytoskeletal machineryof local ATP generation. Glass-Marmor and Beitner (1997) found that the CaMinhibitors, which they had previously shown to reduce the levels of certain glycolyticenzymes and ATP and cause loss of viability (Glass-Marmor

et al.

1996), also detachthese enzymes from their association with the cytoskeleton.

It is possible that CaM could be influencing cell invasion by altering the expres-sion of ECM-associated enzymes. Some years ago A. Ito

et al.

(1991) suggestedthat CaM could be differentially modulating the expression of TIMP and prometal-loproteinases 1 and 3 in fibroblasts derived from human cervical carcinoma. Theexpression of these enzymes is known to markedly alter the invasive behaviour ofcancers (Sherbet and Lakshmi, 1997b). Possibly, therefore, modification of theexpression of ECM-associated enzymes and the properties of the ECM as a meansof modulating cell behaviour, ought to be seriously considered. S100A4 does seemto operate through such a mechanism (Merzak

et al.

1994b; Lakshmi

et al.

1997).It would seem, therefore, that CaM might involve more than one target enzyme inthe modulation of invasive behaviour.

RECOVERIN SUBFAMILY OF NEURAL CALCIUM BINDING PROTEINS AND THEIR FUNCTION

Recoverin (RCN) and related proteins belong to a four-EF-hand recoverin family ofneural calcium-binding proteins (NCBPs) (Table 5). Two types of NCBP can bedistinguished. Type A proteins, e.g., recoverin, have two canonical EF-hands. TypeB proteins, e.g., VILIP (visinin-like protein) and NCS-1, possess three regular EF-hands. Flaherty

et al.

(1993) have elucidated the three-dimensional structure of RCN.According to them, the four EF-hands are arranged in a compact array. The tertiarystructure and arrangement of the EF-hand may control calcium binding. Althoughgenerally described as NCBPs, individual NCBPs do show characteristic differencesin their localisation within the neural tissue and the retinal component itself.

T

HE

G-P

ROTEIN

S

IGNALLING

PATHWAY

Many extracellular modifiers of biological response transduce their signals via het-erotrimeric guanine nucleotide regulatory proteins (G-proteins), which consist of

α

,

β

, and

γ

subunits (Koelle, 1997; Dohlman and Thorner, 1997; Berman and Gilman,1998). These G-proteins are coupled to cell surface receptors. Several agents, suchas epinephrine, norepinephrine, cytokines, and nonsteroid hormones, activate G-protein receptors (Hein and Kobilka, 1997; Watson

et al.

1996). Upon activation


80


these receptors promote guanine nucleotide exchange of guanosine diphosphate(GDP) to GTP on the G

α

. This in turn leads to the dissociation of G

βγ

complex fromG

α

. These then regulate the activity of the target protein, leading eventually to themobilisation of second messengers. A family of proteins called RGS (regulators ofG-protein signalling) can preferentially bind to activated G

α

. RGS proteins appearto function as guanosine triphosphatase (GTPase) activating proteins (GAP) (Berman

et al.

1996; Watson

et al.

1996; Hunt

et al.

1996) and attenuate or block the signallingpathway (Popov

et al.

1997; Tesmer

et al.

1997; Hepler

et al.

1997).The G-protein-dependent and calcium-signalling pathways are often closely

allied. For example, neurotransmitters inhibit calcium channel current, which seemsto be regulated by the photoreceptor G-protein called transducin (Jeong

et al.

1999;also, see below). Similarly, dopamine-coupled receptors inhibit voltage-activatedcalcium channels (Wolfe and Morris, 1999). Dolphin

et al.

(1999) have identifiedthe calcium channel-protein domains that might be involved in the modulation ofcalcium channel currents by G-proteins. In the signalling events associated with theacrosome reaction in spermatozoa, G-proteins require calcium together with PLA2to induce acrosomal reaction (Dominguez

et al.

1999).

R

ECOVERIN

AND

I

TS

F

UNCTION

The transduction of the extracellular sensory signal involves the interaction ofactivated rhodopsin with the photoreceptor G-protein, transducin. The rhodopsinintermediate meta II activates transducin by catalysing the exchange of GDP to GTP.The RGS protein down-regulates this signalling pathway by promoting Gα GTPaseactivity and additionally by down-regulating cGMP phosphodiesterase, which effec-tively enhances GTPase activity. RCN (a homologue of this from the frog is knownas S-modulin) is a 23-kDa protein that plays a specific role of phototransduction inmammalian retinal photoreceptors. The transduction of visual signals requiresrhodopsin to be activated by phosphorylation. Upon illumination rhodopsin is phos-phorylated by rhodopsin kinase at several serine and threonine residues. The acti-vated rhodopsin is dephosphorylated by phosphatases to return it to the basal state.

TABLE 5Recoverin Family of Neural Calcium Binding Proteins

Protein Designation Occurrence Ref.

Recoverin (S-modulin) Photoreceptor cells; mammalian bipolar cells

De Raad et al. (1995)

Rem-1 Retinal cells; haematopoietic cells; gut cells

Kraut et al. (1995)

Visinin Photoreceptor cellss26 Photoreceptor cells (frog retina) Kawamura et al. (1996)VILIP Inner retina De Raad et al. (1995)NCS-1 Photoreceptor inner segments; inner

plexiform layer; ganglion cellsDe Raad et al. (1995)


The Calmodulin Family of Calcium Binding Proteins 81

RCN is involved in the process of light and dark adaptation by rod cells by regulatingrhodopsin phosphorylation, thereby controlling photoreceptor light sensitivity, whichis Ca2+ dependent. Several mutant forms of RCN, with mutations in EF-hands 2 and3, and others with mutation of EF-hand 4, have been isolated recently. Of these, EF-hand 4 RCN mutants were found to be able to inhibit rhodopsin kinase moreeffectively than could wild-type RCN (Alekseev et al. 1998). Apparently, a decreasein cytoplasmic calcium levels is necessary for light adaptation (Figure 11). BesidesRCN, other calcium-binding proteins are involved in this process. Among them arethe photoreceptor-specific GCAP and calmodulin, together with their targets, namelyrhodopsin kinase, guanylate cyclase, cGMP-gated channel, and nitric oxide synthase(Koch, 1995; Gorczyca et al. 1995).

The RCN gene has been mapped to human chromosome 17p13.1 (J.F. McGinniset al. 1995). Interestingly, the autosomal-dominant progressive cone dystrophy(CORD5) gene maps to chromosome 17p12–p13. The genes, coding for retinalguanylyl cyclase and pigment epithelium-derived factor, and the retinitis pigmentosa(RP) genes also occur in the RCN region (Balciuniene et al. 1995).

Recoverin occurs predominantly in mammalian photoreceptor cells. It may befound in other retinal cell types and may therefore subserve other functions besidesphototransduction (McGinnis et al. 1997). RCN appears to be a highly conservedprotein, as suggested by its occurrence in the photoreceptor cells of the lamprey(Dalil-Thiney et al. 1998). Its expression may be developmentally regulated (Yanand Wiechmann, 1997). It is found to be transiently expressed in developing folli-cular and parafollicular pinealocytes in the developing chick embryo (Bastianelliand Pochet, 1994). Four isoforms of RCN occur and each of these shows N-terminalmyristoylation. RCN not only participates in phototransduction, a role suggested byits localisation in photoreceptor cells, but it is also associated with the pathogenesis

FIGURE 11 The pathway of sensory signal transduction involving recoverin and RGS(regulator of G-protein signalling) protein.



of the autoimmune state of cancer-associated retinopathy and uveoretinitis. However,it is uncertain whether RCN is involved with autosomal recessive RP.

MODE OF ACTION OF RECOVERIN

Recoverin regulates photoreceptor response by inhibiting rhodopsin phosphoryla-tion. Rhodopsin kinase is active in the absence of RCN (Gorodovikova et al. 1994a).Rhodopsin phosphorylation and consequent cGMP hydrolysis are Ca2+- and ATP-dependent processes. When the free Ca2+ level is raised, phosphorylation of rhodop-sin is reduced and there is an increase in the lifetime of phosphodiesterase. ThisCa2+ effect is negated by anti-RCN antibodies, which has been interpreted as sug-gesting that the calcium effects observed are a result of the inhibition of rhodopsinkinase (Gorodovikova et al. 1994b). Upon addition of RCN, rhodopsin kinasebecomes sensitive to free Ca2+. Calcium-dependent interaction between RCN andrhodopsin kinase is indeed necessary for the inhibition of rhodopsin phosphorylationby RCN (C.K. Chen et al. 1995). All four isoforms of RCN inhibit rhodopsinphosphorylation in the same free calcium range (0.3 to 0.8 µM), but they differ withrespect to the magnitude of inhibition achieved, which appears to be related to theirhydrophobicity (Sanada et al. 1995).

POST-TRANSLATIONAL MODIFICATION OF RECOVERIN

Four isoforms of RCN have been identified; all appear to be posttranslationallymodified at the N-terminal glycine residue with myristic acid or related lipids.Myristoylation is believed to be essential for the function of RCN. Senin et al. (1995)compared the inhibitory effect on rhodopsin phosphorylation of myristoylated andnonmyristoylated forms of recombinant RCN. They found that both forms of RCNinhibit rhodopsin kinase in the presence of Ca2+, but myristoylated RCN was moreefficient in inhibiting the kinase. However, others believe that myristoylation is notnecessary for the kinase inhibitory effect of RCN and that it only induces a coop-erative Ca2+ dependence of the process (Kawamura et al. 1994; Calvert et al. 1995).Ames et al. (1994) have suggested, on the basis of the flexibility of the N-terminalhelix in myristoylated calcium-free and nonmyristoylated calcium-bound form, thatcalcium binding to the EF-hand 3 domain induces EF-hand 2 to adopt a conformationthat promotes calcium binding to RCN.

Covalent attachment of a myristoyl or related N-acyl group to the N-terminalglycine appears to promote the binding of RCN to the optic disc membrane whenfree Ca2+ is raised. The so-called calcium–myristoylation switch is believed to playan essential role in the targeting of RCN to the cell membrane. The addition of themyristoyl group has been found to reduce the calcium affinity of RCN and inducecooperative calcium binding. Two conformational states have been recognised viz.the T and R states. In the T state the myristoyl group is sequestered inside the protein,whereas in the R state it is exposed, and furthermore, calcium binds to the R stateseveral thousand-fold more strongly than to the T state (Ames et al. 1995). Calciumbinding to the myristoylated RCN induces its translocation to the membrane. The



nonmyristoylated RCN is not translocated in this way (Tanaka et al. 1995). It wouldappear, therefore, that posttranslational modification of RCN is required for targetingto and interaction of RCN with the membrane. The RCN family protein VILIP alsoshows calcium-dependent targeting to the cell membrane. It has been found tointeract with the actin component of the cytoskeleton in its recruitment to the cellmembrane (Lenz et al. 1996). Braunewell et al. (1997) have confirmed that myris-toylated VILIP can be shown to be associated with membranes, whereas nonmyris-toylated VILIP is not. Membrane association may stabilise the RCN–rhodopsinkinase complex, as suggested by experimental demonstration of a preferential asso-ciation of RCN with cell membrane with concomitant increase in rhodopsin kinaseinhibition (Sanada et al. 1996). However, Johnson et al. (1997) found that confor-mational changes and distribution of RCN in the cell were not influenced greatlyby calcium concentration and suggest, therefore, that the calcium–myristoylationswitch may not be the only mechanism involved in the targeting of RCN to the cellmembrane.

RECOVERIN AND CANCER-ASSOCIATED RETINOPATHY

Recoverin and Cancer-Associated Retinopathy in Small Cell Lung Cancer

A degeneration of the retina is infrequently associated as a paraneoplastic conditionwith some forms of cancer. This condition, often described as cancer-associatedretinopathy (CAR), is an autoimmune syndrome involving the degeneration of thephotoreceptor cells of the eyes. Injection of RCN had previously been shown toinduce degeneration of photoreceptor cells of the eyes of Lewis rats, and this wascorrelated with high titres of RCN antibodies in the circulation (Adamus et al. 1994).There are several reports of incidence of CAR in patients with small cell lungcarcinoma (SCLC) (Polans et al. 1995; Matsubara et al. 1996). Polans et al. (1995)showed RCN is expressed in lung tumour biopsies of patients who had CAR butnot in tumour samples of patients without CAR. They further identified two regionsof RCN that were associated with its immunogenicity. Amino acid residues 64–70and 48–52 were the major determinants. Immunisation of Lewis rats with peptide64–70 caused photoreceptor degeneration in the animals. This 64–70 region is inclose proximity to the EF-hand 2 calcium-binding domain. The autoimmune reac-tivity has been found to depend on changes in the conformation of the 64–70 stretchof amino acid residues induced by calcium binding to the EF-hand 2 present in theneighbourhood (Adamus and Amundson, 1996). Murphy et al. (1997) have encoun-tered high titres of antibodies to a 60-kDa retinal protein, as well as high titres ofRCN antibodies, in association with SCLC. The titres of both declined with treatmentand Murphy et al. (1997) have therefore suggested that the detection of RCNantibodies alone should not be used as the sole criterion for diagnosing CAR. Arecent report described a patient with non-SCLC with recoverin antibodies in theserum (Salgia et al. 1998).



Retinopathy Associated with Other Forms of Human Cancer

Retinopathy is associated also with other neoplasms such as breast and cervicaltumours (Holz et al. 1997) and metastatic melanoma (Kirati et al. 1997). Again, thishas been attributed to autoimmune responses to retinal proteins.

RCN is expressed in cell cultures derived from retinoblastoma. Using antibodiesraised against recombinant RCN, Weichman (1996) demonstrated that RCN isexpressed in the cytoplasm of retinoblastoma cell line Y79. The expression wasgreatly increased by treating cells with 2 mM butyrate, and to a lesser degree bydb-cAMP, together with the formation of neurite-like cellular processes indicatinga state of induced differentiation. This possible link-up between RCN expressionand differentiation is worthy of further investigation.

In the sera of patients with CAR, heat shock protein (HSP) 70 has been detectedin addition to recoverin (Ohguro et al. 1999). The involvement of bacterial HSPs inthe induction of autoimmunity has been recognised with the demonstration thatHSP65 from the tubercle bacillus mycobacterium can induce autoimmune diseasein certain animal tumour models. Certain peptides derived from mycobacterialHSP65 and their homologous peptide obtained from patients with Behcet’s uveitisor iridocyclitis induce uveitis in Lewis rats. High antibody titres against thesepeptides are found in rats that developed uveitis as compared with those that hadnot (Uchio et al. 1998). This suggests that HSPs do function as autoantigens.Therefore, the detection of HSP70, together with recoverin, might suggest that boththese antigens stimulate humoral autoimmunity in the pathogenesis of CAR. Thisview would be in line with the implication of HSPs in other autoimmune diseasessuch as rheumatoid arthritis (RA) (Winfield, 1989) and SLE (Minota et al. 1988;Conroy et al. 1994). Antibodies against human HSP60 and E. coli HSP60 have beenfound in patients with RA and SLE and in Reiter’s syndrome which combinesarthritis with conjunctivitis, but the antibody titres are far higher than the titres ofantibodies against mycobacterium HSP65 (Handley et al. 1996). This seems tosuggest that the immune system recognises certain epitopes of these HSPs (Van derZee et al. 1998). Although these observations suggest that HSPs might play a rolein autoimmune conditions, they do not explain the coexpression of RCN with HSPs.It may be that some other HSP function, such as protein folding, may be involvedhere. As we have seen above, certain critical conformational changes are requiredin the proper functioning of RCN. Although, in the realms of speculation, thepossibility that HSPs influence RCN conformation in such a way that sequesteredautoimmune epitopes might be exposed cannot be excluded.

It is unclear whether RCN forms complexes with HSPs. This is an interestingavenue to approach; one can envisage a situation where HSPs regulate RCN functionby forming a complex. HSPs are known to form complexes with important biologicalmacromolecules such as p53, rb proteins, and S100A4, and possibly influence theirfunction as regulators of the cell cycle (Sherbet and Lakshmi, 1997b).



Is Recoverin Involved in Retinitis Pigmentosa?

Retinitis pigmentosa (RP) is an autosomal-recessive disease involving degenerationof photoreceptor cells. RP has been attributed to mutations in the genes coding forrhodopsin and for the rod cGMP-gated channel. Naturally, therefore, the possibilityof RCN implication in RP has received some attention. However, mutations in theRCN gene have been ruled out in 42 Spanish families with autosomal-recessive RP.Furthermore, the study by Parminder et al. (1997) did not encounter any mutationsin the RCN gene in RP or allied heritable retinal diseases.

GUANYLATE CYCLASE-ACTIVATING PROTEINS

The mammalian retina contains two GCAPs: GCAP1 and GCAP2. They are pho-toreceptor-specific calcium binding protein. GCAP1 is expressed at a high level inthe outer segments of rods and cones. GCAP2 occurs mainly in the cone innersegments and is less intensely expressed in inner retinal neurons. Therefore, whereasGCAP1 has a phototransduction function, GCAP2 may not (Otto-Bruc et al. 1997).Furthermore, the expression of GCAP1 and 2 gene transcripts is substantiallyreduced in retinal degeneration (rd/rd) mutant chicken. Here, GCAP2 protein seemedto be normal, but the expression of GCAP1 proteins was down-regulated by morethan 90% and this seems to be consistent with the loss of phototransduction (Semple-Rowland et al. 1996).

GCAP1 has four EF-hand motifs. When not bound to calcium, GCAP is ableto activate GC. Calcium binding inactivates GCAP. This seems to be a consequenceof Ca2+ binding by EF-hands 3 and 4 and the conformational changes in the molecule(Rudnicka-Nawrot et al. 1998). The involvement of EF-hand 3 is also indicated byan inactivating mutation A → G in codon 99, exon 2 of the GCAP1 gene (Payne etal. 1998). This mutation is reported to occur in the GCAP1 gene in a family withautosomal-dominant cone dystrophy.


87

7

The Structure of Contractile Proteins

THE ACTIN COMPONENT OF CONTRACTILE MACHINERY OF THE CELL

The physical alterations of the shape of the cell, its structure, motility and contractileability are an attribute of the structure and organisation of the cytoskeletal machinery,of which the intermediate filaments (IF) (10 nm diameter), actin microfilaments (7nm diameter), and microtubules are the important components. Actin microfilamentsare polymers of F- and G-actin. These filaments are flexible, double-helical poly-meric structures made up of F-actin strands. The G-actin subunits participate inbinding to the myosin globular heads. G-actin also possesses ATP- or ADP-bindingdomains. Actin filaments have a defined polarity, which flows from the fact that themonomers themselves show a distinct polarity. In association with actin-bindingproteins, the actin filaments form specialised structures. To these organised structuresthe cell owes its faculty of modulation of shape, motility, and other adhesion-mediated phenomena. The reorganisation of actin cytoskeleton is also involved withplant cell division, differentiation, and light-induced plastid migration, among othercellular features (Staiger

et al.

1997).

A

CTIN

I

SOFORMS

Four actin isoforms have been identified, of which two, the

α

and

γ

isoforms, aresmooth muscle specific and the other two are the cytoplasmic

β

isoforms. Thetransfection of mutated

β

-actin genes in normal diploid cells alters their morphol-ogy and growth characteristics, and at the same time mutated

β

-actin protein isseen to be incorporated into the cytoskeleton (Leavitt

et al.

1987a). Leavitt

et al.

(1987b) have further shown that the transfectant cells that expressed mutant

β

-actin possessed greater tumorigenic potential than those with lower levels ofmutant actin. Cells derived from these tumours had a shorter latency period fortumour formation as compared with the original transfectant cells. None of theseinteresting studies seem to have been followed up to examine motility, invasive-ness, and metastatic potential, which, in retrospect, seems highly desirable in lightof the changes in cell morphology and growth features engendered by the expres-sion of mutant actin. A natural extension would be to investigate whether wild-type

β

-actin possesses tumour suppressor function. The suppressor function of

α

-actin has been demonstrated recently. The rat fibroblast cell line 3Y1 expresses

α

-actin, but this is down-regulated when the cells are transformed by RSV. In

0942/C07/frame Page 87 Thursday, October 26, 2000 9:59 AM

88


contrast, transfection of

α

-actin into RSV-transformed fibroblasts resulted in thereduction of tumour growth and invasiveness (Okamoto-Inoue

et al.

1999).

R

EGULATION

OF

A

CTIN

D

YNAMICS

Actin polymerisation and depolymerisation occurring in the dense actin cytoskeletonat the leading edge of a cell is instrumental in achieving locomotion (Tilney

et al.

1983; Zigmon, 1993). The polymerisation of actin provides the protrusive powerthat allows the formation of lamellipodia or filopodia in the direction of cell move-ment. There is net polymerisation at the front edge while net depolymerisation occursat the rear of the lamella. The relative rates of polymerisation and depolymerisationare at the basis of cell motility regulation. Actin dynamics are affected and may,indeed, be regulated by many cellular proteins. The actin-binding proteins regulatecell motility by influencing the rates of actin polymerisation and depolymerisation.Actin-binding proteins such as gelsolin thymosin, and profilin are cytosolic proteinsthat inhibit the process of actin polymerisation and profoundly influence cell mor-phology and motility. It follows, therefore, that these proteins could be indirectdeterminants of the altered cell motility and metastasis that often accompany neo-plastic transformation. Among other notable cellular proteins that affect cytoskeletaldynamics is the S100A4 calcium-binding protein. S100A4 has been shown to beclosely associated with the invasive and metastatic behaviour of cancer cells, andalso to promote depolymerisation of the cytoskeletal structures (Lakshmi

et al.

1993,1997). It would be worthwhile, therefore, to discuss the role played by thymosin,cofilin, profilin, and fimbrin in altering cytoskeletal dynamics and assess their impacton cell behaviour.

The currently held view is that the actin-binding proteins alter the ratio ofmonomeric G-actin and F-actin in cells. G-actin-binding proteins such as thymosin

β

4 (T

β

4) and profilin sequester G-actin from the cell pool and thereby prevent theprocess of polymerisation, whereas F-actin-stabilising factors such as myosin sub-fragment I and phalloidin can promote repolymerisation from the T

β

4/G-actin com-plex (Ballweber

et al.

1994) (Figure 12). In continuous cellular locomotion orinherent invasive ability, the cell has to maintain a net high level of actin polymer-isation and a constant F-actin content. Actin subunits derived from depolymerisationoccur at the rear of the lamella, are assembled into filaments at the front of thelamella (Y.L. Wang, 1985; Small 1995). It would be of considerable interest to reviewhow the expression of these actin-binding proteins affects cell behaviour and espe-cially if any one of them shows any correlation with the aberrant behaviouralproperties manifested by neoplastic cells.

C

OFILIN

IN

THE

R

EGULATION

OF

A

CTIN

D

YNAMICS

Recently it has been shown that the phosphoprotein cofilin, also known as the actindepolymerisation factor, is actively involved with actin depolymerisation and incellular processes that require cyclical changes in the actin cytoskeleton (Abe

et al.

1996; Lappalainen and Drubin, 1997; Theriot, 1995). Cofilin is a 20-kDa proteinfound in muscle as well as nonmuscle cells. Two isoforms of this protein occur in



89

mammals: the muscle (M) type and nonmuscle (NM) type. M-type cofilin expressionis up-regulated in the myogenesis of C2 cells

in vitro

(Ono

et al.

1994). Goode

etal.

(1998) identified a 37-kDa protein in budding yeast, and they named it twinfilinbecause it contains two cofilin-like regions. Twinfilin is able to sequester actinmonomers, but it does not inhibit actin polymerisation. Homologues of this proteinmay occur in man, mouse, and

Caenorhabditis elegans

, as sequence data searcheshave revealed, and Goode

et al.

(1998) suggest twinfilin may be highly conservedin evolution.

Cofilin regulates actin polymerisation in a pH-dependent manner. Rapid cyclesof assembly and disassembly of actin depend on this protein (Lappalainen andDrubin, 1997). Overexpression of cofilin leads to disorganisation of actin filaments(Ono

et al.

1996). Its intracellular distribution as well as its interaction with actinare regulated by phosphorylation (Obinata

et al

. 1997). Cofilin is phosphorylatedby LIM kinases (LIMK-1 and -2) (Arber

et al.

1998; Yang

et al.

1998; Sumi

et al.

1999). Phosphorylated cofilin loses its normal function of depolymerising actinfilaments. Cellular response in the form of alterations in cellular morphology, mem-brane ruffling, and neuronal outgrowths results from the growth factors being ableto activate cofilin. In the case of neurite extension, the dephosphorylation of cofilin

FIGURE 12

Schema of the participation of actin-binding proteins in the regulation of actindynamics T

β

4, thymosin

β

4.

Profilin

G-actin ≈ F-actin

Sequestration ofG-actin

Tβ4/G-actin complex Profilin/G-actin

Prevention of polymerisation

Myosin Fragment I

Phalloidin

Repolymerisation

Tβ4


90


has been found to correlate with its translocation to the membrane (Meberg

et al.

1998). Actin-binding peptide derived from cofilin has been reported to compete withgelsolin segments 2 and 3 for actin-binding. These binding sites of cofilin andgelsolin have structural homologies and therefore they possibly share the samebinding domain on the actin filament (Van Troys

et al.

1997). How this structuralrelationship between cofilin and gelsolin translates in functional terms is yet uncer-tain. However, Van Troys

et al.

(1999) seem to have made a beginning by providinga framework based on the structure of the actin-binding modules that occur in actin-binding proteins, the similarities and differences between these binding domains,and how, overall, they might impinge on actin dynamics.

P

ROFILIN

IN

THE

R

EGULATION

OF

A

CTIN

D

YNAMICS

Profilin is another protein that participates in and regulates actin dynamics in cellularresponses to external as well as internal signals (Staiger

et al.

1997). It is a 14 to15-kDa protein that has been found to sequester monomeric actin, to promotenucleotide exchange occurring on actin monomers, and to be capable of inducingactin polymerisation when barbed ends of actin filaments are free. Profilin also bindsto poly-l-proline and phosphoinositol lipids. In mammalian cells two isoforms ofprofilin have been described and these also may be differentially expressed indifferent tissues (Witke

et al.

1998). Profilin forms a high-affinity complex withactin (Di Nubile and Huang, 1997). The importance of this complex formation inthe cellular function of actin filaments is demonstrated by the effects of deletingresidue proline 96 and threonine 97 that occur near the major actin-binding site.Deletion of these amino acids reduces the ability of profilin to bind to actin and itsability to promote nucleotide exchange on actin monomers. When injected into Swiss3T3 fibroblasts, the mutant profilin failed to affect microfilament organisation, whichthe wild-type was capable of achieving (Hajkova

et al.

1997). A diminution ofprofilin expression has been shown to result in abnormalities of cytokinesis and theformation of multinucleate cells (Cao

et al.

1997). Profilin is also a strong competitoragainst T

β

4 in binding to actin (Ballweber

et al.

1998; also, see below). It wouldbe reasonable to accept that profilin is directly involved in actin polymerisation andin this way influences morphological features and biological behaviour of cells.

R

HO

GTP

ASES

IN

A

CTIN

D

YNAMICS

AND

S

IGNAL

T

RANSDUCTION

The Rho family of GTPases are ras-related GTPases. In the yeast

Saccharomycescerevisiae

, certain members of the family are known to control the process of buddingand the determination of cell polarity (Johnson and Pringle, 1990). The mammalianRho proteins have been implicated in cytoskeletal remodelling, and cofilin could bethe terminal target that brings this about. Rho proteins also have been associatedwith the control of microfilament assembly

in vitro

and the formation of cytoskeletalstructures such as filopodia, lamellipodia, adhesion plaques, and intercellular junc-tions (Ridley and Hall, 1992; Nobes and Hall, 1995, 1999; Peterson

et al.

1990; VanAelst and D’Souza-Schory, 1997). Intercellular adhesion is another cellular facultyin which Rho proteins are involved (Morii

et al.

1992; Tominaga

et al.

1993). The



91

processes of cell spreading and alterations in cell morphology and the transductionof growth factor signals also require the participation of and are regulated by Rhoproteins (G.A. Murphy

et al.

1999).At the fundamental level in all these cell functions is the contractile ability of

the actin cytoskeleton, which has been attributed to the activation of Rho GTPasesin response to microtubule depolymerisation (B.P. Liu

et al.

1998). The Rho GTPasesseem to activate a host of signalling molecules such as tyrosine kinases, serine/thre-onine kinases, and lipid kinases. These kinases phosphorylate downstream targetproteins, directly or indirectly

via

the phosphorylation of other kinases. Rho andCdc42 induce the reorganisation of the actin cytoskeleton and promote the formationof stress fibres. This process appears to be related to the activation of LIMK-2.LIMK-2 phosphorylates cofilin and inhibits its normal function of cytoskeletondepolymerisation (Sumi

et al.

1999). The Rho-activated serine/threonine kinases(ROCK) seem to be important elements in cancer invasion. Itoh

et al.

(1999)transfected a dominant active mutant of ROCK cDNA into rat MMI hepatoma cells.These transfectant cells were found to possess greatly enhanced invasive ability, incomparison with cells that had been transfected with a dominant negative ROCKcDNA. The MLCK of endothelial cells is another kinase that seems to be regulatedby Rho (Garcia

et al.

1999). The possibility that Rho proteins might regulate thecontractile apparatus of endothelial cells and alter the permeability of endothelia asa consequence has serious implications for cancer cell dissemination, in which thepermeation of the endothelial barrier is an essential event.

The induction of membrane ruffles has been attributed to Rac, a GTPase thatbelongs to the Rho family. Rho GTPases have been implicated in Rho kinase-mediated phosphorylation of transmembrane adhesion proteins, such as the CD44splice variants, and their interaction with the cytoskeletal protein, ankyrin (Bourgui-gnon

et al.

1999). This interaction between CD44 and ankyrin is manifested aschanges in membrane activity in the form of ruffling. In this particular instance, RhoA GTPase stimulates ROCK, which phosphorylates CD44. This leads to an enhance-ment of CD44–ankyrin interaction. Bourguignon

et al.

(1999) also demonstratedthat the induction of CD44-associated membrane ruffling can be achieved by inject-ing the catalytic domain of ROCK and, furthermore, that the membrane activity isinhibited by anti-CD44 antibodies. A more indirect route might be adopted in theRho-mediated reorganisation of the cytoskeleton involving cofilin. Here ROCKphosphorylates and activates LIMK, which then phosphorylates cofilin (Maekawa

et al.

1999).Some members of the S100 protein family are known to be able to modulate

and possibly regulate actin dynamics. Among them is S100B, which has recentlybeen shown to bind and activate the serine/threonine kinase Ndr (Millward

et al.

1998). The Ndr kinase has structural and possibly also functional similarities withROCK. It is suggested, therefore, that there might exist a general and commonmechanism by which S100 proteins could regulate actin dynamics and the cellularprocesses and functions upon which the modulation of actin dynamics and cytosk-eletal organisation might strongly impinge.

A family of proteins called WASP [Wiskott-Aldrich syndrome (WAS) protein]is regarded as the regulators of actin reorganisation. WAS is an X-linked condition


92


that affects male children. The syndrome is generally described as including immu-nodeficiency, thrombocytopenia, and eczema, but the actual phenotype may bevariable. The WAS gene is mutated in patients and the type of mutation seems torelate to the phenotype of the disease. WASPs are cytoplasmic proteins. One WASPisoform is found only in the lymphocytes. Another isoform called the N-WASP,which was isolated from neural cells, shows a more ubiquitous distribution. Theseproteins are known to bind to several cellular proteins and kinases and also to RhoGTPases, such as Cdc42 and Rac. They interact directly or indirectly with the actincytoskeleton. The haemopoietic cells derived from patients with WAS also have beenreported to show abnormalities of the actin cytoskeleton. These observations suggestthat WASPs play an important part in the transduction of extracellular signals to thecytoskeleton (O’Sullivan

et al.

1999). They are believed to function downstream ofRac. The association of WASP with the induction of membrane activity has beendemonstrated recently. Castellano

et al.

(1999) showed that experimentally inducedrecruitment of activated Cdc42, or its downstream effector WASP, to the cell surfaceresults in actin polymerisation and the formation of filopodia.

Other actin-binding proteins, such as profilin, that are associated with actindynamics may therefore be expected to enter into this picture of WASP- and Rho-mediated regulation and organisation of the actin cytoskeleton. Suetsugu

et al.

(1998)generated a mutant profilin, H119E, that is defective in actin-binding. This mutantprofilin suppressed actin polymerisation induced by N-WASP. Under normal cir-cumstances profilin associates with N-WASP, and is essential for a rapidly poly-merising actin by N-WASP; this cannot happen with mutated profilin.

I

NTERACTION

OF

F

ORMIN

WITH

P

ROFILIN

AND

R

HO

GTP

ASES

The formin family of proteins constitutes another component of the actin regulatorysystem. Formin and related proteins, which are expressed ubiquitously in severalorganisms, seem to knit together profilin and Rho GTPases in their function ofregulating cytoskeletal dynamics. Members of the formin family share many struc-tural features such as the coiled-coil molecular organisation, the occurrence ofhomology sequences known as formin homology (FH) 1 and FH2 domains, thecollagen-like domain, nuclear localisation signals, and phosphorylation sites. Theformin gene was identified by mutations in the gene associated with recessive limbbud deformity (Maas

et al.

1990, 1991; Chan

et al.

1995). The gene has 24 exonsencompassing 400 kb of genomic DNA (C.C. Wang

et al.

1997).The formins are predominantly nuclear proteins participating in cytokinesis and

cell polarisation (Zeller

et al.

1999). They are essential for the correct orientationand alignment of the cell division spindle (L. Lee

et al.

1999). Formin also partic-ipates in morphogenesis, e.g., in the yeast budding process. The importance of theirrole in morphogenesis is also indicated by the fact that mutation in the formin geneleads to disruption of epithelial–mesenchymal interactions, which in turn leads toabnormal limb development. This has suggested the possibility that it may participatein the transduction of morphogenetic signals (Zeller

et al.

1999). Formin has severalligands. Prominent among them is profilin (Mittermann et al. 1998). Rho GTPaseis a downstream target of formin (Watanabe et al. 1997). It seems, therefore, that


The Structure of Contractile Proteins 93

formin is an important element in profilin/Rho-dependent actin polymerisation ofcytoskeletal dynamics and in this way might regulate cytokinesis, cell morphology,and adhesion-dependent processes such as cell motility and invasion.

THE ROLE OF THYMOSIN FAMILY ACTIN-BINDING PROTEINS IN ACTIN DYNAMICS

SEQUESTRATION OF ACTIN BY THYMOSINS

The beta thymosins constitute an important family of actin-binding proteins, whichare recognised as major participants in the regulation of actin dynamics. Among itsmembers are Tβ4, Tβ10, and Tβ15, which are the most intensively investigatedforms of thymosin. Tβ4 is a 4.5-kDa protein consisting of 43 amino acid residues.Tβ14 is another protein isolated from the sea urchin, that has 40 amino acid residuesand a molecular size of 4.53 kDa and bears a high degree of sequence homology toTβ4 (Stoeva et al. 1997). Tβ15 is a 5.3-kDa protein. Thymosins seem to occurubiquitously. Actin-binding proteins showing a high degree of homology to mam-malian thymosins have been isolated from invertebrates. The thymosins from inver-tebrates have 40 amino acid residues, but they appear to differ with respect to theiraffinity for binding to rabbit muscle actin (Safer and Chowrashi, 1997).

The thymosins function by sequestering monomeric actin and thereby diminish-ing actin polymerisation (Safer, 1992). Tβ4 binds to actin monomers by means ofits N-terminal 5 to 20 amino acid residue sequence (Van Compernolle et al. 1992;Czisch et al. 1993; Van Troys et al. 1996). In vitro this peptide adopts a foldedconformation for achieving high-affinity interactions with actin (Feinberg et al.1996a). Feinberg et al. (1996b) have discovered homologous short sequences in Tβ4and gelsolin that bind to the C-terminal region of actin. They have also commentedon the ability of these short sequences to form secondary structures and its relation-ship to their biological function. However, Safer et al. (1997) found that Tβ4 cross-links to both barbed and pointed ends of actin, and this requires that the C-terminaldomain, which participates in the binding, is in an extended conformation.

EFFECTS OF THYMOSINS ON CELL PROLIFERATION

The ability of Tβ4 to inhibit actin polymerisation is well documented. Also ade-quately demonstrated is the involvement of thymosins in proliferation, motility, celldifferentiation, and angiogenesis, all of them important features of embryonic devel-opment and cancer development and progression. Recently, Sanger et al. (1995)experimentally enhanced the intracellular levels of Tβ4 and studied the structuralintegrity of actin bundles of stress fibres and cytokinetic furrows. They microinjecteda viral vector carrying Tβ4 cDNA, transfected the vector into PTK2 cells, or injectedpurified Tβ4 protein into these cells. Both microinjection and transfection of cDNAproduced a disassembly of stress fibres. The effects of injection of the pure proteinwere even more marked; a disassembly of stress fibres occurred within 10 min afterinjection, resulting in delayed cytokinesis. However, although Tβ4 reduces actin



polymerisation by sequestering monomeric G-protein, at higher concentrations itsability to depolymerise F-actin appears to decrease (Carlier et al. 1996).

The effects of thymosins on cell proliferation are therefore inevitable. Sangeret al. (1995) demonstrated that an overexpression of Tβ4 delays cell division. TheN-terminal part of the protein contains an acetylated tetrapeptide sequence ofAc–N–Ser–Asp–Lys–Pro that is known to inhibit haemopoiesis. This peptide hasbeen shown to inhibit the growth of normal bone marrow progenitor cells. It mark-edly reduces the growth of granulomacrophagic and erythroid progenitor cells andthe size of their S-phase fraction (Bonnet et al. 1996). However, Bonnet et al. (1996)also found that although the inhibitory effect of the Tβ4 is similar to that of thetetrapeptide, Tβ4 from which this sequence is deleted retains the inhibitory effect.

THYMOSINS AND CELL MOTILITY AND DIFFERENTIATION

Needless to say, the amply confirmed effects of thymosins on the structural integrityof the cytoskeleton are bound to find expression in altered cell motility. Tβ4 hasbeen shown to enhance strongly the migratory behaviour of an established cell lineof endothelial cells derived from human umbilical vein (Malinda et al. 1997). Ofthe primary human cell lines that Malinda et al. (1997) examined, the migration ofonly human coronary artery cells seemed to respond to Tβ4. They also reported anincrease in the production of metalloproteinases upon treatment with the thymosin.These enzymes can remodel the extracellular matrix and aid migration by alteringcell adhesion to the substratum. NIH3T3 cells induced to overexpress Tβ4 have beenreported to be more adherent than corresponding control cells (Golla et al. 1997).This enhanced adhesion appears to be due to adhesion-mediating proteins such asα-5 integrin as well as the transmembrane proteins such as vinculin that link cellmembrane proteins to the cytoskeleton.

There are also important implications of thymosin overexpression for cell migra-tion in the context of cancer invasion, because thymosins have been reported to beoverexpressed in neoplastic cells (see below). Especially significant are the reportedspecificity of response by endothelial cells to Tβ4 and the associated overexpressionof metalloproteinases. Metalloproteinases have been closely associated with breach-ing of the endothelium and aiding the diapedesis of cancer cells into the vascularcompartment (Sherbet and Lakshmi, 1997b). Tβ4 is reported to be highly expressedin the intrinsically highly motile embryonic mesenchymal cells (Carpintero et al.1996). The expression of Tβ15 seems to be up-regulated in the highly motileDunning rat prostate cancer cell lines. Besides, the transfection of antisense con-structs of Tβ15 gene apparently alters the motility of these cells (Bao et al. 1996).

EXPRESSION OF THYMOSINS IN EMBRYONIC DEVELOPMENT

Differential cell adhesion and migration patterns are an integral feature of celldifferentiation and morphogenesis. The expression of Tβ4 and Tβ10 shows somerelationship to specific developmental stages (Carpintero et al. 1996), although thereare as yet no indications that it is developmentally regulated. Carpintero et al. (1996)found a marked increase in Tβ10 mRNA in early postimplantation mouse embryos.



This mRNA is also found in certain tissues, including mesenchymal tissues and thespinal cord, in 9.5 to 12.5-day old embryos. These authors have also encountereddifferences in the distribution of Tβ4 and Tβ10 mRNAs, which could conceivablysuggest differences in their function in embryonic development. Tβ4 has beendetected in early chick embryo cells but not in adult skeletal muscle cells. Cofilinalso is associated with muscle development, but its relative contribution may changeduring muscle development (Nagaoka et al. 1996). These observations are consistentwith the notion of development-related expression of thymosins.

Gomez-Marquez et al. (1996) showed that Tβ4 mRNA occurs in mouse embry-onic stem cells, and further that Tβ4 is able to induce embryonal P19 cells todifferentiate into neuronal- and glial-type cells or into cardiac and skeletal muscle-type cells. Furthermore, Tβ4 mRNA was demonstrable around blood vessels and inheart tissues.

POTENTIAL ROLE OF THYMOSINS IN CANCER PROGRESSION

As stated before, the putative association of overexpression of Tβ4 with increasedproduction of metalloproteinases in normal cells together with the relationshipbetween Tβ4 and high invasive ability of the Dunning rat prostate cancer cell linespoint to the relevance of thymosins in cancer invasion and metastasis.

Both Tβ10 and Tβ15 have been investigated for their possible relationship withcancer progression. Tβ10 protein was reported to occur at high levels in the malignantcell rather than the normal tissue component of human breast cancer, and the levelof expression increased with tumour grade (Verghese-Nikolakaki et al. 1996). Theexpression of Tβ10 gene was higher in five thyroid carcinoma cell lines as comparedwith normal thyroid-derived primary cells. Expression of the gene was higher inanaplastic tumour tissue (Califano et al. 1998).

Tβ15 levels have been examined in several tumour cell lines of various histo-logical origins. An up-regulation of the gene has been found in highly metastaticmouse lung and human breast cancer cell lines. This study also involved an exam-ination of the levels of Tβ15 protein in human breast and prostate cancer tissues.Higher expression of Tβ15 has been correlated with the Gleeson grade in prostatecarcinomas (Bao et al. 1996). In breast cancer, higher levels of the protein correlatedwith greater metastatic potential (Bao et al. 1998). This is in contrast with theirearlier studies (Gold et al. 1997). Gold et al. (1997) had encountered higher Tβ15expression in nonmetastatic breast cancer, which suggested the involvement ofthymosins in the early stages of breast cancer development rather than in the latestages of the disease. Although much attention has been focused on β-thymosins,Tsitsilonis et al. (1998) found α-thymosins to be of prognostic value in breast cancer.They determined levels of prothymosin α and parathymosin α in breast carcinomatissue as well as in tissues from benign breast disease, and found that levels of bothare much higher in carcinomas than in benign tumour tissue and normal breast tissue.They also reported that these levels correlated with overall survival of patients.

As stated before, Tβ4 expression has been found to be very high in Dunningrat prostate carcinoma cells, which possess high motility. When Tβ4 antisenseconstructs were transfected into these cells the invasive ability was suppressed,



suggesting that the high levels of Tβ4 expression might be associated with highinvasive ability. Contrary to this, transfection of adenovirus E1A and E1B geneshas been carried out in a highly metastatic human melanoma cell line by VanGroningen et al. (1996) who reported that a number of markers for tumour pro-gression were down-regulated as a consequence. Among them was Tβ10. Admit-tedly, E1A gene products can inhibit oncogene-mediated cell transformation as wellas invasion and metastasis in certain animal models. E1A protein is known to beable to induce and stabilise p53 phosphoprotein, which controls the G1-S checkpointtransition of cells, and this is accompanied by apoptic loss of cells. On the otherhand, E1B protein can bring p53 levels to normality (Sherbet and Lakshmi, 1997b).However, the raft of changes produced by the transfection of the adenovirus genesmakes it a rather inappropriate model for the study of tumour progression. Althoughthe transfected cells may have shown a reduction in tumorigenicity upon implan-tation into compatible hosts, under these circumstances it would be difficult, evenunacceptable, to try to extrapolate as to the significance of the suppression ofindividual markers in relation to tumorigenicity.

Some of the difficulties are inherent in the interpretation of data obtained fromexperimental studies such as gene transfer in vitro and on tumour tissues frompatients. Therefore, it is imperative that further studies be undertaken to assess thepotential value of thymosins in determining the state of tumour progression.

THE FIMBRIN FAMILY OF ACTIN-BINDING PROTEINS

MOLECULAR FEATURES OF FIMBRIN

Fimbrins are EF-hand calcium-binding proteins that actively participate in bindingto and bundling of actin. In actin filaments, one molecule of fimbrin might bindeight actin monomers under optimal conditions (Namba et al. 1992). Fimbrins arehighly conserved in evolution, with regard to both their structure and function.Fimbrin from diverse origins, e.g., from Dictyostelium discoideum to humans, sharestructural and biochemical properties. Fimbrin-like proteins have also been isolatedfrom plants, such as wheat (Triticum aestivum) and Arabidopsis thaliana (Cruz-Ortega et al. 1997). The Dictyostelium fimbrin is a 67-kDa protein containing twoEF-hands that are followed by two actin-binding sites (Prassler et al. 1997). The N-terminal actin-binding domain is a highly conserved domain that fimbrin shares withother actin-binding proteins. This N-terminal domain contains two tandem calponinhomology (CH) domains which are implicated in the binding of F-actin (Goldsmithet al. 1997). Fimbrin-mediated cross-linking and bundling of actin is regulated byfree calcium, which it binds with high affinity and selectivity. However, the optimalconcentration for cross-linking of actin was determined to be below 0.15 µM. Thisprocess was progressively inhibited at higher free calcium concentrations and half-maximal inhibition of cross-linking occurred at 1.6 µM Ca2+ concentration (Pacaudand Derancourt, 1993). Pacaud and Derancourt (1993) also noted that calcium-binding of fimbrin, in the concentration range of 0.15 to 1.5 µM, produced confor-mational changes in the molecule, to which they attribute the calcium-mediatedregulation of fimbrin function. It may be that the conformational alterations actuated



by calcium at different concentrations could render actin cross-linking reversible. Afurther possibility has arisen from the studies of Hanein et al. (1997a) that fimbrinmight induce conformational changes in actin itself. Both types of change willinevitably contribute to cell behaviour.

Fimbrins form a large family of proteins. Four genes encode fimbrin in Salmo-nella (Collinson et al. 1996). Three isoforms of fimbrin have been identified anddesignated as I-fimbrin, L-fimbrin, and T-fimbrin. I- and L-fimbrin are characteris-tically associated with intestinal and kidney epithelia, leukocytes, and tumours,whereas T-fimbrin shows a more general distribution in a variety of cells and tissues(Chafel, 1995).

FUNCTION OF FIMBRIN IN CYTOSKELETAL ORGANISATION

A major function of fimbrin appears to be in the assembly of actin filaments. Fimbrinas well as actin capping protein (CP) are required for proper assembly of thesefilaments in the yeast Saccharomyces cerevisiae. There is a reduced filament assem-bly and fimbrin in the CP mutants of the yeast. Actin obtained from CP mutantsshows defects in polymerisation as well as in its binding to fimbrin (Karpova et al.1995). In mammalian cells, Rho GTPases may mediate actin filament assembly andbundling. A constitutive expression of the Rho GTPase Cdc42Hs causes impairmentof cytokinesis of HeLa cells. This seems to be a consequence of a reorganisation ofF-actin with which, among other actin-binding proteins, T-fimbrin is associated(Dutartre et al. 1996). Functional conservation of fimbrin isoforms has been amplydemonstrated by Adams et al. (1995), who found that human T- and L-fimbrin cansubstitute for the yeast fimbrin called Sac6p. T- and L-fimbrin were both able tocomplement the temperature-sensitive growth defect that is seen in sac6 null mutants,and they could also restore normal cytoskeletal organisation and cell shape in thesemutants. The null mutants show defective sporulation, which is restored by humanT- and L-fimbrin (Adams et al. 1995).

Fimbrin isoforms show not only a tissue-specific distribution, but the specificityappears also to extend to differentiation and morphogenesis. Fimbrin shows a defin-able temporal and spatial pattern of expression in the course of the development ofthe cochlea and may be involved in the formation of the inner and outer stereociliaof the hair cell (Zine et al. 1995). The differentiation of intestinal epithelium isassociated with the expression of T-fimbrin in the apical area of the cell and L-fimbrin in the basal area, until 14.5 days of development. Both isoforms are said tobe totally down-regulated in expression by day 16.5, but instead, I-fimbrin appearson day 14.5 of development to give the epithelium a brush border-like localisation.These findings, reported by Chafel et al. (1995), suggest possible differences in theirfunction in the course of differentiation. A number of the events occurring inmorphogenesis and differentiation require changing patterns of cell adhesion, forwhich fimbrin seems to be ideally placed. Babb et al. (1997) studied the localisationof fimbrin in mature as well as differentiating osteoclasts. Fimbrin is a componentof the osteoclast adhesion complexes called podosomes. During migration podo-somes are found at the cell periphery. Microfilament organisation and podosomeassembly is important in the rapid modulation of adhesion to the substratum, and



in the motility of cells. T- and L-fimbrin, but not I-fimbrin occurred as an integralcomponent of the core of the podosomes throughout the process of monocyte-derivedosteoclast differentiation. The levels of T-fimbrin increased with and appeared to berelated to the formation of podosomes.

The involvement of fimbrin in podosomes is compatible with the association offimbrin with the adhesive organelles called fimbriae that have been described inbacteria. Fimbriae are fibre-like structures that mediate the attachment of bacteriato host cells. These are assembled from fimbrin subunits (Smyth et al. 1996).Frederick et al. (1996) investigated the adhesive interactions between lymphokine-activated killer (LAK) cells and came to the conclusion that fimbrin might be animportant factor in intercellular adhesive contacts. They also showed that contactbetween LAK cells and target tumour cells, namely SK-Mel-1 human melanomacells and Raji lymphoma cells, leads to the phosphorylation of L-fimbrin of the LAKcells. S.L. Jones et al. (1998) have also suggested that induction of fimbrin phos-phorylation is an important step in fimbrin function. However, as discussed below,the question of whether phosphorylation leads to fimbrin activation must be regardedas sub judice at present. Podosomes that participate in cell adhesion and locomotionhave been found to contain other cytoskeletal linking proteins such as talin andvinculin, besides fimbrin. L-fimbrin has been shown to regulate integrin-mediatedadhesion of leukocytes (S.L. Jones et al. 1998). It is possible that fimbrin is instru-mental in the organisation of physical pathways, such as fimbriae and podosomes,by which extracellular signals for intercellular and cell–substratum adhesion aretransduced to the cell.

Another membrane organelle with which fimbrin might be associated in organ-ising a signal transduction machinery is the caveola. Caveolae are plasma membraneinvaginations, of 50 to 100 nm dimension, that occur in many cell types. Caveolaehave been attributed with many functions, notably transport of molecules acrossendothelia and signal transduction (Lisanti et al. 1995). A major component ofcaveolae is a 21- to 24-kDa protein called caveolin. Caveolin is said to function asa scaffolding protein that organises the signalling molecules in the caveolae. Thatcaveolae contain essential components of the signal transduction machinery may bedeemed to be firmly established. Thus caveolin has been shown to interact directlywith signal transduction molecules such G-protein α subunits and the ras protein(Lisanti et al. 1995; Song et al. 1996). The receptors for PDGF and EGF are locatedin caveolin-rich microdomains, and caveolin has been shown to interact directly withthe EGF receptor (G.X. Liu et al. 1996; Couet et al. 1997). The interaction ofcaveolin negatively regulates RTK activities associated with activated EGFr and c-erbB2 (Couet et al. 1997), and in mammary tumours expressing c-erbB2, caveolinexpression is down-regulated (Engelman et al. 1998). Therefore, this interactionbetween growth factor receptors and caveolin may deregulate the transduction ofthe growth factor signals. Compatible with this view are recent reports that overex-pression of caveolin results in growth inhibition of tumour cells (S.W. Lee et al.1998; T. Suzuki et al. 1998). On the other hand, caveolin has been associated withcancer progression. It is reported to be overexpressed in infiltrating ductal carcinomaof the breast and prostate. In prostate cancer, caveolin is expressed in both primaryand metastatic tumours (N. Yang et al. 1998). These two sets of data may appear to



contradict each other, but it is possible to envisage caveolin functioning as a growthinhibitor in the early stages of tumour development. Caveolin might function in laterstages of tumour progression merely by virtue of its ability to promote cell–cell andcell–substratum adhesion, which are essential requisites for a successful transitionof the cancer cell along the metastatic cascade. In this connection, it would beworthwhile to note that T-cadherin, an adhesion-mediating molecule, also occurs incaveolin-rich plasma membrane microdomains (Philippova et al. 1998). After aseemingly long digression, it should also be noted that fimbrin is a component ofthe caveolae. Fimbrin occurs together with other proteins, e.g., src and ezrin, in cellmembranes that are rich in caveolin (Mirre et al. 1996). The available evidence,albeit circumstantial in nature, may be interpreted as implicating fimbrin in tumor-igenesis and tumour progression by deregulating signal transduction pathways andcell adhesion mechanisms.

REGULATION OF FIMBRIN EXPRESSION

The apparent tissue-specific distribution of fimbrin isoforms has led inevitably toinvestigations directed toward understanding the mechanisms of fimbrin expressionand whether there is any tissue-specific regulation of its expression. C.S. Lin et al.(1997) have characterised the promoters of human and murine L-fimbrin. Theydescribed considerable similarity in the organisation and found that both promotersfunctioned with equal efficiency in most cell types. Therefore, the apparent tissue-specific and differentiation-related expression of fimbrin could be due to posttran-scriptional modification of fimbrin rather than to its regulation at the transcriptionallevel. Phosphorylation could be a mechanism of regulation of fimbrin function, asdemonstrated for L-fimbrin. The serine residues of the head-piece region of L-fimbrinare phosphorylated (Messier et al. 1993), although earlier, Namba et al. (1992) foundunphosphorylated L-fimbrin of human T cells to be quite effective in F-actin bun-dling. Shinomiya et al. (1995) reported that LPS stimulated the phosphorylation ofL-fimbrin in macrophages. Phosphorylation occurred on amino acid residues at theN-terminal region close to the first Ca2+-binding domain. Shinomiya et al. (1995)also noticed that the phosphorylated region contained motifs that are phosphorylatedby CK II, protein kinase A (PKA), and PKC, but not motifs specific for MAPK.Frederick et al. (1996) also have recently implicated PKC in fimbrin phosphorylationand have confirmed further that only serine, not tyrosine, residue is phosphorylated.The possible linkage of fimbrin function with phosphorylation is indicated by theability of cytokines and phorbol esters, among other agents, to induce fimbrinphosphorylation. Polymorphonuclear leukocytes (PMN) stimulated by IL-8, IL-1,neutrophil-activating proteins, monocyte-derived neutrophil chemotactic factor, andTNF have been reported to stimulate the phosphorylation of I-fimbrin. Phosphory-lation was also influenced by phorbol 12-myristate 13-acetate (PMA) (Shiroo et al.1988; Shibata et al. 1993a, 1993b). Fimbrin of T cells is phosphorylated in responseto IL-2 (Zu et al. 1990). The adhesion of neutrophils to immune complexes inducesL-fimbrin phosphorylation. Furthermore, it appears that phosphorylation-mediatedregulation may be distinct from calcium-mediated regulation of fimbrin (Jones andBrown, 1996). It seems likely that the transduction of extracellular signals involves



this important component of the membrane cytoskeleton. However, despite the cleardemonstration of a fimbrin phosphorylation response to extracellular signals, thereis little direct evidence that these features are functionally related. Messier et al.(1993) provide circumstantial evidence for this. They found phosphorylated fimbrinwas mainly associated with the insoluble cytoskeleton. When this is read with theirobservation that the serine residues of the head piece were specifically phosphory-lated, one might be justified in concluding that phosphorylation could regulate theprocess of actin-binding and bundling by fimbrin.

Whether the isoforms have different functions in the regulation of cell morphol-ogy is a question that has been addressed, albeit superficially, by Arpin et al. (1994).In CV1 fibroblast-like cells, both T- and L-fimbrin cause change of cell shape andreorganisation of the actin stress fibres, and only L-fimbrin is associated withmicrofilaments. In epithelial cells such as LLC-PK1 cells, T-fimbrin remains asso-ciated with actin filaments of microvilli and produces shape changes in them. L-fimbrin has no effect on these structures. These observations might suggest functionaldifferences between the isoforms. The invasion of Shigella flexneri, a bacterium thatcauses dysentery in humans, involves the formation of heavy actin polymerisationand actin bundling near the site of host cell contact with the bacterium. These bundlesform protrusions with which the bacterium coalesces. Adam et al. (1995) experi-mentally overexpressed T- and L-fimbrin in HeLa cells and demonstrated that T-fimbrin might be preferentially recruited to the zone of bacterial entry.

IS FIMBRIN INVOLVED IN CANCER?

The potential of fimbrin to modulate cell behaviour is obviously quite considerable.There are references in the literature since the early 1980s that fimbrin expressionincreases with cell transformation, especially in fibroblast cells. However, littleincisive investigation seems to have been carried out. More recent investigationshave pursued the worthwhile aim of establishing a role for fimbrin in neoplastictransformation and tumour progression. The expression of L-fimbrin has been inves-tigated in cell lines derived from carcinoma of the prostate. L-fimbrin has been foundin many carcinoma cell lines, but not in normal epithelial cell lines derived fromthe prostate. L-fimbrin occurs at higher levels in prostate cancer tissue as comparedwith normal prostate tissue. Immunohistochemical staining has suggested that theincrease of fimbrin occurs predominantly in the glandular epithelial cells of thecarcinoma, whereas in normal prostate tissue, it is found mainly in the fibromuscularstroma. It would have been interesting to explore the fimbrin expression pattern ofbenign prostatic hyperplasia (BPH). Nonetheless, the differences shown to existbetween normal prostatic epithelium and carcinomatous glandular epithelium arepersuasive enough to accept that fimbrin expression may be related to the carcino-matous changes.

Interestingly, fimbrin expression was also reported to be rapidly up-regulated inLNCaP cells by dihydrotestosterone and oestradiol (J.P. Zheng et al. 1997). Thisobservation does not per se contribute much to the argument that fimbrin is involvedin the transformation of normal epithelium to carcinoma. Testicular hormones arethe major factors that regulate the growth and development of the prostate. It is



possible that changes in hormonal milieu are responsible for the development ofBPH and onward to the development of prostatic carcinoma. Androgen receptors(ARs), oestrogen receptors (ERs), and progesterone receptors (PgR) occur in BPHand in prostate cancer (Srinivasan et al.1995). Indeed, AR occurs in all histologicaltypes and all clinical stages of prostate gland cancer. Because androgens are involvedin the development of the prostate itself, it would be reasonable to suppose that ARoccurs in the developing prostate as well. Therefore, one would expect that theandrogen-sensitive cell line LNCaP would respond by an altered level of fimbrin.What is crucially important is to find out whether normal epithelial cells responddifferently from carcinomas. Testicular hormones bind to the appropriate receptorsand stimulate the transcription of androgen-responsive genes, including those thatregulate the growth of prostate cells. It should be borne in mind, further, that theprogression of prostate cancer is associated with a change from androgen-dependentto androgen-independent state. The fimbrin gene may be a steroid-regulated gene,and it is important to determine whether this is the case, and whether androgendifferentially stimulates fimbrin transcription in normal prostatic epithelium andprostatic carcinoma cells.

Another isolated observation, which may, nonetheless, be significant in thecontext of drug resistance of tumours, is the relationship discovered by Hisano etal. (1996) between cisplatin resistance and fimbrin expression. They observed thatcisplatin-resistant cells possessed severalfold greater levels of T-fimbrin comparedwith sensitive cells. They transfected T-fimbrin antisense cDNA into cisplatin-resis-tant cells and showed that reduction of fimbrin expression resulted in increasedsensitivity to the drug.

MODULATION OF ACTIN DYNAMICS AND CANCER CELL DISSEMINATION

Conceptually there can be no difficulties in accepting the proposition that the mod-ulation of actin dynamics should be involved in some way with tumour dissemina-tion. A reasonable body of experimental evidence, in the form of changes in theexpression of gelsolin, thymosins, and fimbrin in cancer progression, has beenadduced in support of this concept. In this context of actin-interacting proteins, thevasodilator-stimulated phosphoprotein (VASP) deserves a mention. VASP is approx-imately 45 to 50 kDa in size and occurs in domains of the plasma membrane thatare involved in the formation of lamellipodia (Reinhard et al. 1992, 1995b). Togetherwith the Drosophila- and murine-enabled (Ena) proteins, VASP belongs to a familyof actin-binding proteins that are actively engaged in the regulation of the actincytoskeleton. VASP has been shown to interact with actin stress fibres via its C-terminal region (Huttelmaier et al. 1999). VASP as well as Ena can interact withthe actin CP profilin (see below) (Reinhard et al. 1995a) and with focal adhesionproteins (Reinhard et al. 1995b, Brindle et al. 1996; Lanier et al. 1999; Ahern-Djamali et al. 1998). This suggests that the VASP family members might be involvedin signal transduction (Huttelmaier et al. 1998). Besides, during in vitro morpho-genesis of human umbilical endothelial cells, VASP, profilin, and gelsolin are mark-edly up-regulated (Salazar et al. 1999). In view of the close association of all three



proteins in actin dynamics, Salazar et al. (1999) have postulated that reorganisationof the actin cytoskeleton is required for the alignment of the endothelial cells duringcapillary morphogenesis. The expression of profilin or VASP in neoplastic transfor-mation has not been studied in great detail. NIH3T3 fibroblasts that have been madeVASP deficient by experimental manipulation have been reported to be able to formtumours in nude mice. In vitro, these cells show loss of contact inhibition and aderegulation of cell proliferation (Liu K.Y. et al. 1999). With the demonstration thatthese proteins are associated with capillary morphogenesis, it would be ofmuch interest to see whether VASP and/or profilin are down-regulated in neoplastictransformation.

One can envisage that perturbation of the actin cytoskeleton could make the cellmembrane malleable and the cell more deformable and in this way aid invasion.Mechanistic perturbations in endothelial cell alignment could generate abnormalfenestration in tumour-associated microvasculature that might serve as ports of entryinto the vascular compartment for tumour dissemination. These observations high-light the potential importance of CBPs in cancer invasion, and also provide a fertileground for extracting information that might be highly relevant in assessing thedegree of malignancy of cancers and in cancer management.

αααα-ACTININ

The actin cytoskeleton is regulated by several actin-binding and cross-linking pro-teins that are themselves regulated by free calcium. α-actinin is an EF-hand proteinapproximately 110 kDa in size and it forms a major component of the cytoskeletonin many cell types. It forms antiparallel, highly stable homodimers. Actinin isoformscan also form heterodimers, both in vivo and in vitro (Y.M. Chan et al. 1998). Y.M.Chan et al. (1998) suggest the possibility that heterodimers formed by differentisoforms could potentially have new functional characteristics. The dimerisation ofsmooth muscle actinin is believed to be mediated by a segment of the C-terminalregion of the molecule (Baron et al. 1987; Imamura et al. 1988; Kahana and Gratzer,1991; A.P. Gilmore et al. 1994). This segment has two cross-linking sites (A andB), and the actinin molecules form cross-links in an antiparallel fashion by thebinding of an A site of one molecule with the B site of the second molecule (Imamuraet al. 1988). Several isoforms of α-actinin have been identified from vertebratecytoskeletal, skeletal, and smooth muscle sources (Duhaiman and Bamburg, 1984;Imamura and Masaki, 1992; Landon et al. 1985; J.P. Bennett et al. 1984). Isoformsalso have been isolated from invertebrate sources such as Dictyostelium discoideum(Noegel et al. 1987) and Drosophila melanogaster (Fyrberg et al. 1990). A cDNAhas been cloned from the nematode Caenorhabditis elegans. The sequence of thisclone predicts that it codes for an α-actinin, which possesses a high degree ofsequence homology to actinin from other sources (Barstead et al. 1991).

MOLECULAR STRUCTURE OF αααα-ACTININ

α-actinin is an EF-hand calcium-binding protein. Two EF-hands are located at the C-terminal region of the molecule. An actin-binding domain occurs at the N-terminal



end, followed by the rod domain, which consists of four spectrin-like repeat elements,followed by the EF-hand to the C-terminus (A. Blanchard et al. 1989; Flood et al.1995). The rod domain is required for stable dimerisation of the molecule, and dimer-isation is reduced markedly if either of terminal repeats 1 and 4 is deleted (Flood etal. 1995). Although all the isoforms possess EF-hands, these have been found to bein a functional state only in the nonmuscle or cytoskeletal isoforms of α-actinin(Burridge and Feramisco, 1981; Duhaiman and Bamburg, 1984). Their significancehas been elucidated by introducing point mutations to make them nonfunctional. Thefirst EF-hand seems to regulate the Ca2+-dependent cross-linking activity of α-actinin(Janssen et al. 1996). Nonmuscle isoforms might themselves differ in calcium-bindingability (Imamura et al. 1994). The EF-hands of α-actinin isoforms 2 and 3 of humanskeletal muscle are not capable of binding calcium, and therefore the binding of theseisoforms to actin might not be calcium sensitive (Beggs et al. 1992). Calcium sensi-tivity or the lack of it can be viewed from a functional viewpoint. The EF-hands appearto undergo conformational changes from a closed position in the absence of calcium,to an open position when calcium is present (Trave et al. 1995). The open conformationmay be conducive to protein–protein interactions, which are required for the transduc-tion of signals via the actin cytoskeleton.

αααα-ACTININ ISOFORMS

Two skeletal isoforms of α-actinin have been designated as α-actinin-2 and -3 andtwo nonmuscle isoforms as α-actinin-1 and -4 (Millake et al. 1989; Beggs et al.1992, 1994; Honda et al. 1998). The skeletal muscle isoform α-actinin-2 is foundin both human skeletal and cardiac muscle, but α-actinin-3 occurs only in limbskeletal muscle (Beggs et al. 1992). The isoforms α-actinin-1 and -4 occur in theactin microfilament bundles and at the adherens junctions. In skeletal, cardiac, andsmooth muscle, α-actinin is localised to the Z-discs and dense bodies, and it par-ticipates in the anchoring of the actin thin filaments and giant titin molecules to theZ-disc (Endo and Masaki 1984; Geiger et al. 1990). α-actinin has been shown tointeract with an N-terminal domain of titin in vitro (Ohtsuka et al. 1997). In the Z-disc, the assembly of the actinin–titin complex seems to involve two types ofinteraction between titin and α-actinin. In the other regions of the Z-disc, titin bindsby means of a single binding site with the outermost pair of α-actinin molecules,but in the middle of the Z-disc the titin binds several α-actinin molecules throughbinding sites at their C-terminal region (Young et al. 1998). Thus α-actinin seemsto play a major part in anchoring actin thin filaments of the two halves of thesarcomere at the Z-disc (see Figure 15). Abnormal expression of α-actinin and otherthin filament-associated proteins are known to interfere with the assembly of Z-discs, which could lead to the formation of so-called nemaline bodies. Two loci areknown to be involved in nemaline myopathy: the tropomyosin-3 locus and thenebulin locus on 2q21.2–q22. Mutations of both genes have been implicated in thisform of myopathy (Laing et al. 1995; Pelin et al. 1999). The formation of thecomplex of tropomyosin, titin, nebulin, α-actinin, and actin can conceivably beaffected by these mutations, leading to abnormalities in Z-disc assembly and theformation of nemaline bodies.



FUNCTION OF αααα-ACTININ

α-actinin functions predominantly in F-actin bundling and anchoring of the filamentsto specific sites within the cell as well as to the cell membrane and in the linking upof the cytoskeletal machinery to the ECM. α-actinin might form a bridge between theactin cytoskeleton and integrin receptors then function as a receptor for componentsof the ECM (Burridge et al. 1990; Geiger et al. 1990). The cytoplasmic tail of β-integrins and intercellular adhesion molecule-I (ICAM-I) interact with α-actinin viabinding sites occurring in the rod domain. Furthermore, α-actinin appears to link thecytoskeleton to the cell membrane (Baron et al. 1987). Kahana and Gratzer (1991)have identified binding sites for long-chain fatty acids in the spectrin-like repeats ofthe α-actinin domain rod. Han et al. (1997) recently have shown that bundling of actinfilaments occurs if diacylglycerol is present in the membrane. It follows, therefore,that α-actinin would be an important component of the signal transduction pathwayas well as an important link in the cytoskeleton-mediated changes in cell shape andmotility. Miyamoto et al. (1995) have delineated the pathway of integrin-mediatedsignal transduction. They reported that integrin aggregation induced the accumulationof several signal transduction molecules, such as Rho A, Rac1, ras, raf, and others, ofcytoskeletal components, such as vinculin, talin, and α-actinin. The tyrosine kinaseinhibitor genistein inhibits accumulation of both the signal transduction molecules andthe cytoskeletal signal transduction components.

ACTININS IN CELL ADHESION, MOTILITY, AND SIGNAL TRANSDUCTION

The cross-linking of actin filaments increases filament elasticity and viscosity andmay be expected to affect the structural properties of actin filaments. It follows fromthis that actinin might, in this fashion, change the properties of the cell membrane,such as intercellular and cell–substratum adhesion, which in turn would be reflectedin alterations in cell shape and motility. These changes will have serious implicationsfor cancer invasion and secondary spread. Furthermore, there are strong indicationsthat actin cross-linking in endothelial cells might result in metabolite transport andpermeability. Although yet to be demonstrated, one would expect that alterations inthe organisation of adherens junctions might effectively alter endothelial integrityand lead to enhanced tumour cell diapedesis across the endothelium. Changes in thestructural properties of the actin cytoskeleton would impugn the link-up between itand the ECM. Such changes would affect seriously the physical pathway of signaltransduction, and effectively lead to its deregulation, and eventually to the deregu-lation of intercellular adhesion and communication and of cell proliferation andgrowth.

THE CADHERIN–CATENIN COMPLEX IN SIGNAL TRANSDUCTION AND CELL ADHESION

A signal transduction complex that occurs in the cell membrane, consisting of thetransmembrane glycoprotein cadherin and other components such as α-actinin, β-catenin, and the adenomatous polyposis coli (APC) protein, has assumed consider-able significance by virtue of its apparent dual function, namely in signal transduction



and cellular adhesion. Intercellular adhesion mediated by cadherin is dependent on theintegrity of the link-up with the actin cytoskeleton (Sherbet and Lakshmi, 1997b)(Figure 13). β-catenin subserves two functions, viz. as a signal transduction moleculeand in the formation of adherens-type junctions. Its participation in signal transductionhas become apparent with the demonstration that plakoglobin and β-catenin are homo-logues of the armadillo protein of Drosophila that is involved in segment polarity.

The armadillo (arm) protein is a component of the signal transduction pathwayof the wg (wingless) molecule (Riggleman et al. 1990). The arm family of proteins,e.g., β-catenin, plakoglobin, and the p120ctn protein, are characterised by a centralarm repeat domain (Riggleman et al. 1989). The p120ctn gene potentially can codefor a large number of isoforms that are generated by alternative splicing, and thesehave been regarded as constituting a subfamily of signalling proteins (Keirsebilcket al. 1998b). Jou et al. (1995) have shown that β-catenin binds to a C-terminal 25amino acid region in the cytoplasmic domain of E-cadherin and to the N-terminaldomain of α-catenin. The p120ctn protein, on the other hand, seems to bind to ajuxtamembrane domain of the cadherin cytoplasmic tail (Yap et al. 1998).

The armadillo and wg proteins exert similar effects on embryonic development(Peifer et al. 1991). A family of wnt proteins has been identified. The wnt proteinis a secreted glycoprotein signalling factor. It is a vertebrate homologue of wg andhas been shown to be a regulator of morphogenesis of Xenopus (Gumbiner, 1996;Miller and Moon, 1996). In a similar vein, β-catenin and plakoglobin are componentsof the signal transduction pathway of the wnt gene as well as being involved in theformation of adherens junctions and Ca2+-mediated cell–cell adhesion (Hinck et al.1994; Peifer, 1995). Cadherin, as a part of this signalling complex, not only regulatesintercellular adhesion, but also seems to negatively regulate the signalling functionof β-catenin. These two functions can be dissociated. Fagotto et al. (1996) noticedthat β-catenin deletion mutants affect cadherin-mediated adhesion but not its sig-nalling function. Sanson et al. (1996) showed that a full length E-cadherin and a

FIGURE 13 Schematic representation of the interaction of the transmembrane adhesionprotein cadherin with the actin cytoskeleton, via β-catenin, α-catenin, and α-actinin. (Basedon van Roy [1992] and Sherbet and Lakshmi [1997b]). Reprinted by permission of thepublisher Academic Press, from The Genetics of Cancer, (Sherbet and Lakshmi, 1997b).

N-terminus

Extracellular Ca binding domains

Adherens junction protein

Plasma membrane

β-catenin

Actin filaments

α-catenin

APC Proteinα-actinin

2+



truncated form of cadherin that have opposite effects on cadherin-dependent adhe-sion, nonetheless function effectively in wg signalling.

Two further elements are involved in the signal transduction complex involvingE-cadherin and catenins. One is the APC protein and the other component is axinor conductin. β-Catenin forms complexes with both axin and APC. The APC proteinis known to compete with cadherin in its binding to the internal repeats of β-catenin.The β-catenin/APC complex is phosphorylated by the glycogen synthase kinaseGSK3β. A consequence of this phosphorylation is that the degradation of β-cateninis greatly enhanced (Munemitsu et al. 1995; Hayashi et al. 1997; Hart et al. 1998).Axin also induces β-catenin degradation and is suggested to function downstreamof APC (Behrens et al. 1998). The GSK3β-mediated phosphorylation is inhibitedwhen the wnt signal transduction pathway is active. This results in the accumulationof β-catenin in the cytoplasm (Hinck et al. 1994; Giarre et al. 1998; Papkoff andAikawa, 1998) and leads to the formation of a complex with the T-cell factor/lym-phoid enhancer factor (Tcf/Lef). The binding between them occurs via the armrepeats. This complex functions as a transcription factor in the wnt/wg signallingpathway (Behrens et al. 1996; Van de Wetering et al. 1997). The pathway might bederegulated by inhibition of β-catenin degradation, leading to a constitutive activa-tion of the transcription complex (Figure 14).

The deregulation that accompanies neoplastic changes seems to be more fre-quently due to mutation of β-catenin or APC protein than to inactivating mutationsof E-cadherin (Morin et al. 1997; Efstathiou et al. 1999). These mutations seem tostabilise β-catenin. They occur at the phosphorylation sites that are essential in theubiquitination and degradation of the protein. β-catenin mutations occur in approx-imately 50% of colorectal tumours. Again, these tend to occur in the serine/threoninephosphorylation sites. They seem to occur more frequently in small adenomas than

FIGURE 14 The wnt signal transduction pathway and cell adhesion signal involving β-catenin, APC, and cadherin. (Based on references cited in the text.)

Wnt/Wg

GSK3b

β-catenin

β-catenin/Tcf-LefTranscription complex

Axin/conductin

APC/β-catenin

APC-P/β-catenin-P

β-catenin degradation

Cell adhesion



in larger adenomatous lesions. Furthermore, a majority of the mutations have beenfound in adenomas rather than in carcinomas (Samowitz et al. 1999). A high rate(61% of 31 patients) of somatic mutation of β-catenin has been encountered inhuman anaplastic thyroid carcinoma (Garcia-Rostan et al. 1999). In a murine hepa-tocarcinoma model, mutations have been reported to occur in the carcinomas butnot in adenomas. These findings suggest that deregulation of signal transductionoccurs as an early event in the pathogenesis and progression of tumours. The β-catenin mutations in colorectal tumours reported by Samowitz et al. (1999) werenot accompanied by APC mutations. The suggestion has been made, therefore, thatmutations of APC and β-catenin might be functionally different. Mutations of APCleading to the loss of its suppressor function have been identified with the familialadenomatous polyposis, and are also closely related to the progression of the disease.Furthermore, mutated APC seems to lack the ability to regulate β-catenin levels.Therefore, APC may be regarded ipso facto as functioning upstream of β-catenin.

The adhesion function of β-catenin flows from its linkage to cadherin, whichspans across the cell membrane, on the one hand, and to the cytoskeleton, on theother, by means of two other elements, namely α-catenin and α-actinin. In fibro-blasts, this linkage has been demonstrated by Knudsen et al. (1995), who also notedthat α-actinin specifically immunoprecipitates with β-catenin and cadherin. Niessetet al. (1997) have identified the binding sites involved in the interaction of α-cateninwith β-catenin on the one hand and with α-actinin on the other. In normal thyroidepithelial cells, the intercellular adhesion junctions show the presence of cadherintogether with the catenins. In contrast, CGTHW-2 cells derived from thyroid carci-noma show a marked alteration in the pattern of distribution of these linking proteinsin the foci of intercellular adhesion.

Further evidence in support of the importance of the integrity of the cad-herin–catenin complex in intercellular adhesion comes from the apparent involve-ment of the Rho GTPase family in the regulation of cadherin-mediated cell adhesion.IQGAP1, which is an effector of two members of the family, namely Cdc42 andRad, has been shown to be able to dissociate α-catenin from the cadherin/β-cate-nin/α-catenin complex. This results in the disruption of intercellular adhesion. BothCdc42 and Rad counteract this process (Kaibuchi et al. 1999). The inhibition of Rhoand Rac (another member of the Rho GTPase family) has been reported to lead toa disruption of E-cadherin localisation at keratinocyte–keratinocyte cell junctions.However, when the GTPases themselves are inhibited, the cadherin-mediated adhe-sive contacts are reestablished (Braga et al. 1999). Rho protein, when microinjectedinto four-cell blastomere-stage embryos, disrupts cortical microfilaments andreduces interblastomere adhesion. Cdc42 in the same way also disrupts the corticalcytoskeleton and interblastomere contacts (Clayton et al. 1999). But the modes ofinvolvement of Rho and Cdc42 are clearly distinguishable in NIH3T3 cells trans-formed by the dbl oncogene, which codes for a supposed exchange factor for RhoAand Cdc42. The transformed cells respond to adhesion to fibronectin substratum bychanging cell shape. This involves the activation of RhoA and the associated ROCKand CRIK, but not Cdc42. In nontransformed cells, however, change of cell shapeseems to require Cdc42 activation (Olivo et al. 2000). Rho GTPases may also beinvolved in the regulation of endothelial cell adhesion involving cadherins. Rho



GTPases are also involved in the regulation of the function of other adhesion-mediating glycoproteins such as CD44. Therefore, prima facie there is a reasonablebasis for investigating their role in maintaining the integrity of the vascular endotheliaand possible implications for metastatic spread of cancer.

Other proteins that link the plasma membrane with the actin cytoskeleton maydisrupt cadherin-mediated adhesion. The proteins ezrin, radixin, moesin, and merlinsubserve such a linking function. Hiscox and Jiang (1999) used antisense nucleotidesto inhibit ezrin expression in colon carcinoma cells and noticed that this resulted ina loss of intercellular adhesion and acquisition of motility. They further noticed thatezrin co-precipitated with E-cadherin and β-catenin. In cells treated with antisenseezrin nucleotides, this could result in a reduced interaction between ezrin and thecadherin complex. Such a reduction indeed occurs when ezrin is phosphorylated orwhen the cells are treated with hepatocyte growth factor.

Huang et al. (1998) found no cadherin or γ-catenin at the adhesion junctions,and β- and α-catenins were distributed diffusely in the cytoplasm of most cells.However, β-catenin, when detected at intercellular junctions, was found to co-localise with α-actinin. Huang et al. (1998) have therefore suggested that the lossof intercellular adhesion could be due to an incorrect assembly of the linkingcomponents. Implicit also in this is the suggestion that such an incorrect assemblymight lead to the acquisition of invasive ability. Although this is an attractivehypothesis, it should be mentioned here that Honda et al. (1998) have reported theoccurrence of a novel actinin, namely actinin-4, whose expression is said to be up-regulated with enhanced cellular migration. This new isoform is said to occur in thecytoplasm. It is reported to be associated with cytoplasmic extensions and is foundin peripheral migrating cells of cell clusters. Actinin-4 appears to be translocatedfrom the cytoplasm to the nucleus, when actin is depolymerised. Honda et al. (1998)have also examined the expression of this novel actinin in breast cancers. It isexpressed in infiltrating breast carcinomas, and the expression was found to correlatewith poor prognosis.

Mamura and Masaki (1996) identified a 115-kDa α-actinin in vascular endot-helial cells. This actinin differed from other known actinins of muscle or nonmuscleorigin in its sensitivity to calcium. However, this isoform of actinin did occur inheart tissue, the pectoralis muscle, and the gizzard. What could be significant aboutthis actinin in relation to cancer progression is its apparent location in the vascularendothelium. It is well known that several inflammatory agents induce a reversiblechange in endothelial cell shape that results in the formation of intercellular gaps.If cancer cells can reduce the endothelial integrity in this way, that could form portsof entry into the vascular system for cancer cells. There is a view that invadingcancer cells take advantage of naturally occurring fenestration of the endothelium.There is no demonstration to date that they might secrete substances that couldinduce the formation of ports of entry. From this point of view it would be interestingto see if cancer cells induce any changes in the expression of actinins of theendothelial barrier.

A new facet that can be added to the story of actinins in invasion and metastasisis their apparent relationship to the expression of metalloproteinases. It was shownsome years ago that the production of these enzymes often paralleled the invasive



and metastatic abilities of cancers, which has suggested the possibility that metal-loproteinases might reconstruct the ECM and provide cancer cells with ECM prop-erties conducive to invasion (Sherbet and Lakshmi, 1997b). Crawford et al. (1999)have made some interesting observations on the expression of matrilysin and β-catenin. They found that β-catenin and matrilysin mRNAs were expressed in parallelin murine intestinal adenomas. A further observation of considerable importance isthat β-catenin significantly up-regulated the matrilysin promoter. Because β-cateninis a structural component of signalling pathway and intercellular adhesion machinery,these experiments bring together, in a unique fashion, transduction of an extracellularsignal that can putatively remodel the ECM and change the invasive behaviour ofcells.

Other cell adhesion molecules such as the ICAM similarly require interactionwith the actin cytoskeleton. A peptide containing a cytoplasmic sequence of ICAM-2 has been shown to interact with α-actinin in vitro. Indeed, interaction occursbetween several sites of α-actinin and ICAM-2. Besides, ICAM-2 co-localises withα-actinin (Heiska et al. 1996). The epithelial ICAM (Ep-ICAM), an adhesion mol-ecule regarded as specific for epithelial cell adhesion, has a cytoplasmic domain thatregulates the cellular adhesion function. It appears that Ep-ICAM achieves this viaα-actinin, with which Ep-ICAM appears to interact directly by means of specificbinding sites (Balzar et al. 1998).

Shigella flexneri, which causes bacillary dysentery, is known to induce changesin the cytoskeleton of epithelial cells. These changes result in the production ofmembrane protrusions that engulf the bacterium. The bacterium secretes a proteincalled IipaA. The cytoskeletal reorganisation, which is essential for this process ofbacterial invasion, involves the recruitment by IipaA of the linking protein vinculinas well as α-actinin (Van Nhieu et al. 1997).

The effects of the loss of α-actinin on cell shape, aggregation, and motility havebeen studied in Dictyostelium. Rivero et al. (1996) generated mutants lacking α-actinin as well as the “gelation factor” and these mutants presented a rounded shaperather than the typical polarised morphology of aggregating cells. The mutants alsoshowed considerable loss of motility. In rat bladder carcinoma cells, the experimen-tally induced loss of motility was associated with the reorganisation of F-actin andα-actinin (Morton and Tchao, 1994). The transmembrane integrin α2β1 functions asthe receptor for the ECM components collagen and laminin. This integrin as wellas α-actinin have been implicated in the invasive ability of melanoma cells in vitro.L.M. Duncan et al. (1996) reported that α-actinin was not detectable in benignmelanocytic naevi and in laterally spreading lesions, but it occurred in all nodularmelanomas and in metastatic melanomas. It would have been of much interestwhether there was an association between α-actinin expression and the verticalgrowth of melanoma.

EGF is known to alter cell morphology as well as promote cell invasion in vitro(Shiozaki et al. 1995). These cellular responses seem to originate from the linkageof the activated EGFr to the cytoskeletal system (Roy et al. 1989, 1991; Van Bergenen Henegouwen et al. 1989). There is some recent evidence that EGFr regulatesintercellular adhesion by interfering with the formation of a complex between theinvasive suppressor transmembrane protein E-cadherin and the actin cytoskeletal



elements, including α-actinin and β-catenin (Hazan and Norton, 1998; see alsoFigure 13). As alluded to earlier, the proliferative signal imparted to the cell by EGFis mediated by PKC. N.R. Murray et al. (1999) generated transgenic mice thatexpress PKC-βII at high levels. The colonic epithelium showed high cell prolifera-tion. Besides, the epithelium was more prone to preneoplastic changes. They foundthat these effects were also accompanied by increased expression of β-catenin anda reduction in glycogen synthase kinase 3β activity. These studies suggest, therefore,that EGFr-mediated signal follows the wnt/APC pathway of transduction leading tocell proliferation. This pathway also may be utilised in neoplastic changes of epi-thelia. Overall, these data now provide a physical basis for previous observationsthat high EGFr expression correlated with poor prognosis, and that EGFr status isa powerful marker of clinical aggressiveness and metastatic potential of tumours(Sherbet and Lakshmi, 1997b).

MYOSIN FILAMENTS

Myosin is a filament protein found in association with actin in both muscle andnonmuscle cells. Like actin, myosin is a bipolar protein. It possesses a globularhead region (subfragment S1), which interacts with actin, and a fibrous region,which promotes aggregation into filaments. The filaments consist of two myosinheavy chain (MHC) subunits and four myosin light chain (MLC) subunits. Theclass II skeletal myosin has two heads whereas other myosins have only one head.The N-terminal globular domain comprises the motor domain. The heavy chainsubunit possesses the ATPase activity that provides the energy for the contractileforce. The process of muscle contraction is driven by a cyclical interaction betweenactin and myosins. Myosins have been described as mechanoenzymes that catalysethe hydrolysis of ATP and release of energy, which is converted into mechanicalforce (Hasson and Moosekar, 1995). Energy is transduced by a series of interac-tions between myosin heads and hydrolytic changes of ATP. This is believed todeform the protein, and this change is transmitted to actin as a mechanical effect.The mechanical displacement and force generated in this way has been measured.The force generated by double-headed myosin is nearly twice as large as thatproduced by single-headed myosins (Tyska et al. 1999). In the skeletal muscle,the two heads of myosin interact when it is bound to filamentous actin (Raymentet al. 1993). The smooth muscle myosin heads interact in the presence of filamentactin and in the absence of ATP (Onishi et al. 1992). Such head–head interactionalso is required in the phosphorylation-dependent regulation of smooth musclemyosin (Matsuura and Ikebe, 1995). The processes of muscle contraction and cellmotility are usually evaluated by an in vitro technique in which actin filamentsare slid over myosin and measured using an electron microscope. Actin sliding aswell as actin activation of myosin-ATPase is dependent on phosphorylation whenmyosin is dimerised, but not while myosin is in the monomeric form. Furthermore,the sliding velocity of the dimer is twice as large as that of the monomer (Sata etal. 1996).



MYOSIN HEAVY CHAIN (MHC) ISOFORMS

A large number of MHC isoforms are known to exist (Table 6). There are preliminaryreports that the pattern of their expression is modulated in embryonic developmentand also in association with certain disease processes. For instance, thyroid hormoneis reported to alter the pattern of MHC isoform expression in embryonic development(Maruyama et al. 1995). During metamorphosis of Xenopus tadpoles, thyroid hor-mone has been reported to down-regulate the expression of embryonic but up-regulate adult isoforms (Sachs et al. 1997). Modulations in MHC isoform expressionhave been reported in the progression of human atherosclerosis (Aikawa et al. 1995).Similar changes have also been described in tumour-associated fibrosis and inneoplastic transformation of breast epithelium (Chiavegato et al. 1995).

The metastasis-associated S100A4 (mts1), which is itself a CBP, has been foundto bind to the myosin rod in the light meromyosin region and could be destabilisingthe myosin filament. Besides, S100A4 binding has been found to inhibit the actin-activated ATPase of myosin (Ford et al. 1997). These interactions could conceivablyalter the faculty of motility, which has been associated with cancer invasion, andthis is especially associated with high expression of S100A4. It would be of interestto note here that the unconventional myosin V could be involved in the mechanismsassociated with filopodial extension of neuronal growth cones. When myosin V isinactivated, cones of dorsal root ganglia show a rapid retraction of the filopodia (F.S.Wang et al. 1996). The distribution of the nonmuscle isoforms of myosin II (A andB) shows a pattern that is compatible with their involvement in cell locomotion.These myosins occur in association with lamellipodia and around the cell nucleus.However, myosin IIA shows a preferential localisation along the leading edge, but

TABLE 6Myosin Heavy Chain (MHC) Isoforms

MHC Isoform Tissue

Myosin I slow MHC 1β, α Skeletal, cardiac, foetalMyosin II fast MHC II a, b, x SkeletalMCH 3 FoetalII c MHC1, MCH 9 Skeletal, immature fibresMyosin II SM1 (204 kDa); SM2 (200 kDa)

Smooth muscle

Nonmuscle MIIA, B1, B2 Fibroblasts, endothelial cells, macrophagesUnconventional myosins V, VI Intestinal brush border, Va in a variety of

cell typesMyosin VIIa Cochlea, retina, testes, lung, kidney

Source: Based on several sources including Kelley and Adelstein (1994); Hei-ntzelman et al. (1994); A. Kimura et al. (1995); Chiavegato et al. (1996); Galleret al. (1997); Hasson et al. (1997); Wu et al. (1998).



myosin IIB occurs along the trailing edge of cells in locomotion (Kolega, 1998). Itis significant that, when injected into the cells, these isoforms show the same patternof localisation as the endogenous variety. This has led to the suggestion that theirlocalisation pattern is an intrinsic property of locomoting cells (Kolega, 1998). Theapparent specificity of localisation may not be a consequence of their synthesis atspecified sites, but could indicate that they are being targeted to those locations.HSPs are believed to behave like molecular chaperones for S100A4. HSPs mighttarget S100A4 to the plasma membrane to be secreted into the ECM, and in thisway endow the cells with invasive ability. Therefore, further effort into the possiblemechanisms involved in the perceived specificity of myosin IIA and B localisationin the locomoting cell would be eminently worthwhile.

Among other myosin-driven biological phenomena that may be cited here iscytokinesis in cell division. The myo2 gene of Schizosaccharomyces pombe encodesa protein called myo2, which shows a high degree of homology to myosin type IIheavy chain. A contractile ring composed of actin and myo2 is formed beforecytokinesis. Disruption of myo2 reportedly leads to an inhibition of cell proliferation(Kitayama et al. 1997). In Dictyostelium the formation of the contractile ring requiresphosphorylation of myosin (Sabry et al. 1997). The unconventional myosin Va hasbeen localised to the microtubule organising centre of interphase cells and to themitotic apparatus in dividing cells. Using specific antibodies against myosin Va, Wuet al. (1998) have been able to demonstrate its association with microtubules in anumber of cell types. The specificity of this association was obvious from experi-ments using cells that were derived from mice with null (dilute gene –/–) phenotype,in which no association with microtubules could be detected.

ACTOMYOSIN ASSEMBLY

Myosin thick filaments are composed of two MHCs of 220 kDa and four MLCs of20 kDa molecular size. The N-terminal end of MHC forms the globular head. Thetail region of the filament is an α-helical coiled coil that constitutes part of the longrigid tail, of which the part proximal to the head region is composed of the C-terminal half of MHC and the light chains form the distal end of the myosin tail.The light chains are of two classes: a phosphorylatable form found in heart muscleand the CNS, but not in skeletal muscle, and a nonphosphorylatable form. Indeed,the N-terminal section of MHC together with two light chains, one of each class,form the globular head. MLCs bind Ca2+ with great affinity and regulate myosin-ATPase activity. The conformation of the myosin globular heads is regulated by thephosphorylation of the MLC by MLCK, which binds to actin via residues 2–42 ofthe N-terminal end of the kinase (Gallagher and Stull, 1997). This phosphorylationis believed to be a Ca/CaM-dependent process. Although reversible phosphorylationof MLC is generally regarded as the regulatory event, other proteins, e.g., caldesmonand calponin, which are capable of binding to the actin–myosin assembly, couldsubserve important functions.

The skeletal and heart muscle cells possess a characteristic striated pattern. Thestriations are due to individual fibrils within the muscle cells. The striations areproduced because the fibrils are arranged in register, which accentuates the striation.



The striations form three distinct zones: the dark and birefringent A-band with alighter zone in the middle called the H-band, which in turn is divided by the M-line; the I-band, which is lighter than the A-band; and the Z-line, which is a darkline in the middle of the I-band (Figure 15). The contractile, regulatory, and structuralcomponents of these zones are summarised in Table 7. The A-band is composed of

FIGURE 15 A sarcomere with its bands. The pattern is a result of the arrangement of actin,myosin, and titin. (Based on Gaub and Fernandez, 1998.)



thick myosin filaments. These are arranged in a parallel array and in register andtherefore they determine the length of the A-band. Thin actin microfilaments formthe lighter I bands. However, the thick myosin filaments form bridges with themicrofilaments, especially in the region of overlap of A and I bands. In transversesection of the muscle, the organisation of the filaments can be described as a lattice.Each thick myosin filament has six thin actin microfilaments and each actin filamenthas two neighbouring myosin filaments. In the thin filaments, tropomyosin occursin a head-to-tail orientation in one groove of actin helix by means of seven actin-binding sites. It is believed that molecular elasticity is regulated by a species of giantrod-like proteins called titin or connectin. Human titin is a ca. 3000-kDa proteinconsisting of variable numbers of immunoglobulin (Ig)- and FN-like repeat domainsand a P (proline), E (glutamate), V (valine), and K (lysine)-rich region of variablelength. Titin overlaps the A-band and the I-band, extending from the Z-line to theM-line (see Figure 15). It is believed that the part of titin that overlaps the I-bandforms the elastic region of the molecule (Linke et al. 1998). Alternative splicing ofthe I-band titin is said to result in changes in the component Ig and FN modulesand in the length of the PEVK region (K. Wang et al. 1991; Labeit and Kolmerer,1995a). Another large protein called nebulin, of approximately 600 to 800 kDamolecular size, spans the whole length of the thin filaments of the I-band. Indeed,nebulin is regarded as the determinant of the length of the thin filaments (Kruger etal. 1991; Jin and Wang, 1991; Labeit et al. 1991; Trinick, 1994). The assembly ofmature Z-discs occurs from precursors called I-Z-I bodies, which contain titin,

TABLE 7Contractile, Regulatory, and Structural Component Proteins of Myofibrils of Vertebrate Skeletal Muscle

ProteinLocation

BandContent % wt.

MolecularWeight (kDa)

Contractile Myosin A 43 ca. 500 Actin I 22 42Regulatory elements Troponin I 5 70 Tropomyosin I 5 M-protein M-line 2 C-protein A 2 α-actinin Z-line 2 ca. 110Structural component Titin A–I 10 3000 Nebulin I 5 600–800

Source: The data in this table have been collated from referencescited in the text.



nebulin, actin, and α-actinin (Holtzer et al. 1997). Tropomyosin seems to beinvolved in nemaline myopathy. Two loci involved in this disease are the tropomy-osin-3 locus and the nebulin locus on 2q21.2–q22 (Laing et al. 1995; Pelin et al.1999). Mutations of both genes have been implicated. The formation of thecomplex of tropomyosin, titin, nebulin, α-actinin, and actin can conceivably beaffected by these mutations, leading to abnormalities in Z-disc assembly and theformation of nemaline.

The C-terminal region of nebulin contains an SH3 domain, which may anchorthe nebulin filament system to the Z-disc (Labeit and Kolmerer, 1995b). This SH3domain has been found to interact with actin, tropomyosin, troponin and calmodulin.This could suggest that nebulin, together with tropomyosin and troponin, might forma complex that is calcium-regulated (K. Wang et al. 1996). In cardiac muscle, anebulin-related protein called nebulette substitutes for nebulin (Millevoi et al. 1998).Nebulette is similar to skeletal muscle nebulin in its organisation and contains fourdomains: an N-terminal domain followed by a domain that contains nebulin-likerepeats, a linker domain connecting the nebulin repeats to the C-terminal SH3domain. The nebulin repeats possess actin-binding ability and the linker zone appearsto target the protein to the Z-line. This is suggested by the binding of recombinantnebulette fragments to actin, tropomyosin, and α-actinin in vitro (Moncman andWang, 1999).

MYOSIN LIGHT CHAIN (MLC) PHOSPHORYLATION AND FUNCTION

As stated earlier, there are two classes of myosin light chain (MLC), viz. a non-phosphorylatable and a phosphorylatable regulatory chain. The reversible phospho-rylation of the regulatory MLC is regarded as the key event in the regulation ofmyosin-ATPase and the generation of the contractile force. The phosphorylation ofMLC is carried out by the MLCK. MLCK is a Ca2+/CaMdependent kinase (Stull etal. 1993) and is itself subject to regulation by kinases and phosphatases (Quadroniet al. 1998). MLCK is known to be able to bind to actin and thereby regulateactin–myosin interaction (Kohama et al. 1992; see also below). Naturally this willinfluence the function of the ATPase. On the other hand, binding of calponin andcaldesmon to actin may inhibit myosin-ATPase and this inhibition may be reversedby calcium-binding S100 proteins and calmodulin (see Fattoum, 1997). Rho kinasealso seems to be able to phosphorylate MLC, together with inactivation of myosinphosphatases (Fukata et al. 1999) (Figure 16).

The regulation of the contractile machinery of the cell has obvious implicationsfor cell motility and diapedesis of cells. The ability of activated PMN to traversethe endothelium appears to be related to the state of MLCs phosphorylation ofendothelial cells. Hixenbaugh et al. (1997) found that activated PMNs increasephosphorylation of serine 19 and threonine 18 of MLCs of endothelial cells. Garciaet al. (1998) have confirmed the enhanced phosphorylation of MLC in endothelialcells and have further shown that inhibitors of MLCK significantly inhibit thediapedesis of activated PMNs across the endothelium. Phosphorylation of MLCKby protein kinase C also inhibits MLC phosphorylation and causes enhanced



epithelial resistance (Angle et al. 1998). The association with actin and the assemblyof myosin II into the cytoskeleton is dependent on the phosphorylation of theregulatory chain. MLC associated with the cytoskeleton of cultured endothelial cellshas been reported to show a fivefold greater phosphorylation than MLC in solubleform. The phosphorylation of serine 19 and threonine 18 appears to regulate theassembly of MLC in vitro. In contradistinction, soluble MLC seems to be phospho-rylated at threonine 9 (Kolega and Kumar, 1999).

It would seem that the phosphorylation of the cytoskeletal machinery couldincrease the intercellular gaps thus allowing cell penetration. It would be worthwhileto investigate whether, like activated PMNs, cancer cells affect the state of MLCphosphorylation of endothelial cells. Unquestionably, vascular permeability is animportant determinant of metastatic spread of cancer. However, invasive ability isan intrinsic property of the cancer cell. Gillespie et al. (1999) have recently shownthat the MLCK inhibitors, ML7 and KT5926, inhibit the invasive ability of gliomacells. MLC phosphorylation in both the invading tumour cell and the endothelialcell barrier might determine the degree of successful entry of the tumour cell intothe vascular compartment (Figure 17).

Some studies have implicated MLC phosphorylation in the regulation of certainother biological features such as cell growth and apoptosis. Mills et al. (1998) havereported that membrane blebbing associated with apoptotic cell death could bereduced by MLCK inhibitors, and, further, that there was increased phosphorylation

FIGURE 16 Control of regulatory myosin light chain (MLC) function in muscle contrac-tion. CaM, calmodulin; MLCK, MLC kinase; PKC, protein kinase C; PP1A/PPiB: proteinphosphatases; RMLC, regulatory MLC.

Muscle contraction

Actomyosin Mg-ATPase

RMLC

MLCK

Ca /CaM

Casein Kinase II

CaldesmonCalponin

S100/S100A4CaltropinCa /Calmodulin

-P

PPIA/PPIB

PKC+P

Myosin Actin

+P

+P2+

2+



of MLCs in membrane blebbing. There are also suggestions based on preliminaryfindings that MLC phosphorylation may also be associated with cell proliferation(Bresnick et al. 1996; Yamakita et al. 1996).

MLCK may affect actin reorganisation by a mechanism that is independent ofits kinase activity. As stated earlier in this section, MLCK is known to be able tobind to actin and influence actin–myosin interaction. Another aspect of MLCKfunction is that it can bind to and result in actin bundling into filaments (Hayakawaet al. 1994). Two sites on MLCK have been identified and characterised; one ofthem is Ca2+/CaM sensitive and the other is Ca2+/CaM insensitive (Ye et al. 1997).These participate in actin filament assembly in vitro (Hayakawa et al. 1999). Byinference from and in common with the properties of other actin-binding and -bundling proteins, it would be reasonable to suggest that MLCK might influencecell shape and flexibility and thereby affect cell migration and diapedesis, quiteindependently of its kinase activity.

FIGURE 17 Possible involvement of MLC phosphorylation in the migration of cells acrossthe endothelial membrane as well as with the intrinsic invasive potential of the tumour cell.MLC, myosin light chain; MLCK, MLC kinase; PKC, protein kinase C.

Diapedesis

MLC-P

+P

MLCK

+P

MLC-P

Cancer Cell Invasion

Inhibitors MLCK kinases(PKC)

+P

�



TROPONINS AND TROPOMYOSINS IN THE REGULATION OF MUSCLE CELL CONTRACTION

THE REGULATORY ROLE OF TROPONINS AND TROPOMYOSINS IN MUSCLE CONTRACTION

Troponins are CBPs that play a major regulatory role in cardiac and skeletal musclecontraction in consort with tropomyosins. Three troponin (Tn) molecules — TnT,TnI, and TnC — bind to each tropomyosin molecule. Many tropomyosin isoformswith a range of molecular sizes have been identified (see below). The high molecularweight isoforms bind actin more strongly than the others (Matsumura et al. 1985).There is a complex and specific form of association between the troponins andtropomyosin that produces conformational changes in actin that lead to musclerelaxation or contraction. TnT binds to the C-terminal region of tropomyosin andforms a link between TnI and TnC, which both bind to tropomyosin. The structuraldomain C and regulatory domain N of tropomyosin interact with specific amino acidsequence motifs of TnI (Pearlstone et al. 1997). Calcium binding to the N domainof TnC can alter the affinity of the N- and C-termini of TnI for TnC. In other words,calcium binding can provide a switch between these two sites for binding with TnC(Tripet et al. 1997). At any rate, the whole complex interacts with actin via TnI. TnIis the myosin-ATPase inhibitory protein. TnC is the calcium binding subunit. In theabsence of Ca2+ binding to TnC, the conformation of actin filament is such that itsinteraction with myosin heads is weak and the muscle fibres are in a relaxed state.The ATP that is bound to myosin is hydrolysed by a myosin-catalysed ATPase. Inthe absence of calcium, the troponin/tropomyosin complex inhibits myosin-ATPase.But when intracellular calcium levels are raised, Ca2+ binds to TnC and myosin-ATPase inhibition is released, ATP hydrolysis occurs and muscle contraction ensues.The contracted state is maintained when Ca2+ and ATP levels are maintained at highlevels (Figure 18). The SR around myofibrils stores Ca2+ as well as the membrane-ATPase that is required for the release of Ca2+ and raises intracellular levels. Thereis therefore a homeostatic mechanism at work, maintaining a 10-7 M Ca2+ level inthe resting muscle, and the increase of this by an order of magnitude produces thecascade of events leading to muscle contraction.

FIGURE 18 Cascade of events leading to muscle contraction upon Ca2+ binding to TnC.

Tropomyosin Muscle contraction

Tnl TnT TnC Myosin-ATPase

Actin conformational change

Ca2+



The calcium–TnC trigger leads to the contraction of cardiac and skeletal muscles.However, different mechanisms may be involved in the activation of myofilamentsof the heart and skeletal muscles. In heart myofilaments, Ca2+ binds to one regulatorysite on TnC but to two sites in skeletal myofilaments. Besides, there are alsodifferences with regard to Ca2+-induced conformational changes between cardiacand skeletal muscle (Spyracopoulos et al. 1997). In the smooth muscle, i.e., invol-untary muscle under CNS control and muscle tissue of invertebrates, there is no SR.In these, intracellular Ca2+ alterations tend to be slower than those found in striatedmuscle. Furthermore, Ca2+ regulation of contraction involves MLCs, which have ahigh affinity for Ca2+. At low calcium levels the interaction of myosin head withactin is weak, but when the light chains bind Ca2+, the myosin head–actin interactionactivates myosin-ATPase leading to muscle contraction.

TROPOMYOSIN ISOFORMS IN BENIGN AND MALIGNANT CELLS

Tropomyosin isoforms are expressed in cells of muscle as well as nonmuscle origin.They are found in many organisms from unicellular ones to mammals (Pittenger etal. 1994). In human fibroblasts, eight isoforms have been identified and character-ised. They are products of four genes (Novy et al. 1993a, 1993b). The isoforms oftropomyosin that have been identified range in molecular size from 32 to 40 kDa(J.J.C. Lin et al. 1985; Matsumura et al. 1983; Novy et al. 1993; Pittenger et al.1994; Warren et al. 1985). The isoforms seem to differ with respect to the strengthof their association with actin and may also differ in their function (Matsumura etal. 1985; Novy et al. 1993a; Gunning et al. 1997). The possible functional differencesbetween the isoforms has been emphasised by the finding that they appear to bespatially sorted out or compartmentalised in neuronal development (Gunning et al.1997). The localisation of tropomyosin isoforms shows a distinctive pattern in thekidney epithelial cells LLC-PK1. Temm-Grove et al. (1998) found that these cellsexpressed both high and low molecular weight isoforms of tropomyosin. The highmolecular weight isoforms were associated with stress fibres but not with adhesionbelts. In contrast, the low molecular weight isoforms were found on adhesion belts.These differences in localisation were also encountered when these isoforms wereintroduced into cells by microinjection or transfection. These observations supportthe view that the different isoforms might subserve different functions.

The loss of cell shape, cell adhesion to the substratum, and the loss of stressfibres in cellular transformation has focused attention on the expression of tropomy-osins in benign and malignant cells and tissues. The expression of tropomyosins hasbeen reported to be frequently down-regulated in transformed cells (Cooper et al.1985; Hendricks and Weintraub, 1981, 1984; Leavitt et al. 1986; J.J.C. Lin et al.1985; Matsumura et al. 1983). In chick embryo fibroblasts transformed by RSV, adifferential repression of tropomyosins has been reported to occur at the transcrip-tional level (Hendricks and Weintraub, 1984). The normal human fibroblast KD cellsexpress four tropomyosin mRNAs. The expression of two of these of 1.1 and 3.0kb was found to be markedly reduced in transformed cells and in a cell line derivedfrom pancreatic cancer (Novy et al. 1993b). Leavitt et al. (1986) have reportedalterations of tropomyosin isoforms that are expressed in human fibroblasts trans-



formed by chemical carcinogens. In these cells, the high molecular weight tropomy-osins appeared to be down-regulated. However, there was a slight down-regulationof one and an up-regulation of another high molecular weight isoform in nontum-origenic immortalised cells. A change in pattern of isoform expression has also beenreported to occur upon cell transformation by viruses (Matsumura et al. 1982, 1983).Oncogenic retroviral genes indeed have been shown to suppress tropomyosin syn-thesis (H.L. Cooper et al. 1985). Furthermore, the suppression of the tropomyosinsappears to have a bearing on cellular morphology. Prasad et al. (1999) transfectedv-src-transformed cells, which express the TM1 isoform at very low levels, withTM1 cDNA. This resulted in higher levels of TM1 expression in the transfectedcells, which also showed a reduction in cell growth and enhanced cell spreading.These effects also were accompanied by alterations in the architecture of microfil-aments. Therefore, differential expression of the isoforms could be seen as suggest-ing differences in their cell function.

A loss of high molecular weight tropomyosin isoforms and MLCs has beenreported in tumorigenic cell lines as compared with nontumorigenic cells derivedfrom prostate epithelium transformed by SV40 or X-irradiation (Prasad et al. 1997).However, TPA, which is a tumour promoter, is able to induce a high molecularweight isoform in human melanocytes (Vogt et al. 1997). Ovarian carcinomas report-edly have lower levels of a high molecular weight isoform of tropomyosin ascompared with benign tumours (Alaiya et al. 1997). Franzen et al. (1996) foundthat the average levels of high molecular weight isoforms were far lower in breastcarcinomas and nonmalignant tumours. The level of expression of one of theseisoforms seemed to relate to the presence of tumour in the axillary lymph nodes,with the primary tumours showing 1.7-fold greater expression as compared withthose that showed no nodal dissemination. The same high molecular weight isoformwas found to be expressed at higher levels in H-ras-transformed fibroblast cell linesthat possessed high metastatic ability than in transformed cells with lower metastaticability. It may be pointed out that this implies that the more malignant the cell thegreater is the level of expression of the high molecular weight isoform. This con-tradicts the relationship subsisting between the normal cell and its transformedcounterpart, where transformed cells show a down-regulation of tropomyosin expres-sion. Nonetheless, such a comparison might lead to oversimplified conclusions. Itshould be recognised that viral transformation of cells does not always lead tometastatic spread. The criterion of tumorigenicity does not contain any element ofmetastasis. For instance, tumours formed by SV40-transformed 3T3 fibroblastsrarely, if at all, metastasise. Furthermore, tumours are too heterogeneous and there-fore association of metastatic ability with specific isoforms requires a detailedinvestigation of specific subpopulations of the tumour. Also desirable would be aninvestigation of primary tumours and the corresponding metastatic tumours. Hash-imoto et al. (1996) compared K-1735 murine melanoma cells with differing meta-static potential. They reported that the β isoform of tropomyosin was expressed onlyin the low metastasis variant of K-1735 melanoma. The differential expression ofthe high and low molecular weight isoforms in adhesion belts and stress fibres(Temm-Grove et al. 1998) underwrites these efforts at exploring differential expres-sion of tropomyosin isoforms in the progression of tumours to the metastatic state.



Cell–cell and cell–substratum adhesion are important factors in tumour cell dissem-ination and their adhesion to metastatic target tissues. However significant might bethe association between tropomyosins and neoplasia, it would be worth recallinghere that TnI, which is the inhibitory component of the troponin/tropomyosin com-plex, might itself be associated with metastatic progression of cancer. As shown byMoses et al. (1999), TnI can inhibit angiogenesis both in vitro and in vivo and alsoinhibit metastatic dissemination.

THE REGULATORY ROLE OF CALDESMON

The cyclical interaction of the globular head domain myosin with thin actin filamentscoupled with the breakdown of ATP drives muscle contraction. Although reversiblephosphorylation of MLC is regarded as an important regulatory event, there isgeneral recognition that not all the events in muscle contraction can be explainedby invoking this mechanism alone. Additional mechanisms have been sought.Caldesmon and calponin are two major proteins, both capable of binding to themajor components such as actin, myosin, tropomyosin, and phospholipids (seeFattoum, 1997; Childs et al. 1992; Szymanski and Tao, 1997; Bogatcheva and Gusev,1995), and both have been seen as offering alternative modes of regulation. Further-more, both proteins interact with calmodulin in the presence of Ca2+ (Medvedev etal. 1996; Zhang and Vogel, 1997; Graether et al. 1997). Whether caldesmon andcalponin have individual roles in the regulation of smooth muscle contraction isbeing debated. Some argue that the two proteins interact with each other. Graceffaet al. (1996) showed that a strong binding occurred between calponin and thecarboxyl domain of caldesmon. They have suggested that this interaction could leadto the stabilisation of the complex. However, Czurylo et al. (1997) subscribe to theview that these two proteins have independent roles in muscle contraction.

Caldesmon is found in both smooth muscle and nonmuscle cells. It is a regulatoryprotein that occurs in the groove of the actin helix. Caldesmon and calponin inhibitactin-mediated activation of myosin-ATPase in smooth muscle fibres. Several seg-ments of the C-terminal region of the caldesmon molecule may be involved inbinding to actin and in its inhibitory function (Heubach et al. 1997). Whereas theC-terminal end binds to actin, the N-terminal end appears to be dissociated frombut present in the close vicinity of actin and seems to mediate the link betweenmyosin and actin filaments (Graceffa, 1995, 1997). In the C-terminal domain encom-passing residues 663-763, three regions have been identified that take part in myosin-ATPase inhibition: a central segment encompassing residues 747–767, a segmentthat is N-terminal, and another segment C-terminal to this central segment. Thecentral segment is essential but not sufficient to produce myosin-ATPase inhibition(I.D.C. Fraser et al. 1997).

Two isoforms of caldesmon arising from alternative splicing of pre-mRNA areknown. The high molecular weight (89–93 kDa) isoform is restricted to adult andfully differentiated smooth muscle cells, whereas the nonmuscle low molecularweight isoform is found in de-differentiated nonmuscle cells. There are severalconserved domains in the caldesmon molecule, which determine its interaction withand binding to actin, calmodulin, myosin, and phospholipids. All the isoforms are



powerful inhibitors of myosin-ATPase, but the specific nature of their distributionsuggests possible differences in function. In nonmuscle cells, caldesmon could bestabilising the microfilament network and in smooth muscle, it may be involved inthe inhibition of muscle contraction (see Huber, 1997). The function of caldesmonin the regulation of cytoskeletal organisation and contractile processes is not limitedonly to animal cells. A caldesmon-like protein occurs in higher plants. A 107-kDaprotein with properties similar and immunologically related to caldesmon has beenisolated from extracts of pollen tubes of Ornithogalum virens. This protein binds toactin in a CaM/Ca2+-dependent manner (Krauze et al. 1998). This is consistent withthe view that actomyosin cytoskeleton participates in the growth and elongation ofpollen tubes. Pollen tube elongation is a calcium-dependent process (Masserli andRobinson, 1997).

Caldesmon also can bind to S100 α and β proteins. The S100 binding site appearsto be in the C-terminal region. Interestingly, the cross-linking of caldesmon viacysteine residues with S100 seems to reduce the inhibitory effect of caldesmon onmyosin-ATPase activity, and S100 seems to be as effective as calmodulin in thisrespect (Polyakov et al. 1998).

There are no implications, as yet, that the different isoforms of caldesmon differfunctionally, because there is extensive sequence homology between isoforms occur-ring in different species (Novy et al. 1991). However, as stated above, the expressionof some isoforms has been related to the state of differentiation. Furthermore, earlystudies by Novy et al. (1991) have shown a two- to four-fold reduction in theexpression of caldesmon in certain transformed cell lines.

CALPONIN: ITS FUNCTION AND REGULATION

Calponin is a 33-kDa protein highly expressed in smooth muscle cells. The humansmooth muscle calponin gene contains seven exons encompassing >11 kb and it hasbeen assigned to chromosome 19p13.2 (Miano et al. 1997).

Calponin is a thin filament-associated TnT-like protein. It is strongly implicatedin smooth muscle contraction. As with caldesmon, calponin binds to actin, myosin,tropomyosin, and CBPs such as calmodulin. Actin possesses two calponin-bindingsites (Kolakowski et al. 1997; Mino et al. 1998). The interaction between calponinand myosin occurs via defined regions of the calponin molecule (Szymanski andTao, 1997). Three isoforms of calponin have been described: the basic h1 isoform,the neutral isoform h2, and the acidic isoform. Two distinct genes encode theisoforms h1 and h2. They are not generated by alternative splicing of pre-mRNA(Fukui et al. 1997). All three isoforms contain the calponin-homology (CH) actin-binding domain in the N-terminal region of the protein. The CH domain comprisesa sequence motif containing approximately 100 amino acid residues. Some actin-cross-linking proteins may contain two distinct CH domains and have contested therather generalised concept that a single CH domain is sufficient to confer actin-binding properties on these proteins (Stradal et al. 1998). CH domains also occurin other actin-interacting proteins. Fimbrin is an actin-cross-linking protein. Itsassociation with F-actin is similar to that of calponin (Hanein et al. 1997b) and,furthermore, it contains two CH domains. The proto-oncogene vav, which is



expressed in haematopoietic cells, codes for a signalling molecule that contains aCH domain (Castresana and Saraste, 1995; Romero and Fischer, 1996).

Nonmuscle isoforms of calponin have been reported in brain tissue. The h2isoform was identified by Fukui et al. (1997) from human skin tissue and humankeratinocytes in culture using antibodies raised against smooth muscle calponin.

Calponin generally has been attributed with the ability to inhibit myosin-ATPase.In vivo, calponin occurs in the ratio of approximately one calponin molecule to sevenactin monomers (Lehman and Kaminer, 1984; Takahashi et al. 1986; Lehman, 1991),but there is a view that higher levels of calponin may be required for an effectiveinhibition of myosin-ATPase (Gimona and Small, 1996). Exogenous calponin hasbeen shown to inhibit Ca2+-responsive muscle contraction (Horowitz et al. 1996;Naka et al. 1998). Calponin serves as a substrate for several kinases and phos-phatases, and its function seems to be regulated by phosphorylation. Pohl et al.(1997) have shown that unphosphorylated calponin is able to inhibit actin sliding,which is demonstrable using in vitro motility assays, but when calponin is phospho-rylated by PKC this effect is inhibited. In vivo, calponin purified from trachealsmooth muscle stimulated with carbachol (carbamyl choline chloride), which is aCa2+-mediated stimulator of phosphorylation, showed two-fold greater phosphory-lation than unstimulated muscle. Naka et al. (1998) also demonstrated a correlationbetween calponin phosphorylation and contraction of porcine coronary artery. How-ever, as discussed below mechanisms other than phosphorylation have been adducedfor calponin regulation, e.g., one involving caltropin.

Aside from being regulated by phosphorylation, it has been reported that calpo-nin co-precipitates with MAPK, but there is no obvious phosphorylation of calponin.The latter also co-precipitated with PKC-ε. A translocation of calponin to the cellmembrane occurs in cells that are stimulated with phenylephrine, when MAPK andPKC-ε are also translocated to the cell membrane. On the basis of these findings,Menice et al. (1997) have implicated calponin in signal transduction. Indeed, thetargeting of these various components to the cell membrane might be a consequenceof the tight binding of calponin to cytoplasmic β-actin (Parker et al. 1998).

Calponin seems also to be able to regulate the amount of free actin availablefor cytoskeletal organisation. The h2 isoform, for instance, appears to be localisedin the cytoplasm of basal cells in situ, whereas in cultured keratinocytes it occursalong the cell–cell contact areas, which suggests that it might play a part in theorganisation of the cytoskeleton. Tang et al. (1997) believe that calponin, beingpolycationic in nature, could promote the formation of F-actin bundles by reducingthe polyanionic repulsive interaction between actin filaments. Calponin can also bindto tubulin, which decreases with increases in Ca2+ concentration and ionic strength(Fujii et al. 1997). These authors also state that calponin has distinct sites for bindingactin and tubulin. Furthermore, calponin seems to bind strongly to desmin and isincorporated into IF and, therefore, it is possible that it might mediate the associationof desmin IF with actin (Mabuchi et al. 1997).

The ability of calponin to promote cytoskeletal organisation may be reflected inits ability also to inhibit cell growth (Z. Jiang et al. 1997). It also has been reportedrecently that the expression of calponin and smooth muscle MHC expression candistinguish between in situ and invasive carcinoma of the breast. Antibodies against



MHC and calponin stain epithelial cells of ducts and acini of normal breast tissueand carcinomas in situ, but these antibodies hardly show any reactivity in invasivecarcinomas (Wang et al. 1997). Calponin is prominently expressed in myofibrils ofleiomyosarcomas (Meyer and Brinck, 1997). Overall, calponin seems to show agenerally reduced expression in human leiomyosarcomas. Compatible with the lackof its expression in invasive breast carcinomas as compared with in situ tumours arethe recent findings of Horiuchi et al. (1999). These authors transfected humancalponin h1 into leiomyosarcoma cells. The transfectants showed a one third reduc-tion in cell proliferation, together with reduction in anchorage-independent growthas well as tumorigenicity. Nonetheless, much further work is required before onecan attribute a tumour suppressor function to calponin.

CALTROPIN-MEDIATED REVERSAL OF MYOSIN-ATPASE INHIBITION BY CALDESMON AND CALPONIN

It is obvious from the above discussion that caldesmon and calponin both inhibitmyosin-ATPase and influence muscle contraction. The binding of caldesmon andcalponin to actin appears to produce this inhibition and this effect is reversible bycalmodulin. Another CBP, i.e., caltropin, appears to be involved in the regulation ofthe inhibitory effects of caldesmon and calponin on myosin-ATPase. Caltropin is an11-kDa protein derived from smooth muscle. It occurs as a dimer in the native form(Willis et al. 1994). Some years ago, Mani et al. (1992) demonstrated that caltropin,like CaM, was able to reverse the inhibition of myosin-ATPase by caldesmon in thepresence of Ca2+. They found that caltropin directly interacted with caldesmon. Thisseemed to influence the interaction of caldesmon with the actin component of heavymeromyosin (Mani and Kay, 1993, 1995a). In vitro, caltropin seems to inhibit thepolymerisation of G-actin (Mani and Kay, 1995b), thus providing further confirma-tion of interference by caltropin in caldesmon–actin interactions. Although Mani etal. (1992) state that caltropin is as effective as CaM in reversing caldesmon-mediatedinhibition of myosin-ATPase, there is evidence that the binding affinity of caltropinto caldesmon is far greater than that of CaM (Zhuang et al. 1995). In consonancewith these observations are the findings of Willis et al. (1994) that caltropin also isable to form a complex with calponin in a calcium-dependent fashion in the ratioof 2:1 and reverse the inhibitory action of caldesmon. Caltropin has been describedas being more efficient than CaM. Thus there seems to be a reasonable body ofevidence, albeit from the same laboratory, that caltropin has considerable implica-tions in the regulation of myosin-ATPase function.


125

8

Structure and Biology of Calbindin

Calbindins D-28K (CBD) and D-9K (CBD-9K) are EF-hand proteins that areregarded as calcium buffer proteins together with parvalbumins (PV). CBD is a typeIII EF-hand protein possessing six EF-hands (see Table 4), all of which seem to bepacked in one globular domain, but the folding and packing of individual domainswithin the globular domain appears to be specific (Linse

et al.

1997). The rat CBD-9K gene consists of three exons and two introns. Exon 2 contains sequences encodingthe first EF-hand and exon 3 for the second EF-hand domain.

CALBINDIN IN NEURONAL POPULATIONS

CBD is a calcium-binding protein that is expressed abundantly in a number of celltypes, such as neuronal cells, mammalian and avian kidney, brain, and pancreas, andin avian intestines. Wasserman and Taylor (1966) isolated this calcium-binding EF-hand protein from the intestine of chicken later found to be a component of the nervoussystem of a wide variety of species (Celio, 1990; Reifergerste

et al.

1993). Theoccurrence of CBD is predominent in long-axon as well as in short-axon neurones(see Baimbridge

et al.

1992; Celio 1990; Frantz and Tobin, 1994; Sequier

et al.

1990).By virtue of its calcium buffering ability as well as its ability to modulate calciumchannel activity, CBD could be regulating calcium homeostasis in neurones (Albritton

et al.

1992). CBD also occurs in the neuroendocrine cells of adult and foetal lung ofhamsters. This has been demonstrated by immunochemical staining together withneuroendocrine markers. In contrast, other CBPs, such as PV and calretinin, are notdetectable (T. Ito

et al.

1998). Calretinin is a protein that bears a high degree ofhomology with CBD and shows similar calcium-binding properties (Cheung

et al.

1993). The occurrence of CBD and calretinin are often mutually exclusive, althoughmany cells may express both proteins at the same time (Rogers and Resibois, 1992).In light of the sequence similarities, it may be suggested by inference that these proteinssubserve similar functions and could define similar neuronal properties.

NEURAL CELL LINEAGE AND THE REGULATION OF CALBINDIN EXPRESSION

It has been argued that clonally related cortical neurones do not express the samecomplement of CBPs,

and therefore their expression may be controlled and influ-enced by extracellular factors rather than be genetically endowed. Pappas and Par-navelas (1997) exposed cultures of developing brain cortex from rat embryos tobFGF and the neurotropins NGF, brain-derived neurotropic factor (BDNF), and


126


neurotropin-3 (NT3). They found an enhanced morphological differentiation of largeGABA-containing neurones together with the expression of calbindin, the effectsbeing dependent on age and maturation of the subpopulations. As stated before,calbindin is highly expressed particularly in Purkinje cells of the cerebellum, but italso occurs in other neuronal cell types in other regions of the CNS. The specificexpression in Purkinje cells seems to be regulated by a 40-bp element, occurring inthe promoter region of the calbindin gene. This regulatory element called PCE1 ispresent also in the promoter of calmodulin II and, obviously, can regulate theexpression of other CBPs as well (Arnold and Heintz, 1997).

Cell type-specific transcriptional regulation mediated by CBPs may underlie thisphenomenon, and it follows from this that different extraneous factors might differ-entially affect the expression of these proteins depending on the neuronal subpop-ulation. These CBPs in turn may regulate the expression of transcription factors thatinduce the expression of other responsive genes. This could rationalise the presenceof calbindin in a wide variety of tissues and their responses to a variety of extracel-lular stimuli. It should be recognised, nevertheless, that other mechanisms such asenhanced stability of transcripts of target genes might also be involved.

CALBINDIN EXPRESSION IN EMBRYONIC DEVELOPMENT AND AGEING

Calbindin expression changes during embryonic development to develop into anadult expression pattern. In the cerebellum, calbindin gene transcripts as well as theprotein increase to reach a peak at 2 weeks postnatal stage (Iacopino

et al.

1990).CBD is transiently expressed during embryonic development (Andressen

et al.

1993). In mouse embryos, cerebellar Purkinje cells appear from 11 to 13 days ofgestation (Miale and Sidman, 1961), and soon after, they begin to express CBD(Enderlin

et al.

1987). CBD is the major component cellular protein of maturePurkinje cells, and the high protein levels maintained in adult life decrease in ageinganimals (Baimbridge

et al.

1982; Lledo

et al.

1992). In developing human cerebel-lum, CBD immunoreactivity is first encountered at 14 weeks of gestation, with CBDexpression being detectable in a variety of cell types. However, the number of CBDstaining cells and the intensity of staining decreases with development. At 21 to 31weeks of gestation, CBD reactivity is restricted to Purkinje and basket cells of thecerebellar cortex (Yew

et al.

1997). A developmental pattern is also discernible inthe appearance of CBD in neuroendocrine cells of foetal hamster lung from days15 and 16 of gestation, but endocrine cells appear on day 13 (Ito

et al.

1998). Inthe hamster brain, CBD transcripts have been reported to decrease markedly (50 to68%) in 19 to 24 month-old hamster as compared with 4- and 9-month-old animals,but, in contrast, calretinin and PV do not show any changes with ageing (Kishimoto

et al.

1998). Calbindin immunoreactivity changes in the cerebellum of frog tadpolesinduced to metamorphose by treatment with thyroxin (Uray

et al.

1998).The potential role of calbindin in cell differentiation has not received much

attention to date. Together with PV, calbindin has been shown to be associated withphenotypic conversion of supporting cells into hair cells (Steyger

et al.

1997).However, the extent of their individual contribution to the nonmitotic regeneration



127

of hair cells is unclear at present. At any rate, the information available is scanty,when compared with the massive evidence available about the effects of S100proteins on cell growth and differentiation.

PHYSIOLOGICAL FUNCTION OF CALBINDIN

Calbindin expression is modulated in a variety of tissues by several extracellularsignals and in an apparently tissue-specific manner. There are strong pointers thatcalbindin may have several functions in normal cellular physiology and that itsexpression in these tissues may be regulated by specific extracellular ligands bydifferent mechanisms resulting in specific outcomes. Therefore, calbindins couldperform tissue-specific functions (Table 8). Among the functions attributed to theseCBPs are facilitation of calcium flux, protection of neurones from degeneration,and as a calcium buffer in restricting calcium signals in nerve synapses and haircells. Neuronal firing patterns may be determined by calbindin (Chard

et al.

1995),and in hippocampal slices it can modify synaptic interactions (Heizmann andBraun, 1992).

The regulation of calbindin expression by VD3 in the kidney and the intestinehas been well documented. In contrast, VD3 does not affect calbindin expression inbrain tissue. Absorption of calcium and lead in the intestines is greatly affected byserum VD3. Conversely, serum levels of VD3 increase markedly with lead ingestionand calcium deficiency. Also apparent, under these conditions, are changes in theexpression of calbindin, which suggests a possible mediation of calcium and leadabsorption by calbindin (Fullmer, 1997). In Madin–Darby bovine kidney (MDBK)cells, TPA has been found to enhance CBD expression, and this is preceded by theactivation and translocation of PKC-

α

. Furthermore, PKC phosphorylates CBD in

TABLE 8Extracellular Biological Signals Influencing Expression of Calbindins

Extracellular Signal Target Tissue Comments Ref.

1,25-dihydroxy vitamin D3

Intestine, kidney Not brain tissue Christakos

et al.

(1989)

Oestrogen Kidney Independently of vitamin D3 Criddle

et al.

(1997)

Adrenal steroids (corticosterone)

Hypothalamic tissue

During prenatal development Lephart

et al.

(1997a)

Growth hormone Intestine Calbindin D-9K mRNA, also induced in parallel, mRNAs for vitamin D3 receptor and insulin-like growth factor (IGF)-1

Salih

et al.

(1997)

Butyrate Rat insulinoma cell line

Parallel production of insulin and its secretion

S. Lee

et al.

(1994)


128


a calcium- and phospholipid-dependent manner. VD3 also increases CBD expressionas well as up-regulates the expression of PKC. PKC-mediated phosphorylation oftwo threonine residues of CBD could lead to the functional activation of CBD, whichis consistent with the transduction of calcium signal by PKC mediation (Gagnonand Welsh, 1997).

Calbindin seems to be involved with insulin secretion by pancreatic

β

cells. Lee

et al.

(1994) found that butyrate is able to induce CBD mRNA as well as proteinlevels in the rat insulinoma cell line RIN 1046-38. This treatment also enhancedinsulin production and its secretion. The involvement of CBD in insulin secretionhas been confirmed in further experiments by Reddy

et al.

(1997), who transfectedthe insulinoma cell line with the CBD gene. In the transfected cells a >20-foldincrease in CBD mRNA was found, and at the same time insulin mRNA showed a>20-fold enhancement. Insulin production and release also increased approximatelysix-fold. It seems possible that this effect of CBD is also mediated by PKC, becauseactivation of PKC can influence insulin secretion. Both CBD and CBD-9K appearto be capable of regulating genetic transcription (Reddy

et al.

1997; Fukushima

etal.

1998). Possibly, calbindin influences the process of calcium-mediated regulationof transcription factors (see Reddy

et al.

1997).It is to be expected that the expression of calbindins themselves would be subject

to regulation at the transcriptional level. That the specificity of their expression mightbe linked with definable transcription pathways is an interesting thought that hasbeen explored experimentally. Arnold and Heintz (1997) have identified a 40-bpelement, the PCE1, that is necessary for the expression of the CBD in Purkinje cells.This element seems to regulate transcription of CBD gene. PCE1 also occurs incalmodulin II. Both CBD and calmodulin II are expressed abundantly in Purkinjecells, and PCE1 might be a common component in the regulation of expression ofthese CBPs in Purkinje cells. CBD and calmodulin II are expressed in other celltypes too. Transcription elements other than PCE1 may be involved in the regulationof the genes expression at these sites (Arnold and Heintz, 1997). Oestrogen-mediatedinduction of CBD expression is attributed to two regions (–1075/–702 and –175/–78)of the mouse CBD promoter (Gill and Christakos, 1995). Similarly, oestrogen canregulate specific regulatory sequences of CBD-9K (Romagnolo

et al.

1996). Thelevels of expression of CBD may be regulated by other means, as indicated by Wangand Christakos (1995), who found that retinoic acid (RA) increased, by 10- to 15-fold, both CBD protein and CBD mRNA in the medulloblastoma cell line D283.The RA effect was mediated by RA-specific receptors, which ordinarily would haveindicated the regulation of transcription of the target gene by RA (see Redfern,1997). However, the regulation in this case did not appear to occur at the transcrip-tional level, but the increased expression of CBD and its mRNA seemed to be dueto an increase in the stability of the mRNA.

CBD seems to play a role in renal reabsorption of calcium. Ovariectomy reducesthe expression of CBD gene transcripts, and this is reversed by the administrationof 17

β

-oestradiol in the ovariectomised animals (Criddle

et al.

1997). These alter-ations in CBD expression show no relation to serum levels of VD3. Thus the roleplayed by CBD in renal absorption may be distinguished from that in intestinal tissue.



129

NEUROPROTECTIVE FUNCTION OF CALBINDIN

CBD is generally regarded, in common with PV and calretinin, as an intracellularcalcium buffer because it has not been found to influence enzyme activity or calciumion channels (see Baimbridge

et al.

1992). Nevertheless, buffer function may mod-ulate intracellular calcium levels and in this way regulate neuronal function. Thus,distinct physiological functions may be identified that are affected by alterations inthe intracellular levels as well as the conformational state of CBD. For instance,calretinin deficiency seems to lead to an impairment of motor coordination andmarked abnormalities in the firing behaviour of Purkinje cells

in vivo

. In calretininnull (cr –/–) Purkinje cells, CBD is detected by anticalretinin antisera. This has beenattributed to the fact that these antisera detect the presence of CBD that has under-gone conformational changes upon being saturated with Ca

2+

(Schiffmann

et al.

1999). These events clearly implicate an impairment of calcium homeostasis in thecr (–/–) cells

in vivo

.The pattern of CBD occurrence in ageing neurones has led to the postulate that

it may function as a neuroprotective agent. Lally

et al.

(1997) found that the sizeand number of CBD-immunoreactive neurones were reduced in Alzheimer’s disease,and this has been suggested to be a consequence of cellular degeneration related tothe reduced CBD. Alzheimer’s nerve cells containing CBD are believed to be lesssusceptible to degeneration than those that have greatly reduced amounts of CBDor no CBD at all. This is supported by experiments with PC12 cells into which CBDcDNA was transfected. These cells were far less susceptible to degeneration causedby serum withdrawal, glutamate, and the neurotoxin 1-methyl-4-phenylpyridinium.However, it would appear that CBD cannot protect these cells from degenerationcaused by calcium ionophores (McMahon

et al.

1998). Calbindin-null mutant miceshow severe impairment of motor coordination (Airaksinen

et al.

1997). However,these null mutants do develop normally without any disturbances in calcium metab-olism and, therefore, Airaksinen

et al.

(1997) believe that CBD expression in periph-eral organs may not be crucial for normal development.

CALBINDIN EXPRESSION AND THE METASTATIC PHENOTYPE

The ability of steroids to influence the expression of calbindins has led to investi-gations aimed at examining their potential involvement in the development andgrowth of tumours, their progression, and prognosis. Watanabe

et al.

(1994) inves-tigated CBD levels of lung cancers. The levels of CBD were low in normal lungtissue, but its expression was higher in lung cancers, with SCLC tissue expressinggreater amounts than non-SCLC (NSCLC). Furthermore, CBD levels correlated withprogression of NSCLC to advanced stages of the disease involving metastaticinvolvement of the lymph nodes. Upon investigating cell lines derived from thesetumours, Watanabe

et al.

(1994) noticed that CBD expression might also relate tothe expression of neuroendocrine-related paraneoplastic properties of these tumours.They found that the CBD could be used to differentiate classical SCLC from variant


130


SCLC. The specificity and sensitivity with which this could be achieved was com-parable to the neuroendocrine marker neurone-specific enolase.

Significant differences also have been noted in the expression of CBD in humancolonic cell lines derived from primary tumours and those derived from metastatictumours. Sampson-Johannes

et al.

(1996) found that SW480 cells obtained fromprimary colorectal tumours did not express CBD, but in sharp contrast, SW620 cellsderived from metastatic tumours did. They also demonstrated that SW480 cells werenot capable of colonising human foetal lung tissue transplanted into SCID-hu mice,but SW620 cells colonised the grafts upon intravenous introduction into the animals.Furthermore, SW620 cells expressed sialyl Lewis-x and Lewis-a antigens which areconducive to metastatic spread at far higher levels than SW480, which were unableto colonise the foetal lung grafts. On the basis of these results, the authors suggestthat the appearance of the metastatic ability could be associated with the acquisitionof CBD expression. It ought to be recognised, however, that this experimental modelis too remote from the spontaneous metastasis. The differences in the abilities ofthe two cell types to localise in foetal lung grafts may simply reflect their adhesivecapabilities, rather than CBD expression. Some preliminary work has been reported(Castro

et al.

1998) that relates the value of CBD expression to prognosis of lungcancer.


131

9

Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker

CALRETININ AND ITS ALTERNATIVELY SPLICED ISOFORMS

Calretinin is a neuronal calcium-binding protein. It shows marked homology withCBD, and presumably on account of this, calretinin and CBD show mutually exclu-sive expression in neuronal populations, although in some cell types both may beexpressed simultaneously (Rogers and Resibois, 1992). The pattern of their expres-sion and marked similarities between them with respect to calcium-binding proper-ties (Cheung

et al.

1993) could indicate that they perform similar functions inneurones. Thus, calretinin seems to share with CBD certain features such as regu-lation of expression by growth factors and involvement in cell proliferation, differ-entiation, and neoplastic transformation. Also, calretinin may possess, in commonwith CBD, a neuroprotective property.

On the other hand, the apparent mutually exclusive nature of their expressionmight be a reflection of the cooperative effects between calretinin and CBD. Thecalcium-buffering function of calretinin might result in CBD not being detected byspecific antisera. As stated in the previous section, in cr (–/–) Purkinje cells, thereis an impairment of calcium homeostasis. As a result, CBD is oversaturated withcalcium. This brings about changes in the conformational state of CBD. As aconsequence, CBD becomes detectable by specific antisera (Schiffmann

et al.

1999).These conformational changes seem to translate into alterations in cell function.Therefore, the experiments of Schiffmann

et al.

(1999) might suggest a cooperationbetween calretinin and CBD in function.

Calretinin is expressed as two splice variant isoforms, encoding two proteins of20 and 22 kDa molecular size, truncated at the C-terminal region. The 20-kDaisoform is a result of splicing of exon 7 to exon 9, and the 22-kDa isoform resultsfrom splicing of exon 7 to exon 10. Both alternatively spliced messages show frameshift and contain stop codons in exon 9 (in calretinin-20K) and exon 10 (in calretinin-22K) (Schwaller

et al.

1995). Calretinin-22K contains the first 178 amino acidresidues of unspliced calretinin, and this sequence is followed by a C-terminal 14amino acid sequence that is not found in the unspliced calretinin (Gander

et al.

1996). Antibodies raised against this 14-amino acid sequence do not recognise full-length calretinin, which indicates the novel nature of the C-terminus of calretinin-22K. However, in spite of their variant secondary structure, the calcium-binding


132


properties of calretinin-20K and -22K appear to be unaffected (Schwaller

et al.

1995). Calretinin-22K occurs in many colon carcinoma cell lines. However, thesignificance of the expression of splice variants remains unclear, especially in viewof the small numbers of cell lines studied and the absence of information on thestability of the variant isoforms and their incidence

in vivo

.

REGULATION OF CALRETININ EXPRESSION

The expression of calretinin is regulated by a number of neurotropic agents andgrowth factors and correlates with the appearance of phenotypic features of differ-entiation. Farkas

et al.

(1997) found that glial cell-derived neurotropic factor (GDNF)increased the number of neurones expressing calretinin in cultures of embryonicstriatal neurones. The numbers of calretinin-expressing cells also is increased bybFGF, alone or in combination with retinoic acid (Pappas and Parnavelas, 1998).The neurotropic factor BDNF induces the extension of neuronal dendrites of hip-pocampal sections maintained in culture. The subpopulations that respond to BDNFappear to be those that express calbindin and calretinin (Marty

et al.

1996). Thereare also indications that there could be some specificity in the morphologicalresponse to the neurotropic factor. No response to BDNF is observed in neuronesthat express PV. Furthermore, the calretinin content of Cajal–Retzius neuronal cellsis regulated by BDNF together with induction of differentiation (Marty

et al.

1996),but the latter is not essential for their survival. Calretinin expression in medial basalhypothalamic regions of male rats is reported to be greater than that of female ratsat 19 and 20 days of gestation, suggesting a developmental and hormonal regulationof calretinin (Lephart

et al.

1997b). There is no question that expression is devel-opmentally regulated as shown by Jiang and Swann (1997), in the evolution of theneuronal populations and the emergence of cells expressing calretinin in the maturehippocampus of the rat. The development and maturation of neurones of the chickdorsal root ganglion are closely related to the expression of calretinin. The gangliabecome calretinin positive after 9 days of incubation, and the number of calretinin-expressing cells increases with the length of incubation (Kiraly and Celio, 1992).Calretinin-staining clusters of cells are also detected in the medullary epithelium ofthe thymus at day 11 of incubation, coinciding with the functional maturation of thethymus (Kiraly and Celio, 1993). It is of interest that PV is detected in the epithelialcells at day 9, but CBD is not detectable. This would suggest that at least in somedeveloping organ systems, calretinin and CBD may be individually associated withfunctional maturation, in spite of their molecular homology to each other.

The expression of calretinin and CBD in the oxytocin (OT)- and vasopressin(VP)-containing neurones of the supraoptic nucleus has been the subject of a detailedinvestigation by Miyata

et al.

(1998). In both types of neurones, there is a markeddifferential distribution of calretinin and CBD positivity. In OT neurones, 72 to 84%of cells were CBD immunoreactive as compared with ca. 50% staining for calretinin.In VP neurones, no calretinin staining cells were detectable and only 25% of neu-rones stained for CBD. Whether this can form a functional differentiation betweenthe two CBPs is uncertain. However, because calretinin and CBD do not differ


Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker

133

substantially with respect to their calcium-buffering ability, the possibility that theymay yet be functionally different in other respects needs to be explored further.

CALRETININ EXPRESSION IN CELL PROLIFERATION AND DIFFERENTIATION

The relationship between calretinin expression and cell proliferation and the stateof differentiation also has been reported in nonneuronal cell types. Indeed, studiesof several tumour cell lines have provided evidence supporting such a relationship.Gotzos

et al.

(1996a) found that 8 of 10 colonic carcinoma cell lines expressedcalretinin, and the protein was found in rapidly proliferating cells. Upon beinginduced to differentiate by treatment with sodium butyrate and hexamethylene bisac-etamide, WiDR colon adenocarcinoma cells showed marked reductions in cell pro-liferation as well as expression of full-length calretinin and its splice variant isoformcalretinin-22K (Schwaller and Herrmann, 1997). HT29-18 colon adenocarcinomacells have been induced to differentiate into enterocytes by using a different strategy— glucose starvation. Again, a marked reduction in calretinin mRNA was found inthe differentiated cells (Cargnello

et al.

1996). These authors read between the linesand suggest that calretinin may maintain an undifferentiated proliferative state char-acteristic of neoplastic transformation.

CALRETININ AND ITS POSSIBLE NEUROPROTECTIVE PROPERTY

In common with CBD, calretinin may be neuroprotective. In Alzheimer’s disease,large pyramidal neuronal cells show a differential susceptibility to degeneration, andspecific subpopulations, which express calretinin, might be resistant to degeneration(Hof

et al.

1993). The presence of calretinin also seems to provide some protectionagainst serum deprivation of rat cerebral cortex organ cultures (Weisenhorn

et al.

1996).A study of the expression of calretinin, together with CBD and PV mRNA, in

hamster brain in relation to ageing, has also helped to focus attention on differencesbetween calretinin and other CBPs. Kishimoto

et al.

(1998) found that whereas CBDtranscript levels fell by 50 to 68%, calretinin and PV transcripts remained unchanged.Implicit in these findings is the suggestion that the down-regulation of CBD expres-sion associated with the ageing process might reflect the neuroprotective action ofCBD, and further, by inference, that calretinin may not function in this way. However,it ought to be recognised that most of the available evidence is too preliminary innature to warrant conclusive interpretation.

CALRETININ AS A POTENTIAL TUMOUR MARKER

The expression of calretinin in a number of colonic carcinoma cell lines and theloss of its expression upon differentiation, which has been discussed in the previoussections, has inevitably led to the suggestion that it may serve as a potential maker


134


of human malignancies. Gotzos

et al.

(1996b) examined a series of mesotheliomasfor the expression of calretinin, PV, and CBD. Calretinin was found in mesotheliomasof the epithelial type and in the epithelial component of mixed tumours. Sarcomatoidmesotheliomas and the sarcomatoid component of mixed tumours did not expresscalretinin. Neither were PV and CBD expressed in these tumours or in normal lungtissue. Furthermore, none of the three CBPs was detected in adenocarcinoma of thelung. Therefore, Gotzos

et al.

(1996b) suggest that calretinin expression couldprovide a useful means of differentiating epithelial-type mesotheliomas from meta-static adenocarcinoma of the lung. Doglioni

et al.

(1996) have claimed a diagnosticsensitivity of 100% in a series of 44 mesotheliomas investigated. Leers

et al.

(1998)have advocated a combination of cadherin and calretinin to distinguish betweenmetastatic carcinomas and mesotheliomas and regard this combination of markersas a powerful tool. Calretinin has been proposed as a marker for serous carcinomaof the ovary (Folpe and Gown, 1997). Also, high levels of calretinin-22K have beenfound in the serum of many cancer patients. Serum levels of calretinin in breast andcolon cancer have been reported to be very high. The protein is detectable inepithelial cells, nerve fibres, connective tissue, and mesothelial cells. Calretinin alsohas been found in ischemic necrosis of the gut (Schwaller

et al.

1998). Needless tosay, there is much scope for investigating the clinical value of serum calretinindetermination in tumour typing. There is virtually no information available aboutits relevance in cancer prognosis.


135

10

Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading

MOLECULAR FEATURES OF CALCINEURIN

Among more than 30 target proteins activated by CaM is calcineurin. Calcineurinis a serine/threonine phosphatase. It is also known as protein phosphatase (PP)-2B.Together with PP-1, PP-2A, and PP-2C, calcineurin forms an important group ofphosphatases that play a critical role in the phosphorylation/dephosphorylationcycles associated with regulation of the activity of a variety of enzymes.

Calcineurin is highly conserved as well as widely distributed. Nevertheless, ithas restricted substrate specificity. Its major substrates are the NF-AT and othermembers of the NF-AT family of transcription factors,

N

-methyl-

D

-aspartate(NMDA) receptors (Barford, 1996; Tong

et al.

1995; also see below), and IP3receptors (Cameron

et al.

1995; Cunningham, 1995). Microtubule-associated pro-teins MAP-2 and the tau protein (Ferreira

et al.

1993; see below for further refer-ences) are also among its substrates. The biochemical and pharmacological signif-icance of these substrates have been discussed below in the context of the variousphysiological functions performed by calcineurin. Calcineurin is a major protein ofbrain tissue, accounting for approximately 1% of its total protein. In brain tissue itis associated with the cytoskeletal structures or is bound to the plasma membrane.It occurs as a major component of neurones, especially of the neurostriatum andcerebellum. Lymphocytes also contain large amounts of calcineurin (Kincaid

et al.

1987).Calcineurin is composed of two subunits: a catalytic subunit known as cal-

cineurin A and a regulatory subunit called calcineurin B. Calcineurin is usuallyisolated with bound iron and zinc ions, but it still requires the binding of exogenousmetal ions to achieve maximum activity. Calcineurin possesses low catalytic activityin the absence of exogenous metal ions. It can be activated by Mn

2+

, Mg

2+

, and Ni

2+

.The activation by Mn

2+

seems to involve the catalytic subunit calcineurin A. Thecalcineurin B sequence is highly conserved, as compared with calcineurin A. Nev-ertheless, in the latter the catalytic domain is highly conserved in evolution. Cal-cineurin A is 59 kDa in size and binds CaM. The regulatory 19-kDa calcineurin Bcontains four EF-hand calcium-binding domains. It is encoded by a single gene and

0942/C10/frame Page 135 Wednesday, October 25, 2000 12:01 AM

136


contains 163 amino acid residues (reviewed by Klee

et al.

1988). However, alterna-tive splicing might result in a number of variant forms of calcineurin B (C.D. Chang

et al.

1994). Three isoforms (

α

,

β

, and

γ

) of calcineurin A and two of calcineurinB have been described (Tumlin, 1997). Calcineurin A-

β

has been mapped to humanchromosome 10q21–22, and calcineurin B to chromosome 2p15–16. The calcineurinA-

α

occurs on chromosome 4 (M.G. Wang

et al.

1996).

CALCINEURIN IN CELL PROLIFERATION AND ADHESION-RELATED PHENOMENA

Calcineurin has been implicated in a number of physiological events, such as cellproliferation, cell death, and signal transduction, and in immunosuppression. It alsohas been implicated in certain functions of the nervous system, e.g., in neurotrans-mission. The discussion here will be restricted mainly to areas pertinent to thebiological behaviour of cancers, although some reference will be made to Alzhe-imer’s disease, in which cytoskeletal abnormalities occur prominently. The regula-tion of physiological activity of a large number of biological macromolecules isdependent on phosphorylation. It is to be expected that, as a phosphatase, calcineurinwould be a key component in the phosphorylation of some of these molecules. Fromthis it should follow, therefore, that calcineurin would impinge significantly on thebiological behaviour of cancers and in other disease states.

P

UTATIVE

R

OLE

OF

C

ALCINEURIN

IN

C

ELL

C

YCLE

P

ROGRESSION

There is a large body of evidence that points to the importance of calcineurin in theprogression of the cell cycle. Calcineurin A gene seems to be essential for the G

1

–Stransition of

Aspergillus nidulans

cells. A disruption of the gene by homologousrecombination results in the arrest of the cell cycle in its early phase. Calcineurin AmRNA accumulates in the G

1

phase well before the G

1

–S transition point (Rasmussen

et al.

1994). The induction of DNA synthesis in response to growth factor stimulationof cells has been shown to depend on Ca

2+

uptake and on the activation of calcineurin(Tomono

et al.

1996). A suggested

modus operandi

is by the regulation of specificcyclins. The initiation of DNA synthesis and mitosis is controlled by two key factors:(1) cyclin-dependent protein kinases (cdk) composed of a cyclin regulatory subunitand a cell division control (cdc2) family kinase and (2) CDK inhibitors (see Sherbetand Lakshmi, 1997b for further information). The involvement of calcineurin in theprogression of the cell cycle is demonstrated by the fact that the inhibition of cal-cineurin leads to an inhibition of fibroblast growth factor FGF-induced expressioncyclins A and E in Swiss 3T3 cells (Tomono

et al.

1998). Calcineurin seems to beassociated also with determining the length of the G

2

phase of the cell cycle and entryof the cell into the mitotic phase. The G

2

–M transition, in

Saccharomyces pombe

forinstance, is dependent on the activation by dephosphorylation of a complex of G

2

-specific cyclin with cdc2 kinase. This process of activation is inhibited by Swe-1 (Wee-1 homologue) tyrosine kinase. The transcription of Swe-1 is regulated by Zds-1.Calcineurin and Mpk-1 seem to regulate the transcription and posttranslational mod-ification of Swe-1, respectively (Mizunuma

et al.

1998).



137

It would be of considerable interest, therefore, to examine whether some of theeffects of calcineurin could be explained by its putative participation in the phos-phorylation events associated with cell cycle progression. A number of cyclins andcdk are expressed in a cell cycle-related fashion. The cyclin D/cdk-4 complexes areactivated by cdk-activating kinases. This activated complex now phosphorylates rbprotein and enables the cell to transit into the S phase. The cyclin B/p34

cdc2

complexis associated with the G

2

–M transition of cells. The phosphorylation of p34

cddc2

attyrosine 161 is believed to activate it, which enables the cell to make the G

2

–Mtransition. Phosphorylation of threonine 14/tyrosine 15 is believed to produce anegative regulatory effect on cell G

2

–M transition (see Sherbet and Lakshmi, 1997bfor references). Calcineurin, being a threonine phosphatase, could conceivably affectthis negative regulation of the G

2

–M transition and allow cells to enter the mitoticphase. It is also possible that calcineurin functions by means other than altering thecyclins associated with the G

1

–S transition. Tomono

et al.

(1998) found thatcyclosporin A, at calcineurin-inhibiting concentration, inhibited cyclins A and E, butnot DNA synthesis.

An interesting aside to this story is that carbachol, a Ca

2+

-mediated stimulatorof tyrosine phosphorylation, induces the formation of actin stress fibres and markedlyincreases the F-actin/G-actin ratio in smooth muscle cells

in vitro

. Tyrosine kinaseinhibitors have been found to inhibit the formation of stress fibres in the cell system(Togashi

et al.

1998). The opposite effects exerted by the phosphorylation ofserine/threonine and tyrosine residues could conceivably suggest a regulatory mech-anism of stress fibre formation by a process of differential phosphorylation. Such amechanism is encountered in the control cell cycle progression, where cyclin func-tion is regulated by differential phosphorylation. As alluded to above, the phospho-rylation of the threonine residues and inactivation of p34 has a negative regulatoryeffect on cell cycle progression. In the light of the crucial role played by thecytoskeleton in cell division, further studies of the potential involvement of cal-cineurin in the cell division cycle and allied areas are clearly warranted and mayturn out to be highly fruitful.

The progression of the cell cycle is controlled at both G

1

–S and G

2

–M transitioncheckpoints by the phosphoprotein p53. The biochemical basis of p53 function is itsability to regulate transcription by binding to DNA. Calcineurin appears to be able tomodulate p53 binding to the long terminal repeats (LTR) of human immunodeficiencyvirus (HIV-1). The compound PD 144795 inhibits HIV-1 transcription, which corre-lates with the inhibition of the phosphatase activity of calcineurin (Gualberto

et al.

1998). This demonstration of p53-mediated transactivation provides another putativemechanism by which calcineurin could influence cell cycle progression.

Calcineurin is known to modulate the function of other transcription factors,e.g., NF-AT3 and GATA-4, by dephosphorylation (Molkentin

et al.

1998). We knowfrom the work of Cai

et al.

(1996) that calcineurin might be involved also in thetranscription of the IL-2 gene. They showed that inactivation of calcineurin leads toan inhibition of the transcription of the IL-2 gene. The expression of fibre-typespecific genes, of slow- and fast-twitch myofibrils of adult skeletal muscles, seemsto be regulated by calcineurin. The transcription of these genes occurs by the


138


mediation of NF-AT and MEF2 transcription factors (Chin

et al.

1998). The cal-cineurin inhibitor FK506 reversibly inhibits insulin secretion by HIT-T15 cells inculture, and in parallel, intracellular levels of insulin mRNA and insulin alsodecrease. The inhibition of insulin gene transcription has also been confirmed usinghuman insulin/CAT reporter gene constructs (Redmon

et al.

1996). Recent reportsshow that IGF, which is involved in muscle growth, regeneration, and hypertrophy,also appears to follow the calcineurin NF-AT, GATA pathway in activating theexpression of the appropriate genes. The transfection of IGF-1 gene into skeletalmyocytes and its expression at the postmitotic stage induce the expression of cal-cineurin mRNA transcripts and the protein. The latter becomes localised in thenucleus. IGF-1 as well as activated calcineurin also induce GATA-2 expression. Thistranscription factor associates with calcineurin and NF-Atc1 (Musaro

et al.

1999).In parallel studies, Semsarian

et al.

(1999) have shown that treatment of skeletalmuscle cells with IGF-1 or dexamethasone activates calcineurin and induces thetranslocation of NF-Atc1 to the nucleus. These observations clearly implicate thecalcineurin/NF-AT signalling pathway in the cellular responses generated by thesegrowth factors.

Finally, the ubiquitous HSPs come into this picture as well. HSPs have gainedmuch coverage as stress-induced proteins. Besides, they are of exceptional impor-tance to the life of the cell, for they take part in processing of nascent protein,transport, and targeting of proteins to various cellular subcompartments. Further-more, not only are HSPs expressed in a cell cycle-related manner, but they alsointeract with many cellular proteins that are involved in cell cycle regulation. It isof much interest, therefore, to cite here some recent work that seems to strengthenthe association of both HSPs and calcineurin in the progression of the cell cycle.Someren

et al.

(1999) have shown that HSP70 and HSP90 both activate calcineurinin the presence of CaM. The requirement of CaM seems to be absolute for theactivation by HSP70. Activation of calcineurin does not occur in the absence ofCaM. Someren

et al.

(1999) have also demonstrated that calcineurin co-precipitateswith HSP. However, in light of much evidence to be discussed in a later section, itis difficult to establish the precise pathway by which either HSPs or calcineurinmight influence the progression of the cell cycle.

T

HE

E

FFECTS

OF

C

ALCINEURIN

ON

C

ELL

A

DHESION

AND

M

OTILITY

The expression of calcineurin A as well as B is up-regulated in HL-60 cells inducedto differentiate by treatment with all-

trans

RA. A progressive increase in calcineurinphosphatase activity occurs in parallel with granulocytic differentiation and inhibi-tion of cell proliferation (Kihara

et al.

1998). Therefore, there may be anotherpathway by which calcineurin influences cell proliferation. RA is a well-knownmodulator of cell adhesion and inhibitor of cell motility and the invasive behaviourof cancer cells (Fazely and Ledinko, 1990; Edward and Mackie, 1990; Edward

etal.

1989, 1992; Wood

et al.

1990).Hendey and Maxfield (1993) found that the motility of neutrophils on vitronec-

tin-coated substratum could be inhibited by using inhibitors of calcineurin. Theformation of filopodia and the motility of neurones of chick dorsal root ganglia are



139

said to depend on calcineurin. The calcineurin inhibitors cyclosporin A and FK506delay the formation of neurites and inhibit neurite extension. A targeted and focalinactivation of calcineurin in regions of growth cones affects filopodial retractionand direction of its subsequent outgrowth (Chang

et al.

1995). FK506 has also beenfound to inhibit the transendothelial migration of human lymphoma cells, Nalm-6,in both

in vitro

and

in vivo

situations (Tsuzuki

et al.

1998). These authors havesuggested that the inhibition of migration might be related to an inhibition of vascularcell adhesion molecule (VCAM)-1 and its

α

4

β

1

integrin receptor VLA-4. TheVCAM-1/VLA-4 system is known to provide the recognition mechanism and adhe-sive facility for certain tumour cell types to the endothelium. VCAM expressionseems to relate to the progression of melanomas (Denton

et al.

1992), but not ofbreast cancer (Fox

et al

1995). Overall, the evidence for any general relationshipbetween the expression of VCAM and its receptor and metastatic potential is notcompelling. However, calcineurin can stimulate the transcription factor NF-

κ

B andenhance its DNA-binding property (Franz

et al.

1994). This transcription factor isalso involved in the induction of cell adhesion molecules, and anti-NF-

κ

B reagentsare able to block cancer cell adhesion to endothelial cells (Tozawa

et al.

1995).Therefore, the effects of FK506 on transendothelial migration described by Tsuzuki

et al.

(1998) could be interpreted as indicating that calcineurin might itself beinfluencing the diapedesis by cancer cells.

Mohri

et al.

(1998) have recently provided some direct evidence that shows thatcalcineurin affects cell adhesion to substratum and, by implication, also modulatesthe invasive behaviour of cells. They used the colon cancer cell line Colo201 anddemonstrated that the protein kinase inhibitors K252a and KT5720 enhanced celladhesion to and their spreading on substratum. This process was accompanied bythe formation of actin stress fibres. The adhesion appeared to be mediated by integrin

α

2

and

β

1

, as indicated by their accumulation at the sites of focal adhesion. Thehigher adhesion mediated by K252a and KT5720 could be blocked by cyclosporinand FK506, which also inhibit calcineurin. An increase in the expression of TGF

β

-1 in peripheral blood mononuclear cells obtained from patients treated withcyclosporin has been reported (Shin

et al.

1998). Other cell types, such as A549cells, seem to respond to cyclosporin in a similar fashion. The increased expressionof TGF

β

is associated with enhanced cell motility, invasive behaviour

in vitro

, andenhanced metastatic behaviour, as indicated by inhibition of the cyclosporin-inducedeffects of anti-TGF

β

antibodies (Hojo

et al.

1999). It would be worthwhile to pointout here that TGF

β

produces a variety of effects. Among these are induction ofangiogenesis, inhibition of cyclin-dependent kinases, and induction of inhibitors ofmetalloproteinases (see Sherbet and Lakshmi, 1997b for references). The invasivebehaviour of trophoblast cells is believed to be controlled by the induction of TIMPsby TGF

β

(Lala and Graham, 1990).These cellular faculties of adhesion to substratum, cell spreading, and cell

motility are closely related to cell proliferation, besides being dependent on cytosk-eletal organisation. Therefore, one should consider whether calcineurin could bealtering cytoskeletal dynamics by altering the phosphorylation status of cytoskeletalcomponents.


140


Calcineurin and the CaM-dependent protein kinase II have been reported toregulate the phosphorylation levels of neurofilament subunits and

β

-tubulin elementsof rat cerebellar cytoskeleton (De Mattos-Dutra

et al.

1998). The tau protein hasbeen shown to be an important requirement for neurite outgrowth and growth conemotility. The inactivation of tau seems to lead to an inhibition of neurite outgrowth,and other microtubule-associated proteins are unable to compensate for the inacti-vated tau (C.W.A. Liu

et al.

1999). In common with many other proteins, thebiological function of tau is regulated by phosphorylation. tau promotes microtubuleassembly and inhibits depolymerisation (Drechsel

et al.

1992). Phosphorylated tauhas a reduced capacity to bind to microtubules (G.V.W. Johnson, 1992). The tauprotein of Alzheimer’s disease paired helical filaments (PHF) has been reported tocontain 21 phosphorylation sites, of which some are serine/threonine–proline resi-dues (Morishima-Kawashima

et al.

1995). The phosphorylation of threonine 231,serine 235, and serine 262 is essential for maximal inhibition of binding of tauprotein to microtubules (Sengupta

et al.

1998). Mandelkow

et al.

(1996) believe thatthe single serine 262 phosphorylation determines the binding of tau to microtubules.This abolishes tau–microtubule binding and greatly affects the ability of tau tostabilise microtubule dynamics.

Because calcineurin is a serine/threonine phosphatase, it may be expected tocontrol tau phosphorylation and in this way also regulate its interaction with micro-tubules. The dephosphorylation of the tau element of the cytoskeleton of PC12 cellsappears to be mediated by calcineurin (H.Q. Xie and Johnson, 1998). CyclosporinA consistently inhibits the phosphatase activity of calcineurin and inhibits axonalextension (Ferreira

et al.

1993). Also, tau protein is found in a hyperphosphorylatedform in calcineurin A-

α

knock-out (–/–) mice (Kayyali

et al.

1997). Furthermore,calcineurin strongly influences F-actin stability in cultured hippocampal neurones.The exposure of these cells to NMDA produces a rapid loss of dendritic spinestogether with a loss of actin filaments. This is prevented by actin-stabilising agentsand calcineurin inhibitors (Halpain

et al. 1998).

CALCINEURIN IN ALZHEIMER’S DISEASE

A characteristic feature of Alzheimer’s disease is the formation of neurofibrillarytangles (NFTs). NFTs are intraneuronal and consist mainly of PHF. It has beenpostulated that the assembly of PHF involves initially a process of antiparalleldimerisation of tau monomers. The dimers then form PHF (Mandelkow et al. 1996).The tau protein is a major component of PHF. Tau is phosphorylated to an abnormallyhigh degree in Alzheimer’s disease. The phosphorylation is three- to four-fold greaterin Alzheimer’s tau protein than in the native protein (Mandelkow et al. 1996;Grundke-Iqbal et al. 1986a, 1986b). This abnormally phosphorylated state isregarded as being responsible for the detachment of tau from microtubules and theconsequent microtubule instability. Iqbal and Grundke-Iqbal (1996) state that abnor-mal phosphorylation might precede the polymerisation of tau into PHF. Furthermore,they claim that dephosphorylation of abnormal tau can restore the ability of tau tointeract with and promote microtubule assembly. However, others believe that nosuch causal relationship exists between tau phosphorylation and the formation of


Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading 141

PHF (Goedert, 1996). Another factor that might be envisaged is genomic mutations.Mutations in the exons as well as introns of the tau gene have been reported in casesof familial dementia and Parkinson’s disease. Mutations in the exons seem to affectthe ability of tau to promote microtubule assembly (Hasegawa et al. 1998). None-theless, this abnormal phosphorylation state has been attributed to the generation ofan imbalance between protein kinases and phosphatases, engendered by a reductionin the levels of phosphatases (Iqbal and Grundke-Iqbal, 1996). More specifically,the high state of tau phosphorylation has been related to calcineurin dysfunction.This is suggested by the findings of Kayyali et al. (1997) that calcineurin A-α (–/–)knock-out mice show hyperphosphorylation of tau. The abnormal tau accumulates,and these mice also exhibit cytoskeletal changes that might affect neuronal function.

The neurodegenerative condition of amyotrophic lateral sclerosis (ALS) is char-acterised by a loss of motor neurones and neurones of the corticospinal tract. Thisneuronal loss has been attributed to a deregulation of NMDA receptors (Krieger et al.1993; Plaitakis, 1990). Glutamate-induced apoptosis and necrosis of cerebellar granulecells is prevented by the calcineurin inhibitors FK506 and cyclosporin (Ankarcronaet al. 1996). Abnormal levels of calcineurin have been reported to occur in ALS. Thisis believed to result in abnormal phosphorylation of NMDA receptors, which in turnhas been suggested to result in the pathogenesis of ALS (Wagey et al. 1997).

Alzheimer’s disease and Down’s syndrome also are characterised by extracellularoccurrence of amyloid deposits in the form of senile plaques and deposits associatedwith the microvasculature. The fibrillary amyloid is composed of the amyloid-β protein(ABP) with 39 to 43 amino acids. ABP is derived from the integral membrane glyco-protein, the β-amyloid precursor protein (βAPP) The latter shows an abnormally highaccumulation in the brain of people with Alzheimer’s disease. Calcineurin seems tobe involved in the formation of this peptide (Desdouits et al. 1996).

CALCINEURIN IN IMMUNOSUPPRESSION

Calcineurin plays a key role in immunosuppression. There has been an unequivocaldemonstration over the past few years that the drugs cyclosporin A, FK506, andrapamycin achieve immune suppression by inhibiting the function of calcineurin.That calcineurin was the target of some of these immunosuppressants was estab-lished some years ago. O’Keefe et al. (1992) transfected the catalytic calcineurinA into cells and showed that this raises the IC50 inhibitory concentration ofcyclosporin and FK506. Besides, Jurkat cells that overexpressed calcineurin wereresistant to the action of cyclosporin and FK506 and showed enhanced genetranscription that depended on NF-AT and related transcription factors (Clipstoneand Crabtree, 1992).

These immunosuppressant drugs interact with specific intracellular immunophi-lin receptors, which results in the formation of a drug–immunophilin complex thatis capable of blocking the function of its target, which, in this instance, is calcineurin(J. Liu et al. 1991; Ho et al. 1996). When T cells are activated by mitogenic signalsor by antigen binding, they proliferate and switch on the expression of cytokinegenes. Antigen binding to the TCR produces a cascade of signalling events (Figure19), which have been elucidated in some detail.


142


Calcineurin participates in this signalling cascade as a key enzyme. It is respon-sible for dephosphorylating the transcription factor called NF-AT and this factorthen translocates to the nucleus. NF-AT1 contains a regulatory domain, which liesN-terminal to its DNA-binding region, that binds to calcineurin (C. Luo

et al.

1996).In this way calcineurin seems to be targeted to NF-AT. The maintenance of NF-ATin the nucleus requires high and sustained levels, not transients, of calcium (Tim-merman

et al.

1996), probably reflecting its continual calcineurin-dependent activa-tion and translocation. The NF-AT proteins are expressed in many cell types of theimmune system, and these constitute a family of proteins that possess this conservedregulatory domain involved in their binding to calcineurin. NF-AT proteins resemblethe Rel family of proteins with respect to their ability to bind to the regulatoryelements of certain genes coding for cytokines (Rao

et al.

1997). In the nucleus,NF-AT forms a complex with the transcription complex AP1

(jun/fos).

The AP1/NF-AT complex then initiates the transcription of a number of genes, such as genescoding for the lymphokine, IL-2, IFN-

γ

, and TNF-

α

, among others. The inhibitionof calcineurin by the immunosuppressants inhibits the dephosphorylation of NF-ATand thereby inhibits its translocation to the nucleus and all the downstream events.Batiuk

et al.

(1997) have fully correlated the effects of cyclosporin A with the various

FIGURE 19

The involvement of calcineurin in a key position in the TCR-signalling cas-cade. See text for details.



143

events of the cascade, namely the dephosphorylation of NF-AT, its translocation,binding of the transcription complex to the DNA, and the eventual activation of genetranscription. They also demonstrated the relationship between the extent of cal-cineurin inhibition, the inhibition of lymphocyte proliferation, and the degree ofIFN-

γ

production

in vitro

.It would not be out of place to briefly mention here that calcineurin is involved

in the autoimmune condition SLE. SLE affects women of child-bearing age. Themenstrual cycle and pregnancy seem to affect disease activity (Lahita, 1993; Jungers

et al

. 1985; Mund

et al.

1963). Rider

et al.

(1998) have shown that T cells fromSLE patients respond to oestradiol by an up-regulation of calcineurin mRNA expres-sion, in a dose-dependent fashion. The effect seems to be specific for oestradiol,because progesterone and dexamethasone fail to elicit a similar response. Theincrease in mRNA expression corresponds with calcineurin phosphatase activity inoestradiol-treated T-cell extracts. As Rider

et al.

(1998) pointed out, the mechanismby which oestrogen influences calcineurin expression is unclear, because the latterhas no identifiable oestrogen response elements in its promoter region. However,they suggest that oestrogen could be exerting control at the level of calcineurintranscription. They cite the findings of H. Becker

et al.

(1995) that there is anincreased NF-AT binding to IL-2 promoter in SLE patients as compared with controlsubjects. As discussed earlier in this section and shown in Figure 19, calcineurin

FIGURE 20

The multiple functions of calcineurin, on the cell division cycle, in the mod-ulation of cell shape, adhesion and motility, and in the regulation of gene transcription. Alsoindicated are its participation in the functioning of immunosuppressants and its possibleassociation with abnormal phosphorylation of tau in Alzheimer’s disease.



plays a key role in the translocation of the NF-AT transcription factor to the nucleus.Therefore, it is conceivable that oestrogen influences the T-cell signal transductioncascade in SLE.

One is fully justified in concluding at the end of this chapter that calcineurin isa highly versatile phosphatase, and its functional repercussions, shown in Figure 20,are discernible in many facets of biological behaviour.


145

11

Centrins (Caltractins) and Their Biological Functions

Centrin (also known as caltractin) is an EF-hand calcium-binding protein of approx-imately 20 kDa molecular size. Errabolu

et al.

(1994) have cloned human centrincDNA, which contains an ORF of 516 bp and a predicted 172 amino acid residuesin the protein. Sequence analysis shows that human centrin is closely related tocentrins from plants, protozoa, algae, and

Xenopus

, and to CDC31 of

Saccharomyces

.The sequence also suggests the occurrence of four calcium-binding EF-hands. Thecentrin gene has been mapped to chromosome Xp28 (Chatterjee

et al.

1995). How-ever, the identification of several isoforms of centrin suggests the possibility of theexistence of a family of centrin genes.

Centrin is a component of spindle pole bodies, basal bodies, and centrosomes.Centrin and

γ

-tubulin are common components of these organelles. A major physi-ological function of centrin is the organisation of microtubules. The contractile fibresassociated with centromeres, for instance, show calcium mediated contraction, andfilament contraction occurs independently of ATP (Chiebel and Bornens, 1995).Using immunofluorescence techniques, researchers have shown that centrin localisesin the centrosome of interphase cells. The centrosome duplicates during this phaseof the cell cycle and centrin then redistributes between the spindle and the polarbodies. Quite obviously, therefore, it plays an important role in the separation ofcentrosomes during mitosis (Errabolu

et al.

1994).Other functions also have been attributed to centrin. It is associated in the yeast

with the duplication of the spindle pole body. Vallen

et al.

(1994) showed that theCDC31 of

Saccharomyces cerevisiae

, a protein that is closely related to centrin,interacts with the KAR1 gene product in the duplication of the microtubule organ-ising centre. Centrin interacts with KAR1 protein with great affinity. This interactionis regulated by changes in cellular calcium levels, which, in their turn, seem to beinfluenced by certain amino acid residues within the KAR protein (Geier

et al.

1996).In fact, antibodies to the centrin expressed by the flagellate

Naegleria gruberi

canrecognise CDC31 of yeast (Levy

et al.

1996). Centrin and related proteins areinvolved in flagellar severance and the retraction of ciliary apparatus. In

Chlamy-domonas reinhardtii

the excision of flagella occurs by a process of microtubuleseverance caused by contraction of centrin-containing fibres (Sanders and Salisbury,1994).

The molecular mechanisms involved in the function of centrins are still poorlyunderstood, but centrosome fractions prepared from haematopoietic cells have been


146


found to contain active Ca

2+

/CaM-dependent protein kinase II.

In vitro

, this enzymehas been shown to phosphorylate several centrosome-associated proteins (Pietromo-naco

et al.

1995). However, there is no evidence at present of centrin phosphorylationin relation to its function, apart from the observation that centrin is phosphorylatedduring prophase and metaphases of mitosis (Lutz

et al.

1995).The H6 gene of the plant

Atriplex nummularia

encodes a protein with a highdegree of sequence homology to algal centrin. Three transcripts of this gene, of 1.3,2.2, and 2.4 kb size, are expressed, and it would appear that these transcripts aredifferentially expressed in relation to mitotic activity and developmental and extra-cellular signals. The 1.3-kb transcript is induced by tactile and hyperthermic signals.Expression of the 2.2-kb transcript is related to the state of mitotic activity. The 2.4-kb transcript is expressed in response to heat shock (J.K. Zhu

et al.

1996). Centrinhas been found in the form of a complex with the high molecular weight HSP70and HSP90 in

Xenopus

oocytes that are arrested by exposure to cytostatic factors.When the oocytes are activated by electric shock or by means of ionophores, thecentrin–HSP70 complexes appear to undergo dissociation (Uzawa

et al.

1995). Thefunctional significance of the formation of these complexes is unclear, although thework of Uzawa

et al.

(1995) appears to suggest a relationship with activation anddivision of the oocytes. It ought to be pointed out that high molecular weight HSPsmay not be involved in the process of cell proliferation, but they may influence thesize of the steady-state cell population by protecting cells from apoptosis. In contrast,the lower molecular weight forms such as HSP28 are indeed so involved. HSP28inhibits cell proliferation. It may form complexes with EF-hand proteins such asS100A4 (see Albertazzi

et al.

1998a). HSP70 has been reported to show a cell cycle-related pattern of expression. Milarsky and Morimoto (1986) found that it isexpressed at the G

1

–S boundary. It can bind also to the cell cycle regulatory p53phosphoprotein (Pinhasi-Kimhi

et al.

1986; Finlay

et al.

1988). So, it is conceivablethat it is involved in the regulation of the cell cycle. On the other hand, HSPsparticipate prominently in protein folding and transport. Whether the formation ofa complex between HSP and centrin alters the molecular folding of the latter is notknown, but such a modulation of molecular shape has the potential to alter theaccessibility and binding of calcium to centrin and thus affect its function. Con-versely, complex formation with HSPs might be an aspect of centrin function. Theformation of the complex could itself depend upon the conformation adopted bycentrin upon calcium-binding.

Centrin also may be involved, together with other components of the cytoskeletalsystem, in maintaining cell polarity and motility (Lingle and Salisbury, 1997).Centrins have been found in nonmotile ciliary structures. Wolfrum (1995) has foundcentrin in photoreceptor cells. There is some preliminary evidence suggesting thatthe central helix of centrin may be important for its biological function. A study ofthe mutations of the

Chlamydomonas

vfl2 centrin gene has revealed that mutationof the glycine residue at 101 to lysine results in the loss of centrin function. However,this suppressor effect is reversed with additional mutations at amino acid residues96 or 104, and all three residues occur in the central helix of the protein (Taillonand Jarvik, 1995).


Centrins (Caltractins) and Their Biological Functions

147

Centrin is highly conserved in evolution. Bhattacharya

et al.

(1993) studied themolecular organisation of centrins of several lower organisms. From the phylogeneticpoint of view, the EF-hand domains of several centrins show congruence and mayhave arisen by gene duplication from an ancestral EF-hand domain. Furthermore,the domains of centrin are congruent with those of CaM. These observations providestrong indicators that centrin and related proteins may have evolved from a commonancestral form by means of gene duplication. A similar conclusion can be reachedfrom the pattern of expression of centrin and related proteins in association with aphylogenetically evolving contractile system (Levy

et al.

1996). In mammals, threecentrin genes have been described: HsCEN1, HsCEN2, and HsCEN3 (Middendorp

et al.

1997). These authors found that HsCEN1 was related to the yeast centrin-encoding gene CDC31 and further, that HsCEN2 and HsCEN3 possessed a greaterdegree of identity to algal centrin than did HsCEN1. This is interpreted as suggestinga divergence in the evolution of centrins.


149

12

Reticulocalbin Family of EF-Hand Proteins

There are two species of calcium binding proteins that are associated with theendoplasmic reticulum. One of them is a non-EF-hand protein, calreticulin. A familyof EF-hand proteins, with variable number of EF-hand domains, has been identifiedover the past few years (Table 9). Among them are reticulocalbin, calumenin, ERC-55 and ERC-fc, and Cab-45, and these may be described as belonging to thereticulocalbin family of CBPs. These proteins are expressed ubiquitously in a varietyof tissues. Human calumenin occurs at high levels in the heart, placenta, and skeletalmuscle, and at lower levels in the lung, kidney, and pancreas. Its levels are very lowin the brain and liver (Vorum

et al.

1998). Scherer

et al.

(1996) have isolated Cab-45 from mouse adipocytes.

TABLE 9The Reticulocalbin Family of EF-Hand Calcium Binding Proteins

Protein Nomenclature Molecular Features Ref.

Reticulocalbin 44-kDa protein; C-terminal HDEL sequence; 6 EF-hands; gene 13 kbp and 6 exons

Ozawa and Muramatsu (1993); Ozawa (1995a,b)

11p13 (WAGR locus) Kent

et al.

(1997)ERC-55 55-kDa protein; 6 EF-hands Weis

et al.

(1994)ERC-pf 40-kDa; 6 EF-hands La Greca

et al.

(1997)DNA-SCF

a

45-kDa cytoplasmic, 30-kDa nuclear M. Kobayashi

et al.

(1998)Calumenin (human) 315 amino acid residues; 7 EF-hands Vorum

et al.

(1998); Yabe

et al.

(1997)

Cab-45 (murine) 45 kDa; 6 EF-hands; Golgi location; no signal for membrane anchoring

Scherer

et al.

(1996)

CBP-50 50-kDa protein; 1 EF-hand motif (?) Hseu

et al.

(1997)Crocalbin 315 amino acid residues; 6 calcium-

binding domains; binds to phospholipase A2

Hseu

et al.

(1999)

a

DNA supercoiling factor, found to show homology to reticulocalbin.


150


MOLECULAR FEATURES OF RETICULOCALBIN HOMOLOGUES

Reticulocalbin and other members of the family possess a variable number of EF-hand domains. Also variable is their affinity for calcium ions. The EF-hand domainsof reticulocalbin bind calcium with low affinity (Vorum

et al.

1998). According toTachikui

et al.

(1997), only EF-hands 1, 4, 5, and 6 bind calcium. The EF-hands 2and 3 do not bind Ca

2+

. Calcium binding produces conformational changes inreticulocalbin.

The reticulocalbins are approximately 40 to 45 kDa in size and show a highdegree of homology among themselves, mostly with respect to sequences outsidethe EF-hand domains. As the name of the family denotes, these CBPs are associatedwith the ER. Cab-45 occurs mainly in the Golgi complex. These proteins contain amembrane localisation and anchoring sequence HDEL of 4 amino acid residues atthe C-terminal region (Ozawa and Muramatsu, 1993; Vorum

et al.

1998; Yabe

et al.

1997). The deletion of these residues from ERC-55 has been shown to result in lossof anchorage to the membrane and secretion of the protein (Weis

et al.

1994).Reticulocalbin also contains an N-terminal signal for its transfer to the lumen of theER (Ozawa and Muramatsu, 1993). Cab-45, which occurs in the lumen of the Golgibodies, does not possess the membrane-anchoring signal (Scherer

et al.

1996).The mouse reticulocalbin gene spans more than 13 kbp of the genome. It contains

six exons. The human reticulocalbin shows more than 95% amino acid sequencehomology with the murine protein, in both EF-hand and non-EF-hand regions ofthe protein. Both human and murine proteins contain six EF-hand motifs. Whereashuman reticulocalbin possesses the HDEL sequence, the mouse homologue containsthe KDEL sequence at the C-terminal end (Ozawa, 1995a, 1995b).

The human reticulocalbin gene has been mapped to chromosome 11p13 in theWAGR locus, between the Wilms’ tumour gene wt1 and the aniridia PAX gene (Kent

et al.

1997).

PUTATIVE FUNCTIONS OF RETICULOCALBIN AND ITS HOMOLOGUES

There has not been sustained effort to study the functions of the reticulocalbin familyof proteins. Three putative functions can be identified. Their association with ERand the evolutionarily conserved ER anchoring sequence suggests that one functionmay be protein trafficking. M. Kobayashi

et al.

(1998) have identified two cDNAclones of 1.6 and 1.8 kb, expressed in

Drosophila melanogaster

. The 1.8-kb mRNAcontained an open reading frame (ORF) that showed marked homology to mousereticulocalbin. The shorter mRNA encoded a protein in which an N-terminalsequence was deleted. Antibodies against the DNA supercoiling factor identified acytoplasmic 45-kDa protein and a 30-kDa protein in the nucleus. They also foundthat the 30-kDa nuclear protein interacted with topoisomerase II, which suggeststhat this protein is the functional form of the supercoiling factor. In early develop-mental stages, such as the blastoderm stage of embryonic development, the antibod-ies stain interphase nuclei. M. Kobayashi

et al.

(1998) have also described the pattern


Reticulocalbin Family of EF-Hand Proteins

151

of immunostaining of the polytene chromosomes of

Drosophila

, which are essen-tially interphase chromosomes. The polytene chromosomes have characteristic con-densed bands harbouring specific genes. These form puffs when the genes aretranscriptionally active. M. Kobayashi

et al.

(1998) found that immunostaining forthe supercoiling factor was associated with naturally occurring puffs or thoseinduced by ecdysone. These data seem to associate the supercoiling factor with geneexpression.

There is a further suggestion that reticulocalbin may be associated with theinvasive behaviour of tumours. It is reported to be overexpressed in breast cancercell lines with high invasive ability, but not in cell lines that are weakly invasive(Z.D. Liu

et al.

1997). Although these authors have carried out

in vitro

invasionassays in parallel, it is difficult to tie the invasive behaviour specifically to theexpression of reticulocalbin. The MBA and MCF7 breast cancer cell lines that theyhave used are known to differ markedly in the expression of S100A4, growth factorreceptors, and plasminogen activator, among others, which are known to affect theinvasive ability of these cell lines. Perhaps some gene transfer experiments arewarranted before any evidence can be regarded as conclusive. Even then, concep-tually, it is difficult to visualise a mode of function at the present stage in ourknowledge of the participation of reticulocalbin homologues in cell physiology.


153

13

Calpains in Normal and Aberrant Cell Physiology

THE CALPAIN FAMILY OF CALCIUM-BINDING PROTEINS

Calpains constitute a family of calcium-dependent proteases showing ubiquitousdistribution in a wide spectrum of animal species. They are intracellular cysteineproteases. They cleave substrate proteins in a manner that might constitute a mech-anism of regulation of the protein substrates (Croall and De Martino, 1991; K. Suzuki

et al.

1992). Therefore, they have been regarded here as regulatory CBPs.Mammalian calpains were known, on the basis of activity, as

µ

- and m-calpains(referred to herein with their gene designation as capn1 and capn2). These are theubiquitously occurring isoforms (Table 10). Other isoforms showing tissue-specificdistribution also are known, and these have been designated as capn3 and capn4.Capn5 and capn6 have been discovered recently and, despite their amino acidsequence homology with other calpains, appear to be distinctive in that they do notpossess the CaM-like calcium-binding domain (Dear

et al.

1997). The tra-3 gene,which encodes the calpain of nematode worms, shows the highest degree of homol-ogy to capn5 and capn6. Thus, in spite of the sequence similarities with otherisoforms, capn5, capn6, and tra-3 are not dependent on calcium for their activityand may indeed be devoid of all proteolytic activity (Dear

et al.

1997; Barnes andHodgkin, 1996). The human homologue of tra-3, known as the htra-3 gene, has beencloned. This encodes a protein that is highly homologous to the calpain 80-kDasubunit. Furthermore, like tra-3, the htra-3 peptide lacks the CaM-like calciumbinding motifs (Mugita

et al.

1997). It would seem, therefore, that calpains might have diverse functions that may

be related to the existence of some mechanism by which they might be activated.This is compatible with the discovery of a family of calpain activator proteins, whichnot only resemble one another but also possess chaperonin-like properties (Melloni

et al.

1998a, 1998b). The 30-kDa activator described by Melloni

et al.

(1998a) hasbeen reported to show a rigorous specificity for capn1 and to be totally ineffectiveon capn2. This activator protein binds to the 80-kDa subunit of calpain and promotesits dissociation from the smaller subunit. The process of activation then takes theform of autoproteolysis, leading to the formation of two forms of calpain of 78 and75 kDa molecular size. The activator described by Melloni

et al.

(1998a, 1998b)bears a high degree of sequence homology with another calpain activator that has


154


been isolated from goat liver and called UK114 (Ceciliani

et al.

1996a, 1996b).UK114 has been regarded as a cancer marker. However, whether an inappropriateactivation of calpains can lead to transformation and acquisition of properties thatcharacterise cancer cells would be a fruitful avenue of approach. The possibility thatcalpains might, however indirectly, control gene transcription certainly makes anexploration of this avenue highly worthwhile.

MOLECULAR ORGANISATION OF CALPAINS

The calpain molecule consists of two subunits: an 80-kDa and a smaller 30-kDasubunit. The gene encoding the large subunit of rat capn2 contains 22 exons, ofwhich exons 3 to 21 encompass 33 kb of the rat genome. The corresponding cDNAof 3.2-kb size codes for a protein consisting of 700 amino acids. This protein showsa high degree (93%) of sequence homology with human capn2 and 61% homologywith human capn1. The cDNA, when tagged to an expression vector, generated an80-kDa protein that was found to be identical to human capn2 (De Luca

et al.

1993).The 80-kDa subunit has four domains. Domain II is the cysteine protease domain

and domain IV is the Ca

2+

-binding domain. The smaller subunit has two domains:

TABLE 10Calpain Family Isoforms

Gene/Protein Designation Chromosome Location Characteristic Features

capn1 (m-calpain) Chromosome 11q13

a

Active at micromolar concentrationcapn2 (m-calpain) Chromosome 1 Active at millimolar concentrationcapn3 (p94; nCL-1) Chromosome

15q15.1–q21.1

b

Skeletal muscle specific

Lp82 (splice variant of capn3)

Rat lens

capn4 (nCL-2) Stomach specificnCL-4 Stomach, intestine, but not uteruscapn5 Chromosome

11q13.5–q14

c

capn6 X chromosome Expressed only in the placentacapn8 Murine calpain, expressed in brain,

kidney, and the digestive tracttra-3


sex determination

htra-3 (human homologue of tra-3) 11q14

d

High expression in colon, small intestine, and testis tissues

Note:

Chromosomal location data from (a) Pang

et al.

(1997); (b) Richard

et al.

(1995); (c)Ollendorf

et al.

(1992); (d) Mugita

et al.

(1997).

Source:

Data collated from Ohno

et al.

(1990); Richard

et al.

(1995); Schuler

et al.

(1996); Matena

et al.

(1998); H.J. Lee

et al.

(1998); Ma

et al.

(1999). Isolation of the new capn8 was recentlyreported by Braun

et al.

(1999b).



155

domain V, the N-terminal glycine-clustering hydrophobic domain, and the Ca

2+

-binding domain VI (Nishimura and Goll, 1991; Crawford

et al.

1993; Minami

et al.

1988). The calcium-binding domains (IV and VI of the two subunits) possesssequence similarities to CaM (Ohno

et al.

1984). These domains contain four EF-hands each (EF-hands 2–5). Two additional EF-hands occur in the 80-kDa subunit,one at the junction between domains II and III and the second one at a position N-terminal to EF-hand 2 (Theopold

et al.

1995; Andresen

et al.

1991). The associationof the subunits occurs through domains IV and VI, and EF-hand 5 may play animportant part in maintaining the dimeric organisation (Y. Minami

et al.

1988;Kretsinger, 1997).

REGULATION OF PHYSIOLOGICAL EVENTS BY PROTEOLYTIC FUNCTION

Calpains are not wide-spectrum proteases. Importantly, the limited number of sub-strates that they cleave are involved with the activation of specific cellular systems.Notable among their substrates are PKC-

γ

, the actin-binding proteins,

α

-fodrin andthe homologous

α

- and

β

-spectrin, and growth factor receptor proteins (Lofvenbergand Backman, 1999; Saido

et al.

1994; Croall and De Martino, 1991; Martin

et al.

1995). It is to be expected that calpains would be involved in physiological eventsmerely by virtue of their proteolytic activity. Several such events can be cited. Oneof these is keratinocyte differentiation. Calpains are actively involved in the pro-teolytic cleavage of profilaggrin into filaggrin in the process of keratin filamentaggregation (Figure 25). They also may regulate the levels of MAPs during neuronaldifferentiation. During early brain development MAPs occur as MAP-1B and MAP-2. MAP-1B occurs as two isoforms differing in the level of phosphorylation, andMAP-2 occurs as two isoforms with different molecular weights. The isoforms ofMAP-1B as well as MAP-2 show equal sensitivity to calpain, but overall, MAP-2is more susceptible to the proteolytic action of calpain than MAP-1B (I. Fischer

etal.

1991). Calpain has been shown to be able to induce apoptotic cell death, andthis is associated with the cleavage of substrate proteins such as

α

-fodrin (spectrin),Ca/CaM-dependent protein kinase IV, etc.

Neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited conditioncharacterised by a disposition to develop certain intracranial tumours such as vesti-bular schwannoma and meningioma. The NF2 gene codes for a protein called merlin,also known as schwannomin, and it is believed to function as a tumour suppressor.Mutations of merlin have been found in a significant proportion of NF2-associatedtumours and to a lesser extent in sporadic schwannomas and meningiomas. However,NF2 tumours that show no mutations of the NF2 gene also have been found. Kimura

et al.

(1998), therefore, examined the possibility that the suppressor protein may beinactivated by mechanisms other than mutation. These authors found that a markedactivation of calpain occurs in sporadic meningiomas. Merlin is cleaved by calpain,and inactivation of calpain restores merlin in meningioma cells in culture. It wouldappear, therefore, that proteolytic control by calpain might be a mechanism by whichthe tumour suppressor function of merlin is regulated. Much of this evidence is


156


correlative, but admittedly proteolytic pathways are involved in the regulation ofexpression of several cellular regulatory proteins such as p53 (see below) and themdm2 protein (L.H. Chen

et al.

1997), which negatively regulates p53 function.Indeed, the interaction of mdm2 with p53 reduces p53 protein levels by targeting itto ubiquitin-dependent degradation (Kabbutat

et al.

1997).The phosphoprotein p53 is intricately involved in the checkpoint control at the

G

1

-M as well as at the G

2

-M transition of cells. p53 has a short half-life. Itsdegradation has been attributed to ubiquitination as well as to proteolysis by calpain.The PEST (Pro, Asp/Glu, Ser, and Thr) motif is generally regarded as a signal inthe substrate that is required for its recognition by an intracellular protease. p53contains the PEST sequence, which is required for proteolysis by calpain. Ubiquit-ination of certain substrates, e.g., STE3 receptor of yeast, has been attributed to aPEST-like sequence (Roth

et al.

1998), although we do not know if this is the casewith p53 ubiquitination. Mutated p53, which has an increased half-life, does notcontain these sequences. However, in some instances, p53 may be degraded bycalpain in the absence of the PEST sequences. Wild-type p53 is also stabilised whencells expressing it are treated with inhibitors of calpain. However, the presence ofthe PEST motif may not be an absolute requirement for calpain function (Carillo

etal.

1996; Van Antwerp and Verma, 1996). The human papilloma virus (HPV) E6protein promotes p53 degradation through the ubiquitin-dependent pathway, and thisis unaffected by inhibitors of calpain. Overall this suggests that the stability of p53might be controlled by the proteolytic action of calpain (Kabbutat and Vousden,1997). Similarly, cyclin D1, another regulator of cell cycle progression, has beenreported to be a target of calpain proteolysis. Choi

et al.

(1997) showed that cyclinD1 is rapidly proteolysed in NIH3T3 cells when deprived of serum, but calpaininhibitors reversed this. These inhibitors also raise the half-life of cyclin D1.

Proteolytic cleavage of the cytoplasmic domain of the cytokine receptor

γ

-chain(

γ

-c) by calpain may represent an important regulatory or control event in

γ

-c-mediated signalling. Noguchi

et al.

(1997) demonstrated that the small subunit ofcalpain cleaves

γ

-c, which contains the hydrophilic amino acid PEST sequence, butnot when the PEST sequence is mutated. Calpain inhibitors inhibited

γ

-c cleavagein TCR-stimulated murine thymocytes. Furthermore, in these cells, anti-CD3 cleaved

γ

-c, but calpain inhibitors inhibited this. Calpain inhibitors enhance the proliferativeeffect of anti-CD3 antibodies, and this is effectively prevented by

γ

-c antibodies.Cytokines and growth factors transduce their signals by using the Janus tyrosinekinase (Jak) and STAT (signal transducer and activator of transcription factors)pathway (O’Shea, 1997; Darnell, 1997). The binding of these ligands to the appro-priate receptors results in the activation of the Jak tyrosine kinase and the activationof latent transcription factors called STAT proteins that occur in the cytoplasm. Infact, STAT-3 appears to regulate G

1

–S transition of cells in response to cytokinestimulation (Fukada

et al.

1998). Calpain inhibitors have been shown to influencecytokine-mediated cell proliferation and STAT protein synthesis (Noguchi

et al.

1997). It would appear, therefore, that the proteolytic function of calpain couldconstitute an intricate part of signal transduction pathways.

These are prime examples of specific modes of regulatory function performedby calpain, in which a specific target is hydrolysed by calpain to bring about the



157

regulation of a particular physiological event. In addition, one should bear in mindthe finding that calpain can degrade transcription factors such as AP1. Hirai

et al.

(1991) reported that the c-

jun

and c-

fos

gene products, which form the AP1 tran-scription factor, contain the PEST sequence that is the target sequence of calpainaction. Calpain inhibitors increase the expression of AP1 (Zhu

et al.

1995). There-fore, one could expect calpain would have a generalised effect on the transcriptionof a variety of genes, merely by virtue of its ability to regulate the function of thetranscription factor itself.

INVOLVEMENT OF CALPAINS IN DEVELOPMENT AND DIFFERENTIATION

There have been no significant investigations into the involvement of calpains indevelopmental processes. However, there is some evidence that could be construedas providing a partial burden of proof. For instance, the tra-3 gene has beenimplicated in the process of sex determination during early development of thenematode

C. elegans

. tra-3 lacks the CaM-like calcium-binding sites (of domainIV), but EF-hand 6 is conserved. Its involvement in sex determination has beensuggested to be a consequence of potentiating the function of another gene, e.g.,tra-2 (Barnes and Hodgkin, 1996). The conservation of EF-hand 6 in tra-3 mightsuggest that the latter is required in calcium signalling in the sex determinationcascade. Calpain homologues of

Drosophila

may be associated with developmentalabnormalities. Mutations of the

sol

gene of

Drosophila

result in neuronal defectsthat cause behavioural abnormalities. Delaney

et al.

(1991) cloned two alterna-tively spliced transcripts of the

sol

locus. The predicted sequences of the proteinsencoded by them showed similarities to different regions of calpain. The carboxylicregion of the larger protein showed similarities to the catalytic domain of calpain,and the N-terminal region contained several zinc finger-like repeats. The smallerpredicted protein did not contain the calpain catalytic domain and had only twozinc finger-like repeats.

Calpains also have been found in the human brain. Their possible role in dif-ferentiation is illustrated by changes in calpain expression in the SH-SY5y neuro-blastoma cells induced to differentiate by retinoic acid (RA) treatment. RA inducesdifferentiation and neurite outgrowth in these cells. After 3 days of RA treatment,the levels of activated and precursor forms of calpain have been found to increaseby >50% and 26%, respectively, in the particulate and cytosolic fractions of thecells. However, the levels of calpastatin, an endogenous inhibitor of calpain, wereunchanged. Thus the process of neurite extension seems to be associated with a netactivation of calpain (Grynspan

et al.

1997). The calpain–calpastatin system seemsto be regulated during processes of proliferation and differentiation of osteoprogen-itor cells in response to the bone morphogenetic protein (Murray

et al.

1997).Further evidence has been derived from the pattern in the expression of capn3

gene that is discernible during early foetal development. This could be interpretedas an indicator of its association with, if not participation in, morphogenesis. capn3expression is detected only in skeletal muscle at this time. At an earlier stage, capn3


158


expression is found in the heart and it disappears subsequently. Variants of capn3generated by alternative splicing are also detected in smooth muscle (Fougerousse

et al.

1998). The requirement of both calpain and the proteasome proteolytic systemsin the differentiation of myoblasts is exemplified by the work of Ueda

et al.

(1998).L8 myoblast cells grown in the absence of mitogen show enhanced levels of creatinekinase together with the differentiation of myotubes. However, when calpain inhib-itor and the proteasome inhibitor lactacystin are added to these cultures, creatinekinase levels are markedly reduced together with inhibition of myotube differenti-ation. Although calpains have been implicated in neurodegenerative diseases, adiametrically opposite view is held by some that calpains may contribute to processessuch as the remodelling of neuronal dendritic structures after neuronal injury. Inmurine cortical cultures subjected to dendritic injury, calpain did not seem to aug-ment the injury. Quite the contrary, calpain appeared to assist dendritic recovery(Faddis

et al.

1997). Similarly, the calpain inhibitor MDL28170 promotes neuronalrecovery from injury by moderate hypoxia (Z.F. Chen

et al.

1997).There are now clear indications that capn1 may be expressed in the early stages

of the development of the skin (Michel

et al.

1998, 1999). Michel

et al.

(1999) haveprovided definitive evidence for this. capn1 appears at day 54 of gestation of the humanfoetus, in the basal layer and in the periderm of the developing dermis. By day 125,capn1 is found in the granular layer. A possible link-up with differentiation is indicatedby a significant reduction of capn1 expression in biopsies obtained from harlequinichthyosis, a condition known to result from abnormal terminal differentiation of theskin. The changes in capn1 expression were apparently specific for harlequin ichthyosisand were not encountered in other skin disorders. These findings are compatible withthe known ability and involvement of capn1 to influence the proteolytic cleavage ofprofilaggrin to monomeric filaggrin during terminal differentiation.

It follows from the association between calpain and changes in cellular mor-phology that the cytoskeleton could be an important target of calpains. Emori andSaigo (1994) studied the expression of calpain in developing

Drosophila

embryos.In the early stages of cleavage of the fertilised eggs, calpain was found in thepresumptive cleavage furrows and was co-localised with actin caps. As stated in anearlier section, calpains might regulate the levels of MAP components of the neuronalcytoskeleton (I. Fischer

et al.

1991). Chakrabarti

et al.

(1993) studied the expressionof calpains during brain development in rats, especially in relation to myelin for-mation. They found low capn2 levels during days 1 to 7 (postparturition), but thesereached a peak between days 16 and 30. In rats that were more than 30 days old,roughly half of the calpain activity in the brain was found in the myelin component,whereas in 1 to 10-day old rats the majority of capn2 activity was cytosolic. Fur-thermore, this contrasted starkly with the pattern of expression of capn1, suggestingthat capn2 is associated with the formation of the myelin sheath.

CALPAINS IN CELL PROLIFERATION AND APOPTOSIS

The involvement of calpain in the cell cycle traverse by proteolytic degradation ofp53, which controls the cell cycle transition checkpoints, has been alluded to earlier.It is to be expected, therefore, that calpain will powerfully influence cell proliferation



159

and growth. Calpeptin, an inhibitor of calpain has been found to inhibit the growthof the breast cancer cell lines MCF7, T47D, and ZR75 (E. Shiba

et al.

1996). Thesecell lines are oestrogen receptor (ER) positive, but in a subsequent report it has beendemonstrated that the calpeptin-mediated inhibition of growth is not due to a sup-pression of the hydrolysis of ER by calpain. This could be due to one or more of avariety of mechanisms that interfere with cell cycle control. There is a significantinverse relationship between the expression of ER and EGFr in some of these celllines. It would be interesting to see if the transduction of EGF signal

via

thecytoskeletal machinery is affected in any way by calpeptin. Aside from that, it ispossible that calpeptin influences p53 stability by inhibiting calpain.

Apoptosis or programmed cell death is a natural physiological phenomenon.Cells undergoing apoptosis display distinctive morphological changes, such as con-densation of nuclear chromatin and shrinkage of the cytoplasm. The cells break upinto membrane-bound apoptotic bodies. The nuclear DNA is fragmented byCa

2+

/Mg

2+

-dependent endonucleases. DNAse I and DNAse II have been regarded asputative candidates in DNA degradation, but recently Enari

et al.

(1998) haveidentified a caspase-activated DNA nuclease that is involved in the DNA degradationthat occurs during apoptosis. The fragmentation of the DNA produces a characteristicladder pattern of 180 to 200 bp of oligomers. Perturbations of the intracellularcalcium levels are an important feature of apoptosis. High intracellular Ca

2+

levelsproduced by calcium influx or its release from intracellular stores can lead toapoptosis (Orrenius

et al.

1996). However, there is conflicting evidence about theinduction of apoptosis by calcium chelators and calcium ionophores. Nevertheless,the bcl2 family of genes, which is composed of inducers as well as inhibitors ofapoptosis is known to regulate intracellular calcium (see Sherbet and Lakshmi, 1997bfor references). Calcium signalling is known to activate a number of proteases.Among the notable ones are calpains and caspase. Both proteases have been impli-cated in apoptosis.

Calpains often have been attributed with the ability to induce apoptotic cell death(Figure 21). They are said to be actively associated with T-cell activation andapoptosis (Squier

et al.

1994; Sarin

et al.

1995; S.J. Martin and Green, 1995). Inmature T lymphocytes the induction of apoptosis by TCR is protease dependent(Sarin

et al.

1995). The binding of the appropriate ligand of the TNF family to theFas receptor initiates the activation of caspases and the down-stream protease cascadethat leads ultimately to DNA degradation (see below). Dexamethasone is known toinduce apoptosis of thymocytes, accompanied by Ca

2+

-dependent proteolytic activity.This is also accompanied by the autoproteolysis of the capn1 proenzyme, whichsuggests that calpain activation is taking place. Calpain inhibitors block apoptoticdeath (Squier

et al.

1994). However, inhibitors of calpains have also been found toinduce apoptosis. W. Zhu

et al.

(1995) found that two calpain inhibitors causedapoptosis of human prostate adenocarcinoma cells. However, in L1210 leukaemiacells, inhibition of calpain did not induce apoptosis (Wojcik

et al.

1997). Apoptoticdeath can be induced in rat hippocampal pyramidal neurones in culture by exposingthem to ABP

and staurosporine. This can be inhibited by the calpain inhibitorMDL28170 (Jordan

et al.

1997). There is a preliminary report that prevention ofcalpain activation produces resistance to necrosis in hepatocellular carcinoma cells


160


(A.S. Arora

et al.

1995). However, necrosis and apoptosis are two distinct processes,and it would not be appropriate to discuss, in the same breath, how they are affectedby calpain.

A possible pathway by which calpain could influence apoptosis has been sug-gested by Meredith

et al.

(1998) who showed that during apoptosis of humanumbilical vein endothelial cells, the cytoplasmic tail of integrin

β

3

undergoes limitedproteolysis. They showed not only that calpain is activated during apoptosis but alsothat calpain inhibitors prevent the proteolysis of the cytoplasmic domain of theintegrin. Needless to say, more work needs to be done in order to establish thepathways of apoptosis that are influenced by calpain.

CALPAINS IN CELL SPREADING AND MIGRATION

As stated often in these pages, the cytoskeletal dynamics are of overriding importancein signal transduction, cell morphology, and cell adhesion and locomotion. Calpain,by virtue of its proteolytic action, is bound to affect the integrity of the cytoskeletonand the adhesive and migratory properties of cells. Cell spreading is inhibited bythe calpain inhibitors, calpeptin and MDL28170 (K.A. Potter

et al.

1998). K.A.Potter

et al.

(1998) cloned a variant NIH3T3 cell line that overexpressed (two- toeight-fold) the calpain inhibitor, calpastatin. These cells markedly differed from the

FIGURE 21

Involvement of the calcium-activated enzymes calpains and caspases in theinduction of apoptosis. Raising intracellular calcium levels leads to activation of theseenzymes and to apoptosis. In many instances it has been shown that when their activity isinhibited apoptosis is also suppressed. The Bcl-2 family has apoptosis-inducing genes as wellas genes that inhibit apoptosis. These regulate intracellular calcium levels and influenceapoptosis through the caspase activation mechanism. The

ced-9

gene of

C. elegans

encodesa protein that has apoptosis-inhibiting activity.


Calpains in Normal and Aberrant Cell Physiology 161

parent cell line with respect to their morphology, spreading ability, and cytoskeletalcharacteristics. The variant cells failed to extend lamellipodia and showed abnormalfilopodial extension and retraction. The calpain activity in these cells was found tobe substantially reduced. Calpain inhibitors have been found to increase cell adhe-siveness and reduce cell migration. A Chinese hamster ovary (CHO) cell line thatexpresses capn1 intrinsically at low levels has a low migratory ability (Huttenlocheret al. 1997). However, cell migration involves transient adhesion to and release fromthe substratum and these interactions require the linkage of the focal adhesionreceptors to the cytoskeleton. The linkage of the cytoplasmic tail of integrin receptorsis of crucial importance in the process of cell adhesion to the substratum. Calpainand similar proteins would affect it by cleaving the cytoplasmic domain of thereceptor and may be regarded to be negatively regulating adhesion (Meredith et al.1998). Palacek et al. (1998) have described migration as a function of cyclic mem-brane protrusion and adhesion at the leading edge and cytoskeletal contraction anddetachment at the rear edge. They go on to demonstrate that calpain is involved indissociation of the integrin receptor linkage with the cytoskeleton. These authorsargue that an inhibition of integrin release by calpain decreases the speed of cellmotility, and, inter alia, this may be due to the increased density of adhesive bondsor the strength of adhesion. Huttenlocher et al. (1997) have stated categorically thatinhibitors of calpain increase cell adhesiveness and decrease the rate of detachment,whereas Meredith et al. (1998) regard calpains as negative regulators of adhesion.Because the adhesion of the leading edge and the retraction of the rear edge willnecessitate a differential regulation of calpain, it would be interesting to see if theendogenous calpain inhibitor, calpastatin, might come into the adhesion dynamics.Calpastatin is itself subject to proteolysis by calpain (Nagao et al. 1994). Forinstance, very little is known about the intracellular localisation of calpastatin inrelation to the direction or speed of cell locomotion. Furthermore, as indicated bythe effects of calpain on integrin-mediated signal transduction, other components,such as talin, that are involved in the linkage of cytoplasmic domain of the integrinreceptors with the cytoskeleton could conceivably form a part of the total picture.

CALPAINS IN INTEGRIN-MEDIATED CELL ADHESION AND SIGNAL TRANSDUCTION

The inhibition of integrin function is believed to be responsible for the down-regulation of these adhesive and migratory abilities. Integrins are transmembraneproteins constituting focal adhesion plaques. Talin is a protein that links integrinsto the cytoskeletal structures. Interestingly, talin has calpain susceptible sites. There-fore, calpain could affect the processes of cell adhesion and signal transduction,which are mediated by integrins. Using antibodies that recognise the calpain-sus-ceptible sites of talin, Inomata et al. (1996) have demonstrated the involvement ofcalpain in integrin-mediated signal transduction. Calpain is able to cleave the cyto-plasmic domain of β3 integrin (Du et al. 1995) and this could interrupt the signallingpathway. The focal adhesion kinase (pp125FAK) is a non-RTK associated with inte-grin-mediated signal transduction. Cooray et al. (1996) have argued that calpain



may have a role of terminating signal transduction in human platelets, by modifyingpp125FAK by proteolysis and reducing its kinase activity as well as its subcellularlocation. Calpain indeed affects several events occurring after platelet aggregation,e.g., the attachment of the integrin tail to the cytoskeleton, as well as tyrosinephosphorylation of cytoskeletal protein and fibrin clot retraction (Schoenwaelder etal. 1997). Paxillin is another protein associated with focal adhesion plaques that isdegraded by calpain (R. Yamaguchi et al. 1994).

CALPAINS IN CANCER GROWTH AND PROGRESSION

Calpain is significantly involved, as discussed above, in a number of physiologicalprocesses that are integral features of the development and progression of cancer.We have seen that the suppressor phosphoprotein p53, which is closely linked withthe control of cell cycle progression, is degraded by calpain. Mutations of p53 geneare a common event in tumorigenesis, and these lead to the abrogation of normalcheckpoint control function exerted by wild-type p53. We have noted also thatcalpain is able to regulate the levels of cyclin D. Deregulation of the cell cycle usingeither target will lead to the deregulation of cell proliferation. Besides, one can alsoenvisage a deregulation of the cell cycle under conditions where calpain is overex-pressed per se or calpain activators are inappropriately expressed. Besides, there issome evidence that the calpains may influence apoptosis in certain cell lines. Thisneeds to be established beyond reasonable doubt, because there are reports to theeffect that both calpain and its inhibitors are capable of inducing apoptosis.

Tumour growth could be seen as a net outcome of these opposing factors ofproliferation and apoptosis. A dynamic equilibrium between these two can result ingrowth stasis. Such a state of equilibrium also can apply to the growth of metastasesand can lead to a state of dormancy of metastatic deposits.

As shown in Figure 22, calpains and caspases may have positive or negativeregulatory effects on tumour growth and metastatic spread. Although the expressionof calpain in cancers has not been the subject of much investigation, there is someinformation on the association of calpain inhibitors with cancers. Squamous cellcarcinomas of the lung have been reported to express a protein — squamous celllung carcinoma antigen (SCCA) — that is able to inhibit capn1 (Kato, 1996).However, because SCCA inhibits other proteinases such as cathepsin L, it is difficultto assess the individual merit of calpain inhibition in squamous cell lung carcinoma.It should be borne in mind that the cathepsin family proteinases play an importantrole in tumour development and progression to the metastatic state, although, admit-tedly, cathepsin L has not been found to be as crucially important as other membersof the family (see Sherbet and Lakshmi, 1997b).

Braun et al. (1999a) have reported an enhanced expression of capn1 mRNA inclear cell renal carcinoma. The level of expression related closely with the presenceof metastatic tumours in regional lymph nodes. Tumours with metastasis expressedcapn1 mRNA at a higher level than tumours that had not spread to regional lymphnodes.

Ceciliani et al. (1996a) isolated three low molecular weight proteins — UK101,114, and 150 — from goat liver. These proteins are membrane associated. Of these,



UK150 has been characterised to be a glycoprotein. UK114 has been suggested tobe a marker for cancer (Ceciliani et al. 1996b). UK114 is a 14-kDa protein madeup of 137 amino acid residues and shows sequence homology with the calpain-activating factor isolated from rat and bovine brain (Melloni et al. 1998a, 1998b).Furthermore, the goat liver UK114 also possesses calpain-activating properties (Mel-loni et al. 1998a).

Bartorelli et al. (1996) demonstrated that administration of UK101 to patientswith breast and colon carcinomas elicited significant immune responses from thepatients. Antibody titres against UK114 and 150 rose by 63 and 87% in breast andcolon cancer patients, respectively. Although there has been very little backgrounddata on the levels of UK114 and 150 in patients with cancer, UK101 therapy appearsto have been reasonably successful (Mor et al. 1997). It was administered to 217patients with metastatic colon cancer. Twenty-five patients in this trial showedreduction in metastatic mass, and 40% of the patients showed static disease. Patientsurvival correlated with both the baseline Karnofsky index score and anti-UK114titre. Bussolati et al. (1997) have suggested that the antitumour effects are due tothe cytolytic action of anti-UK114 antibodies. The latter were found to be cytolyticin vitro and were able to inhibit the growth of human tumours as xenografts innude mice.

CALPAINS IN MYELODEGENERATIVE DISEASES

During early stages of brain development, calpains show a pattern of expression thatis compatible with the view that they are mainly associated with the formation ofthe myelin sheath. Subsequent to this, they are found in the cytosol rather than inthe myelin sheath. Myelin basic protein (MBP) and myelin-associated glycoprotein

FIGURE 22 Putative pathways of the involvement of calpain and caspase in the growthof the primary tumour and its metastases. The basic position is that these proteases can alterthe steady state of population growth in both primary and secondary tumours. Caspase, butnot calpain, has been credited with the ability to induce apoptosis of endothelial cells thatcan assist the entry of tumour cells into the vasculature. Endogenous activators of the enzymeshave been suggested as a possible mode of immunising the host that can conceivably lead toloss of enzyme activation and potential loss of growth.

Tumour growthMetastases

Apoptosis Deregulation of Endothelial cell proliferation cell apoptosis

Calpain/Caspase

Antigenicresponse in host

Endogenous activators



are substrates of calpain. Banik et al. (1994) have shown that component I of humanMBP is more susceptible to proteolysis by calpain than components II and III. Theyhave identified two major and several minor cleavage sites in MBP. Because MBPis degraded in demyelinating diseases such as multiple sclerosis (MS), the potentialrole of calpains in the pathogenesis of MS has been studied in several laboratories.

The presence of calpains 1 and 2 in myelin sheath was known for some time.High levels of capn1 and capn2 activity in myelin and premyelin were found tocorrelate with delayed myelination and high myelin turnover in the demyelinatingparalytic tremor (PT)-mutant rabbit (Domanskajanik et al. 1992). Calpain activityhas been reported to increase in MS tissue and cerebrospinal fluid. Macrophages,lymphocytes, and reactive glial cells are the sources of the enhanced calpain activity.Calpains are found also in myelin-forming oligodendrocytes and Schwann cells(Banik et al. 1994). Both capn1 and capn2 occur in human lymphoid cell lines.Lymphocytic cell lines predominantly expressed capn1, but in monocytic cells thecapn2 tended to be the major isoform (Deshpande et al. 1993). Activation of lym-phoid cells with PMA and the calcium ionophore A23187 enhances the expressionof both calpain protein and the corresponding mRNA (Deshpande et al. 1995a).Deshpande et al. (1995b) demonstrated further that activation of calpains was accom-panied by the degradation of MBP and rat CNS myelin in vitro. A large proportion(60 to 80%) of the degradative activity was attributable to calpain, together withcontributions from other proteases. It is conceivable that this degradation results inthe exposure of antigenic epitopes. On the other hand, calpains occur in normalmyelin and are associated with myelin formation during developmental stages.Therefore, calpains could be a normal constituent of myelin and may be involvedin turnover of myelin proteins in the normal course of physiological events (Z.H.Li and Banik, 1995). Possibly, activated infiltrating lymphoid cells generate a higherlevel of myelin breakdown and greater quantities of antigenic products. The statusof the calpain–calpastatin system has been studied in experimental allergic enceph-alomyelitis (EAE), which is an animal model of MS, mainly in the laboratory ofShields and Banik. In Lewis rats with EAE, the optic nerves have been reported toshow a marked increase in calpain content, although RT-PCR did not reveal anychanges in calpain messenger RNA. Calpain-specific degradation of fodrin increasedby 46% and the myelin-associated glycoprotein decreased by 25%. The endogenousinhibitor of calpain, i.e., calpastatin, was unaltered (Shields and Banik, 1998a,b).Human MS plaques reportedly show very similar alterations when compared withwhite matter from normal subjects (Shields et al. 1999a). Interestingly, alterationsin calpain expression occur in peripheral lymphoid organs before the onset ofsymptomatic EAE (Shields et al. 1999b). MS plaques are characterised by reactivegliosis, infiltration of inflammatory cell, and a focal destruction of myelin andoligodendrocytes. An enhanced expression of calpain is said to occur in glial andinflammatory cells (Shields et al. 1998a, 1998b). These observations putativelyimplicate calpain in the autoimmune-mediated demyelination process that occurs inEAE and MS.

The regulation of the activity of this normal calpain component deserves seriousstudy. Growth factors such as NGF, PDGF, and acidic as well as basic FGF exertdifferent effects on capn1 and capn2 expression in transfected Schwann cells



(Neuberger et al. 1997). Neuberger et al. (1997) reported that cAMP and NGFinhibited both capn1 and capn2 activity. Both aFGF and bFGF enhanced capn1activity by 37% and capn2 by 58%. In contrast, PDGF down-regulated both isoforms.Interestingly, the enhanced calpain activity following FGF treatment was associatedwith markedly reduced levels of calpastatin activity. In comparison, in cells exposedto cAMP and NGF, calpastatin levels were nearly comparable with those of controlcells. These observations suggest that growth factors strongly affect the dynamicequilibrium between calpains and their endogenous inhibitor. The experiments ofNeuberger et al. (1997) also indicate the possibility of their intracellular transloca-tion. The expression of calpain isoforms as well as calpastatin isoforms has beenstudied in the EAE model (Shields and Banik, 1998). The transcription of neitherthe calpain isoforms nor calpastatin isoforms showed an up-regulation in EAE, butthe expression of calpain protein increased more than fourfold. Calpastatin transla-tion was also increased in EAE. This suggests another putative mechanism by whichthe calpain–calpastatin dynamics could be altered in myelodegenerative conditions.

CALPAINS IN MUSCULAR DYSTROPHY

Several forms of muscle myopathy are known. The major conditions are Duchennemuscular dystrophy (DMD), Becker muscular dystrophy (BMD), and limb girdlemuscular dystrophy (LGMD). LGMD is recognised to be of two types: the autoso-mal-dominant type 1 (subtypes 1A–1D) and the recessive type 2, with severalsubtypes. Calpain has been implicated in the pathogenesis of LGMD-2A.

ASSOCIATION OF CALPAINS WITH DUCHENNE MUSCULAR DYSTROPHY

Calpains are activated in many pathological and aberrant physiological conditionsthat result in muscle wastage. There is much evidence that calpains degrade a numberof myofibrillar proteins, and there is consensus that calpains participate actively inthe early stages of myofibril breakdown (F.C. Tan et al. 1988). Calpains as well ascathepsins have been associated with muscle fibre degradation occurring in inflam-matory muscle myopathy (Kumamoto et al. 1997).

The characteristic muscle weakness and the progressive degradation of striatedmuscle encountered in DMD and BMD appear to be attributable to calpain activity,besides the abnormalities in the DMD and BMD gene loci that alter the function ofdystrophin and the dystrophin–glycoprotein complex. DMD is generally associatedwith a loss of dystrophin, which links the sarcolemma with the actin cytoskeleton.Although the precise function of dystrophin is unclear, its loss is associated withincreased calcium influx and high intracellular levels of calcium. Being calcium-activated enzymes, calpains were implicated in the pathogenesis of this chronicdegenerative disease many years ago (Arahata and Sugita, 1989). Kumamoto et al.(1995) studied the expression of calpains and calpastatin in DMD and BMD.Calpains occurred at markedly elevated levels in muscle fibres that had atrophied.In necrotic fibres, both calpain and calpastatin were expressed at moderate levels,but hypertrophic and opaque fibres manifested no activity at all. Ueyama et al. (1998)have recently confirmed the increase of capn1 and capn2 proteins and their mRNAs



in progressive muscular dystrophy as well as in ALS. However, capn3 appeared tobe unaffected. These findings suggest a possible deregulation of the calpain–calp-astatin system as the cause of progressive muscle degeneration.

Fasting has serious consequences in terms of calpain activation. A two-foldincrease in the degradation of myofibrillar protein of rabbit skeletal muscle occurredin response to fasting, together with an increase in mRNA levels encoding capn1and capn2 as well as calpastatin mRNA levels (Ilian and Forsberg, 1992, 1994).Fasting raised the mRNA levels of cathepsin D and proteasome. These changesappear to be specific for skeletal muscle. Ilian and Forsberg (1994) did not find anychanges in the protease mRNA levels in other tissues such as liver, lung, or kidney.The transcription of the genes seems to be up-regulated to meet the need to mobilisemuscle protein in response to the physiological insult. Therefore, Ilian and Forsberg(1992) suggest this explains why they did not encounter any increase in capn1 andcapn2 protein levels. However, it is essential to know the stability of the messages,as well as the half-life of the enzymes, before relating the findings to the continuedrequirement for protein mobilisation. It should be borne in mind also that otherproteases would have contributed to the process of protein mobilisation as well. Atany rate, Kumamoto et al. (1992) confirmed the earlier findings of Ilian and Forsberg(1992) relating to the increase of calpains in response to fasting. They also demon-strated that capn1 and capn2 occurred in the Z-band, where the degradation ofmyofibrils is initiated. Immunostaining studies showed that the Z-band containedtwice as much calpain and calpastatin as the I-band or the A-band.

CALPAINS AND LIMB GIRDLE MUSCULAR DYSTROPHY

Three autosomal dominant and several autosomal-recessive loci have been identified.With the view of placing the following discussion in the proper perspective, but atthe risk of a minor digression, details about the LGMD subtypes and the molecularfeatures associated with them have been provided in Table 11. LGMD-2A is anautosomal-recessive form of muscular dystrophy. The skeletal muscle-specific homo-logue capn3 has been implicated in LGMD-2A. The capn3 gene is located in theregion of chromosome 15q15.1–q21.1 (Fougerousse et al. 1994). Five distinct geneshave been identified with LGMDs, and capn3 as a putative LGMD-2A candidategene (Chiannilkulchai et al. 1995; Beckmann et al. 1996). Recently, capn3 mutationshave been found to co-segregate with the disease in families with LGMD-2A, andthis has led to the suggestion that the disease is due to a defect in the enzyme ratherthan to abnormalities of any structural proteins (Richard et al. 1995). This has beenconfirmed by recent findings that mutations of the gene result in the loss of pro-teolytic activity of capn3 (Y. Ono et al. 1998). Therefore, there seems to be a linkbetween this loss of proteolytic activity of capn3 and the pathogenesis of LGMD-2A. Spencer et al. (1997) did not find capn3 in muscle biopsies from LGMD-2Apatients, but did detect it in control subjects as well as in non-LGMD-2A patients.Capn3 is said to undergo rapid autolysis and it has a half-life of less than 1 hr, whichcould be one of the reasons for the failure of detection. Sorimachi et al. (1995)found that capn3 also interacts with connectin (titin), which spans the M- to Z-linesof muscle sarcomeres. They have, therefore, suggested that connectin may regulate



capn3. Obviously, there is a need for much further work to elucidate the role ofcapn3 in LGMD-2A, especially with a view to relating it to the significance of theassociation of sarcoglycans with LGMD. As Spencer et al. (1997) have pointed out,capn3 was regarded as the agent responsible for the posttranslational generation ofα- and β-sarcoglycans, but it may not be so involved.

TABLE 11Limb Girdle Muscular Dystrophy Types and Associated Molecular Features

LGMD Type/Subtype Associated Molecular Features Ref.

Autosomal DominantLGMD-1ALGMD-1BLGMD-1C Caveolin-3 mutations, 3p25 Minetti et al. (1998); McNally

et al. (1998a)Also in DMD and mdx mice Vaghy et al. (1998)

LGMD-1D

Autosomal RecessiveLGMD-2A Calpain, 15q15.1 Chiannilkulchai et al. (1995);

Moreira et al. (1997) LGMD-2B (Myoshi myopathy)

Dysferlin mutations, 2p13 J. Liu et al. (1998); Bashir et al. (1996, 1998)

LGMD-2C γ-Sarcoglycan missense, α-sarcoglycan mutation and loss, 13q12

Van der Kooi et al. (1998); Jung et al. (1996); Moreira et al. (1997)

LGMD-2D ε-Sarcoglycan mutation, 17q12-q21 Ettinger et al. (1997); McNally et al. (1998b); Moreira et al. (1997)

LGMD-2E β-Sarcoglycan missense mutations, 4q12 Bonnemann et al. (1996); Moreira et al. (1997)

LGMD-2F δ-Sarcoglycan mutations, 17q11-q12 Nigro et al. (1996); Duggan et al. (1997); Moreira et al. (1997)

Note: Jung et al. (1996) state that LGMD-2A–2E all show abnormal expression of α, β, γ, and δ-sarcoglycans.


169

14

Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia

CASPASES IN APOPTOTIC CELL DEATH

Calcium-mediated activation of caspases is also associated with apoptosis. Theapoptotic pathway involves a cascade of proteolytic events mediated by caspases,their activators, and their repressors. Caspases are cysteine proteases of a family ofinterleukin-1

β

-converting enzymes (ICE). A number of proteases —

ced-3, ced-4,

and

ced-9

— have been identified in the nematode

C. elegans

. Of these,

ced-3

is acaspase. A human homologue of the

ced-4

protein, called

apaf-1

, has been described(Zou

et al.

1997). Three new forms of

apaf-1

have since been identified in mam-malian cells (Hahn

et al.

1999). The

apaf-1

protein appears to promote cytochrome

c

-dependent activation of caspases (Zou

et al.

1997; Kluck

et al.

1997; P. Li

et al.

1997). Cytochrome

c

has been found to be involved in caspase activation as an earlyevent in many forms of apoptosis. However, it may not be involved in at least Fas-induced activation of caspase and apoptosis. (Vier

et al.

1999). The chromatincondensation and nuclear fragmentation that occur in apoptosis involve, besides thecaspases, caspase-activated DNAase (Enari

et al.

1998; X.S. Liu

et al.

1998).When

apaf-1

is stably transfected into HL-60 cells, not only is

apaf-1

overex-pressed but there is also a marked induction of apoptosis (Perkins

et al.

1998).According to two recent reports,

apaf-1

(–/–)

null

mice died at day 15 of embryonicgrowth and showed reduced apoptotic cell loss and overgrowth of the brain. The

null

mutants also manifested marked craniofacial abnormalities. Markedly enhancedproliferation of neuronal cells was also encountered in these embryos.

Apaf-1

(–/–)cells seemed to resist apoptosis, with an attendant impairment of caspase activation(Yoshida

et al

. 1998; Cecconi

et al.

1998). In oncogene E1A-dependent inductionof apoptosis, in which the caspase is activated by the oncogene product, the activatedcaspase-9 forms a complex with

apaf-1

and cytochrome

c

(Fearnhead

et al

. 1998).As shown in Figure 21, some members of the

bcl-2

family of genes can inhibitapoptosis. A general model has been proposed that the bcl-2 proteins inhibit apop-tosis by binding to

apaf-1

, thereby preventing the activation of caspases. However,this model has been disputed by Morishi

et al.

(1999), who believe that the inhibitionmight not be a direct effect of the sequestration of

apaf-1

by bcl-2 proteins.


170


Caspase-9 might initiate the programme of apoptosis or activate other caspasesdownstream and ultimately cleave downstream target proteins, which brings aboutapoptotic disintegration of the cell (Thornberry, 1997; Thornberry and Lazebnik,1998). Cytochrome c-mediated activation of capase-9 seems to be followed by theactivation of a string of other caspases. When cell extracts are depleted of caspase-9, the activation of the downstream caspases does not occur. This indicates thatactivation of capsase-9 is obligatory for the activation of other caspases by cyto-chrome

c

(Slee

et al.

1999). In Jurkat cells induced into apoptosis, caspase-3 occurswith caspase-6 in the activation complex. HSP60 is also found in this complex(Xanthoudakis

et al.

1999); these authors have suggested that this HSP may partic-ipate in protein folding and aid in the proteolytic activation of caspase-3.

In vitro

,HSP60 aided the activation of the caspase by several caspase promoters.

As many as ten caspases (caspases-1 to -10) have been identified. These formthree natural groups based on their mode of activation from the corresponding pro-caspase form. Caspases-1, -2, -4, and -5 show autocatalytic activation from theirpro-caspase form. The pro-caspases are believed to bind to activator molecules andlead to their oligomerisation and autoactivation. The second group composed ofcaspases-3, -6, -7, and -9 do not show autocatalytic activation. Lysosomal cysteineproteases may be involved in the activation of some caspases (Ishisaka

et al.

1998).The overexpression of caspases-3 and -7 leads to apoptosis of the prostate cancercell lines called LNCaP. In this system, caspase-7 seems to be activated by proteolysis(Marcelli

et al.

1999). Caspases-8 and -9 undergo TNF-R1/Fas (CD95) mediatedactivation. The Fas ligand is a member of the TNF family. It is expressed in activatedT cells. The apoptosis of target cells is induced by the binding of this ligand to itsreceptor (Nagata and Golstein, 1995; Nagata, 1997). The occupancy of Fas has beenfound to lead to the activation of caspases (Enari

et al.

1995; Longthorne andWilliams, 1997; Armstrong

et al.

1996; Boldin

et al.

1996) and of downstreamproteases (Nagata, 1997; Fraser and Evan, 1996). The outcome of the function ofthis proteolytic cascade initiated by Fas binding is DNA fragmentation. The activa-tion is believed to be due to the binding of caspase to the activated membranereceptor complex.

Another line of evidence that strongly supports the role of caspases concernsthe resistance of some cell systems to the induction of apoptosis by Fas receptorbinding or other factors. Perara and Waldmann (1998) have used peripheral bloodmonocytes, which undergo spontaneous apoptosis unless the culture medium issupplemented with serum, growth factors, bacterial LPS, or cytokines. This inductionof resistance to apoptosis is accompanied by a marked down-regulation of caspase-8. Deficiency of caspase-8 also makes the Jurkat cell line JB-6 resistant to Fas-mediated apoptosis (Kawahara

et al

. 1998). T.S. Zheng

et al.

(1998) deny any rolefor caspases in the induction of Fas-mediated apoptosis of liver cells, but recognisethat caspase-3 may be instrumental in bringing about morphological changes bycleaving substrate proteins. Equally, as suggested by Kawahara

et al

. (1998), cellkilling triggered by Fas binding could take two pathways. One of these is

via

caspase-8, and the other is more akin to necrosis that does not involve caspases. It has beensuggested also that HSPs may interfere with apoptotic cell death. HSP70, forinstance, seems to ensure cell survival by inhibiting caspase-3-dependent events of



171

apoptosis (Jaattela

et al.

1998). However, the involvement of HSPs must be inter-preted with some caution, especially in light of HSP60 being shown to be possiblyassociated with the activation complex of caspases.

Caspases have several substrates such as poly (ADP-ribose) polymerase (PARP)(see below), lamin A, and several other cytoplasmic and nuclear proteins (see reviewby Zhivotovsky

et al.

1997). The actin-binding proteins,

α

-fodrin and gelsolin, arecleaved by caspase-3. Actin itself is cleaved by caspase. According to Mashima

etal.

(1999), this results in the generation of two actin fragments of 15 and 31 kDa.When introduced into cells, the 15-kDa actin fragment brings about morphologicalchanges that are akin to apoptosis.

The retinoblastoma susceptibility

rb

protein is also degraded by caspase.Caspase-3 cleaves CAMPK within its catalytic site (McGinnis

et al.

1998). Nedd4,a ubiquitin–protein ligase, is cleaved by several caspases (Harvey

et al.

1998). Thecleavage of these proteins is associated with apoptosis, but the significance of theprocess is not understood. The proteolytic cascade involves endonucleases down-stream of caspases and calpain in the apoptotic pathway. Because caspase inhibitorsalso result in the inhibition of DNA fragmentation (X. Liu

et al.

1997), it wouldappear that caspase may expose the DNA to endonuclease function by cleaving thechromatin-associated proteins such as lamin B, histone H1, and topoisomerase II(Neamati

et al.

1995; Kaufmann, 1989). In the same vein, nuclear scaffold proteaseinhibitors block not only the cleavage of lamin but also DNA fragmentation. It seemsthen, that the cleavage of chromatin scaffold proteins by either pathway results inDNA fragmentation (Lazebnik

et al.

1995). Not all caspases are inducers of apop-tosis. Caspase-11 is a pro-inflammatory caspase and is activated by the lysosomalprotease cathepsin B (Schotte

et al.

1998).Apoptotic changes triggered in nuclei by CaCl

2

are inhibited by the inhibitionof caspase-3 and to a lesser extent by caspase-1 (Juin

et al.

1998). Hydrogen peroxide(at 50

µ

M

, but not at higher concentrations) induces apoptosis of Jurkat T lympho-cytes and, in parallel, an enhancement of caspase activity is also detected. However,at H

2

O

2

concentrations greater than 50

µ

M

, cells undergo necrosis (Hampton andOrrenius, 1997). Caspase seems to be involved in neuronal apoptosis followingischemic injury, and again caspase inhibitors appear to be able to protect neuronesfrom undergoing apoptosis (Gorman

et al.

1998). Overall, it seems reasonable toconclude that caspase plays a significant role in the cell apoptosis occurring inresponse to a variety of extracellular signals. The evidence is based, as noted, mainlyon the protective function of caspase inhibitors and also on the inhibition of thecaspase substrate PARP. It ought to be stated, however, that in certain cell types,activation of caspase-3 does not automatically lead to apoptosis (Well

et al.

1998).There could be other components in the pathway, e.g., the p36-MBP kinase (MBPK),the activation of which has been demonstrated recently by Kakeya

et al.

(1998).These authors showed that cytotrienin A, isolated from

Streptomyces

, induces apo-ptosis of HL-60 cells by activating MBPK. The broad-spectrum caspase inhibitor,Z-Asp-CH2-DCB, inhibited activation of MBPK as well as apoptosis. However, itdid not inhibit the activation of other kinases such as c-jun N-terminal kinase/stress-activated protein kinase and p38 MAPK, which were also activated by cytotrieninA, albeit with a different kinetic pattern from MBPK activation. This suggests that


172


activation of MBPK is a component of the apoptosis-signalling pathway. The path-ways of caspase and calpain involvement in apoptotic cell death are shown in Figure21. It is fairly obvious from the above discussion that at present our knowledge ofthe mechanisms involved is rather limited.

POLY (ADP-RIBOSE) POLYMERASE AS A MARKER OF APOPTOSIS

The process of recovery from DNA damage involves the synthesis of poly (ADP-ribose) in response to strand breaks. The nuclear enzyme poly (ADP-ribose) poly-merase (PARP) catalyses the cleavage of NAD

+

into nicotinamide and adenosine 5

′

-diphosphoribose (ADP-ribose). PARP further catalyses the covalent linkage of poly(ADP-ribose) to the damaged sites in the DNA. PARP is activated upon binding toDNA strand breaks. It binds to DNA

via

its zinc finger domains (De Murcia andDe Murcia, 1994; De Murcia

et al.

1997). It links poly (ADP-ribose) to the nicksin the DNA and maintains its structural integrity until DNA excision repair is carriedout by DNA polymerases (Lindahl

et al.

1995). Caspases cleave this 113-kDaenzyme into an N-terminal DNA-binding fragment and a C-terminal catalytic frag-ment. Apoptosis is characterised by the cleavage of PARP and the appearance ofthe 89-kDa catalytic fragment is regarded as a marker event of apoptosis.

There is a large body of evidence that PARP is involved in apoptosis. Theionophore A23187 has been reported to induce apoptosis in PC12 cells, when theionophore is applied at low concentrations. This is accompanied by the activationof caspase-3. When caspase-3 is inhibited the process of apoptosis is also inhibited(Takadera and Ohyashiki, 1997). Dexamethasone and thapsigargin both induce apo-ptosis of WEHI mouse lymphoma cells, albeit by different pathways. Nevertheless,the activation of caspase-3 and cleavage of PARP accompanied the induction ofapoptosis by both these agents. Both latter events were inhibited by overexpressionof the

bcl-2

gene (McColl

et al.

1998). The protein encoded by the

ced-9

gene of

C.

elegans

is an inhibitor of apoptosis belonging to the

bcl-2

gene family (Reed,1997). A similar effect on apoptosis and PARP cleavage by caspase inhibitors hadbeen demonstrated earlier in a different cell system by Bonfoco

et al.

(1996).The nature of PARP involvement is increasingly being appreciated. Caspase-3

activation and the triggering of PARP cleavage may be described as key events thatoccur in the induction of apoptosis. The poly (ADP-ribosylation) of nuclear proteins,followed by caspase-3-mediated PARP cleavage, is an early event of the apoptoticprocess, and it may indeed be an important requirement for apoptosis to proceed.The cleavage of PARP, internucleosomal DNA fragmentation, and morphologicalchanges in nuclei do not occur if cells are depleted of PARP by using antisensestrategy. These apoptosis-associated changes take place in cells with wild-type PARP(+/+) genotype, but are conspicuously absent in PARP (–/–) mutants (Simbulan-Rosenthal

et al.

1998). Simbulan-Rosenthal

et al.

(1999) have since argued thatPARP is also a component of the DNA replication complex, which includes severalimportant proteins. The expression of these might be affected by PARP depletion,inhibit DNA replication, and commit the cells to the apoptotic pathway.



173

Notwithstanding these studies, the precise role played by PARP is not fullyunderstood. The inhibition of PARP affects apoptosis. The activation of PARP byDNA damage results in NAD

+

depletion and a depletion of ATP in the restorationof NAD

+

levels. Therefore, cell death can be a result of the loss of ATP. In PARP–/– cells, ATP levels seem to be maintained and the cells are protected from necroticcell death. However, in both PARP +/+ and PARP –/– mutants, cells can be equallysusceptible to apoptosis (Ha and Snyder, 1999). In renal epithelial cells, hydrogenperoxide-induced PARP activation results in necrotic death due to NAD

+

/ATP deple-tion (Filipovic

et al.

1999). Walisser and Thies (1999) found that endothelial cellsexposed to hydrogen peroxide undergo cell death. Presumably, here too cell deathmay have been caused by NAD

+

and consequent ATP depletion. However, whenPARP is inhibited these cells switch to apoptosis. Other studies, however, suggestPARP inhibition may indeed protect cells from apoptosis (Richardson

et al.

1999;Guo

et al.

1998).A putative link-up between the function of caspases and PARP in apoptosis has

emerged recently. Aoufouchi

et al.

(1999) transfected a PARP (–/–) cell line withthe DNA-binding domain fragment of PARP (DBD), or mutants: DBDbd (–) whichis defective in binding to DNA strand breaks, and DBDcl (–), which resists cleavageby caspases. DBD transfected PARP (–/–) cells showed no changes in staurosporine-induced apoptosis. Cells that had been transfected by both mutants showed markedinhibition of apoptosis. Furthermore, the mutant DBDs inhibited the cleavage of thecatalytic subunit of DNA-dependent protein kinase by caspase-3. These resultssuggest that PARP might be involved in the events leading to caspase activation. Itseems, therefore, that it is possible to dissociate the DNA-binding properties ofPARP from caspase activation. Aoufouchi

et al.

(1999) have suggested the possibilitythat PARP might interact with components involved in caspase activation.

CASPASE-MEDIATED APOPTOSIS AND CELL GROWTH INHIBITION IN TUMOUR EXPANSION

The relevance of caspase-mediated apoptosis in tumour development has beenemphasised recently by the finding that enhanced expression of caspase-2 leads toa reversion of

ras

-induced transformation in NIH3T3 cells (Hiwasa and Nakagawara,1998). Hiwasa and Nakagawara (1998) transfected caspase cDNAs into c-Ha-

ras

-transformed cells. Enhanced expression of caspase-2 alone, but not caspases 1 and2 together, resulted in a reduction in the ability of the transfected cell to grow insoft agar. This was accompanied by

ras

protein degradation, suggesting that theapparent reversion of the transformed phenotype was due to the degradation of thetransforming gene product by caspase-2. However, Hiwasa and Nakagawara (1998)have not presented any evidence relating to the tumorigenicity of the

ras

-transformedcells, or any data on possible inhibition of tumorigenicity by the transfection ofcaspase-2 cDNA into these transformed cells. It is essential to recognise that celltransformation is a process quite distinct from tumorigenicity. Whether caspase-induced apoptosis occurring in the developing tumour can control the growth ofthe tumour is still an open question. As stated elsewhere, apoptosis subserves an


174


important function in maintaining the kinetics of cell expansion in metastatic growth.By inference, there is no reason why a similar role may not be played by apoptosisin the control of primary tumour growth, or, to put it more precisely, primary tumourexpansion. The apoptosis-mediated restriction of a cell population can be differen-tiated from control by inhibition of growth, although these might not represent totallyindependent mechanisms. This has been demonstrated in the case of TGF-

β

, whichinfluences apoptosis as well as cell growth. However, in WEHI 231 cells, the caspaseinhibitor BD-fmk can effectively inhibit TGF

β

-induced apoptosis but not counteractTGF

β

-mediated inhibition of cell growth (T.L. Brown

et al.

1998). This apparentdichotomy of mechanisms is obvious also from the pathways of radiation-inducedapoptosis. Ionising radiation induces the activation of caspase-3, which is followedby the induction of apoptosis. Ionising radiation induces rapid apoptosis in humanlymphoblast cells expressing wild-type p53, but in cells where p53 is mutated orabrogated by viral oncoprotein, apoptosis is delayed as well as reduced (Yu andLittle, 1998). In this instance apoptosis seems to occur in conjunction with growthcontrol mediated by wild-type p53. On the other hand, caspases have been shownto cleave p53-induced cyclin-dependent kinase inhibitor p21

waf1/cip1

and induce cellsto undergo apoptosis (Y.K. Zhang

et al.

1999). In the latter case, apoptosis seemsto depend on proliferation. In contrast with p53-mediated apoptosis, VD3 has beenreported to induce apoptosis of certain breast cancer cells independently of both p53and caspases. This report is based on the observation that VD3 produces growtharrest and apoptosis of both MCF7 cells, which are p53 positive, and T47D cellsthat are p53-negative. Furthermore, this apoptotic induction is not inhibited byinhibitors of caspase, whereas TNF- or staurosporine-induced apoptosis is inhibited(Mathiasen

et al.

1999).Whatever pathway apoptosis might take, it is inevitable that tumour growth and

subsequent processes should be shaped by balancing forces of apoptosis and cellcycle control factors. Nonetheless, it is worthwhile to note here that ICE-like proteaseexpression has been reported to correlate with progression and prognosis of neuro-blastoma. The frequency of expression of ICE mRNA was markedly reduced inadvanced-stage neuroblastomas as compared with early-stage tumours (Ikeda

et al.

1997). The expression of caspase-3 in normal gastric mucosa, gastric adenomas andadenocarcinomas has been studied in some detail by Hoshi

et al.

(1998). They foundthat caspase-3 expression decreased from a high level (42% cells staining forcaspase) in nonneoplastic gastric mucosa to a lower level (33%) in adenomas, andto a still lower level (17%) in adenocarcinomas. These differences were statisticallysignificant in spite of the large standard deviations of the mean. The caspase-3positivity, of the three groups, correlated directly with apoptotic indices determinedby the TUNEL method and inversely with proliferative indices provided by Ki67labelling. These data are consistent with the view that a loss of apoptosis-mediatedcontrol over cell turnover is an important feature of tumour growth. Conversely,there is an implicit suggestion that ICE-like proteases may be involved in apoptosis-mediated regression of tumours, in the demonstration by Ikeda

et al.

(1997) thatICE protease staining could be co-localised in the nucleus with DNA fragmentation.In this context, one should also take note of the recent report that caspase-3 activitywas found to increase in colonic carcinomas and adenomas as compared with normal



175

mucosa (Leonardos et al. 1999). Obviously, these observations may serve to confirmthat the expression of the caspase is related more to the degree of apoptosis takingplace in the tumour than to the degree of tumour progression. Donoghue et al. (1999)reported a difference in the pattern of caspase-3 distribution in B-cell diffuse largecell lymphoma. In immunohistochemistry, caspase-3 showed a diffuse distributionin the cytoplasm or a punctate or spotty pattern. The diffused pattern appeared torelate to poor prognosis, and the punctate pattern was associated with completeresponse to therapy. The authors have stated further that where the percentage ofcaspase-3-expressing cells was low, prognosis was poor. Not only are some of thesefindings not compatible per se, but the study seems to raise more questions than itsuccessfully answers. It should be conceded, however, that it would be unhelpful toattribute all apoptotic activity to caspases. Donoghue et al. (1999) found no corre-lation between apoptosis and caspase expression in the cells, but the degree ofapoptosis was associated with poor prognosis. A finding of potential significance isthat the caspase distribution pattern might be significant in terms of enzyme activity.Whether the punctate distribution might reflects sequestration of the enzyme is worthfurther investigation.

It should be recognised, nevertheless, that caspase expression may reflect cancerprogression and that caspases may actively promote metastatic deposition by a moredirect route. Thus, a high proportion of in situ and invasive (58 and 90%, respectively)carcinomas of the breast stain strongly for caspase-3. Caspases-6 and -8 also areexpressed at high levels more frequently in carcinomas than in hyperplasia. Theenhanced expression correlated well with apoptotic indices in the samples. Further-more, enhanced apoptosis was associated with poor prognosis (Vakkala et al. 1999a,1999b). The formation of metastatic lesions depends on a cascade of events; prom-inent among them is the invasion of the vascular and endothelial systems by cellsof the primary tumour. The entry into the circulatory system has been attributed toan active process of transmigration or diapedesis across the endothelial layer, aswell as to the inherent structural defects often found in the endothelium. Recently,Kebers et al. (1998) found that several breast cancer cell lines, among them MCF7,MDA-MB231, T47D, and HT1080, induced a four-fold increase in apoptosis ofhuman umbilical vein endothelial cells (HUVEC), with an attendant enhancementof caspase-3 activity. The interaction of MCF7 cells with HUVEC caused a transientincrease in intracellular calcium levels (Lewalle et al. 1998), which, presumably,may have led to caspase activation. The induction of apoptosis required cell–cellcontact, because media conditioned by the growth of these cells were ineffective.Kebers et al. (1998) have further observed that lymphocytes do not induce apoptosis,suggesting that the apoptosis of endothelial cells might constitute a specific mech-anism in the diapedesis of tumour cells. Whether caspase-mediated apoptosis ofendothelial cells occurs in vivo is yet to be demonstrated.

The outcome of tumour progression has often been assessed in relation to asingle given variable as a prognostic factor. As noted above, in the context of caspaseswe have a paradoxical situation that in both caspase-positive and caspase-negativecircumstances some relationship is noticed with tumour progression. As observedearlier, tumour growth and progression are a net outcome of the balancing forcesof apoptosis and cell cycle control factors and cell proliferation. Perhaps it would



be more rewarding to reexamine the question of caspases in tumour progression inthis light. For this viewpoint, the recent findings of Volm and Koomagi (2000) aremost encouraging. They have examined the relevance not only of caspase-3 but alsoof c-myc expression in the prognosis of non-SCLC. Volm and Koomagi (2000) havereported that caspase-3-negative patients had a median survival time of 41 weeksas compared with 79 weeks for caspase-3-positive patients. They then looked at c-myc expression and its influence on prognosis. Patients who were c-myc negativehad a median survival time of 89 weeks, whereas the median for c-myc-positivepatients was 43 weeks. Not only did these two factors correlate inversely withsurvival, but, myc–/caspase+ patients showed a median survival time of 102 weeksas compared with only 22 weeks for myc+/caspase– patients. This clearly makes thepoint that more than one factor might influence a given cellular feature and therebydetermine the outcome of the disease. The study also serves to further emphasisethat it would be unhelpful to try to evaluate a single prognostic factor, while otherfactors might be present that would impinge on the direction of cellular changes.

CASPASE-MEDIATED PROTEOLYSIS OF FODRIN: IMPLICATIONS FOR APOPTOSIS, CELL ADHESION, CELL MIGRATION, AND NEOPLASTIC TRANSFORMATION

Among the several substrates of caspases is the membrane protein called fodrin.The erythroid homologue of fodrin is known as spectrin, with which fodrin sharessubstantial amino acid sequence homology. Fodrin is attributed with the function ofmaintaining the structural integrity of the plasma membrane. Fodrin and spectrinform a major component of the skeletal network that underlies the plasma membrane(Levine and Willard, 1981; W.J. Nelson et al. 1990; Bennett and Lambert, 1991;Bennett and Gilligan, 1993). Fodrin (alias spectrin) isoforms are also found in themembranes of the Golgi apparatus (Devarajan et al. 1996, 1997; Beck et al. 1994,1997; Godi et al. 1998; Fath et al. 1997; Stankewich et al. 1998), lysosomes (Hoocket al. 1997), and intracellular vesicles (Malchiodi-Albedi et al. 1993; Stankewich etal. 1998). Fodrin is an actin-binding protein; therefore, two further putative functionsshould also be considered. One of these is a presumptive involvement in the processof signal transduction, because fodrin has been found to be able to inhibit phospho-lipases A2 (PLA2), C, and D (Lukowski et al. 1996, 1998). In comparison, othercytoskeletal proteins such as actin and vimentin are far less efficient than fodrin(Lukowski et al. 1996). Phospholipases are closely associated with the generationof DAG and IP3 from PIP2. These are involved in the activation of downstreampathways of signal transduction that, in turn, involve the activation of appropriateprotein kinases, such as PKC, and Ca2+ release from intracellular stores.

Another potentially important function that can be attributed to fodrin is ininfluencing cell adhesion and migration. Spectrin is required for neurite extensionin neuroblastoma cells. Sihag et al. (1996) were able to inhibit neurite extension inNE2a/dl neuroblastoma cells with an antibody directed against the N-terminaldomain of spectrin. The latter is known to interact with actin. The protein called


Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia 177

ankyrin mediates the link-up between membrane adhesion proteins and the spectrincytoskeleton. The expression of some of these adhesion molecules has been shownto cause cell aggregation in which process both ankyrin and spectrin are recruitedto the foci of adhesion (Dubreuil et al. 1996).

In wound healing of corneal epithelium, fodrin becomes redistributed from itssubplasma membrane location to a cytoplasmic location. This redistribution occurssoon after wounding of the epithelium. A similar redistribution occurs in responseto PMA treatment and is inhibited by PKC inhibitors (Amino et al. 1995). Presum-ably these events are related to the cell migration that follows, although there is littledirect evidence linking these.

As stated above, fodrin (spectrin) is a substrate for caspases. Apoptosis inducedby several different pathways has been shown to be accompanied by proteolysis offodrin. Inhibition of apoptosis also results in the inhibition of fodrin proteolysis(Martin et al. 1995). This proteolysis seems to be produced by ICE/ced-3 proteases(Cryns et al. 1996; Vanags et al. 1996). Kouchi et al. (1997) believe that calpainsare not associated with the cleavage of the 240-kDa α subunit of fodrin in theapoptosis of rat thymocytes both in vivo and in vitro. However, Porn-Ares et al.(1998) do implicate calpains. Fodrin occurs in a variety of cell types includingkeratinocytes, chromaffin cells, and renal epithelium, and in a variety of epithelialand fibroblast cell lines. Fodrin shows a homogeneous cytoplasmic and a discontin-uous membrane distribution in benign melanocytic tumours, whereas normal mel-anocytes at the basal layer of the epidermis only faintly stain for fodrin at the plasmamembrane. Overall, neoplastic cells show greater amounts of fodrin than theirnonneoplastic counterparts (Tuominen et al. 1996). This observation has been con-firmed in an immunohistochemical study of a variety of adenocarcinomas andsquamous cell carcinomas by Sormunen et al. (1997). However, malignant melano-mas contain subpopulations that do not express fodrin (Tuominen et al. 1996). Thisdoes not detract from any putative relationship between fodrin expression and malig-nancy, because malignant tumours are notoriously heterogeneous with respect to alarge spectrum of cellular characteristics. Although much work needs to be done inthis area, already there are clear indications that caspase-mediated alterations infodrin expression and function might be involved in biological processes (e.g.,apoptosis, cell adhesion, motility, and modulation of cell shape) that are inherentfeatures of tumour development, dissemination, and metastasis.

CASPASES AND NEURONAL LOSS IN ALZHEIMER’S DISEASE

Alzheimer’s disease is characterised by massive neuronal loss, which has beenattributed, in recent years, to apoptotic cell death (Barinaga, 1998). The participationof caspases in inducing apoptosis has naturally led to the investigation of theseproteinases, together with the bcl-2 family genes, in the pathogenesis of Alzheimer’sdisease (Figure 23). Kitamura et al. (1998) showed that the expression of severalbcl-2 family genes was up-regulated in Alzheimer’s disease. Caspases are involvedin the induction of neuronal apoptosis (Bambrick and Krueger, 1999). Two genesknown as PS (presenilin)-1 and PS2 have been associated in a mutated form withearly onset of Alzheimer’s disease. The presenilins are integral proteins of the ER.


178


Their mutation could lead to a perturbation of calcium homeostasis in the cell withattendant aberrations of calcium-mediated processes of apoptosis as well as theprocessing of the amyloid precursor protein. Wild-type PS has been shown topromote neurite outgrowth in neuroblastoma cells grown

in

vitro

(Dowjat

et al.

1999). It is conceivable that mutations would affect this process as well. The involve-ment of PS proteins in apoptosis has been demonstrated by several experiments. Forexample, overexpression of the mutated PS protein can enhance apoptosis inducedby several agents. PS2 antisense mRNA inhibits apoptosis. A new facet might beadded to this story of PS2 involvement in neuronal apoptosis. A CBP has beensuggested to play a part in this process. Stabler

et al.

(1999) have reported that aCBP, which they call calmyrin, interacts preferentially with PS2. Calmyrin consistsof 191 amino acid residues and possesses two C-terminal EF-hands. It has beenshown to interact with the cytoplasmic domain of the platelet integrin

α

II b

β

3

, andis regarded as a putative regulator of the function of the platelet integrin (Naik

etal.

1997). Stabler

et al.

(1999) found that calmyrin is myristoylated, and, in themodified form, it interacts with PS2. In HeLa cells, calmyrin is said to co-localisewith PS2. The posttranslational modification of the protein could be required fortargeting it to the membrane. This is reminiscent of myristoylation of recoverin andits function. Although, in the context of calmyrin, we can only draw inferences asto the significance of complex formation between calmyrin and PS2, some prelim-inary observations made by Stabler

et al.

(1999) make interesting reading. Theyobserved an enhancement apoptosis upon co-transfection of HeLa with PS2 andcalmyrin.

The PS proteins are substrates for caspases. Caspases cleave these at specificsites, e.g., aspartic acid 345/serine 346 and aspartic acid 329/serine 330. Caspaseinhibitors as well as mutations at these sites inhibit cleavage of the proteins (Loet-scher

et al.

1997). Phosphorylation has been found to regulate the caspase sensitivity

FIGURE 23

Possible pathways by which caspases may regulate neuronal apoptosis asso-ciated with Alzheimer’s disease. PS, presenilin proteins; PS-P, phosphorylated form of PSprotein.


Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia 179

of PS2. Two phosphorylation sites, at serine residues 326 and 329 have been iden-tified in PS2. Both occur close to the site of cleavage by caspase-3. Cleavage of theprotein is inhibited by phosphorylation of these residues, and the loss of this abilityseems to inhibit apoptosis (Walter et al. 1999). However, there have been no attemptsto date to determine if the phosphorylation of PS is related to disease activity. Wedo know that the sensitivity of the mutated PS1 and PS2 proteins to cleavage bycaspases is not affected, which suggests that caspase-mediated modification of PSproteins might not be associated with the disease (Van de Craen et al. 1999). Also,PS proteins carrying mutations at the caspase cleavage site have been found to befunctional (Brockhaus et al. 1998). Furthermore, the participation of PS genesthemselves might have been overemphasised. Nonetheless, it has emerged fromrecent studies that caspases-2 and -3 show a marked increase in the brains ofAlzheimer’s patients (Shimohama et al. 1999). In that study, Shimohama et al. (1999)also examined the expression of these caspases in developing rat brain. Their datahave suggested some differential regulation of expression of these proteases. Forinstance, caspase-3 is highly expressed from 19-day-old embryos to 96-week-oldembryos. However, high expression of caspase-2 begins only in approximately 4-week old embryos. Although it does seem that there is a developmental regulationin rat embryos, the relation between the expression of these caspases and the processof ageing is not quite obvious. In any event, these data are at present insufficient tobase any conclusions about whether the pathogenesis of Alzheimer’s disease isrelated to changes in the regulation of caspase expression. Another observationworthy of note is that caspase-3 can cleave PS2 at arginine 329 and generate a C-terminal peptide that might protect cells against apoptosis (Vito et al. 1997). Thus,caspase-mediated promotion of apoptosis by the modification of PS proteins couldcontain a self-regulatory mechanism. Vito et al. (1997) have suggested that theapoptosis-promoting function and the negative feedback signal provided by the C-terminal fragment generated by caspase activity could eventually determine theoutcome.

As discussed elsewhere Alzheimer’s disease is also characterised by abnormal-ities of the tau protein. This protein have been identified as a substrate for bothcaspases and calpain. The cleavage and dephosphorylation of tau has been shownto occur in neuronal apoptosis (Canu et al. 1998). Similarly, the amyloid precursorprotein (APP), again a prominent feature of the disease, seems to be cleaved bycaspase-3 (N.Y. Barnes et al. 1998). Not only does caspase-3 occur at high levelsin Alzheimer’s brains, but the product of APP degradation co-localises with thecaspase in degenerating neurones (Gervais et al. 1999). These observations representanother pathway involving caspase function in the pathogenesis of Alzheimer’sdisease.


181

15

Parvalbumins in Neuronal Development, Differentiation, and Proliferation

The neuroepithelial ventricular zone of telencephalic ventricles gives rise to themajority of neurones and the glial component, which form the mammalian cerebralcortex. It has become increasingly obvious over the past few years that the variousneuronal CBPs, such as calretinin, calbindin, neurocalcin, and parvalbumins, mayeach uniquely identify neuronal subpopulations across the cortical regions. Subpop-ulations with different CBPs may indeed represent metabolically distinctive cellularsubtypes, and the presence of specific proteins may hold functional implications forthe particular subtypes. These proteins may be playing different calcium-signallingroles in different cell types under different biological parameters.

Parvalbumins are CBPs with three EF-hand domains. Three isoforms of PV maybe distinguished:

α

,

β

, and CPV3.

α

-PV, with its high affinity for Ca

2+

is regardedas a calcium buffer. Furthermore, its distribution is much wider than that of the otherisoforms. PVs occur in a neurone-specific fashion and are used as markers in thedevelopment and differentiation of neurones. A study of the localisation of PV inextraocular neurones has revealed moderate anti-PV immunoreactivity in motorneurones, but their axons show heavy staining, suggesting an intracellular segrega-tion of PV (De La Cruz

et al.

1998). De La Cruz et al. (1998) also state that PV isthe only marker for extraocular motor neurones.

The expression of PV seems to be related to differentiation. PV, among othercalcium binding proteins such as calbindin D-28K and calretinin, shows age-relatedchanges in the developing brain (Kishimoto

et al.

1998; Majak

et al.

1998). It hasbeen reported that rhabdomyosarcoma cells that have been induced to differentiate,not only exhibit characteristic dendritic processes but also show a parallel increasein PV together with small increases in vimentin, desmin and neurone-specific enolase(Pappas

et al.

1996). The regeneration of hair follicle cells, not involving mitosis,is another system in which the differentiation of supporting cells into hair cells hasbeen shown to be accompanied by changes in the expression of CBPs, e.g., calbindinand parvalbumin (Steyger

et al.

1997). Whether there are any tissue specific mech-anisms regulating PV expression is unclear at present. Some preliminary work by


182


Castillo

et al.

(1995) does suggest this. These authors produced transgenic mice toexpress PV under the control of specific promoters. It is of some significance thatPV expression driven by a neurone-specific enolase promoter was at the highestlevel in the brain, but lower levels of expression were detectable in other tissues. Itis also notable that ectopic PV expression in these transgenic mice did not producedevelopmental or behavioural abnormalities.

β

-PV is referred to often as the onco-developmental parvalbumin or oncomodulin(OM). OM occurs in trophoblast cells, preimplantation embryos and neoplasms. Itsdistribution is far more restricted than that of

α

-PV. The

β

-PV isoform known asCBP-15, which has been reported to occur in the guinea pig organ of Corti (innerear) (Thalmann

et al.

1995), is identical to OM with respect to its 30 N-terminalamino acid residues. The identity between OM and CBP-15 has now been confirmedby the demonstration that isoform-specific anti-OM antibodies do cross-react withCBP-15 (Henzl

et al.

1997). The OM gene has been mapped to the long arm ofchromosome 5 of the mouse (Staubli

et al.

1995). The calcium ion affinity of OMis lower when compared to that of

α

-PV. OM may therefore have a calcium-dependent regulatory function.

Some investigators have addressed the question of a putative role for PV in cellproliferation. Blum and Berchtold (1994) have reported the expression of OM mRNAtranscripts as well as the protein increases at the G

1

–S boundary in two neoplasticcell lines (T14 and T10) that they tested. The increase was less marked in the T14line when mitotically synchronised. However, because CaM shows a similar rise atthe G

1

–S interphase, the significance of the rise in the levels of OM expressioncannot be evaluated at present. It may be pointed out, however, that the expressionof CaM and OM appeared to be differentially affected by the levels of extracellularcalcium. When extracellular calcium levels were reduced, CaM expression increasedby up to 60%, whereas OM levels decreased and the effects on both were reversedwhen the extracellular Ca

2+

levels were increased (Klug

et al.

1994). It is noteworthythat Klug

et al.

(1994) also state that the low extracellular calcium and the highCaM levels associated with it appeared to be best suited for cell growth. Andressen

et al.

(1995) transfected PV cDNA into a human ovarian carcinoma cell line butfound a reduced mitotic rate in the cell lines carrying the exogenous PV cDNA.Furthermore, there were distinct alterations in cellular morphology and motility.Needless to say, further investigations into the influences exerted by PV on theseaspects of cellular behaviour seem worthwhile.

CPV3 is an avian PV whose expression seems to be restricted to the thymus(Hapak

et al.

1994a, 1994b). CPV3 has an isoelectric point (pI) of 4.6, which issomewhat lower than the pI of muscle PV. A full-length clone of CPV cDNA has671 bp, and the protein product bears 68% sequence homology to OM. The CPV3molecule contains cysteine residues at positions 18 and 72 and hence can formoligomers by means of disulphide bonds (Hapak

et al.

1994b; Henzl

et al.

1995).


183

16

Osteonectin in Cell Function and Behaviour

MOLECULAR STRUCTURE OF OSTEONECTIN

Osteonectin is a secreted glycoprotein associated with the extracellular matrix. It isalso known by other epithets, such as SPARC (

s

ecreted

p

rotein

a

cidic,

r

ich in

c

ysteine)and BM (basement membrane)-40. The protein is encoded by a single gene (Mason

et al.

1986). Human osteonectin has a predicted molecular size of approximately 32kDa (Swaroop

et al.

1988). Human osteonectin has been cloned by PCR from anendothelial cell (EC) cDNA library, and this soluble biologically active monomerexpressed in

E. coli

has 293 amino acid residues (Bassuk

et al.

1996). Several homo-logues of osteonectin have been isolated, which share a high degree of amino acidsequence homology, with 61 to 65% of the 200-odd residues at the C-terminal regionbeing identical. But they do show great deviation in the secondary structure of the N-terminal region (McKinnon

et al

. 1996; Soderling

et al.

1997). According to McKinnon

et al.

(1996) the mouse homologues possess an exon structure similar to that of theosteonectin gene. Hafner

et al.

(1995) have reported the occurrence of a purine-richstretch in the 5

′

end of the osteonectin gene. This region of bovine and murine genesshows marked sequence similarities, and it contains two GGA boxes with an inter-vening pyrimidine-rich spacer sequence. Hafner

et al.

(1995) also have shown thatthis region contains several regulatory domains. The GGA box 1 of the bovine promoterappears to be sufficient for maximal transcription of the gene, and the pyrimidine-richspacer element seems, in contrast, to down-regulate gene expression. However, theGGA box does not seem to regulate gene expression, as in bovine cells. The transcrip-tional machinery obviously needs much further characterisation, especially to elucidatewhether the regulatory domains described by Hafner

et al.

(1995) contribute to celltype-specific expression of osteonectin.

Osteonectin has a C-terminal EF-hand domain (Yost and Sage, 1993; Maurer

etal.

1995). Maurer

et al

. (1995) have identified two distinct domains (i.e., an acidicdomain and a follistatin like [FC] domain), besides the EF-hand domain. Follistatinis a regulatory protein with multiple functions, notably inhibition of follicle-stimu-lating hormone. Follistatin has a characteristic and highly conserved amino acidsequence motif known as the follistatin domain. This domain has been shown tooccur in a number of proteins, and these have been deemed as forming the follistatinfamily of proteins. Because osteonectin contains a follistatin domain, it has beenregarded as a member of the follistatin family (Maurer

et al.

1995; Phillips and


184


De Kretser, 1998). The EF-hand domain binds calcium with high affinity. Osteonec-tin from

C. elegans

, however, binds calcium with far less affinity as compared withthe mammalian counterpart (Maurer

et al.

1997). Calcium binding results in con-formational changes in the molecule (Bassuk

et al.

1996; Maurer

et al.

1995; Takitaand Kuboki, 1995). The EF-hand domain also mediates the Ca

2+

-dependent interac-tion of osteonectin with cells and the ECM. Other calcium-binding sites, which bindthe cation with far less affinity, are also known to occur (see Table 13).

FUNCTIONS AND FUNCTIONAL DOMAINS OF OSTEONECTIN

Osteonectin is a highly conserved protein and is known to be expressed in a varietyof organisms, from invertebrates to mammals (Damjanovski

et al.

1997;Schwarzbauer and Spencer, 1993; Lane and Sage, 1994). It is not surprising thereforethat, despite its restrictive nomenclature, it participates in a variety of cellularfunctions and cell behaviours (Table 12).

Several individual molecular domains that participate in this physiological pan-oply have been identified with specific functions, by using peptide segments derivedfrom different regions of the osteonectin molecule and antibodies raised againstthese peptides (Jendraschak and Sage, 1996). These domains and their putativefunctions are shown in Table 13.

REGULATION OF OSTEONECTIN EXPRESSION

The expression of osteonectin is regulated by several growth factors that can beclosely identified with specific cellular properties such as cell proliferation, angio-genesis, etc. For instance, TGF

β

induces the expression of osteonectin mRNA andprotein in fibroblasts and other cell types (Wrana

et al.

1991; M.J. Reed

et al.

1994;

TABLE 12Osteonectin in Cell Function and Behaviour

Embryonic differentiation and developmentRemodelling and rebuilding of the ECMAngiogenic and anti-angiogenic functionCell spreadingModulation of cell shapeIntercellular and cell–substratum adhesionCell proliferationTumour development and progression

Source

: Based on references discussed inthe text.



185

Blazejewski

et al.

1997). Also, PDGF, cytokines, and IGF-1 stimulate osteonectinsynthesis and secretion (Chandrasekar

et al.

1994). However, S. Nakamura

et al.

(1996) found that, in rabbit articular chondrocytes, IL-1

β

and -1

α

decreasedosteonectin levels. In fact, IL-1

β

decreased osteonectin mRNA levels as well asglycosylation of the protein. S. Nakamura

et al.

(1996) also have shown that severalother factors, among them TNF-

α

, PMA, and bFGF, can down-regulate osteonectinexpression. The down-regulation of its expression by bFGF might be related to adestabilisation of the osteonectin mRNA (Delany and Canalis, 1998; Delany

et al.

1996). H. Shiba

et al.

(1995) found that bFGF reduced osteonectin synthesis as wellas osteonectin mRNA in dental pulp cells. They suggested that the effects of thegrowth factor on odontoblast differentiation might occur at least in part at somepretranslational stage. In this context, it might be of interest to note that bFGF hasbeen implicated in cell motility, proliferation, and angiogenesis, all features in whichosteonectin is also putatively implicated. Furthermore, different growth factors andcytokines seem to affect the expression of ECM components, such as fibronectin,laminin, and collagens, in a differential manner (Reed

et al.

1994; H. Shiba

et al.

1998). This provides additional support to the view that the effects on osteonectin

TABLE 13Functional Domains of the Osteonectin Molecule

Domain

Amino Acid

Stretch Subdomain Sequence Putative Function

I 3–51 1.1 QTEVAEEIVEEETVVEETGV Low-affinity Ca

2+

binding; inhibition of cell adhesion, spreading; increase of PAI-1 expression

II 52–132 2.1 QNHHCKHGKVCELDESNTP Ca binding; inhibition of EC proliferation; loss of focal adhesion; effect on cell cycle progression

2.3 TLEGTKKGHKLHLDYIG Ca binding; follistatin homology; stimulation of EC proliferation and angiogenesis; plasmin sensitivity

III 132–227 3.2 KNVLVTLYERDEGNNLLTEK Induction of MMP expressionIV 4.2 DLDNDKYIALEEWAGCFG EF-hand domain; inhibition of

cell spreading, proliferation, and focal adhesion; binding to endothelial cells

Note

: Domains III and IV might be involved in binding collagens (see text for references). PAI-1,plasminogen-activator inhibitor; EC, endothelial cell; MMP, matrix metalloproteinases.

Source

: Based on Jendraschak and Sage (1996).


186


produced by the various biological response modifiers might be identifiable withtheir individual responses. It is small wonder, therefore, that osteonectin expressionin cancers has been investigated with some vigour, albeit not with the degree thatcould be deemed appropriate in the context of the multifarious biological effects ofosteonectin.

OSTEONECTIN IN THE REMODELLING OF THE EXTRACELLULAR MATRIX

The significant role that is played by components of the ECM in cell motility andmorphogenesis has been keenly appreciated by the scientific community. A secretedglycoprotein, osteonectin has been a prime candidate for participation in theseprocesses. It was reported to be closely associated with the ECM in several normalas well as abnormal tissues (Dahlback

et al.

1986). Several enzymes that are prom-inently associated with the membrane, refashion and remodel the ECM and in thisway influence cell behaviour. Among these are plasminogen activator (PA) and itsinhibitor PAI, and a host of cathepsins MMPs (see Sherbet and Lakshmi, 1997b forreview) (Figure 24). That osteonectin could be responsible for pericellular proteolysisand ECM remodelling is supported by several lines of evidence. Osteonectin isknown to bind to a number of ECM components, such as collagen types IV and Vand plasminogen (Mayer

et al.

1991; Kelm

et al.

1994; R.L. Xie and Long, 1996).Mayer

et al.

(1991) reported that osteonectin bound mainly collagen type IV, withother collagens (I, III, V, and VI) bound far less competitively. These bindinginteractions may involve specific regions of the molecule, as shown for collagensIV and V binding by osteonectin (Mayer

et al.

1991; R.L. Xie and Long, 1996).Binding to ECM components could be a means of anchoring osteonectin to theECM, so that it can participate in ECM remodelling. It also could serve as a co-factor in the formation of plasmin mediated by tissue plasminogen activator (tPA)(Kelm

et al.

1994). On the other hand, osteonectin can induce the expression ofPAI-1 as it has been shown to do in the case of bovine aortic endothelial cells(Hasselaar

et al.

1991). A possible relationship between the expression of PAI-1 andosteonectin also has been encountered in senescent human diploid cells, where theyappear to be up-regulated in parallel (S. Wang

et al.

1996), although no causal link-up can be suggested on this basis. This would lead to a conservation of ECMcomponents. The proteolytic feedback loop is completed by the demonstration thatplasmin itself is capable of hydrolysing osteonectin in a limited way (Sage

et al.

1984). Tremble

et al.

(1993) found that osteonectin up-regulated the expression ofseveral MMPs, which included MMP1, MMP3, and MMP9, in rabbit synovialfibroblasts. Tremble

et al.

(1993) also identified domains of osteonectin that wereinvolved with the regulation of MMP1 expression. It is unclear if up-regulation ofMMPs alone is sufficient for ECM remodelling. In some systems, MMPs requireactivation. In human glioma U251.3 and fibrosarcoma HT1080, the MMP2 proen-zyme is activated by MMP membrane type 1 (Deryugina

et al.

1998). Whatever thedownstream events may be, MMPs would be expected to refashion the expressionand disposition of a number of ECM proteins. Furthermore, MMPs have been found



187

to produce a limited cleavage of osteonectin. Such cleavage is accompanied by agreatly enhanced binding of osteonectin to collagens I, IV, and V (T. Sasaki

et al.

1997). The involvement of a specific site of cleavage (in helix C of the C-terminalcalcium-binding module) has prompted T. Sasaki

et al.

(1997) to postulate that thisenhanced affinity for collagens might be akin to a process of activation.

A remodelling of the ECM partly mediated by MMPs and their inhibitor TIMPshas recently been shown to be related to the expression of S100A4 in B16 murinemelanoma and human astrocytoma-derived cell lines. The enhancement of invasivebehaviour and metastatic spread, which occurs upon up-regulation of S100A4expression, has been putatively linked with these events associated with the ECM(Merzak

et al.

1994b; Lakshmi

et al.

1997). It is not surprising therefore that, inlight of the ECM changes produced by osteonectin, its role in embryonic develop-ment and differentiation, and other cell membrane-associated phenomena, such ascell adhesion, modulation of cellular morphology, cell proliferation, and woundhealing, should have been investigated. It is also not surprising that such studiesshould be extended to neoplastic development, invasion, and dissemination.

OSTEONECTIN IN EMBRYONIC DEVELOPMENT AND DIFFERENTIATION

Osteonectin is secreted by a variety of tissues and its expression appears to bedevelopmentally regulated. Osteonectin is highly expressed in the germ layers ofthe early mouse embryo, and the modulation of its expression follows a definitepattern in the course of embryogenesis (Holland

et al.

1987; Nomura

et al.

1988;

FIGURE 24

Feedback loop involving serine proteinases and matrix metalloproteinases(MMPs) in the remodelling of the extracellular matrix (ECM) by osteonectin. PAI, plasmi-nogen-activator inhibitor; tPa, tissue plasminogen activator; uPa, urokinase-type plasminogenactivator. (Based on references cited in the text.)


188


Sage

et al

. 1989a). Interference with this temporally controlled activation ofosteonectin results in developmental abnormalities. Thus, when osteonectin antibod-ies are introduced into the blastocoel of

Xenopus

embryos, defective neurulation andorganogenesis occur (Purcell

et al.

1993). Microinjection of specific peptides derivedfrom osteonectin induces abnormalities in the establishment of the embryonic axisin morphogenesis. The injection of osteonectin peptides containing FC domains andcopper-binding sequences did not affect this process, but peptides containing thedisulphide-bonded Ca

2+

-binding domain profoundly inhibited axial formation, lead-ing to ventralisation of the embryos. The disulphide bonding seemed to be essentialfor axial inhibition, because peptides lacking cysteine residues were unable to induceaxial abnormalities (Damjanovski

et al.

1997). However, the recent work of Gilmour

et al.

(1998) suggests that osteonectin deficiency may not damage developmentalprocesses. They disrupted the osteonectin locus in murine embryonic stem cells andshowed that the osteonectin-deficient mice developed normally and were fertile.However, severe abnormalities of the eye, especially cataract formation and ruptureof the lens capsule, developed around the age of 6 months. S.Y. Kim

et al.

(1997)have described the association of osteonectin in the sequence of events occurringduring the development and maturation of chicken retina.

Osteonectin is not only widely present in embryonic tissues, but it is also closelyassociated with organogenesis. In foetal rat lung, osteonectin is associated withepithelial cells of the airways during the pseudoglandular stage of lung morphogen-esis. It is also found in the canalicular and saccular stages of branching morphogen-esis of the lung (Strandjord

et al.

1995). Using an

in vitro

model, Strandjord

et al.

(1995) were able to demonstrate that osteonectin antibodies inhibited the process ofbranching morphogenesis, resulted in the formation of dilated airways. The patternsof expression of bone matrix proteins, including osteonectin, suggest that thesematrix proteins play different biological roles in the development and differentiationof mineralised tissues (Sommer

et al.

1996). Distraction osteogenesis (bone length-ening) has afforded an excellent model that has indicated the involvement ofosteonectin in various stages of differentiation (M. Sato

et al.

1998). In cartilagedifferentiation, tissue transglutaminase catalyses the cross-linking of osteonectin intooligomers

in situ

, and this has been suggested as a major mechanism in the stabili-sation of the cartilage matrix (Aeschlimann

et al.

1995). Glycine residues 3 and 4have been identified as the major amine acceptor sites, of which at least one isconserved in vertebrate osteonectin (Hohenadl

et al.

1995).

MODULATION OF CELLULAR ADHESION, CELL SHAPE, AND MOTILITY BY OSTEONECTIN

It is to be expected that any modulation of the character of the ECM should bereflected in changes in cellular features such as cell–substratum and intercellularadhesion, as well as in changes in cell shape and motility. It was reported severalyears ago that osteonectin inhibited cell spreading and consequently altered cellshape, which indicated that cell–substratum interaction was being inhibited (Sage

et al.

1989b). Subsequently, Lane and Sage (1990) identified the osteonectin domains



189

responsible for this inhibition of spreading to be the N-terminal domain I and asequence occurring in the C-terminal EF-hand domain. They raised antibodiesagainst these peptides and demonstrated that the antibodies blocked the ability ofosteonectin to inhibit cell spreading. Experimental overexpression of osteonectin,produced by transfection, in F9 embryonal carcinoma cells correlated with a roundedcell morphology, whereas transfection of antisense osteonectin cDNA constructswas found to restore cell spreading (Everitt and Sage, 1992). However, transfectionof human osteonectin cDNA into human 293 and HT1080 cell lines has givensomewhat contradictory results. Nischt

et al.

(1991) found no changes in cell spread-ing, proliferative rate, or adhesion behaviour of the transfected cell clones, eventhough they expressed osteonectin mRNA at high levels and secreted large quantitiesof the protein. Osteonectin can induce a loss of focal adhesion of bovine aorticendothelial (BAE) cells, and it appears to achieve this by a reorganisation of thesubmembranous cytoskeleton (Goldblum

et al.

1994; Murphy-Ullrich

et al.

1995).It would be a natural extension of the efforts described above to inquire into the

mechanism by which osteonectin inhibits cell adhesion and spreading. As discussedearlier, osteonectin can alter the structural and functional characteristics of the ECMby inducing proteolytic degradation of its components, and thereby modulatecell–substratum interaction. Some clues are provided for the presence of anothermechanism by the observation that osteonectin brings about changes in the submem-branous cytoskeleton. Extracellular signals may be transduced to the cytoskeleton

via

integrin receptors. Integrins are heterodimeric transmembrane glycoproteins.They are composed of two subunits: the

α

and

β

subunits. A large number of thesesubunits has been identified. They combine to form a vast array of integrins thatfunction as receptors for ECM components such as fibronectin, laminin, tenascin,osteopontin, thrombospondin, and vitronectin, as well as cell adhesion moleculessuch as VCAM and ICAM (Dedhar, 1990). These integrins form a link between theligand and the cytoskeleton.

There are at present no indications that the function of osteonectin is mediatedby integrins, although we do know that it can bind to certain ECM components suchas collagens and vitronectin, which do function through the agency of integrinreceptors. Vitronectin is an adhesion-mediating protein. It is a secreted glycoproteinof predominantly hepatocyte origin. It occurs in the ECM of blood vessels and skinand has also been reported to occur in many tumours (Dahlback

et al.

1986). It bindsto

α

v

β

3

integrin. The expression of this integrin has been associated with the pro-gression of cutaneous tumours and some neuroectodermal tumours (see Sherbet andLakshmi, 1997b). In light of these observations, it is significant that osteonectin caninteract with vitronectin and modulate the adhesive properties of the latter (Rosen-blatt

et al.

1997). Interaction seems to occur between the heparin-binding region ofvitronectin and the C-terminal calcium-binding domain of osteonectin, which isregarded as the region actively involved in the inhibition of cell adhesion andspreading. Rosenblatt

et al.

(1997) have demonstrated also that PAI-1 induces thebinding of osteonectin with vitronectin. As stated previously, osteonectin is able toinduce the expression of PAI. Thus the inhibitor seems to participate in the modu-lation of cell adhesion by two distinct pathways: by a direct route of inhibiting


190


PA-mediated excision and modification of ECM components, and by an indirectroute of inducing the interaction between osteonectin and vitronectin.

Whether other ECM components are similarly targeted by osteonectin largelyremains to be investigated. Some ECM components such as fibronectin andosteopontin can be eliminated. The binding of these to their integrin receptors isdependent on the presence of the RGD (arginine–glycine–aspartic acid) motif, whichappears to function as a recognition signal for the binding of these ligands to theirrespective receptors. Sage (1992) has shown that inhibition of cell spreading byosteonectin is not affected by the RGD peptide. It may be suggested on the basisof this finding that neither fibronectin nor osteopontin enters into any interactionwith osteonectin. However, one cannot yet exclude this possibility, because theadhesion of some cell types, such as osteocytes and osteoblasts, to osteonectin andosteopontin is strongly inhibited by the RGD peptide (Arden

et al.

1996).

MODULATION OF CELL PROLIFERATION BY OSTEONECTIN

Osteonectin can inhibit or stimulate cell proliferation. This faculty appears to dependon the cell type and possibly also on the physiological environment. Osteonectinexpression has been found in some instances to closely correlate with the processesof cell proliferation and differentiation, upon exposure to hormonal or growth factormilieus. The modulation of endothelial cell proliferation by osteonectin, pursuedrather relentlessly by the research group of Sage, is a prime example of its function.Some of their work is discussed below. Osteonectin can inhibit proliferation ofmicrovascular endothelial cells induced by VEGF (Kupprion

et al.

1998). In sharpcontrast with effects on ECs, in dental pulp mesenchymal cells, bFGF enhancesosteonectin expression and also acts as a potent mitogen (H. Shiba

et al.

1995). Inosteoblasts, oestrogen-induced differentiation and inhibition of cell proliferation istotally unrelated to the levels of osteonectin (Robinson

et al.

1997). However, woundhealing, where cell proliferation and migration are important processes, does involveosteonectin. Osteonectin is a component of the alpha granules of platelets, and alongwith thrombospondin it participates in platelet aggregation (Kelm and Mann, 1990;Clezardin

et al.

1991). A high level of osteonectin immunoreactivity has beenobserved in the healing epithelium of the cornea, during a 6-day period followingwounding, and then osteonectin reactivity falls off (Latvala

et al.

1996).As shown in Table 13, two domains have been identified that correspond with

these putative functions. Osteonectin has been shown transiently to inhibit theprogression of vascular endothelial cells at the mid-G

1 phase of the cell cycle. Asimilar inhibition is produced also by the peptide 2.1 (Funk and Sage, 1991). Thisinhibition was not accompanied by a rounding-up of cells. This is not surprising,because rounding-up of cells is a prelude to cell division, and cessation of divisionis usually followed by a phase of cell spreading. Peptides from domain IV containingthe EF-hand motifs produced strong growth inhibition, e.g., peptide 4.2, whichseemed to cooperate with peptide 2.1 (Sage et al. 1995). Funk and Sage (1991)noticed further that the inhibition of cell spreading was produced by peptide 1.1,


Osteonectin in Cell Function and Behaviour 191

which did not inhibit cell cycle progression. However, peptide 2.1 stimulated theproliferation of fibroblasts derived from human foreskin and foetal bovine ligament.This stimulation was strictly concentration dependent and occurred only in the rangeof 0.1 to 0.4 mM of the peptide. Higher levels of the peptide resulted in growthinhibition (Funk and Sage, 1993). Another peptide; i.e., peptide 2.3, stimulated theproliferation of fibroblasts. Overall, osteonectin appears to be able to control theprocess of proliferation in a cell type-specific fashion, in which different domainsof the molecules participate. Although the function of different peptides in vitrocannot directly form the basis for inferring the function of osteonectin in vivo, it ispossible that differential effects on cell proliferation, adhesion, and spreading couldbe seen as an attribute of different configurations of the molecule assumed uponcalcium binding. There will be significant changes in the α-helical content of themolecule in relation to calcium binding. As pointed out by Maurer et al. (1996)conformational changes may occur within a domain. Furthermore, different distantdomains may be linked and thereby alter their orientation relative to one another.One also needs to take into account the posttranslation changes, such as glycosylationand phosphorylation, which profoundly affect biological activity. Glycosylation, forinstance, has a regulatory effect on the binding of osteonectin to collagen V (R.L.Xie and Long, 1995, 1996).

EFFECTS OF OSTEONECTIN ON ANGIOGENESIS

Neovascularisation is a complex process consisting of several events, such as theendothelial cell proliferation and their migration toward the source of the angiogenicfactor. The endothelial cells align themselves end to end to form a sprout, whichthen develops a lumen (reviewed by Sherbet and Lakshmi, 1997b). The currentlyavailable evidence for the induction or inhibition of angiogenesis by osteonectin isstill somewhat indirect and rather scanty. This is based on the observation thatosteonectin influences the proliferation of endothelial cells. Further, certain growthfactors that are known to be angiogenic also enhance osteonectin expression, andfactors that have been shown to be able to inhibit angiogenesis also inhibit osteonec-tin expression. Thus, TGFβ induces both angiogenesis and osteonectin expression,whereas IL-1 inhibits both. Some factors such as PA, PAI, and TIMPs might indi-rectly affect osteonectin expression and also influence angiogenesis. The uncertaintyarises when one considers the effects of factors such as bFGF and TNF. bFGF is aninducer of angiogenesis. However, it down-regulates osteonectin expression in endot-helial cells, although its effect is manifestly the opposite in mesenchymal cells. Asdiscussed above, there is evidence that VEGF, which is demonstrably angiogenic(Zhang H.T. et al. 1995), inhibits osteonectin expression. Similarly, TNF inducesangiogenesis but down-regulates osteonectin. The presence of osteonectin in theECM of vascular structures and its proven ability to induce loss of focal adhesionof endothelial cells, and to reorganise the submembranous cytoskeleton, are clearindications of a positive effect on endothelial cell motility (see Table 14).

Unfortunately, there is very little direct evidence that osteonectin induces angio-genesis. Some findings could be construed as direct evidence. For instance, thepresence of osteonectin in enhanced quantities has been found to accompany the



formation of capillary-like structures by endothelial cells both in vitro and in vivo(Iruela-Arispe et al. 1991a, 1991b; Lane et al. 1994). Mendis et al. (1998) noticedan enhanced expression of osteonectin mRNA in the early stages of the formationof blood vessels following injury to adult rat cerebral cortex. However, one does notknow whether changes in osteonectin levels occur as a consequence of induction ofangiogenesis by some other factor, and whether osteonectin itself is a causative agentor merely an epi-phenomenon. One could concede that, prima facie, osteonectinseems to be involved in the regulation of angiogenic processes, but there is an acuteneed for more substantive data, such as, for instance, by transfecting osteonectininto appropriate recipient cancer cells and determining if the expression altersmicrovascular density. Technically this could be demanding, notably becauseosteonectin contains domains with both angiogenic and anti-angiogenic properties,but it is certainly an achievable goal.

TABLE 14Correlation between Angiogenesis and Modulation of Osteonectin Expression by Growth Factors and ECM Components

Effector Agent Effect on Angiogenesisa

Effect on Osteonectin Status/Effect of Osteonectin

on Effector Agent

TGFβ + +PA + +2b

IL-1 – –TIMPs –c –b

Thrombospondin – –PAI – –VEGF – –b

bFGF + –d

TNF + –

Note: The table provides a summary of the evidence that links, directly or indirectly,angiogenesis with osteonectin expression. Although osteonectin contains domains thatputatively possess both angiogenic and anti-angiogenic properties, the table, con-structed on the basis of work discussed in the text, underlines the need for moredefinitive experiments in order to establish whether osteonectin is involved in theregulation of angiogenesis.

a +, up-regulation; –, down-regulation of expression.b Osteonectin influences the expression of the effector agent.c TIMPs 1–4 are known to inhibit angiogenesis (Sherbet and Lakshmi, 1997b; Blavieret al. 1999).d bFGF is believed to up-regulate osteonectin in mesenchymal cells.



OSTEONECTIN EXPRESSION IN CANCER DEVELOPMENT AND PROGRESSION

Osteonectin seems to be able to influence most of the biological properties of thecell that are highly relevant in the context of cancer development, growth, anddissemination. The most obvious reasons for this are that osteonectin is able toinitiate changes in the ECM, cell adhesion and spreading, and also in the cytoskel-eton. It has been found to influence cell proliferation, and apparently, it can regulatevascularisation. The elucidation of these properties has inevitably led to the studyof its expression in cancer development and progression.

Osteonectin reportedly occurs in a wide spectrum of cancerous tissues fromhuman subjects. Porter et al. (1995) found high osteonectin immunoreactivity ininvasive tumours of the GI tract, breast, lung, kidney, ovary, brain, and adrenal cortex.They state that normal tissues show low levels of reactivity. It is somewhat para-doxical that trophoblast cells, which are a highly invasive cell component of theplacenta, show low levels of reactivity. Nevertheless, bone extracts and osteonectinitself have been found to enhance the motility in vitro of prostate epithelial cells aswell as prostate cancer cells (Jacob et al. 1999). The presence of osteonectin hasalso been demonstrated in normal as well as adenoma of human thyroid (Burgi-Saville et al. 1997). However, there is a reasonable body of evidence that suggestsa close association of osteonectin expression with the progression of human mela-nomas. Osteonectin is not expressed by normal melanocytes and it is weaklyexpressed in a small proportion (4/25) of nevocellular nevi. The level of osteonectinexpression is moderate in a majority (13/14) of dysplastic nevi. However, the expres-sion occurs invariably and at a very high level in both primary (7/7) and metastatic(29/29) melanomas (Ledda et al. 1997).

A few studies have also been carried out on the relevance of osteonectin as amarker for the progression of breast cancer. According to Bellahcene and Castronovo(1995), osteonectin is only weakly expressed in benign breast disease, but theexpression is very strong in both in situ and invasive carcinomas. The presence ofoestrogen receptors in breast cancer is regarded as an indicator of differentiationand good prognosis, and their absence is suggestive of clinically aggressive disease.Apparently, there is an inverse relationship between ER status and osteonectinexpression. Tumour samples that were low in ER content tended to contain four-fold higher levels of osteonectin mRNA as compared with tumours with high ERlevels (Graham et al. 1997). It would be of much interest, in this context, to examinewhether osteonectin expression relates directly to the presence of EGFr, becauseER-negative breast cancers often tend to be EGFr positive. There is thus an apparentrelationship of osteonectin levels to disease progression. This has been confirmedin another study, in which Podhajcer et al. (1996) found that the osteonectin geneis expressed at a high level in invasive human breast carcinoma and also in metastaticlymph nodes. Osteonectin transcripts were found in the fibroblast stroma. Further-more, high levels of expression of stromelysin-3 also accompanied high osteonectinexpression. Osteonectin seems to be able to activate MMP2 in the invasive breastcancer cell lines MDA-MB231 and BT549, but not in the noninvasive MCF7 cells.



This ability was associated with the osteonectin peptide 1.1 (Gilles et al. 1998).Although Gilles et al. (1998) also found some attenuation of TIMP-2, they havesuggested that the invasive propensity of the breast cancer cell lines is most likelyattributable to osteonectin-mediated activation of MMP2.

There can be little doubt that metalloproteinase expression and the expressionof their inhibitors corresponds closely with invasive potential of cancers (see Sherbetand Lakshmi, 1997b). The apparent relationship between osteonectin and MMP doesnot necessarily reflect a causal connection. There is the distinct possibility that acommon effector, such as bFGF, may regulate both. Nonetheless, the outcome couldbe an alteration in the invasive nature of the tumour.

Bellahcene and Castronovo (1995) have stated also that high expression ofosteonectin was associated with calcification of the lesions. However, contrary tothe findings of Bellahcene and Castronovo (1995), Hirota et al. (1995) reported nocorrelation between osteonectin expression and the development of foci of calcifi-cation. It ought to be stated, nonetheless, that in other cellular systems a relationshipdoes seem to subsist between osteonectin levels and calcification. Human dentalpulp cells maintained in tissue culture express osteonectin, and they also containcalcified nodules. The level of osteonectin closely correlates with the level of cal-cification. When these cells are treated with bFGF, osteonectin expression is reducedand calcification of the ECM is abolished (H. Shiba et al. 1995). M. Sato et al.(1997) used a human salivary cancer cell line and have reached similar conclusions.These cells produce tumours when implanted into nude mice. When treated withVD3, tumour growth rate was reduced and calcified foci appeared in the tumours.In parallel, M. Sato et al. (1997) also found the expression of osteonectin mRNAin these treated tumours.

Whether the expression of osteonectin, together with other bone matrix compo-nents such as osteopontin and bone sialoprotein, could have some bearing on thepropensity of breast tumours to metastasise to the bone, is currently being debated.Osteopontin has been implicated in tumour cell motility (Xuan et al. 1995; Sung etal. 1998). Oates et al. (1996, 1997) transfected Rama-37, a rat mammary epithelialcell line, with genomic DNA fragments from a human mammary carcinoma cellline. The transfectants were found to produce tumours with metastasising ability.They then isolated a cell line from a metastatic tumour and compared its mRNAprofile with a control cell line, by subtractive hybridisation. One of the mRNAsstrongly expressed, (nine-fold greater in the metastatic cell line as compared withthe nonmetastatic parent line), was that for osteopontin. An increase in the level ofexpression does not constitute irrefutable evidence of a relationship to metastaticability. Oates et al. (1996) did show that similar transfection of Rama-37 cells withDNA from benign tumours did not result in elevated expression of osteopontin.Some of these early studies have been confirmed recently. The levels of osteopontinhave been found to be low in nontumorigenic cells and tumour cells with lowmetastatic ability. The osteopontin-transfectant cells as well as cells exposed toexogenous osteopontin have been reported to make marked gains in invasive ability(Tuck et al. 1999). What is even more interesting is the observation by these authorsthat, under both experimental conditions, the gain in invasive ability was accompa-nied by increases in the expression of uPA mRNA as well as the uPA protein. This



has marked similarities with the effects of osteonectin on cell invasion. One shouldrecognise, nonetheless, that VD3 can act independently of osteopontin, becausealthough it can enhance the expression of both osteonectin and osteopontin, VD3indeed inhibits cell proliferation and invasion. Its ability to inhibit invasion can bedirectly linked with the down-regulation of PAs and MMPs together with an up-regulation of the endogenous MMP inhibitors. These arguments simply emphasisethe need to include osteopontin in studies of osteonectin involvement in cancer cellinvasion and metastasis.

It would be relevant to cite here the observations of Jacob et al. (1999), whofound that bone extracts as well as osteonectin enhanced the motility of tumour cellsthat normally metastasised to the bone, e.g., breast and prostate cancer. However,cell lines derived from tumours that do not normally metastasise to the bone did notrespond in this way. Jacob et al. (1999) postulate, in consonance with the discussionabove, that this apparent differential effect of osteonectin on cell motility could bedue to the ability of osteonectin to induce tumour-associated metalloproteinaseactivity. An activation of uPA together with enhanced proliferative potential has beenreported in MCF7 cells exposed to soluble factors secreted by the osteogenic cellline SaOS-2. MCF7 cells are normally only weakly invasive, but they appeared tobecome more invasive when cultured on an ECM produced by SaOS-2 cells. Fur-thermore, this acquisition of invasive potential seemed to be related to the ability ofthe ECM to induce uPA expression in MCF7 cells (Martinez et al. 1999). Althoughthese findings are of considerable significance in the context of cancer invasion, itis necessary to take into account a number of related facts and factors, lest one beled into an alley of overinterpretation of the data. It should be recognised that bothMCF7 and MDA cells do synthesise uPA, although MCF7 cells do so at a far lowerlevel than do MDA-MB231 cells. It should be recognised also that ability to syn-thesise uPA is not itself directly related to the invasive ability of cancer cells.Urokinase receptors as well as PAIs enter into the equation. Undoubtedly there is alarge body of correlative evidence derived from the study of plasminogen activatorexpression in a host of human tumours. Nonetheless, all potential interacting factorsneed to be checked before one can be certain that one is, indeed, dealing with uPAas the major instigator of invasive potential.

Martinez et al. (1999) state that the enhancement of invasive behaviour wasfound only in the ER-positive MCF7 cells, but not in the ER-negative MDA-MB231cells. This is consistent with the experiments described by Hachiya et al. (1995),who found that oestrogen enhanced uPA as well as tPA expression and also enhancedthe invasive ability of breast cancer cells. They also demonstrated that PA expressionis regulated by oestrogen, because tamoxifen blocked the production of PA and alsoinhibited the invasive ability. Among other factors that come into the reckoning isthe hepatocyte growth factor (HGF). HGF bears sequence homology to PA. PA isknown to activate HGF (Mars et al. 1993). Furthermore, HGF is a potent mitogenand can induce angiogenesis as well as vascular invasion (Hildebrand et al. 1995).It would be worth recalling here that the ER status correlates inversely with EGFrin these cell lines. EGF is another modulator of the invasive behaviour of cancercells. Whether the changes in the invasive behaviour might have been mediated byEGF receptors is a moot point.



As stated previously in this section, osteonectin expression is said to be inverselyrelated to ER status, with high expression of osteonectin being found in ER-negativecells. Furthermore, it also has been claimed that osteonectin stimulates MMP expres-sion in MDA cells, but not in MCF7 cells. It may be that uPA is more relevant inthe context of breast cancer cell invasion than are MMPs. Overall, there is a rea-sonable body of evidence to suggest the ectopic expression of these bone matrixproteins might have serious implications for the osteotropic metastasis of breastcancer. However, the mechanisms involved remain to be elucidated.

Although the above discussion suggests a direct relationship between malig-nancy and levels of osteonectin expression, in ovarian epithelial cells an inverserelationship has been reported. Mok et al. (1996) found high osteonectin expressionin normal ovarian epithelial cells, but this was markedly reduced in ovarian carci-noma cells. They also transfected osteonectin cDNA into SKOV3 cell, which is anovarian carcinoma cell line. This resulted in reduced growth rate in vitro, andfurthermore, these cells were less tumorigenic when implanted into nude mice. Theseresults suggest and impute a tumour suppressor property to osteonectin. This issupported by recent work by Vial and Castellazzi (2000). The expression of osteonec-tin is down-regulated when cells are transformed by oncogenes. Such a down-regulation is noticed in chick embryo fibroblasts transformed by v-src or v-junoncoproteins. When the protein is reexpressed, these cells retain the transformedphenotype but lose their ability to form fibrosarcomas in vivo (Vial and Castellazzi,2000). In other words, in the experimental model osteonectin does seem to behavelike a tumour suppressor. Nonetheless, it would be reasonable to expect furtherconfirmation of the possible tumour suppressor function. Some of these uncertaintiesare compounded by the view expressed in some quarters that osteonectin might notbe a reliable marker for osteosarcoma (Grundmann et al. 1995; Park et al. 1996).

An overall view of the present status of osteonectin in relation to cancer pro-gression ought to be ambivalent. There is a need for far more extensive investigationof human tumour types. Above all, much more experimental work is needed toestablish the various postulates that bring together the putative functions of osteonec-tin with the altered biological properties of the cancer cell. In a sense, therefore, itwould be premature to dive into investigations of clinical material without appro-priate groundwork. This is especially important with respect to osteonectin function,because the protein contains domains that reputedly possess antagonistic functions.The scientific community has not even begun to unravel the mechanisms by whichthese antagonistic functions become expressed in the physiological setting. Equally,it would be unreasonable to delay the investigation of the relevance of osteonectinin tumour classification, or its potential in clinical management of patients, if indeedit has even the semblance of predictive value.

OSTEONECTIN INVOLVEMENT IN OTHER DISEASE STATES

A number of nonneoplastic diseases have been linked with osteonectin. Among theseare rheumatoid arthritis (RA) and osteoarthritis (OA). Immunostaining studies of



cartilage and synovial specimens from patients with RA and OA have been reported(S. Nakamura et al. 1996; Nanba et al. 1997). Osteonectin immunoreactivity is foundin chondrocytes in the superficial and middle zones of the cartilage from RA andOA joints, but these zones of normal cartilage are either devoid of any activity orstain only too weakly. Osteonectin expression is greatly increased in synovial cellsfrom these patients. S. Nakamura et al. (1996) have also reported that, on average,the osteonectin content of synovial fluid from RA patients was ten-fold greater thanthat of synovial fluid from OA patients. Nanba et al. (1997) have made the interestingand valuable observation that osteonectin is detected in the early stages of OA, andthey point out that this could be significant in terms of disease progression, becausecartilage calcification is closely related to the stage of the disease.

Liver fibrosis appears to be associated with enhanced levels of osteonectin.According Inagaki et al. (1996), osteonectin is virtually undetectable in normal liver,but osteonectin gene transcripts were abundantly expressed, mainly in the lipocytes,which markedly increase in number in fibrosis. Similar increases in osteonectinlevels have been described by Blazejewski et al. (1997). These authors reported thatcultured myofibroblasts from liver synthesised osteonectin. They further observedthat in normal liver myofibroblasts occured in small numbers, but that they prolif-erated markedly in fibrosis and synthesise ECM proteins. Consistent with this,osteonectin mRNA expression was low in normal liver but was markedly enhancedin fibrotic liver.

Osteonectin expression is reported to decrease at the early stages of diabetes-related renal enlargement in experimental animals (Gilbert et al. 1995). As notedby Gilbert et al. (1995), renal enlargement is a mixture of several events occurringin the ECM along with cell hypertrophy and hyperplasia, and one can see howosteonectin might be implicated in the pathogenesis of the disease.

OSTEONECTIN HOMOLOGUES AND THEIR PUTATIVE TUMOUR SUPPRESSOR PROPERTIES

Osteonectin is probably a typical member of a large family of proteins whosemembers share several important structural features and perform similar functions.In recent years several cDNAs have been isolated that show a high degree ofstructural homology to osteonectin. Hevin (MAST9, SC1), QR1, testin, andtsc36/FRP are members of the osteonectin family. Hevin was first identified inendothelial venules of lymphoid tissue (Girard and Springer, 1995, 1996). The hevingene is located on chromosome 1 (Claeskens et al. 2000). The hevin cDNA encodesa protein that shows a substantial sequence homology to osteonectin, SC1 (of murineorigin), and QR1 isolated from quail (Girard and Springer, 1995). Monomeric hevinis 75 kDa in size and possibly forms dimers in vitro. Bendik et al. (1998) havecharacterised the full-length cDNA, which has 2808 nucleotides and an ORF of1992 nucleotides corresponding to a 75-kDa protein. Overall, all osteonectin homo-logues identified thus far show a molecular organisation similar to that of osteonectin,and, in particular, possess the characteristic FC domain and EF-hand motifs in thecalcium-binding domain.



Hevin is expressed ubiquitously in most normal tissues and in diseased tissuesof nonneoplastic origin. There may be differences in the patterns of their distributionin normal tissues, as demonstrated for SC1 (Soderling et al. 1997). The expressionof hevin has been reported to be down-regulated in many neoplasms, e.g., in non-small cell lung carcinome (NSCLC) (Bendik et al. 1998) and adenocarcinoma ofthe prostate (P.S. Nelson et al. 1998). Claeskens et al. (2000) transfected hevincDNA into HeLa-35 cells, which do not express indigenous hevin. The transfectedhevin cDNA negatively regulated proliferation and seemed to block G1–S transitionof cells. Therefore, Claeskens et al. (2000) suggest that hevin may be a putativesuppressor gene. However, it might be premature to label a gene as a suppressorgene merely on the basis of possible inhibition of cell proliferation. It ought to bestated, in defence of the postulate, that hevin does possess anti-adhesion properties,can inhibit focal adhesion of cells and confers a rounded morphology on cells (Girardand Springer, 1996). Needless to say, that this area is worthy of pursuit and shouldprovide interesting results relating to the relevance of these molecules in cell migra-tion, diapedesis, and cancer dissemination.


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17

S100 Proteins: Their Biological Function and Role in Pathogenesis

There has been an unremitting effort by the scientific community in the past twodecades aimed at identifying the genetic determinants of the invasive and metastasisingproperties of cancer cells. This has been based on the premise that the acquisition ofthese seemingly aberrant abilities flows from the expression of a candidate gene or agroup of genes that might be connected with the process of metastasis. However, onecan muster very little direct evidence for the “metastasis gene” concept. Indeed, areview of recent research would suggest that alterations of the proliferative status,motility, and cell–cell and cell–substratum adhesion of cells, may be seen as the basicrequirement for cancer growth, invasion, and metastasis, as well as other nearly anal-ogous processes such as cell differentiation, growth, and morphogenesis. Quite obvi-ously, therefore, a vast array of genes can be identified whose expression profoundlyaffects cancer cell behaviour as well as cell differentiation, growth, and morphogenesis(Sherbet and Lakshmi, 1997b). It can be argued that neoplastic transformation of cells,accompanied by the acquisition of enhanced proliferative potential, motility, and het-erotypic adhesion properties, can be attributed to an overexpression or inappropriateexpression of genes that are associated with extracellular signal transduction, and thoserequired in normal physiological function.

The S100 proteins, which form the main focus of this section, have been shownto be capable of modulating enzyme function and altering cytoskeletal dynamics.They can bind to a variety of cellular target proteins, and possibly by this meanscontrol cell cycle progression. Furthermore, much evidence has accumulated thatshows these proteins are associated with terminal cell differentiation; they canpromote remodelling of the ECM, alter cell shape, motility, and enhance the invasivebehaviour, and metastatic spread of cancer. Here we attempt to link the physical andphysiological alterations occurring in a cell consequent to overexpression of S100proteins with the salient phenotypic aspects of biological behaviour of cells.

The S100 family of proteins comprises a large number of CBPs. As can be seenin Table 15, a majority of them are found in a 2.05-Mbp segment of human genomicDNA of chromosome 1q21 region (Marenholz

et al.

1996; Mischke

et al.

1996; Schafer

et al.

1995). The S100 family of genes, including those coding for profilaggrin (FLG)

0942/C17-A/frame Page 199 Wednesday, October 25, 2000 7:30 AM

200


TABLE 15S100 Family of Calcium-Binding Proteins: Their Chromosomal Location and Putative Physiological Function

S100 NomenclatureChromosomal

Location (Human)Putative Physiological Function and in

Pathogenesis

S100A1 (S100

α

1) 1q21 (Dorin

et al.

1990; Engelkamp

et al.

1993; Schafer

et al.

1995)

Associated, together with S100B, with cardiomyopathy; differentially modulated in tissues in experimental diabetes (Zimmer

et al.

1997); see text

S100A2/S100L 1q21 (Engelkamp

et al.

1993)Putatively associated with tumour suppression; see text

S100A3/S100E 1q21 Specifically expressed in the skin; participation in the differentiation of hair follicles; found in glial tumours

S100A4 (18A2/mts1, CAPL, p9Ka, metastasin, calvasculin, FSP1)

1q21 See text

S100A5/S100D 1q21S100A6 (calcyclin, 2A9, CACY, caltropin)

1q21 Associated with acute myeloid leukaemia (Calabretta

et al.

1986a,b) and melanomas; see textS100A7 (psoriasin) 1q21 (Hardas

et al.

1996)

Psoriatic skin and other skin diseases; regarded as a potential marker for squamous cell carcinoma of the bladder; see text

S100A8 (MRP8)

a

S100A9 (MRP14)

a

1q21 Together with MRP-14, associated with cystic fibrosis, rheumatoid arthritis (Odink

et al.

1987; Fanjul

et al.

1995); inflammatory bowel disease, allograft rejection, recruitment of neutrophils and monocytes to delayed-type hypersensitivity inflammatory sites (Dunn

et al.

1996); might be involved in the regulation of the inflammatory process, transendothelial migration of monocytes (Kerkhoff

et al

1991a, 1999b)S100A10 (p11, calpactin light chain, 42C)

S100A11 (S100C) 1q21 (Mooglutz

et al.

1995; Wicki

et al.

1996a)

Differentially expressed in uveal melanomas and cell lines derived from them (Van Ginkel

et al.

1998).

S100A12 (calgranulin C, P6, CGRP, CAAF1)

1q21 (Wicki

et al.

1996a; Yamamura

et al.

1996)

Gene with 3 coding exons; exons 2 and 3 encode peptide of 92 amino acid residues (Wicki

et al.

1996a, Yamamura

et al.

1996a); 2 EF-hand domains; isolated from neutrophils, bovine amniotic fluid (Guignard

et al.

1995; Hitomi

et al.

1996, 1998); also found in foetal epidermal keratinocytes, squamous epithelia; oesophageal epithelium; differentiation-related expression, but no known association with cell proliferation; associated with Mooren’s ulcer



201

and trichohyalin (THH), which are associated with terminal differentiation of kerati-nocytes, are arranged in the following order: 1 cen–S100A10–S100A11–THH–FLG–IVL (involucrin)–LOR (loricrin)–S100A9–S100A12–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A13–S100A1–1qtel.S100B is an exception in that it occurs on chromosome 21q22 (Duncan

et al.

1989).

TABLE 15 (CONTINUED)S100 Family of Calcium-Binding Proteins: Their Chromosomal Location and Putative Physiological Function

S100 NomenclatureChromosomal

Location (Human)Putative Physiological Function and in

Pathogenesis

CAAF2 2 EF-hand domains; ca. 30% sequence homology with CAAF1; 63% homology with S100A7 (Hitomi

et al.

1996)

S100A13 1q21 (Wicki

et al.

1996b)

ca. 50% homology to S100A5 and ca. 60% to S100A12; mouse A13 shows ca. 87% homology to human A13; mRNAs expressed at high levels in skeletal muscle, heart, kidney, ovary, pancreas (Wicki

et al.

1996b)S100B/S100

β

21q22 (Duncan

et al.

1989)Associated with Alzheimer’s disease; Down’s syndrome, neurite extension factor; occurs in glial and Schwann cells, cytoskeletal disruption; possibly influences cell cycle progression via activation of Ndr protein kinase

S100P 4p16 (Schafer

et al.

1995)

Isolated from human placenta (Becker

et al.

1992; Emoto

et al.

1992); cutaneous sensory signal transduction; progression of prostate cancer; see text

FLG (profilaggrin) 1q21 (Marenholz

et al.

1996; Mischke

et al.

1996)

Filaggrin IF monomers, aggregation of filaments in keratinocyte differentiation; reduced expression in and possible aberrant interaction with keratin filaments in ichthyosis; rheumatoid arthritis

Trichohyalin (THH) 1q21 (Fietz

et al.

1992)

Repetin (rptn) (mouse) Mouse chromosome 3 (Krief

et al.

1997)

Overexpression in mouse skin tumours (Krief, P., personal communication)

Calbindin D-9K (intestinal calcium-binding protein)

Xp22.2

a

MRP8 and 14 are regarded as myeloid-specific proteins. Shapiro

et al.

(1999) have identified a B-cell-specific antigen, which appears to possess N-terminal sequence homology with human MRP8. The antigenalso shows homology to other S100 proteins too, albeit at a far lower level. Shapiro

et al.

(1999) haveargued, on rather tenuous grounds, that because MRP8 is not expressed in lymphocytes, this antigenmight represent a new member of the S100 family.

Source

: Data collated from Heizmann (1996), Sherbet and Lakshmi (1997b), and references cited in thetable and the text.


202


A similar clustering and synteny of S100 genes is found in mouse chromosome 3, andthe organisation shows a great resemblance to that of the human S100 gene cluster.Eight mouse S100 genes are organised in the same way as the human genes. Thelinkage between S100A8 and S100A9 and the linkage of the arrayS100A3–S100A4–S100A5–S100A6 are conserved. However, S100A1 and S100A13are separated, unlike as in the human gene cluster. Therefore, much of the organisationseems to be conserved in evolution, with a degree of rearrangement of some of thegenes (Ridinger

et al.

1998).S100 proteins are in general of a small molecular size. Although some of them,

e.g., S100B(

β

) and S100A(

α

), have been known for a long time and much infor-mation has accumulated over the years, the function of the majority is still largelyunknown. There have been significant advances in recent years in our understandingof the involvement of some S100 proteins in normal and aberrant physiology.Especially noteworthy are the close association some of these proteins show in theabnormal behaviour of cancer cells, and the effects of their elevated expression intumours and the consequent modulation of cytoskeletal dynamics, cell mobility, andcell proliferation.

S100 PROTEINS IN CELL DIFFERENTIATION, MOTILITY, AND CANCER INVASION

P

ROFILAGGRIN

(FLG)

IN

K

ERATINOCYTE

D

IFFERENTIATION

The consortium of genes at the 1q21 region, whose physical mapping has beenreferred to above, includes a number of genes associated with the terminal differ-entiation of human epidermis. This collection of genes has been called epidermaldifferentiation complex (EDC) (Mischke

et al.

1996). This complex has been mostlucidly described by Marenholz

et al.

(1996) as being composed of three groups ofgenes: (1) structural genes such as IVL, LOR, and others; (2) genes coding for theIF-associated proteins FLG and THH; and (3) S100A1–S100A10.

FLG and THH are synthesised in the granular layer of the epidermis and par-ticipate in the aggregation and aligning of keratin IF during the terminal differenti-ation of keratinocytes. Both proteins contain EF-hand calcium-binding domains andare functionally associated with Ca

2+

-dependent processing (see below). Krieg

et al.

(1997) have cloned a novel gene, the repetin gene, from the mouse. This gene hasmarked similarities to FLG and THH with respect to genomic organisation and EF-hand calcium-binding domain characteristics. Repetin, together with FLG and THH,could represent a subfamily of S100 genes (Table 16).

The Molecular Characteristics of Profilaggrin

Human FLG is a 400-kDa phosphoprotein that is found in the granular cells of theepidermis. Its N-terminal region contains two calcium-binding motifs similar to theEF-hand calcium-binding domains found in the S100 family of proteins. The twocalcium-binding sites differ markedly with respect to their calcium-binding affinity(Presland

et al.

1995). The FLG molecule consists of several units of filaggrin linked



203

together. The molecule undergoes proteolytic cleavage at the sites of linkage duringterminal differentiation, generating monomeric filaggrin units with an averagemolecular size of 42 kDa. The filaggrin units also acquire specific C- and N-terminalsequences as a result of this proteolytic processing (Resing

et al.

1993a; Thulin andWalsh, 1995). The processing of FLG is a two-stage process that generates inter-mediate products with filaggrin repeats. An endopeptidase, FLG endopeptidase 1(PEP1), involved at this first stage of the cleavage of the linkers has been identified.The functional specificity of this enzyme has been suggested to be due to phospho-rylation of FLG (Resing

et al.

1995). PEP1 is a serine protease. Also involved inthis proteolytic processing are leupeptin-sensitive cysteine protease and proteinphosphatases. The former has features resembling calpain (Resing

et al.

1993b,1995). The calpains are Ca

2+

-activated cysteine proteases. Yamazaki

et al.

(1997)have shown that

µ

-calpain is capable of processing FLG (Figure 25).

TABLE 16Genomic and Molecular Features of Profilaggrin, Trichohyalin and Repetin

Feature FLG THH rptn

Exons/introns 3 exons, 2 introns 3 exons, 2 intronsEF-hands Two N-terminal exons I

and IITwo N-terminal Two N-terminal exons I

and IIRepeat elements Filaggrin units Involucrin-like Involucrin-like

Source:

Based on Markova

et al.

(1993); S.C. Lee

et al.

(1993); Presland

et al.

(1995); Krieg

et al.

(1997).

FIGURE 25

The generation of filaggrin monomers from profilaggrin, with enzymaticcleavage of linking peptides occurring between filaggrin repeats. One of the enzymes impli-cated in this process is

µ

-calpain. This enzyme is made up of a large 80-kDa subunit and a30-kDa subunit. Calpain is activated by the binding of calcium to CaM-like domains thatoccur in both subunits. (Based on Resing

et al.

1993a, 1993b, 1995; Saido

et al.

1993;Yamazaki

et al.

1997; Kam

et al.

1997).

0942/C17-A/frame Page 203 Friday, October 27, 2000 9:26 AM

204


The expression of FLG is markedly reduced in ichthyosis. This has been attrib-uted to its regulation at the posttranscriptional level. A decimation of FLG levels inkeratinocytes from ichthyosis-affected patients has been reported, but the decreasein FLG mRNA expression amounted to only 30 to 60% of controls (Nirunsuksiri

etal.

1995). Another reason for this could be the stability of the gene transcript. TheFLG transcripts in keratinocytes of ichthyosis subjects were found to be far moreunstable and possessed a shorter half-life than the transcripts in normal keratinocytes(Nirunsuksiri

et al.

1998).The normal function of FLG might also depend on the state of its phosphory-

lation. It has been proposed that phosphorylation may prevent FLG from causingpremature aggregation of keratin filaments and their packaging into storage granules.It has been suggested also that phosphorylation might be involved in the proteolyticprocessing of FLG into filaggrin monomers (see Figure 25). The conversion of thehighly phosphorylated FLG into nonphosphorylated filaggrin appears to depend onprotein phosphatases PP-2A and PP-1. Keratinocytes show a high level of PP-2Aand PP-1 activity, but in harlequin ichthyosis, the activity of these phosphatases isgreatly reduced, with a resultant disruption of filaggrin generation (Kam

et al.

1997).It has been reported that PKC-

δ

can mediate FLG phosphorylation. This, togetherwith the solubilisation of FLG, indicates that phosphorylation may be involved inthe generation of filaggrin monomers (Old

et al.

1997).Filaggrin monomers are formed during terminal differentiation of keratinocytes.

A major process occurring at this stage of differentiation is the aggregation of keratinfilaments. This occurs in association with FLG monomers. It is believed that profil-aggrin itself is unable to accomplish this. Haydock

et al.

(1993) transfected antisenseFLG constructs into rat epidermal cell lines. In transfectants expressing the antisensemRNA, the processing of FLG was suppressed, and the differentiation

in vitro

ofkeratinocytes was markedly affected as a consequence. The generation of filaggrinalso produces marked changes in cell morphology and affects nuclear integrity. Celladhesion to substratum also appears to be affected (Dale

et al.

1997). FLG occursin keratohyalin granules, and a recent study by Dale

et al.

(1997) suggests that theinsoluble native form of FLG is required in their formation, which indicates that thelinker peptides have a role to play in this process.

Profilaggrin and filaggrin might be involved in the pathobiology of rheumatoidarthritis (RA). They show a distribution pattern similar to the antigens recognisedby antibodies that are used as markers for RA. These so-called anti-keratin antibodieshave been shown to recognise epidermal filaggrin and are demonstrably anti-filaggrinantibodies (Simon

et al.

1995). Antibodies against filaggrin have been employed asa diagnostic aid, and their use might complement that of anti-keratin antibodies indiagnosing RA (Vincent

et al.

1998). The possibility has been envisaged that thepathogenesis of RA could involve a loss of immunological tolerance to filaggrin(Berthelot

et al.

1995).Abnormal interaction between keratin filaments and filaggrin may be involved

in some cases of epidermolytic ichthyosis. Here, although profilaggrin/filaggrin areexpressed at an enhanced level, no abnormalities of this protein have been encoun-tered (Ishida-Yamamoto

et al.

1994). Abnormal expression of FLG as well as K6and K16 filaments is found in keratinocytes from the congenital condition of



205

harlequin ichthyosis. FLG expression is either severely down-regulated or totallyundetectable in ichthyosis vulgaris. The possible reasons for this have been discussedabove.

T

RICHOHYALIN

(THH)

Trichohyalin is another protein that is associated with IF. THH is a 248-kDa proteinthat is found in trichohyalin granules and possesses two EF-hand-like calcium-binding domains at its N-terminal end. The protein could occur as a flexible rodapproximately 215 nm in length (S.C. Lee

et al.

1993). It associates with and formsregular and precise arrays of keratin IF in the medulla and inner root sheath cellsof hair follicles (Oguin

et al.

1992). THH appears to undergo specific sequentialposttranslational modifications to produce highly cross-linked rigid filaments. Tarcsa

et al.

(1997) have proposed that the insoluble cytoplasmic THH is first modified bypeptidyl arginase to convert it into a less organised and soluble molecule. TheseTHH molecules are then cross-linked by the agency of Ca

2+

-dependent transglutam-inases to generate rigid filaments that interact with keratin filaments. THH is alsofound in nonfollicular epithelia, e.g., filiform papillae of tongue epithelium (E.J.O’Keefe

et al.

1993). It occurs, together with FLG, in epithelia that possess IFcontaining K6/K16 keratins, which has led to the suggestion that THH may specif-ically mediate aggregation of IF containing these keratins (Manabe and Oguin,1994).

The association of THH with a possible differentiation pathway has promptedstudies into THH expression in skin tumours (Manabe

et al.

1996). Preliminaryfindings also are available with regard to the possible involvement of repetin (rptn)in the progression of skin tumours in the mouse (P. Krieg, personal communication,1998). Apparently, rptn mRNA is expressed at low levels in normal mouse epidermis.Treatment with TPA, which is a tumour promoter, results in a transient overexpres-sion of the mRNA. In benign tumours this overexpression becomes constitutive.Benign papillomas show far higher levels of rptn mRNA than normal epidermis.During progression into carcinomas, the steady-state levels of rptn mRNA arereduced. Compatible with this, in a few squamous cell carcinomas investigated, rptnmRNA has been found to occurs at lower levels as compared with papillomas. Thereis a hint of the possibility that rptn gene expression may be involved as an earlyevent in the progression of skin tumours. These studies are too preliminary in natureto merit serious discussion at present. However, the recognition of the potential ofthese genes in the context of cancer development does merit citation. Needless tosay, they could lend themselves as possible candidates for markers of differentiationin the development and progression of skin tumours.

EFFECTS OF S100 PROTEINS ON CELL DEFORMABILITY AND CELLULAR MORPHOLOGY

The ability to alter the malleability of the membrane and cell shape and regulationof intercellular adhesion are two essential requirements for facilitating cell motility,diapedesis, and invasiveness. Changes in cellular morphology also occur in terminal


206


differentiation and when differentiation is experimentally induced. For instance, inneuroblastoma cells exposed to retinoic acid, changes occur in cell shape withattendant changes in S100 family gene calcyclin (Tonini

et al.

1991). Calcyclinappears to be associated with the tangential migration of proliferating cells of thesubventricular zone of the lateral ventricle into the olfactory lobe and their differ-entiation into interneurones. A subpopulation of astrocytes in the tangential migra-tion pathway seems to express calcyclin (Yamashita

et al

. 1997). The migration ofsubventricular zone (SVZ) cells is believed to be organised by the polysialylatedneural cell adhesion molecule (PSA-NCAM). Neural cell adhesion molecules(NCAM) and cadherins have been studied extensively in the context of neuriteregeneration and neuronal adhesion. Whereas cadherins are Ca

2+

dependent for theirfunction, NCAMs are not. It is of considerable interest, therefore, that the migrationof the SVZ cells mediated by an NCAM is associated with calcyclin expression.

Intracellular calcium levels regulate cellular morphology. Cells appear to altertheir shape in response to subtle changes in intracellular Ca

2+

. Thus calcium tran-sients produced by the mobilisation of calcium from intracellular stores can haveeffects that differ profoundly from those induced by influxed calcium. Caffeineinduces transient increases in calcium in the dendrites and spines of hippocampalneurones. An increase in the size of existing dendrites and possibly also the formationof new dendrites occurs in association with these calcium transients (Korkotian andSegal, 1999). In contrast, glutamate receptor activation by NMDA and glutamateitself produces a collapse of dendritic spines, together with an infux of calcium.This leads to comparatively large changes in the levels of intracellular calcium. Theloss of dendritic spines corresponds with the loss of filamentous actin. Actin-stabi-lising agents counteract the effects of NMDA (Segal, 1995; Halpain

et al.

1998).Influxed calcium and calcium mobilised from intracellular stores might have inde-pendent roles (Haverstick

et al.

1991). It also has been recognised that calciumreleased from intracellular stores might activate specific components of the signaltransduction pathway (Parker and Sherbet, 1992). One can then hardly overempha-sise the potential influence CBPs can exert on cellular morphology.

S100 proteins alter cell shape and motility apparently by virtue of their abilityto alter cytoskeletal dynamics. Inhibition of S100B, which is predominantlyexpressed in glial and Schwann cells, has been shown to result in changes in cellularmorphology as well as in the organisation of the cytoskeleton of glial cells in culture(Selinfreund

et al.

1990). Selinfreund

et al.

(1990) introduced antisense S100Boligonucleotides, which were placed under the control of a dexamethasone-induciblepromoter, into rat C6 glioma cells. When the antisense constructs were induced toexpress, the levels of S100B decreased with concomitant changes in cell morphology.They assumed a flattened morphology and displayed an organised network ofmicrofilaments. The neurite extension factor, which bears sequence homology toS100B, is a homodimer of S100B-like subunits. This factor actively promotes neuriteextension (Kligman and Marshak, 1985). S100 proteins are expressed in the dendriticcells in human transitional cell carcinoma of the bladder, and the invasive potentialof these tumours has been found to correlate with the presence of cells expressingS100 protein (Inoue

et al.

1993). The occurrence of S100 proteins together with



207

smooth muscle actin and vimentin has been reported in a metaplastic carcinoma ofthe breast invading the chest wall as well as in recurrent tumour (Harb

et al.

1995).Reeves

et al.

(1994) generated transgenic S100B mice in which S100B mRNAexpression was two- to seven-fold greater than that in control mice. They foundenhanced expression of glial fibrillary acidic protein (GFAP) and also enhancedneurite proliferation in the transgenic mice. Similarly, the expression of S100A4appears to correlate with the

in vitro

invasive potential of glioma cells in culture(Merzak

et al.

1994b). The ability of S100A4 to interact with nonmuscle myosinsupports the view that it takes part in cellular motility (Takenaga

et al.

1994a, 1994b).S100A4 is highly expressed during embryonic development in the highly invasivemesenchymal elements (Klingelhofer

et al. 1997).These effects appear to be due to an interaction of the S100 proteins with the

cytoskeletal machinery of the cell. NGF markedly enhances calcium uptake (seeKozak et al. 1992). Many of the effects of NGF on PC12 cells, for instance, areaccompanied by alterations in the expression of S100 proteins. An S100 proteincalled p11 (S100A10), identified some years ago, which shows a high degree ofsequence homology to p42C. The latter is induced in PC12 cells by treatment withNGF (Masiakowski and Shooter, 1988). p11 binds to and inhibits the phosphoryla-tion of a tyrosine residue of annexin (p36). It has been found that a complexcomposed of p11 dimer and two subunits of annexin p36 is involved with the processof actin bundling and in the linkage of the actin component to the cell membrane(Kligman and Hilt, 1988). Zimmer et al. (1998) transfected S100A1 cDNA inantisense orientation into PC12 cells. These transfectants showed a marked reductionin S100A1 expression. When these transfectant cells were exposed to NGF, therewas a marked increase in neurite formation. In parallel, Zimmer et al. (1998) alsofound increased levels of α-tubulin. The antisense S100A1 clones also showedreduced anchorage-dependent growth. S100B has been reported to form a complexwith the cytoskeletal tau protein, and this complex appears to inhibit microtubulepolymerisation (Baudier et al. 1982; Baudier and Cole, 1988).

S100 proteins show a substantial expression in brain tissues; they are found inastrocytes and oligodendrocytes, and their expression seems to be functionally linkedwith their morphology and differentiation. These functions may be associated withchanges in the cytoskeletal elements, their organisation, and disposition. S100 pro-teins have several putative target cellular proteins; interactions with these mightinfluence a panoply of cellular features (Table 17). S100B has been shown to inhibitthe polymerisation of GFAP (Bianchi et al. 1993). It appears to interfere with theearly stages of GFAP polymerisation, reducing the rate of assembly of GFAP sub-units, besides actively promoting depolymerisation of GFAP polymers (Bianchi etal. 1994). A specific domain in GFAP interacts with S100 proteins, and a peptideknown as TRTK-12, which bears homology to this domain of GFAP, has been shownto be able to counteract the inhibition by S100 of the polymerisation of GFAPmonomers (Bianchi et al. 1996). Bianchi et al. (1996) have suggested further thatS100B achieves this by sequestering GFAP monomers. This view is in sharp contrastwith a recent observation that S100B promotes the extension of MAP-2, and mightindeed promote the reassembly and stabilisation of the cytoskeleton (Nishi et al.1997). S100A4 also has been shown to be associated with cytoskeletal elements



(Davies et al. 1993; Gibbs et al. 1994; Takenaga et al. 1994a). In S100A4 transfectedRama-29 cells, the S100A4 staining pattern markedly resembles that of phalloidin,which binds to F-actin filaments (B.R. Davies et al. 1993; Gibbs et al. 1994). Suchan association seems to result in cytoskeletal disorganisation (Watanabe et al. 1992,1993), possibly related to the ability of the protein to promote microtubule depoly-merisation (Lakshmi et al. 1993).

Takenaga et al. (1994c) demonstrated that the binding of S100A4 to nonmuscletropomyosin was calcium dependent and identified amino acid residues 39–107 ofS100A4 as the binding site. They showed further by immunoblotting that part ofS100A4 associated with Triton-insoluble cytoskeletal components. Indeed, in situ,S100A4 could be localised together with tropomyosin in microfilament bundles. InNIH3T3 and 3Y1 tissue culture cells S100A4 is found diffusely distributed in thecytoplasm, but some of the protein is also found in association with actin stress

TABLE 17S100 Proteins and Their Intracellular Targets

S100 Protein Target Molecule Functional Association Ref.

S100A1 Phosphoglucomutase Stimulation Zimmer et al. (1995); Landa et al. (1996)

S100A4 Myosin II Cell locomotion Ford et al. (1997)Nonmuscle tropomyosin

Cell locomotion Takenaga et al. (1994b)

F-actin Cell locomotion Davies et al. (1993); Gibbs et al. (1994)

Tubulin monomers Cell proliferation; locomotion

Lakshmi et al. (1993)

p53 Cell proliferation Parker et al. (1994a, 1994b)

S100A8/A9 (MRP8/14)

Membrane arachidonic acid

Translocation to cell membrane

Klempt et al. (1997)

S100A10 (p11)

Annexin II Cytoskeletal reorganisation

Kaczanbourgois et al. (1996)

S100A11 (S100C)

Binds to actin filaments and inhibits Mg2+-ATPase; annexins

Reorganisation of the cytoskeleton; putative participation in growth and differentiation

Zhao et al. (2000); Naka et al. (1994); Mailliard et al. (1996); see text

S100B Phosphoglucomutase Stimulation Landa et al. (1996)GFAP Inhibition of GFAP

polymerisationBianchi et al. (1993)

CapZ actin-capping protein

Actin polymerisation Kilby et al. (1997)

Ndr nuclear serine/threonine kinase

Influence on cell cycle progression by the activation of Ndr kinase

Millward et al. (1998)

Calponin Interaction with membrane phospholipids

Fujii et al. (1995)


S100 Proteins: Their Biological Function and Role in Pathogenesis 209

fibres (Takenaga et al. 1994b). S100A4 also has been shown to interact specificallywith myosin II at the rate of 3 moles of S100A4 per mole of MHC. However, itdoes not interact with myosin I or II. Further, this interaction might destabilisemyosin filaments (Ford et al. 1997). The S100A4-binding site has been identified.It occurs in the rod region and is mapped to a sequence, between residues 1909 and1937, at the C-terminal end of MHC (Kriajevska et al. 1998). These authors haveshown that S100A4 inhibits PKC-mediated phosphorylation of MHC at serine res-idue 1917. It seems possible that S100A4 might influence tropomyosin-assistedmyosin–actin interactions. The distribution of A and B isoforms of nonmusclemyosin II, as discussed earlier, shows a marked relationship to processes associatedwith cell locomotion. This might afford a mechanism by which S100A4 couldregulate the cytoskeletal machinery and influence locomotion.

S100 protein has been reported to show calcium-dependent binding to calponin,a protein that can bind to CaM, actin, and tropomyosin. An N-terminal 22-kDadomain of calponin was involved in the interaction with S100 as well as with actin,tubulin, CaM and tropomyosin (Fujii et al. 1994, 1997). Calponin also interacts withmembrane phospholipids, and these interactions are inhibited by S100 (Fujii et al.1995). Most of these observations come from in vitro studies and thus no inferencescan be drawn at present as to their relevance to in vivo situations, especially in lightof the finding that calponin does not affect tubulin polymerisation (Fujii et al.1997).Nevertheless, Kilby et al. (1997) showed that certain C-terminal amino acid residuesand N-terminal residues valine 8 to aspartic acid 12 of S100B may be the domainsthat are involved in its interaction with the actin-capping protein CapZ. This lendsitself strongly in favour of S100 interactions with their target proteins as being aconsistent feature in determining cell behaviour.

There is, therefore, a complex series of interactions between S100 proteins andcytoskeletal structures, which in turn may be related to membrane fluidity andflexibility, and this could well influence the shape and motility of cells. Theseinteractions may also be indicative of terminal differentiation, as shown experimen-tally by Tonini et al. (1991). These authors found an enhancement of S100 expressionin neuroblastoma cells that were induced to differentiate using retinoic acid. Zimmerand Landar (1995) studied the expression of S100A and B in the differentiation ofPC12 cells and L6S4 skeletal muscle cells. Both proteins are expressed in theundifferentiated as well as the differentiated state. S100A1 protein expression washigher in differentiated cells, but the expression of its mRNA showed no change indifferentiated L6 cells, and in fact mRNA levels decreased in PC12 with differen-tiation. This would suggest that S100 expression is regulated at the posttranscrip-tional level.

The S100 protein MRP14 (S100A9) is phosphorylated during monocytic differ-entiation. Two phosphorylated isoforms of MRP14 have been detected. MRP14seems to respond to enhanced Ca2+ levels by undergoing a selective translocationfrom the cytoplasm to the cell membrane (Van den Bos et al. 1996). In fact,heterodimers of MRP8 and 14 are capable of binding specifically to membranearachidonic acid (Klempt et al. 1997). The association of S100 proteins with syn-cytioblast maturation and differentiation in the placenta has been reported recently.S100A10, which was formerly known as p11, forms a heteromeric association with



annexin II that is linked to cytoskeletal structures, and both annexin II and p11 showa progressive increase in association with placental differentiation (Kaczanbourgoiset al. 1996). Apart from the obvious implications for membrane-related activities ofthe cell, there may also be other functions subserved by the association of S100proteins with cytoskeletal components. MRP8 and MRP14 are both activated byPKC phosphorylation before they are secreted, and the activated proteins associatethemselves with tubulin filaments before release, which seems to suggest that suchan association could provide a route for the release of MRPs (Rammes et al. 1997).

CELL ADHESION AND INVASIVE POTENTIAL OF CANCER CELLS

Adhesive interaction between cells as well as between cell and substratum is animportant ingredient of cellular motility. This cellular faculty is dependent on notonly the cell membrane-associated adhesive macromolecules but also their temporaland spatial distribution in the cell membrane. It follows, therefore, that alterationsin the overall expression of adhesion-mediating components of the cell membraneas well as their topographical distribution should entail alterations in the adhesiveproperty. As a converse of this, one would expect to see changes either in the natureor in the pattern of distribution of cell membrane components in association withthe acquisition of invasive potential. In inquiries into the question of whether S100proteins influence the adhesive abilities of cells, a testable postulate would be to seeif they regulated the expression on cell membrane components that participate incell adhesion.

Historically there is much evidence that calcium signalling is an important eventin the induction of cell motility. Calcium signalling in processes such as the pene-tration of the oocyte by the sperm cell involves G-protein-mediated increases inintracellular calcium levels (Florman et al. 1989). The migration of neutrophilsappears to depend on the enhancement of intracellular calcium levels (Marks andMaxfield, 1990). Chemoattractants have been known to induce calcium influx (Milneand Coukell, 1991). They produce rapid changes in the intracellular Ca2+ levelstogether with cell spreading, formation of pseudopodia, motility and its direction,and phagocytosis. However, blocking of the intracellular Ca2+ transients seems tohave little effect on these biological features, indicating the possible involvement ofother factors (Hendey and Maxfield, 1993). Nonetheless, Hendey and Maxfield(1993) report that cell motility on physiological substrates such as fibronectin andvitronectin are dependent on intracellular calcium levels. Cell surface integrin recep-tors possess calcium-binding domains similar to EF-hands (Tuckwell et al. 1992).The calcium-binding motifs of the α subunit of αMβ2 integrin of leukocytes showsimilarities to as well as differences from EF-hand motifs (Oxvig and Springer,1998). Savarese et al. (1992) found that type IV collagen, which induces cell motility,also brings about increases in intracellular calcium levels, presumably by the releaseof Ca2+ from intracellular stores. Therefore, it would seem that intracellular calciumtransients closely relate to the cellular interaction with substratum mediated by ECMcomponents such as fibronectin and collagen. These interactions could be importantevents in the initiation of calcium signalling. The S100 proteins MRP8 (S100A8)and MRP14 (S100A9) are expressed abundantly in the cytoplasm of neutrophils.



These proteins associate with vascular endothelia and presumably aid in the diaped-esis of cells across the endothelium. Newton and Hogg (1998) have found thatMRP14 regulates the function of β2-integrin, an adhesion-mediating glycoprotein.The function of MRP14 appears to be restricted to just this regulatory process. Thefunction of MRP14 itself may be regulated by MRP8. Apparently, the formation ofa heterodimer between MRP8 and MRP14 can inhibit the function of MRP14.However, the nature of β2-integrin regulation by MRP14 remains to be elucidated,although Newton and Hogg (1998) do state that integrin does not function as areceptor for MRP14.

Many components of the ECM are associated with and known to participateactively in cell motility and cancer invasion (see Sherbet and Lakshmi, 1997b). Morepertinent to the topic under discussion here is the observation by Savarese et al.(1992) that type IV collagen, which stimulates motile behaviour in melanoma cells,also increases intracellular calcium levels. The effect seems to be mediated by therelease of Ca2+ from intracellular stores rather than by an influx of extracellularcalcium.

Another ECM component, the transmembrane glycoprotein CD44, has been thesubject of much debate with regard to its putative role in cancer invasion andmetastasis (see Sherbet and Lakshmi, 1997b). CD44 is a membrane-associatedadhesive glycoprotein that mediates intercellular adhesion by virtue of its functionas a hyaluronate receptor. It has been attributed with enhancing the invasive abilityof cells (Radotra et al. 1994; Merzak et al. 1994a). In this context may be citedrecent studies of CD44 expression in B16 melanoma cells in which the expressionof S100A4 was experimentally altered (Lakshmi et al. 1997). CD44 expression didnot increase when S100A4 expression was up-regulated. However, an enhancedexpression of S100A4 produced a redistribution of CD44 into a patchy focal pattern.Because S100A4 is able to depolymerise cytoskeletal elements, it has been suggestedthat the redistribution of the glycoprotein could be a result of cytoskeletal depoly-merisation and the resultant enhanced lateral mobility of CD44 molecules leadingto the aggregation and patching of CD44 receptors (Lakshmi et al. 1997). Fullycompatible with this line of argument are the recent findings that CD44 moleculeslocalise in cholesterol-rich domains of the plasma membrane. These molecules enjoyrestricted lateral mobility because of the cholesterol-rich nature of the membranedomain in which they are localised and also the integrity of the actin cytoskeleton.A disruption of the integrity of the actin cytoskeleton results in an increase in thepartitioning of CD44 to the cholesterol-rich domains (Oliferenko et al. 1999).

Hyaluronic acid, which is a ligand for CD44, induces such patching, and theseCD44 patches are associated with a preferential accumulation of and binding toplaques of the cytoskeletal protein called ankyrin (Bourguignon et al. 1993; Welschet al. 1995). The interaction of CD44 with ankyrin occurs when CD44 is phospho-rylated by Rho kinase, which, in turn, is activated by the GTPase activity of Rho Abound to CD44v. The phosphorylation of CD44 enhances its interaction withankyrin. Bourguignon et al. (1999) have shown that microinjection of the catalyticdomain of Rho kinase produces membrane ruffling. This can be inhibited by CD44antibodies and by the microfilament inhibitor cytochalasin D.



It is obvious from these observations that the induction of cell motility by CD44is a complex process. The redistribution of CD44 into patches occurring underconditions of high S100A4 could provide the tumour cells with discrete and stronglyadhesive foci that may promote their invasive behaviour by increasing and strength-ening anchorage and intercellular interaction (Lakshmi et al. 1993). Overall, thesedata indeed suggest that S100A4 may be linked with the cytoskeleton.

Further evidence, albeit circumstantial, that S100A4 might associate with thecytoskeleton, has come from recent work by Keirsebilck et al. (1998a). Theyinvestigated the status of E-cadherin and S100A4 in two murine tumour cell linesand found tha E-cadherin expression correlated inversely with S100A4 expressionin these cell lines. E-cadherin is a transmembrane CBP, which is linked to theactin cytoskeleton via a number of linking elements (see Figure 13). E-cadherinis a putative suppressor of invasive and metastatic abilities (see Sherbet andLakshmi, 1997b). Low levels of E-cadherin expression have been reported tocorrelate with poor prognosis of a variety of human cancers. Therefore, the inverserelationship between it and S100A4 is compatible with the invasion- and metasta-sis-promoting ability of the latter. The exact mechanics of this relationship haveyet to be elucidated. Keirsebilck et al. (1998a) found an alteration in the intrac-ellular distribution of α- and β-catenins, which link the cytoplasmic tails of E-cadherin to the actin cytoskeleton. Some cellular proteins, such as the adherencejunction protein, are known to bind to this cytoplasmic domain of E-cadherin.APC protein, which is believed to function as a tumour suppressor protein, isknown to compete with E-cadherin for binding to β-catenin (Hulsken et al. 1994).Arguably, the suppressor effect of cadherin does not depend on merely the presenceof cadherin per se, but also on its being present in a functional state in its entiretyas a complex with the linking proteins. Three forms of cadherin–catenin complexesmay occur in cells. The form with one cadherin molecule linked to a β-catenin/α-catenin or plakoglobin/α-catenin is the conventional from depicted in Figure 13.In the second type of complex, the extracellular domains of cadherin dimerise ina parallel fashion. A proposed third type is one in which the extracellular domainsof two conventional complexes belonging to two neighbouring cells associate inan antiparallel fashion (Chitaev and Troyanovsky, 1998). Further, Chitaev andTroyanovsky (1998) have suggested that an antiparallel association of dimerisedcomplexes of neighbouring cells is responsible for intercellular adhesion, and thatthis is dependent on not only extracellular calcium but also the presence of thecatenin components. It is apparent from this discussion that suppression of invasionby cadherin is a complex process, and at present it is difficult to envisage howS100A4 and cadherin antagonise each other. If the mechanism proposed by Chitaevand Troyanovsky (1998) were valid, one would not expect an inverse relationshipbetween cadherin and S100A4 expression, for the latter would be expected topromote the dimerisation of cadherin complexes by enhancing the lateral mobilityof the complexes in the cell membrane. Nonetheless, it would be of considerableinterest to see if S100A4 affects in any way the interaction of E-cadherin with thecatenins.



S100 PROTEINS IN REMODELLING OF THE EXTRACELLULAR MATRIX

ECM undergoes constant changes in terms of remodelling and renewal in a host ofbiological processes, such as morphogenesis, cell differentiation, wound healing,angiogenesis, cell motility, and also in the invasive and metastatic behaviour ofcancers. A wide variety of proteinases are involved in the remodelling of the ECM.These enzymes and their inhibitors are known to be genetically regulated (seeSherbet and Lakshmi, 1997b). That ECM remodelling might involve S100A4 wasdemonstrated some time ago by Merzak et al. (1994). In glioma cell cultures,S100A4 expression appears to be inversely related to the expression of TIMP-2gene, which encodes an inhibitor of tumour-associated metalloproteinases. Interest-ingly, S100A4 is highly expressed in cell lines derived from invasive gliomas, ascompared with cell lines obtained from noninvasive localised brain tumours or foetalbrain cells. On the basis of these observations, it has been suggested that the invasiveproperty of glioma cells could be a result of the uninhibited function of tumour-associated proteinases and that this lack of regulation of the proteinases concernedmight be due to inhibition of TIMP-2. In a recent report, Bjornland et al. (1999)claimed that TIMP-2 up-regulation can occur independently of the state of S100A4expression. They isolated several clones from a highly metastatic osteosarcoma cellline, which expressed S100A4 at different levels on account of being transfectedwith an anti-S100A4 ribozyme. It is somewhat intriguing that in the clones with lowor intermediate S100A4 expression, both MMPs and TIMP-1, were down-regulated.With a complex experimental system such as this, it is difficult to visualise howMMP homeostasis could be altered by parallel modulation, in the same direction,also of the MMP inhibitor TIMP-1 (also see below).

Several other studies also have suggested that the expression of S100A4 mightcorrelate with that of MMPs. De Vouge and Mukherjee (1992) found that bothS100A4 and transin-2 are up-regulated in parallel in rat kidney cells transformedby Ki-ras. K. Andersen et al. (1998) found that IL-1α down-regulated the expressionof TIMP-1 in osteosarcoma cells that express S100A4 at high levels. In contrast, incells with low level of S100A4 expression, IL-1α did not change TIMP expression.K. Andersen et al. (1998) have suggested that the effects of bFGF, which also affectsTIMP, might not be related to the levels of S100A4 expression. They suggest thatIL-1α functions synergistically with S100A4. It should be borne in mind, however,that both bFGF and IL-1α effects on TIMP could have been mediated by osteonectin,because bFGF is known to regulate the expression of osteonectin. In mesenchymalcells, bFGF is known to up-regulate the expression of osteonectin (Shiba et al. 1995).IL-1α, on the other hand, down-regulates the expression of osteonectin. Osteonectinhas been shown to modulate MMP expression, and possibly also that of its inhibitor.There is no evidence at present for the latter. However, in many systems studied todate, the MMP/TIMP system is regulated as a unit, with MMP up-regulation moreoften than not being a result of down-regulation of TIMP or vice versa. None of theexperiments described here provide any indication of how S100A4 achieves TIMP-2 inhibition. This remains to be elucidated.

It should be recognised that the remodelling of the ECM and the downstreameffects of such changes on cell adhesion and motility might be directly linked with



the reorganisation of the cytoskeleton. We know from the work of Irigoyen et al.(1997) that cytoskeletal reorganisation leads to, probably among other things, anup-regulation of uPA. Irigoyen et al. (1999) have further shown that this up-regula-tion occurs via the ras/erk-signalling pathway. The ability of S100A4 to alter theorganisation of the cytoskeleton has been satisfactorily demonstrated. Besides, DeVouge and Mukherjee (1992) have shown that cell transformation by the ras onco-gene results in the up-regulation of S100A4 as well as an MMP. Furthermore, heatshock can down regulate not only S100A4 expression (Albertazzi et al. 1998a) butalso the expression of membrane type I MMP together with an inhibition of in vitroinvasive ability (Sato et al. 1999). Under these circumstances one would be amplyjustified in considering this to be a putative link between S100A4 expression andECM remodelling and the associated changes in membrane properties and cellbehaviour.

S100A4 is generally believed to be a cytosolic protein. However, its putativeparticipation in ECM remodelling suggests that it might be secreted into the ECM.The findings of Duarte et al. (1999) seem to confirm this view. They reported thatS100A4 is secreted under both in vivo and in vitro conditions. They further reportedthat S100A4 inhibits the process of mineralisation and bone nodule formation inbone marrow cell cultures. It is possible, therefore, that the apparent modulation ofTIMP activity and any consequential effects on the character of the ECM describedby Merzak et al. (1994) could have resulted from the action of the S100A4 secretedinto the ECM.

The ECM-modulating effects of S100A4 are also implicit in a set of observationsrelating to the epithelial–mesenchymal transformation that occurs in vertebrates. Animportant feature of this transformation is that it is associated with the acquisitionof the ability to invade and migrate into the ECM (Hay, 1995). Several genes areswitched on during this transformation, among them S100A4, which was called thefibroblast-specific protein 1 (FSP1). Although the fibroblast specificity is debatable,it has been shown that antisense S100A4 suppressed the expression of the gene andat the same time suppressed epithelial–mesenchymal transformation (Okada et al.1997). Okada et al. (1997) also have gone on to demonstrate that EGF and TGFβ−1, both capable of inducing cellular motility, also induce S100A4 expression inepithelial cells.

A somewhat simplified conclusion that flows from the above discussion is thatS100 proteins play a very significant role in cell motility, cell adhesion, and theinvasive behaviour of cancer cells. It is also obvious that many questions remainunanswered, especially those regarding the interaction of these proteins with thecytoskeletal elements, i.e., the mode of their function as agents that promote cytosk-eletal disassembly or inhibit the polymerisation of cytoskeletal monomeric elements.We do, however, have some significant leads as to how S100 proteins might beinvolved in the apparent regulation of tumour-associated proteinases.

S100 PROTEINS IN CELL PROLIFERATION

There is a general acceptance of the concept that cancers possess a high proliferativepotential and that this apparent potential is more a consequence of the loss of



homeostatic growth control than of an increase in the rate of proliferation. Inevitably,S100 genes, whose expression correlates remarkably well with invasive and meta-static potential of cancers, have also been investigated for their relationship to growthderegulation in cancers. It is significant, therefore, that some of the major S100proteins are expressed in a cell cycle-specific manner. Indeed, some members of theS100 family such as S100A4 were originally cloned from highly proliferative tissuesand were described as proliferation-related proteins.

Two questions can be posed in order to elucidate the relationship between growthregulation and the expression of S100 proteins. One question is whether the presenceof S100 proteins correlates with the growth fraction and proliferative behaviour oftumours, or with tumour cells or normal cells in culture. Recently, S100B has beenattributed with a role in the regulation of the cell cycle. The evidence takes the formof the demonstration of the ability of S100B to bind to and activate in vitro thenuclear serine/threonine protein kinase called Ndr, in response to changes in theintracellular calcium levels. An N-terminal regulatory region of Ndr seems to beinvolved in the Ca2+-dependent binding of S100B, and this region contains sequencesthat are characteristic of CaM/S100 binding (Millward et al. 1998). S100B alsoseems to be able to regulate the intracellular activity of Ndr. Millward et al. (1998)have proposed a biological role for this interaction between S100B and Ndr and theregulation of the activity of the latter. In melanomas that overexpress S100B, theCaM inhibitor W7 inhibits the activation of Ndr, but not in those that lack S100Bexpression. The Ndr kinase was identified by Millward et al. (1995). It seems tobelong to a subfamily of protein kinases that shows 40 to 60% sequence similaritiesamong the members of the family. These kinases have been implicated strongly inthe progression of the cell cycle as well as in the regulation of cell morphology.Therefore, Millward et al. (1998) have argued that an overactivation of Ndr by S100Bmight be related in some way to the development and progression of melanomas.However, as Millward et al. (1998) have pointed out, some melanomas do overex-press S100B without also showing an overactivation of Ndr. Therefore, they suggestthat a mechanism of negative control of Ndr might exist. This postulate requires thatthis negative regulation is inactivated by loss-of-function mutations of the Ndrregulator.

S100B has been reported to stimulate sciatic nerve regeneration and may func-tion as a growth factor for peripheral nerve axons (Haglid et al. 1997). Growth maybe regulated by a homeostasis of cell proliferation and apoptotic loss of cells. S100Bhas been reported to rescue motor neurones from apoptotic death (Iwasaki et al.1997). The ability of S100B to influence actin dynamics might be involved in thiseffect. The status of actin polymerisation has been found to regulate apoptosis.Induction of polymerisation leads to apoptosis, whereas inhibitors of polymerisationblock apoptosis (J.Y. Rao et al. 1999). As alluded to above, S100B can inhibit actinpolymerisation. Therefore the findings of Iwasaki et al. (1997) are compatible withthose of J.Y. Rao et al. (1999). On the other hand, it has been found that S100Bmay lead to apoptotic death by releasing nitric oxide (Hu et al. 1997).

The calcyclin gene is expressed more frequently in epithelial-type or Schwanncells than in neuroblastic cells. This is compatible with the difference in their growthpotential. A consequence of the induction of differentiation of neuroblastoma cells



by retinoic acid is an enhancement of calcyclin expression together with arrest ofcell growth (Tonini et al. 1991). It is yet uncertain if calcyclin indeed negativelyregulates growth, because, in total contrast, a higher proportion of melanoma cellsshows the presence of calcyclin in the vertical growth phase (Weterman et al. 1993).It may be pointed out, however, that S100B expression has been reported to increasein neuroblastoma differentiation (Tsunamoto et al. 1988). Tonini et al. (1991) foundan inhibition of growth rate in cells where S100B levels had been experimentallyreduced. This is not compatible with the findings of Tsunamoto et al. (1988) thatreduction of growth rates invariably accompany the induction of differentiation.Nonetheless, S100A4 expression is up-regulated by positive proliferative responsessuch as those imparted by growth factors and down-regulated by negative responseselicited by hyperthermia, with parallel changes in the size of the S phase fraction.For instance, human tumour cells that are high expressors of EGFr also tend tooverexpress S100A4 (Sherbet et al. 1995). In murine BL6 melanoma cells and humanHUT cells, which are a heat-resistant variant derived from HepG2 cells, hyperthermiadown-regulates S100A4 gene expression. In parallel, there is a decrease in the sizeof the S phase fraction and an increase in the doubling time of cells (Sherbet et al.1996; Albertazzi et al. 1998a).

Hyperthermia, which is known to increase intracellular calcium levels (Furukawaet al. 1997), induces the synthesis of several heat shock (cognate) proteins. Amongthe most prominent in terms of their relevance to cell proliferation are HSP70 andHSP28. The microtubule-interacting protein MIP-90 has been reported to showextensive sequence homology to HSP90 (Cambiazo et al. 1999). HSP70 has beenknown for some time to show a cell cycle-related expression. This HSP seems tobe essential for mitotic division in the early developmental stages of the sea urchinParacentrotus lividus. HSP70 has been reported to accumulate in the mitotic appa-ratus, and further, antibodies raised against HSP70 have been shown to inhibit mitosis(Sconzo et al. 1999). It would seem that HSP70 is an important ingredient of celldivision and could play a role in the cell division machinery. In sharp contrast,HSP28 seems to exert an inhibitory effect on cell proliferation, and furthermore,HSP28 function might have a genetic basis. This is obvious from the experimentalwork described by Albertazzi et al.(1998a), where there was a remarkable differentialincrease in HSP28 as compared with the non-HSP28 component of total cellularHSP. Thus, in the BL6-derived HTG murine melanoma cells exposed to hyperther-mia, HSP28 expression increased by 3.5-fold as compared with control BL6 cells.The corresponding increase in the non-HSP component of the HTG cells was only22%. The enhancement of HSP28 together with a down-regulation of S100A4 (mts1)is compatible with previous observations that HSP28 is a growth-inhibitory protein.HSP28 is induced by hyperthermia in normal lymphocytes and macrophages as wellas in leukaemia cells. Its induction has been reported to coincide with peak prolif-erative activity and the onset of growth arrest (Spector et al. 1992, 1993, 1994).HSP28 is highly expressed in quiescent keratinocytes as compared with their pro-liferating counterparts (Honore et al. 1994).

A low molecular weight heat shock protein, HSP27, has been reported to beexpressed abundantly in normal squamous epithelia of the oesophagus, but is mark-edly down-regulated in Barrett’s metaplasia and adenocarcinoma of the oesophagus



(Soldes et al. 1999). The HSP27 gene has been transfected into A375 melanomacells and A431 cells. In transfectant cells obtained from both cell lines, overexpres-sion of the HSP was associated with reduced proliferative rates (Kinder-Mugge etal. 1996). Presumably, HSP27 and 28 are the same molecular species. Antibodiesagainst HSP27 have been detected in sera of breast cancer patients but not of normalsubjects. Furthermore, their presence in sera correlated with improved patient sur-vival (Conroy et al. 1998). It may be suggested that this reflects less aggressivedisease on account of the growth-inhibitory properties of the HSP. It is interestingto note also that cells over-expressing HSP27 show a delay in tumorigenicity(Kinder-Mugge et al. 1996). This delay is compatible with inhibition of cell prolif-eration. However, after this initial delay tumours did appear, but these were devoidof HSP27 expression and they showed growth properties similar to control tumours.Again, this supports the close link between HSP27 expression and proliferativepotential. It would be interesting to study how HSP28 functions as an intermediaryin the inhibition of proliferation accompanying the S100A4 down-regulation. Thereare two pointers to the possible mode of action of this low molecular weight HSP.HSP27 binds both α- and β-tubulin and is also found in association with microtubules(Hino et al. 2000). We know that S100A4 markedly influences cytoskeletal dynam-ics. Therefore, Albertazzi et al. (1998a) have postulated HSP28 might be involvedin direct interaction with S100A4. There are several reports that HSP can bind top53. Normal human cell lines exposed to hyperthermia show G1 arrest together withan increase in the expression of p53, but not in rb protein phosphorylation (Miyakodaet al. 1999). In light of these findings, the suggestion by Albertazzi et al. (1998a)that there might exist a complete regulatory loop involving S100A4, HSP, and p53in the control of the G1–S checkpoint of the cell cycle, seems highly credible(Figure 26).

CELL CYCLE-RELATED EXPRESSION OF S100 PROTEINS

The progression of the cell cycle involves a sequence of events that includes calciumsignalling. Calcium signalling involves both an influx of extracellular calcium andan elevation of intracellular calcium levels by the release of Ca2+ from intracellularstores. Mitogenic agents generate calcium signals via the IP3-signalling pathway.These calcium signals might control the entry of resting cells into the cell cycle,G1–S and G2–M transitions as well as the exit of the cells from mitosis. Mitogensactivate calcium influx into the cells, which does seem to control the progression ofthe cell cycle (Barbiero et al. 1995). In the mitotic phase, transient increases of Ca2+

occur between metaphase and anaphase. An increase in cytosolic calcium is asso-ciated also with the process of cell division, i.e., cytokinesis. Calcium channelblockers can inhibit cell proliferation by arresting cells in the G0G1 phase (Zeitleret al. 1997). Fertilisation of the ovum results in a rapid rise in Ca2+. This is requiredfor the cleavage of the fertilised egg (Whitaker and Patel, 1990), which can beblocked by chelating Ca2+ (Zucker and Steinhardt, 1978). Unexpectedly, mobilisationof cell calcium and calcium channel activation also have been found to inhibit thepassage of cells at the G1–S and G2–M checkpoints (Vanolah and Ramsdell, 1996).It may be that calcium sequestration by CBPs controls intracellular levels of calcium



and thereby regulates the role of calcium signalling in cell cycle progression. There-fore, much attention has been focused on the potential role of S100 proteins andindeed other calcium-sequestering proteins in cell proliferation. Furthermore, muchinformation is currently available identifying individual CBPs with intervention andfunction at specified stages of the cell cycle. Ca2+ has been regarded also as playingan important part in the cycle of meiotic cell division.

The question of whether S100 proteins regulate growth can be answered in twoparts. The first part of the answer is the unequivocal association between the expres-sion of certain S100 proteins and specific stages of the cell cycle. It was noticedsome years ago that S100B and calcyclin genes were expressed predominantly inthe G1 phase of the cell cycle (Hirschhorn et al. 1984). Selinfreund et al. (1990)showed that intracellular levels of S100B can be reduced by introducing antisenseS100B oligonucleotides into cells. This resulted in a decrease in the growth rate ofthe cells. The induction of differentiation of neuroblastoma cells by retinoic acidenhances the expression of calcyclin mRNA. This is accompanied by arrest of cellproliferation, and cells appear to accumulate in the G1 phase of the cell cycle (Toniniet al. 1991). The mechanism by which calcyclin might be regulating the cell cycleis not fully understood at present. Calcyclin could indirectly affect cell cycle pro-gression. It has been found to interact with great specificity with the annexin CAP-50 (Minami et al. 1992; Hidaka and Mizutani, 1993). As discussed elsewhere, theexpression of annexins is closely associated with cell cycle progression and,

FIGURE 26 Hypothetical view of interaction between S100A4, HSP28, and p53 in theregulatory loop that might control G1–S transition of cells. (From Albertazzi et al. 1998a.)Reprinted by permission of the publisher Mary Ann Liebert Inc.



furthermore, CAP-50 shows a predominant nuclear location (Hidaka and Mizutani,1993). Minami et al. (1992) have suggested, in light of the apparent specificity ofcalcyclin binding to CAP-50, that this annexin could be regulated by calcyclin.Therefore, one cannot exclude the possibility that calcyclin might influence andregulate the progression of the cell cycle and cell growth by an indirect route.

S100A4 (mts1) was cloned by Jackson-Grusby et al. (1987), who showed thatit could be induced to express by serum stimulation of growth-arrested cells andthat its expression increased during the S phase of the cell cycle. The NGF inducesthe synthesis of two proteins in rat PC12 cells, which were originally referred to as42C and 42A. 42C and 42A are regarded as the rat homologues S100A10 andS100A4, respectively. Not only does NGF-induced neuronal differentiation induce42C and 42A, but the cells concomitantly exit from mitosis and enter the G0 phase(Masiakowski and Shooter, 1988). From this it seems not only that some S100proteins may be specifically expressed in relation to the stage of progression of thecell division cycle, but they could conceivably also regulate cell cycle progressionitself. Some definitive evidence is now available that shows that S100A4 mayregulate cell cycle progression. As stated previously, in B16 and human HUT cellsexposed to hyperthermia, the S100A4 is down-regulated, with a reduction in thesize of the S phase fraction (Albertazzi et al. 1998b). Parker et al. (1994b) transfectedan S100A4 gene, which was placed under the control of a dexamethasone-inducibleMMTV (murine mammary tumour virus) promoter, into B16 murine melanomas.They showed that the transfected cells, in which the gene was switched on byexposure to dexamethasone, contained substantially larger S phase fractions ascompared with the control cells. These observations suggest that the S100A4 genecould be driving the cells into the S phase.

Another line of evidence is provided by the work of Parker and Sherbet (1992),who found that verapamil, a specific blocker of L-type calcium channels, downregulated the expression of S100A4. Subsequently, several investigators have dem-onstrated that verapamil can inhibit not only cell proliferation (Brocchieri et al.1996; Zeitler et al. 1997; Hoffman et al. 1998) but also invasion and metastasis(Farias et al. 1998). Interestingly, verapamil reduces the expression of certain mem-brane-associated metalloproteinases and urokinase-type PA. An enhanced expressionof S100A4, on the other hand, is associated with a remodelling of the ECM in away that is conducive to enhanced cell motility and invasiveness.

POSTULATED MECHANISM OF CELL CYCLE CONTROL BY S100A4

The mechanisms by which S100A4 exerts control over the progression of the cellcycle are yet unclear. However, much circumstantial evidence has been adduced tosupport the concept that this protein might form complexes with certain cellulartarget proteins, such as the suppressor p53 phosphoprotein that has been regardedas the guardian of the genome. When cellular DNA is damaged p53 protein isexpressed, and this appears to block the transition of the damaged cells from G1 intothe S phase, until DNA repair takes place. In B16 melanoma cells, up-regulation ofS100A4 (18A2/mts1) expression is associated with an enhanced level of p53, asdetected by immunohistochemical methods. This has been attributed to the formation



of a complex of S100A4 with p53 (Parker et al. 1994a, 1994b). Such a complexformation would have the effect of stabilising and enhancing the half-life of p53,and as a consequence p53 becomes detectable by immunohistochemical stainingprocedures. The formation of a complex between S100A4 and p53 could effectivelysequester p53 and abrogate its G1–S checkpoint control, resulting in an increase inthe size of the S phase fraction. One might then ask why the cells do not continueto accumulate in the S phase, but successfully transit from G2 into mitosis. It hasbeen shown recently that p53 is also involved in the control of the G2–M transitionof cells (Michalowitz et al. 1990; Stewart et al. 1995; Pellegata et al. 1996). Oneof the proposed mechanisms of this involves the participation of a protein calledstathmin.

Stathmin is a 19-kDa cytosolic phosphoprotein. It is up-regulated in manyneoplasms as well as in highly proliferating normal tissues (Luo et al. 1994; Row-lands et al. 1995; Friedrich et al. 1995; Bieche et al. 1998). Even immortalisation,an event regarded as a prelude to neoplastic transformation, of primary embryonicfibroblasts, results in a four-fold enhancement of stathmin expression and a substan-tial increase in the rate of proliferation. However, in this experimental system therewere no changes in stathmin expression upon oncogenic transformation (Mistry andAtweh, 1999). It would seem, therefore, that modulation of stathmin expression isa feature of cell proliferation rather than of neoplastic transformation. This statementis amply substantiated below.

The expression of stathmin is down-regulated when wild-type p53 is expressed.Using inducible p53 constructs, it has been demonstrated that when p53 is switchedon the stathmin gene is down-regulated together with the arrest of cells in the G2

phase. Indeed, p53 may be capable of down-regulating the stathmin promoter. Ahnet al. (1999) have shown that a decrease in the expression of stathmin occurs inimmortalised human and murine cells in parallel with induction of wild-type p53by DNA-damaging agents. Of interest in the context of the function of S100A4 isthe observation that stathmin destabilises the cytoskeleton (Marklund et al. 1996;DiPaolo et al. 1997). It sequesters free tubulin and inhibits tubulin polymerisation(Andersen et al. 1997; Curmi et al. 1997, 1999). Thus S100A4 and stathmin seemto share several properties, of which the most prominent are the influence they exerton cell proliferation and their postulated interaction with p53. A conundrum, in theobserved association between S100A4 and cell cycle progression, is why cells thatare induced by S100A4 to enter the S phase do not accumulate in the S phase butsuccessfully negotiate the G2–M checkpoint into mitosis.

Some recent work has shown that stathmin expression closely parallels that ofS100A4. Indeed, changes in S100A4 expression seem to be tightly coupled withcorresponding changes in the expression of stathmin gene. Thus, stathmin expressionchanges when cell proliferation is inhibited by hyperthermia. This happens alsowhen cells are growing exponentially, i.e., with increasing growth rates, or whenfull serum supplement is restored to serum starved cells (Sherbet and Cajone, 2000;Cajone and Sherbet, 2000) (Table 18).

There are two schools of thought about how stathmin might be involved withcell proliferation. One school believes that the G2–M transition of cells is associated



with enhanced stathmin expression (Eustace et al. 1995; Jones et al. 1995). Theother proposes that phosphorylation of stathmin is the critical event that allows thecells to make the transition (Marklund et al. 1994; Larsson et al. 1995; Duraj et al.1995; Beretta et al. 1995; Lawler et al. 1998). In the studies cited above, Sherbetand Cajone (2000) and Cajone and Sherbet (2000) have not investigated the stateof stathmin phosphorylation, for the correlation between stathmin gene expressionand cell proliferation was highly significant. S100 proteins have been shown to beable to modulate PKC-mediated phosphorylation of proteins (Kriajevska et al. 1998).S100A4 might function by inhibiting the phosphorylation of stathmin, which isregarded by some as essential for stathmin function. Therefore, the potential signif-icance of posttranslational modification of stathmin cannot be excluded.

As stated earlier, previous work has demonstrated that the interaction of S100A4and p53 might sequester the latter and abrogate its checkpoint control at the G1–Scheckpoint (Parker et al. 1994a, 1994b). However, after this successful transitioninto the S phase, one would expect them to accumulate in the S phase. This doesnot seem to happen. Although the size of the S phase fraction does increase uponinduction of S100A4 expression, cells do not appear to be held back at the G2–Mcheckpoint.

It is in this context that the parallel modulation of the expression of S100A4and stathmin genes assumes some significance. Cajone and Sherbet (2000) havepostulated that S100A4 might directly induce an up-regulation of stathmin expres-sion, enabling the cell to enter into mitosis. Because wild-type p53 has been knownto down-regulate stathmin expression, it has been suggested that S100A4 mightsequester p53 and abrogate its control over stathmin function, and thereby enablecells to make the G2–M transition (Figure 27). There is simplicity about this concept,which emphasises the possibility of S100A4 being involved at both G1–S and G2–Mtransition checkpoints. This postulate needs to be tested rigorously, for this is thefirst CBP that has been shown not only to have close links with cell cycle progressionbut also to be intricately involved in the two main control checkpoints of thecell cycle.

TABLE 18Coupling of S100A4 and Stathmin Gene Expression in Relation to State of Proliferation

Experimental Condition S100A4 StathminGrowth

Rate

Hyperthermia (HUT cells) (decreased growth rate) ↓ ↓ ↓HeLa cells, 24-, 48-, and 72-hr cultures ↑ ↑ ↑

Source: Based on Sherbet and Cajone (2000) and Cajone and Sherbet (2000). Reprintedby permission of the publisher Kluwer Academic Publishers.



S100A ISOFORMS

S100A protein occurs as two isoforms made up of homo- or heterodimers of thetwo subunits called the α and β subunits. These two subunits comprise 93 and 91amino acid residues respectively, and share 58% amino acid sequence homology(Isobe and Okuyama, 1978, 1981). S100A0 is a homodimer of two α subunits, andS100A is a heterodimer of an α and β subunit. S100B is made up of two β subunits.The S100A isoforms as well as S100B occur predominantly in the brain. S100A0

forms a minor component of S100 proteins in the neurones and peripheral nerves(Isobe et al. 1984). S100A0 is also associated with the sarcolemma and the SR(Haimato and Kato, 1987; Donato et al. 1989). Because the SR holds the intracellularstores of calcium, S100A0 could be involved in the mobilisation of calcium fromthese intracellular stores. Fano et al. (1989) have shown that S100A0 does inducecalcium mobilisation from SR-terminal cisternae, which were isolated from ratskeletal muscle.

The levels of S100A0 in the bloodstream of patients with acute myocardialinfarction have been investigated on the premise that the protein may be releasedinto the bloodstream from damaged heart muscle. In a group of patients investigated,Usul et al. (1990) found a nearly fourfold increase of S100A0 in the serum thateventually increased to roughly 20-fold higher than the levels of the protein foundin control subjects. However, there was considerable variation between patients.Nevertheless, such increases were not encountered in patients with angina pectoris.Usul et al. (1990) have, therefore, suggested that this might provide a method fordifferentiating between myocardial infarction and angina pectoris.

FIGURE 27 The putative involvement of S100A4 in G1–S and G2–M transition in the cellcycle. As discussed in the text, S100A4 may drive the cells into the S phase by sequestrationof p53. Stathmin is up-regulated as well as phosphorylated during the G2–M transition. Wild-type p53 is said to down-regulate stathmin and might block the G2–M transition by thismethod. Because stathmin expression parallels that of S100A4, the figure represents thepostulate that S100A4 might itself up-regulate stathmin and promote G2–M transition, or thiseffect is routed through p53 sequestration.

M

G1G2

S100A4

Stathmin

+

P

p53

+

p53

++

S100A4

p53

S

-



223

S100A2 AS A PUTATIVE TUMOUR SUPPRESSOR

It is evident from the foregoing discussion that a majority of S100 family proteinsappear to possess the ability to promote tumour progression by enhancing theproliferative and invasive potential of tumours. One member of the S100 proteinfamily, namely S100A2, is believed to function as a tumour suppressor. S100A2was isolated more than a decade ago by Glenney

et al.

(1989). It is found in certaincell types in the kidney, lung, and breast epithelium. It is moderately expressed inthe liver, and cardiac and skeletal muscle, but not encountered in the adrenal gland,the intestine, or the brain. Lee

et al.

(1991) identified several cDNA clones that werehighly expressed in normal tissues but suppressed in corresponding tumour-derivedcells. Among these clones was a transcript of an S100 family gene. Subsequently,its expression was reported to be down-regulated in breast tumour cells. Exposureof these cells to 5-aza-2

′

-deoxycytidine produced a reexpression of this gene, whichsuggested that its expression was normally suppressed in the tumour cells by hyper-methylation (Lee

et al.

1992). That hypermethylation of this gene is responsible forthe loss of its expression in breast cancer cell lines has been confirmed recently(Wicki

et al.

1997). A loss of S100A2 expression has been reported in several breastcancer cell lines (Pedrocchi

et al.

1994). A marked loss of expression has been foundin human sarcomas (E. Horvig, personal communication, 1998). S100A2 expressionhas been found in only 7% (of 107) of human sarcomas. In contrast, S100A4 andA6 expression was detected in 38 and 48%, respectively, of the specimens. Thissuggests a preponderant loss of S100A2 expression. However, there was no obviouscorrelation with clinical features or patient survival. In human astrocytomas alsothere is a conspicuous loss of S100A2 expression, whereas, in contrast, several otherS100 proteins, notably S100A1, A4, and A6, are markedly expressed (Camby

et al.

1999).The suppressor function of S100A2, however, is not so clear-cut either in normal

melanocytes or in melanomas. Thus, in normal melanocytes S100A2 is expressedat very low levels or is virtually undetectable. Neither is its expression up-regulatedin malignant melanoma (L.B. Andersen

et al.

1996). S100A2 staining has beenreported in the basal layer of the epidermis and in hair follicles, but none has beenfound in naevi. Also, only a small proportion (4/39) of primary cutaneous melanomasand none of 14 metastatic lesions stained for S100A2 (Boni

et al

. 1997). A furtherreport has appeared on S100A2 expression in epidermal cell types and epithelialtumours of the skin. Again the basal cells, epithelial cells of the sebaceous glands,and epithelial cells of hair follicles stained positive for S100A2. Also immunoreac-tive were basal cell as well as squamous cell carcinomas (Shrestha

et al.

1998).Overall, the evidence available to date does not lend itself to a firm interpretationthat S100A2 has a suppressor function or that its expression is associated withadvanced stages of tumour progression. This view is also supported by the datapublished by Maelandsmo

et al.

(1997). The differences in the levels of S100A2expression between naevi and cell lines derived from primary tumours seem to bemore marked than those between the primary and metastatic lesions. This suggeststhat a down-regulation of the gene could occur in the early stages of developmentof these tumours. Xia

et al.

(1997) have cast further doubts about the suppressor

0942/C17-B/frame Page 223 Wednesday, October 25, 2000 7:46 AM

224


function of S100A2. They found this gene to be highly expressed in basal andsquamous cell carcinomas of the skin and oral cavity, although

in situ

hybridisationstudies have indicated that the amounts of S100A2 occurring in the tumour cellsthemselves were limited. The majority of the protein was found in the hyperplasticepidermis around the tumour. Xia

et al.

(1997) found no differences in the expressionof the protein in primary tumours and metastatic lesions.

Ilg

et al.

(1996) studied the binding of antibodies against several S100 proteins,including S100A2, and noticed a marked difference in their intracellular localisation.S100A2 was located predominantly in the nucleus, but S100A6 occurred mainlyaround the nucleus. They also found that the binding pattern of S100A2 antibodiesdiffered from that of antibodies against S100A4, the expression of which has beenwidely reported to promote tumour-progression (see below). Although there is someevidence supporting the putative tumour suppressor function of S100A2, most ofthe research deals with its expression in tumour-derived cell lines, and there are nosignificant studies on the status of its expression in human tumours themselves.

The observation made by Xia

et al.

(1997) relating to the presence of S100A2in hyperplastic epidermis incidentally serves to emphasise a putative relationshipbetween the expression of this protein and the proliferative state of cells. Thisrelationship now will seem more secure with the finding that EGF up-regulatedS100A2 expression in organ cultures of human skin. EGF also markedly up-regulatedthe expression of S100A2 mRNA in immortalised human keratinocytes in culture.In both cases, the EGF effects could be blocked by using PD153035, which is aspecific inhibitor of EGFr tyrosine kinase (Stoll

et al.

1998). These experimentsdemonstrate the requirement of EGFr activation for the up-regulation of S100A2expression, and therefore, suggest the presence of a direct relationship betweenS100A2 and mitogenic stimulation.

With this background of great ambiguity regarding the suppressor function ofS100A2, one should look to some recent evidence that S100A2 function might bemediated by wild-type p53. Tan

et al.

(1999) have identified putative p53-bindingsites in the promoter of S100A2.

In vitro

, wild-type p53 seems to transactivateS100A2. This transactivation is blocked by dominant negative p53 mutants. Thiscan be deemed as evidence that S100A2 might influence cell proliferation with themediation of p53. Thus p53 mediation may yet prove to be an underlying mechanismin the regulation of cell proliferation by S100 proteins.

S100A3 EXPRESSION IN CELL DIFFERENTIATION AND NEOPLASIA

M

OLECULAR

F

EATURES

OF

S100A3

S100A3 is a cysteine-rich CBP that binds to Ca

2+

with low affinity but binds withhigh affinity to Zn

2+

(Engelkamp

et al.

1993; Fohr

et al.

1995). As many as 10 outof 101 amino acids of the protein are cysteine residues. A subfamily of S100 proteinsthat binds Zn

2+

with high affinity can be identified. These proteins bind four Zn

2+

ions per protein monomer (Fohr

et al.

1995). S100A3 may bind two Zn

2+

ions permonomer. One zinc atom binds to four cysteine residues and the second atom binds



225

to one histidine residue. Therefore, a commonality of the zinc-binding domain andits organisation and stabilisation seems to be evident, and this has obvious similaritiesto the GAL4 zinc finger transcription factor from yeast. However, S100A3 binds toZn

2+

with far less affinity than that displayed by zinc finger transcription factors.The binding of zinc by S100A3 seems to be a well-coordinated process (Fritz

et al.

1998), and might have effects on its conformational and functional states. TheS100A3 gene occurs in the S100 gene cluster on the human chromosome 1q21 andon mouse chromosome 3.

S100A3 E

XPRESSION

IN

C

ELL

D

IFFERENTIATION

AND

H

UMAN

G

LIOMAS

S100A3 seems to be expressed specifically in skin and derivative tissues (Kizawa

et al.

1996, 1998; Boni

et al.

1997) and shows a specific pattern of localisation inthe subcompartments in human and murine follicles. High expression of S100A3has been found in the cuticle sheath and cortical cells undergoing terminal differ-entiation. This has led to the suggestion that it might participate in the differentiationof the cuticle cell and the formation of the hair shaft (Kizawa

et al.

1998; Takizawa

et al.

1999).The expression of S100A3 has been studied in a series of human astrocytomas

of various grades of malignancy (Camby

et al.

1999). These authors have reportedthat they were able to differentiate between grade I (WHO) pilocytic astrocytomasand grades II–IV tumours on the basis of their expression of S100A3. Further, onthis basis they suggest grade II–IV astrocytomas form a group of astrocytomas thatare biologically distinct from WHO grade I tumours. On the other hand, the expres-sion of S100A3 could be merely an indicator of the degree of anaplasia.

S100A4 IN CANCER DEVELOPMENT AND PROGRESSION

S100A4 E

XPRESSION

AND

M

ETASTATIC

P

OTENTIAL

OF

C

ANCERS

The considerable influence that S100A4 exerts over cell adhesion, motility, and cellproliferation has prompted the investigation of the possible relevance of its expres-sion in tumour development and their progression to the metastatic state.

S100A4 is found in a variety of normal tissues of both murine and human origin(Grigorian

et al.

1994; Gibbs

et al.

1995). It occurs in normal adult rat tissues suchas smooth muscle, brown adipose tissue, and the liver. Other tissues, e.g., normalbreast tissue, endothelia, absorptive and keratinised epithelia, neuronal, as well assome cells of the haematopoietic system, also contain S100A4. S100A4 is predom-inantly intracellular in distribution, but Gibbs

et al.

(1995) believe that in breasttissue, it may occur as an extracellular secreted protein. There could be some species-specific differences in tissue distribution of the protein (M. Davies

et al.

1995).In spite of this apparently generalised expression of S100A4, it ought to be

recalled here that there are reports that S100A4 (FSP1) is expressed in a fibroblast-specific manner. Not only this, S100A4 (FSP1) is described as one of the genes


226


whose expression is associated with the epithelial–mesenchymal transformationencountered in chordates (Hay, 1995). The fibroblast-specific expression has beenattributed to the presence of a

cis

-acting element in the 5

′

-flanking region of the firstintron of the gene. This sequence is believed to be active in fibroblasts but not inepithelial cells. Okada

et al.

(1998) have shown further that the 5-bp sequenceTTGAT (–177 to –173) interacts specifically with nuclear extracts obtained fromfibroblasts.

In addition to its wide distribution in normal tissues, high levels of S100A4 havealso been recorded in human as well as in murine tumours with high metastaticpotential (Ebralidze

et al.

1990). A clear correlation seems to exist between the levelof S100A4 mRNA and protein and the metastatic potential of Dunning rat prostatecarcinoma cell lines (Ke

et al.

1997). Indeed, some of this evidence might beconstrued as suggesting that S100A4 (FSP1) is a developmentally regulated gene.The aberrant expression of S100A4 in neoplasia could be viewed as reflectingabnormalities in the process of de-differentiation associated with cancer developmentand progression.

In addition to the empirical correlation, substantial experimental work in recentyears has allowed a strong link to be established between the levels of S100A4expression and metastatic potential. In the B16 murine melanoma, modulation ofits expression levels alters metastatic behaviour (Parker

et al.

1991). Upon transfec-tion with the S100A4 gene, benign rat mammary epithelial cells have been foundto become more tumorigenic and acquire a higher potential for metastasis (B.R.Davies

et al.

1993). Although these results were unambiguous, the process oftransfection of exogenous genes has been subjected often to the criticism that itcould lead to genomic perturbation as a consequence of a stable integration of anexogenous gene. Kerbel

et al.

(1987) noted that calcium phosphate-mediated DNAtransfer also changed cellular behaviour. They reported that when CBA/J mouseadenocarcinomas were transfected with a vector carrying only a marker gene butnot transforming genes, 17% of transfectant cells showed lung colonisation. Equally,there are reports that transfection of extraneous DNA does not alter biologicalbehaviour in any way (Jamieson

et al.

1990a, 1990b). Nonetheless, it is imperativethat the expression of S100A4 is linked with changes in the biological behaviour ofcells. Therefore, to obviate this potential criticism, B16 melanoma cell lines havebeen transfected with S100A4 (18A2

/

mts1) and placed under the control of thedexamethasone-inducible MMTV promoter. In these transfectants, switching on theexogenous gene markedly enhances localisation of the transfectant cells in the lung(Parker

et al.

1994b). In the same investigation, Parker

et al.

(1994b) also transfectedthe inducible construct into a dexamethasone receptor negative B16 cell line anddemonstrated that the exogenous gene is not switched on. These experiments haveestablished conclusively that S100A4 enhances lung colonisation by B16 tumourcells. It is worthy of note, however, that Parker

et al.

(1994b) did not determinewhether S100A4 transfectants were capable of spontaneous metastasis. As is oftenpointed out, much of this work is still open ended. There are one or two points ofcriticism that need to be taken into account. One of these is that dexamethasone isknown to up regulate the expression of other EF-hand proteins such as calcineurin.Because the S100A4 transfectants were exposed to dexamethasone to switch on



227

S100A4 expression, these cells should have been tested for calcineurin expression.Furthermore, tail vein injection of cells often allows them to bypass the initial hurdlesthat cells released from a primary tumour have to face, and they have direct accessto the lung. Thus, although one can accept that S100A4 enhances the localisationof cells in the lung, there are no data on the effects of S100A4 on the release ofcells from the primary tumour, their ability to gain access to the blood vessels, andtheir ability to withstand or escape the immunological surveillance by the host.

The recent studies of Lloyd

et al.

(1998) have answered some of these questions.They transfected the human S100A4 gene into the rat benign mammary tumour cellline, Rama-37. Transfectant cells that expressed S100A4 mRNA at high levels werecapable of forming primary tumours in syngeneic rats and were also able to metasta-sise spontaneously. In contrast, those transfectant cells that expressed S100A4 atlow levels were unable to form secondary tumours. Furthermore, they have shownfor the first time that metastatic deposits that expressed S100A4 mRNA were ableto form primary tumours that were also capable of metastasising spontaneously. Inother words, a constitutive expression of the gene does confer metastasising prop-erties. Another piece of documentation, which supports the metastasis-promotingability of S100A4, is the generation of S100A4 transgenic lines, e.g., Tg463 andTg507, of GRS/A mice by Ambartsumian

et al.

(1996). The GRS/A primary strainrather characteristically shows a high incidence of mammary tumours that do notappear to possess metastatic ability. In contrast, the S100A4 transgenic strainsdeveloped metastatic tumours in approximately 40% of the animals. Ambartsumian

et al.

(1996) also demonstrated the presence of S100A4 in both primary and meta-static tumour. Simultaneously, M.P.A. Davies

et al.

(1996) carried out similar exper-iments on transgenic S100A4 mice. In the S100A4-expressing animals, mammarytumours were palpable earlier than in corresponding control animals, and macro-scopic deposits of tumours were detectable in the lungs. There is thus adequate proofthat high expression of S100A4 is conducive to metastatic dissemination.

Conversely, the transfection of antisense constructs into malignant cell lines hasresulted in the reduction of metastatic potential (Grigorian

et al.

1994). The abilityof antisense S100A4 mRNA to suppress metastatic potential has been confirmed infurther experiments with highly metastatic lines of the Lewis lung carcinoma. Severalclones with transfected antisense S100A4 mRNA have been isolated, and all of themproved to have very low metastatic potential (Takenaga

et al.

1997a). In a novelexperimental approach, Maelandsmo

et al.

(1996) transfected human osteosarcomacells that expressed S100A4 at a high level with a ribozyme directed against thegene transcript. With the destruction of the S100A4 mRNA by the ribozyme, theosteosarcoma cells showed no skeletal metastases when injected intracardially intonude mice. They also found that the degradation of S100A4 mRNA had no effecton the proliferation of the cells either

in vitro

or

in vivo

. On the basis of the latterresults, Maelandsmo

et al.

(1996) seem to be suggesting that metastatic ability maybe dissociated from proliferative ability. This is an interesting postulate, but, so faras S100A4 is concerned there is little doubt that its high level of expression leadsto high growth rates.

Compatible with the association of high S100A4 expression with malignancy isthe recent demonstration that the S100A4 gene may be down-regulated in benign


228


mammary epithelial cells. It would appear that S100A4 transcription is inhibited bya

cis

-acting sequence, related to the consensus recognising GC factor, occurring 1.3kbp upstream of the transcription start site. An inverse relationship has been notedbetween the GC-factor and S100A4 mRNA levels (Chen

et al.

1997).In conclusion, one may justifiably regard the currently available body of evi-

dence, relating to the conferment of invasive and metastatic abilities by S100A4, asvirtually irrefutable. It ought to be stated, nonetheless, that there has appeared asolitary report to the effect that S100A4 shows no relationship to cell proliferation,invasion, or metastasis. This report described the transfer of S100A4 sense constructsinto MCF7 cells, with no apparent effects on

in vitro

invasion or proliferation. Thetransfectants produced no metastasis in mice, but there was marked tumour necrosisand abnormalities in tumour vasculature (Onischenko

et al.

1996). There are anumber of points to be taken into account with regard to this work. Grigorian

et al

.(1996) also used MCF7 cells for transfection of S100A4 and found that the trans-fected cells acquired marked metastatic properties. This is in sharp contrast with thefindings of Onischenko

et al.

(1996). MCF7 cells are known to express the putativemetastasis suppressor gene

nm23

at a high level (Sherbet

et al.

1995).

S100A4

and

nm23

genes might be coordinately regulated (Parker

et al.

1991; Hsu

et al.

1997).Admittedly, the significance of

nm23

expression in breast cancer cells may not berated high. However, Albertazzi

et al.

(1998b) have reported recently that the expres-sion status of both

S100A4

and

nm23

taken together correlated far more powerfullywith the clinical aggressiveness of breast cancer than when the status of

S100A4

alone was considered. In other words, it would have been advisable for Onischenko

et al.

(1996) to look also at nm23 expression in their experiments. There have beenno other reports so far that S100A4 has any effects on neovascularisation or celldeath by necrosis or apoptosis. Without prejudice to their findings, however, it wouldbe worth remarking that these latter effects could be collateral changes associatedwith the integration of the exogenous gene into MCF7 cells. The chances of thishappening have been obviated by Grigorian

et al.

(1996) by adopting a differentexperimental strategy of transfecting the gene under the control of an induciblepromoter.

Some ambivalent views have been expressed also by Chiaramonte

et al.

(1998).These authors used a murine mammary adenocarcinoma cell line, TS/A, and twomurine melanoma cell lines, B16-A and B78H1. All three cell lines responded toIFN-

γ

treatment with an increase in metastatic ability. However, when they testedthese IFN-

γ

-treated cells for

S100A4

expression, no changes were noticed in TS/Acells. Further, of the two melanomas only B16-A showed an enhancement of

S100A4

expression. The effects of IFN-

γ

seem to be somewhat variable. It has been foundto enhance transcription in human macrophages (Grigorian

et al.

1994). On the otherhand, IFN-

γ

down-regulates S100A4 expression in human colon adenocarcinomacell lines. This effect is dependent on membrane-associated IFN-

γ

receptors and isnot shown by IFN-

γ

or -

β

(Takenaga, 1999). Chiaramonte

et al.

(1998) have, there-fore, quite legitimately argued that different genes may be associated with themetastatic behaviour of different tumour types. It would be useful to pursue theseobservations, even in the face of the virtually overwhelming evidence for a majorrole for S100A4 in tumour progression.



229

Whether S100A4 promotes the metastatic spread of tumours by influencing cellularproperties at a specific stage of the metastatic cascade is not clear at present. In lightof the alleged inhibition of the microvasculature by S100A4, Cajone and Sherbet (2000)have employed heme oxygenase-1 (HO-1) gene as a marker in a series of experiments.Heme oxygenase (HO-1) is a stress–response protein. The induction of HO-1 gene isbelieved to play a protective role against both heme- and nonheme-mediated oxidantinjury. Many agents such as hyperoxia, bacterial LPS, hydrogen peroxide, interleukin,prostaglandins, TPA, and hyperthermia, among others, are known to induce HO-1 (Choiand Alam, 1996). The HO-1 gene expression is regulated by the presence of responsiveelements that can bind these agents. Another agent called 4-hydroxynonenal (4-HNE)is also a potent inducer of HO-1 expression. 4-HNE is a product of the peroxidationof membrane lipids and it has been shown to be able to induce the synthesis of HSPs(Cajone et al. 1989; Cajone and Bernelli-Zazzera, 1989). One of the HSPs induced by4-HNE is a 32-kDa protein (Allevi et al. (1995). This HSP32, apparently associatedwith oxidative stress, seems to be none other than HO-1. Although total identity betweenthese two proteins has not been fully established, HO-1 has been referred to, notinfrequently, as HSP32 (Choi and Alam, 1996).

Cajone and Sherbet (2000) modulated the levels of S100A4 expression by usingagents that are known to up-regulate HO-1 expression. They found that the HO-1expression was up-regulated irrespective of the effects of the agent on S1004 expres-sion (Table 19). Grigorian et al. (1994) found that LPS has a variable effect onS100A4 expression in inflammatory macrophages. In these cells it reduced S100A4expression in the first 3hr but then up-regulated it over the next four hours.

In other words, there was a total dissociation of the expression of the two genes.This might throw some light on the possible function of this protein in cancermetastasis. HO-1 has been reported to have a marked effect on tumour angiogenesis.

TABLE 19The Lack of Relationship between Expression of S100A4 and Heme Oxygenase (HO-1) Genes

Cells/Treatment S100A4 HO-1

HUT 37,a – 42°C ↓ ↑3T3 normal serum + 4-HNE ↓ ↑3T3 serum-starved cells + 10% FCS ↑ ↑ 3T3 + TPA ↑ ↑ 3T3 + 4-HNE ↑ ↑a HUT is a heat-resistant clone of cells derived from

HepG2 cells.Source: Based on data from Cajone and Sherbet

(2000). (Reprinted by permission of the publisher Kluwer Academic Publishers.



Transfection of human HO-1 gene has been found to produce a two-fold increasein neovascularisation (Deramaudt et al. 1998). Indeed, some agents that induce HO-1 gene expression are also inducers of angiogenesis. For instance, the HO-1 genecontains response elements for LPS, IL-6, and prostaglandins (Camhi et al. 1995,1996; Kozumi et al. 1995), all of them known inducers of angiogenesis.

These are powerful indicators that that S100A4 might not be associated withinduction of neovascularisation. However, the effects of S100A4 expression on thecellular properties that are highly relevant in other compartments of the metastaticcascade, such as the phase of expansive growth of the primary tumour, phase ofinvasion, and possibly also the growth of overt metastases, are virtually indisputable(see also Figure 28 and Table 21).

CLINICAL POTENTIAL OF S100A4 AS A MARKER FOR CANCER PROGNOSIS

S100A4 has attracted a great deal of attention in recent years because of the widevariety of physiological events and parameters of biological function that it appearsto influence. However, in contrast with several other putative markers of malignancy,investigations into the possible clinical value of this gene are of recent origin. Thisis because much of the proposed work has been dampened by the somewhat ill-informed criticism and reservations expressed about it. Fortunately, a great deal ofresearch into the basic aspects of S100A4 function has been carried out, which hasgreatly emphasised the importance of S100A4 in the clinical assessment of thedisease. Some of the work, such as that of Pedrocchi et al. (1994), Grigorian et al.(1996), and Maelandsmo et al. (1996), used tumour cell lines derived from humantumours, and these authors have shown that the levels of S100A4 expression wererelated to invasive and metastatic ability. Sustained studies of human tumours aimedat determining the relevance of S100A4 in predicting progression of the diseasehave followed. These have been carried out mainly in melanomas, breast cancer,and astrocytic tumours.

S100A4 IN HUMAN BREAST CANCER

Pedrocchi et al. (1994) first described the importance of S100A4 in breast cancercell lines. Recently, Albertazzi et al. (1998b) have published a detailed study ofS100A4 expression, not only in relation to the degree of aggressiveness of thedisease, but also as to how it relates to other clinical markers such as the status ofoestrogen and progesterone receptors and the degree of tumour differentiation.

Albertazzi et al. (1998b) also employed another marker, nm23-H1. nm23 is aputative metastasis suppressor gene identified in murine melanomas (Steeg et al.1988a, 1988b), of which two human homologues have been cloned (Rosengard etal. 1989; Stahl et al. 1991). Several reports in the literature suggest an inverserelationship between the level of expression of nm23 mRNA or the gene product,nucleoside diphosphate (NDP) kinase, and metastatic potential. Such an inverserelationship has been described in some forms of human cancer, e.g., melanoma(Florenes et al. 1992; Caligo et al. 1994). In colorectal and gastric carcinomas, nm23



expression has been reported to be down-regulated substantially in metastatic tumouras compared with the primary carcinoma (Ayhan et al. 1993; H. Nakayama et al.1993). An inverse relationship between nm23/NDP kinase expression and metastaticpotential has been reported to occur in hepatocellular carcinoma (T. Nakayama etal. 1992). Low levels of NDP kinase expression have been correlated with reducedsurvival of patients with infiltrating ductal carcinoma of the breast (Barnes et al.1991; Royds et al. 1993). However, several studies repudiate this inverse relationship(Haut et al. 1991; Hailat et al. 1991; Keim et al. 1992; Zhou et al. 1993; Sawan etal. 1994; Saitoh et al. 1996; Srivasta et al. 1996; Aldersio et al. 1998). A study ofa large series of breast carcinomas has shown that NDP kinase expression is neitherrelated to disease relapse or patient survival, nor does it correlate with other prog-nostic factors such as tumour grade, oestrogen and progesterone receptor status andp53 expression (Sawan et al. 1994). Stephenson et al. (1998) examined 412 casesof lung carcinoma for nm23/NDP kinase expression. They found no relationship ofits expression to metastatic disease or prognosis. Similarly, overexpression of nm23-H1 in epithelial ovarian carcinomas is associated with lower survival and signifi-cantly poor prognosis in early stage and well-differentiated carcinomas (Schneideret al. 2000).

Some of the problems associated with the observed lack of inverse correlationbetween nm23/NDP kinase expression and metastatic progression might be due tothe tacit assumption that NDP kinase function and the putative metastasis-suppressorproperties are interrelated and inseparable. Contrary to this, it has been argued thatthese properties can indeed be dissociated. H.Y. Lee and Lee (1999) transfected acDNA coding for a mutant form of nm23-H1 that lacked NDP kinase activity intohuman prostate carcinoma cells. Nonetheless, the transfectant cells showed reducedinvasive ability, in the same way as cells that had been transfected with wild-typecDNA. Although these observations do suggest that the two properties of nm23 aredissociable, conceptually the putative metastasis-suppressor function of nm23becomes even harder to appreciate.

This somewhat equivocal correlation, between nm23/NDP kinase expression andtumour progression, has led to the suggestion and actual demonstration that thisgene might be coordinately regulated with the metastasis-promoting S100A4 genein the B16 murine melanoma (Parker et al. 1991). These authors used MSH and RAto modulate the levels of S100A4 expression and found that the expression of nm23levels showed corresponding changes. As a result, the ratio of their expressionremained virtually constant. Similar results have been described more recently byHsu et al. (1997) in CH27 human lung cancer cells, which essentially confirm thefindings of Parker et al. (1991). Indeed, there is evidence that S100A4 expressionpromotes the depolymerisation of tubulin, whereas nm23 expression has the oppositeeffect (Nickerson and Wells, 1988; Lakshmi et al. 1993).

Whether there is a direct regulatory link between S100A4 and nm23 remains tobe established. It is unclear whether S100A4 is itself involved in some way with thenegative regulation of nm23. The 1q21 locus, which harbours the S100A4 gene, alsocontains the human homologue of the PRUNE gene of Drosophila melanogaster.The prune eye colour phenotype of Drosophila is attributed to null mutations of thePRUNE gene. The occurrence of one mutant copy of awd (abnormal wing disc) (the



Drosophila homologue of nm23-H1) with the prune genotype is lethal. This suggeststhat nm23-H1 and the prune protein might interact. The prune protein has indeedbeen found to interact with nm23-H1 and to negatively regulate the latter. In furtherconfirmation, it has been noticed that the interaction is impaired when nm23-H1 ismutated (Reymond et al. 1999). The emergence of this negative regulation of nm23-H1 by the prune protein does not explain the inverse relationship shown to subsistbetween S100A4 and nm23-H1. Perhaps it would be fruitful in future studies relatingto the expression of these genes also to measure PRUNE expression as well.

With this background, Albertazzi et al. (1998b) focused on the expression ofS100A4 (h-mts1) as well as that of nm23 in human breast cancer. This was with aview to determining their individual abilities to serve as markers, and further tocheck whether a combination of the status of their expressions might enable one toobtain a more accurate assessment of the metastatic potential of the tumours.

The data presented by Albertazzi et al. (1998b) show that high S100A4 expres-sion is associated with metastatic spread to the regional lymph nodes. The expressionof nm23 on its own did not show a statistically significant inverse correlation withnodal spread. However, the expression status of the two genes, taken together,strongly correlated with nodal metastasis. Furthermore, the correlation was moresignificant when S100A4 and nm23 were considered in combination,as comparedwith S100A4 on its own (Table 20). This suggests that, although nm23 did not seemrelevant to nodal metastasis, a more accurate assessment of nodal status could bederived by looking at the expression of both S100A4 and nm23 genes.

A further indication that S100A4 expression status might be related to the clinicalaggressiveness of the tumour is provided by another line of evidence. Albertazzi etal. (1998b) have reported that breast cancers with no detectable expression of S100A4were ER and PgR positive. With increased expression of S100A4, the tumours couldpossibly progress toward a hormone-independent state. Such progression has beendemonstrated in vitro. MCF7 breast cancer cell lines that were transfected with andproducing S100A4 have been reported to acquire hormone-independent growth(Grigorian et al. 1996). This is compatible with the generally held view that ER/PgR-negative breast cancers are clinically more aggressive and also tend to be EGFrpositive. The ER/PgR status of breast cancers generally correlates inversely withEGFr status, which has been regarded by some as an indicator of poor prognosis(Sainsbury et al. 1987a, 1987b; Bolla et al. 1990; Hainsworth et al. 1991). This fitswith the findings of Sherbet et al. (1995) that breast cancer cell lines that were highexpressors of S100A4 tended also to be high expressors of EGFr. Furthermore, therecent finding that EGF is able to induce the expression of S100A4 in epithelialcells (Okada et al. 1997) may be deemed as supporting the relationship betweenEGFr expression and cancer prognosis. A correlation between the expression of type1 growth factor receptor family and prognosis has also emerged from the recentwork. Rudland et al. (2000) found that S100A4 positivity of breast cancer correlatedsignificantly with the expression of c-erbB2 and c-erbB3. The prognostic signifi-cance of c-erbB2 has been well established and overexpression of this receptor isassociated with poor prognosis. However, there are no data relating to the prognosticvalue of c-erbB3 nor c-erbB4 (Angus et al. 2000). Albertazzi et al. (1998b) alsolooked at tumour differentiation, but this did not correlate with S100A4 expression.



The clinical data, together with the state of expression of steroid receptors andthe expression levels of S100A4 and nm23 genes, were analysed using artificialneural networks (ANNs) for accuracy of prediction of nodal spread of the carcino-mas. Naguib et al. (1997) previously had demonstrated that ANNs could be used topredict nodal involvement in breast cancer. In that study several established as wellas experimental cancer markers had been analysed, and ANN techniques were foundto be capable of dissecting and identifying the most powerful predictors of nodalmetastasis. The ANN analyses, provided by Albertazzi et al. (1998b) as well as byNaguib et al. (1998), have also supported the conclusion that, overall, S100A4expression is associated with and indicative of more aggressive forms of the disease.The investigation of Albertazzi et al. (1998b) does emphasise the view that whencomplemented with nm23, S100A4 could provide a powerful marker for predictingbreast cancer prognosis. On the other hand, S100A4 might be a significant andindependent marker for prognosis. An investigation of a large series of breast cancerover a period of 16 years has led Rudland et al. (2000) to conclude that S100A4expression correlates strongly with patient survival. The Rudland group foundS100A4 mRNA in both epithelial and stromal components of breast cancers and,further, that the levels of S100A4 are higher in carcinomas as compared with benignbreast tumours (Nikitenko et al. 2000). They have also reported that 80% of patientswho were S100A4 negative were alive after 19 years of follow-up, whereas only

TABLE 20Relationship Between S100A4, S100A4v, and nm23 Expression to Nodal Spread of Breast Cancer

Gene Expressed Relationship to Nodal Spreada

S100A4 +nm23 –S100A4+nm23 ++S100A4v+b –S100A4–/S100A4v–b +

a +, statistically significant relationship (p < .05); ++, strongrelationship (p < .01).b The expression of S100A4v (the splice variant form) alonedid not relate to nodal spread; however, tumours that expressneither S100A4 nor S100A4v do not tend to spread to theregional lymph nodes. Only 27% of carcinomas expressingneither h-mts1 nor h-mts1v showed metastatic spread to thelymph nodes; 57% (4/7) of carcinomas in which only thevariant isoform was detectable showed nodal metastasis.This apparent difference did not reach statistical signifi-cance.

Source: Based on Albertazzi et al. (1998a, 1998b). Reprintedby permission of the publisher Mary Ann Liebert Inc.



11% of S100A4-positive patients were alive at the end of this period. The correlationbetween S100A4 negativity and survival was highly statistically significant.

Interestingly, Rudland et al. (2000) found that S100A4 expression correlatedonly marginally with axillary lymph nodal involvement. Nevertheless, S100A4-positive patients who were also node positive had poorer survival than S100A4patients with no nodal involvement. The involvement of regional lymph nodes isregarded as the most consistent predictor of prognosis (Angus et al. 2000). Thepoorer prognosis of the S100A4+/node+ group may be related to whether or notS100A4 is expressed by tumour cells in the regional lymph nodes. Overall, the workreviewed here leaves little room for doubt that S100A4 is associated with tumourmalignancy and prognosis. With the large body of evidence of the active participationof S100A4 in a wide spectrum of cellular functions, a demonstration of its associationwith metastatic cells of human breast cancer would be the coup de grace that islong awaited and that might satisfy the scientific cognocenti.

As stated previously, two splice variants of S100A4 have been reported so far(Ambartsumian et al. 1995; Albertazzi et al. 1998c). The larger variant, describedby Ambartsumian et al. (1995), occurs in many tissues, although with differencesin the levels of expression. Albertazzi et al. (1998c) found only the shorter splicevariant (h-mts1v) in the series of breast cancers that they had investigated. They didnot detect the expression of either variant in a number of tumour cell lines. Theapparently highly specific nature of its expression is somewhat inexplicable. None-theless, Albertazzi et al. (1998c) found that h-mtsv expression did tend to correlatewith nodal spread of breast cancers, albeit the correlation was not as persuasive asin the case of the unspliced S100A4 transcript.

Notwithstanding the positive nature of these findings, it ought to be stated herethat assessing the state of expression of the S100A4 protein, not merely that of itsmRNA, is equally important for providing a total picture of the relevance of thegene to progression of cancer. It is the occurrence of the functional protein thatwould determine the nature of the downstream events that define the degree ofaggressiveness of the disease. As Ambartsumian et al. (1999) observed recently,S100A4 mRNA is expressed in all organs of S100A4 transgenic mice that they haddeveloped. However, the protein is not expressed in organs that do not normallyexpress the S100A4 gene in the wild-type animals. Therefore, it is imperative thatinformation concerning the expression of the protein is collated at the same time asthe mRNA levels are measured. This would take into account the fact that there mayexist mechanisms that regulate the translation of the mRNA transcripts and possiblyalso decay of the protein in the normal course of cellular events.

S100A4 IN OTHER FORMS OF HUMAN CANCER

Among other forms of human cancer that have been studied for S100A4 expressionis human melanoma. Maelandsmo et al. (1997) detected high S100A4 levels onlyin approximately 50% of melanomas, but benign nevi also showed roughly similarlevels of S100A4. Quite obviously, S100A4 bears no relationship to the clinical stateof disease in this case. A criticism that can be made of this study is the method ofassessment of gene expression as undetectable, low, moderate, and high. It would



have been helpful if the authors had given the actual values of signal densities andprovided the range of values of each group. The findings of Shrestha et al. (1998)generally support those of Maelandsmo et al. (1997). Shrestha et al. (1998) foundlittle or no S100A4 in several cell types derived from normal skin. Furthermore,neither did basal cell carcinomas or squamous cell carcinomas show any S100A4immunoreactivity. The apparent lack of correlation of S100A4 with melanomaprogression is in sharp contrast with the high degree of correlation found in B16melanoma cell lines. One should state, notwithstanding, that a direct comparison ofexpression of the gene in tissue culture cell lines and tumour material, which isheterogeneous in cell composition, is probably not helpful, nor can it lead to mean-ingful conclusions.

In human gliomas, the expression of S100A4 was found to correlate with thedegree of malignancy as indicated by the World Health Organization (WHO) histo-logical grading. However, the changes in S100A4 were far less striking than thoseoccurring in S100A1, which significantly correlated with malignancy (Camby et al.1999). It would be worthwhile to bear in mind that the histological grading of thesetumours is a differentiation-related feature, and often tumour behaviour may not strictlyconform to characteristics defined for a particular histological group. Nonetheless,intrinsically the degree of differentiation would be inversely related to malignancy,and therefore the correlation reported between S100A4 and gliomas of different gradesmay be seen as generally supporting the involvement of S100A4. With the addedknowledge that S100A4 expression is higher in cell lines derived from higher gradegliomas as compared with lower grade tumours, it would not be premature to concludethat S100A4 may play an important role in the malignancy of gliomas.

The expression of S100A4 in colorectal tumours seems to show a clear corre-lation with disease progression. The expression of the gene is low in colonic fibro-blasts. Normal mucosal tissue and adenomas have been described as showing com-parable levels of expression, but expression level is markedly higher in adeno-carcinomas. Cells lines derived from adenocarcinomas also show high levels ofS100A4 expression. Immunohistochemical studies have generally confirmed theseNorthern analyses of S100A4 expression. Of much greater interest is the observationthat although adenomas are negative, carcinomatous foci within the adenomas havebeen found to stain for S100A4 (Takenaga et al. 1997b). Quite obviously, theexpression of S100A4 correlates well with the progression of colonic tumours.

S100A6 (CALCYCLIN) IN CANCER

The pattern of expression of calcyclin, now carrying the new nomenclature ofS100A6, has been studied in several forms of human cancer, e.g., melanomas(Weterman et al. 1992, 1993), salivary gland tumours (J.W. Huang et al. 1996),chondro-osseous tumours (Muramatsu et al. 1997), and squamous cell carcinomasof the oral mucosa (Berta et al. 1997). Muramatsu et al. (1997) have reported thatcalcyclin, together with S100A1 and A4, was particularly strongly associated withthe development of chondro-osseous tumours. Enhanced calcyclin expression hasbeen reported in squamous cell carcinoma of the oral mucosa but not in benign



mucosal lesions. This was accompanied also by an increased expression of the H-ras oncogene (Berta et al. 1997).

Several genes are differentially expressed in rat mammary tumours, which areinduced by the carcinogen N-methyl-N-nitrosourea (NMU). L.H. Young et al. (1996)detected several differentially expressed cDNA clones. Of these, one clone showedstrong homology to calcyclin. The expression of this clone was ten-fold greater intumour tissue than in normal tissue. Pedrocchi et al. (1994) investigated severalbreast cancer cell lines and noticed marked differences among S100 proteins in theirrelationship to aggressive behaviour. Calcyclin did not appear to bear any relationshipto tumour aggressiveness. However, its expression was higher in the vertical phaseof growth of melanomas and correlated with metastatic potential of melanomas(Weterman et al. 1992, 1993). Van Ginkel et al. (1998) found S100A6 to be differ-entially expressed between normal uveal melanocytes and uveal melanomas and celllines derived from them. Also differentially expressed were annexins V and VI, CaM,S100A11, and S100B. Although they have suggested that S100A6 expression mayrelate to the malignancy of uveal melanomas, it would be difficult to dissociate theeffects of S100A6 from that of other CBPs. As Sudo and Hidaka (1999) have shown,the CBPs may interact with one another. The correlation of calcyclin expressionwith metastatic potential has been confirmed by two recent studies (Boni et al. 1997;Maelandsmo et al. 1997). In the first study, all of 39 primary cutaneous melanomastested showed intense cytoplasmic staining for calcyclin. Also 9 of 14 metastatictumours were found to be calcyclin positive. In the second study, S100A6 expressionwas reported to correlate with patient survival times (Maelandsmo et al. 1997). Ina series of human astrocytomas, Camby et al. (1999) found the expression of S100A6to be clearly related to their WHO grading. Thus, grades I and II tumours could bedistinguished, on the basis of S100A6 expression, from the more malignant gradeIII and IV astrocytomas. However, as discussed previously, there is some ambiguityabout the relationship between calcyclin expression and tumour growth. Some inves-tigators regard calcyclin as a negative regulator of growth on the basis that inductionof differentiation in tumours can enhance calcyclin expression. It may be that theinvolvement of this S100 protein has other facets in its relationship with tumouraggressiveness than are currently appreciated and investigated. For example, it hasbeen shown to bind to annexin XI-A (Sudo and Hidaka, 1999). This interactionseems to be of a highly specific nature. This may have some functional implicationfor S100A6, because annexin expression shows a marked relationship to cell cycleprogression and, furthermore, the expression of annexins is linked with deregulationof growth of cancers.

THE BIOLOGICAL PROPERTIES OF S100A7 (PSORIASIN)

STRUCTURE AND MOLECULAR PROPERTIES OF S100A7

Psoriasin (S100A7) has been traditionally associated with psoriatic skin lesions. TheS100A7 gene coding for this protein has been described as the psoriasis-suscepti-bility gene. It spans 2.7 kb of the genome and consists of three exons and twointrons. The gene has been mapped to the S100 gene family cluster at chromosome



1q21 (Hardas et al. 1996). The S100A7 protein has been isolated from psoriaticskin. It is a ca. 11-kDa cytoplasmic protein with PI of 6.2. It is also secreted intothe extracellular compartment. S100A7 is structurally similar to other S100 proteins.However, there are some important differences, especially with regard to the N-terminally located EF-hand calcium-binding domain. The latter contains a distortedloop, and notably, in S100A7 this EF-hand lacks an important calcium-bindingresidue. Consequently, it binds no more than one calcium ion per monomer (Brod-ersen et al. 1998). S100 family proteins are also known to bind other divalent cationsbesides Ca2+, and their binding to the proteins can often be functionally moresignificant than their binding of Ca2+. Recombinant S100A7 indeed binds Ca2+, Zn2+,and Mg2+. It can bind seven Ca2+ ions in the presence of KCl and several in thepresence of NaCl. Therefore, in the secreted state, S100A7 could bind Ca2+ and, inthe cellular compartments, possibly binds higher amounts of calcium. It can bindeight Zn2+ ions in the presence of KCl and four in the presence of NaCl (Vorum etal. 1996). The binding of these cations produces conformational changes in theprotein. A subsequent picture of calcium-binding described by Brodersen et al.(1998) suggests a greatly reduced calcium-binding ability, and moreover, their find-ings are not compatible with the view that calcium-binding brings about conforma-tional changes in S100A7. It may be that insofar as S100A7 is concerned, calcium-binding may be functionally less significant than Zn2+. Recently, Brodersen et al.(1999) have identified a Zn2+-binding site in S100A7 that contains three histidineand one aspartate residues. It is of much interest to note that these residues presentthe Zn2+ in a way characteristic of certain metalloproteinases, which are importantcomponents of the ECM. It also seems from the work of Brodersen et al. (1999),that the absence of Zn2+ may have collateral effects on the organisation of thedistorted EF-hand loop. This might result in reduced calcium-binding by S100A7.Although the zinc-binding site occurs in other S100 proteins as well, the reason whycalcium-binding is functionally the most significant event in a majority of theseproteins could be the absence of collateral Zn2+-induced changes.

S100A7 IN SKIN PATHOLOGY

S100A7 (psoriasin) was originally isolated from psoriatic skin and seen to be asso-ciated clearly with inflammatory disease of the skin. Retinoic acid treatment hasbeen shown to induce S100A7 expression in skin but not in other tissues. Thissuggests a tissue-specific regulation of its expression. Expression occurs at a lowlevel in untreated epidermal keratinocytes but not in fibroblasts or melanocytes(Tavakkol et al. 1994). S100A7 has been shown to function as a chemotactic factorfor CD4+ T lymphocytes and to mediate inflammation. However, it is structurallydistinct from conventional lymphokines (J.Q. Tan et al. 1996). S100A7 has beenshown to form complexes with the epidermal-type fatty acid-binding protein(EFABP). EFABP, which shows an increased expression in psoriatic skin, is believedto be involved in the transport of cytosolic fatty acids. S100A7–EFABP complexesoccur in the cytoplasm of differentiating keratinocytes derived from psoriatic skin(Hagens et al. 1999). The functional significance of the complex is not understood



at present. Two possibilities have been suggested: keratinocyte differentiation and/orthe transport and targeting of cytosolic fatty acids.

Semprini et al. (1999) have suggested that the S100A7 gene may not be acandidate gene for familial psoriasis susceptibility. Several other genes occurring inthe 1q21 cluster are said to be overexpressed in psoriasis (Hardas et al. 1996). Thismay support the contention that S100A7 is not a candidate gene for psoriasis.Furthermore, the 1q21 gene cluster contains a collection of genes associated withthe terminal differentiation of epidermis. As discussed in an earlier section, genesin this cluster are also actively involved in the control of cell proliferation. Primaryhuman keratinocytes exposed in vitro to growth factors show an up-regulation ofnot only S100A7 but also MRPs 8 and 14, S100A11. Also expressed in these cellsare the actin-binding non-EF-hand proteins, gelsolin and annexin (Olsen et al. 1995).Because of the wide-spectrum genetic modulations involved, the relative significanceof the overexpression of individual genes of this cluster, is very difficult to assessin relation to pathogenesis of psoriasis. It is inevitable that the overexpression ofother CBP genes at the locus will have repercussions on the expression of S100A7,because not only does calcium bind S100A7, but calcium is also able to up-regulatethe synthesis of S100A7 (Hoffmann et al. 1994). It follows, therefore, that a signif-icant sequestration of intracellular calcium by other CBPs would affect S100A7expression.

S100A7 IN NEOPLASTIC DISEASE

The possibility that S100A7 expression in keratinocytes might reflect the state oftheir proliferation and differentiation has made it easier to appreciate why A7 isexpressed in both normal and malignant keratinocytes in culture (P.H. Watson et al.1998). Early studies indicated that S100A7 is expressed in breast cancers as wellas cell lines derived from breast cancers, but it was not detectable in control tissues(Moog-Lutz et al. 1995). Leygue et al. (1996) compared its expression in ductalcarcinoma in situ and invasive ductal carcinoma. They detected S100A7 mRNA onlyin the in situ carcinomas. It would be inappropriate to extrapolate from these limitedstudies on the possible relationship between expression of the gene and invasivebehaviour of the carcinoma. In this context it might be interesting to note thatS100A7 has been found to enhance the adhesion of neutrophils to epidermal cells(Von den Driesch et al. 1998). Unhappily, most of these studies are very preliminaryin nature. Nonetheless, the results are significant enough to warrant further investi-gation and confirmation.

There have been tentative investigations of the possibility that S100A7, as asecreted protein, could be a potential marker in certain tumour types. In squamouscell carcinoma of the bladder, urinary S100A7 has been suggested to be a usefulnoninvasive marker for follow-up of patients (Ostergaard et al. 1999). S100A7 isexpressed, together with markers for keratinised stratified squamous epithelia, insquamous cell carcinoma of the bladder. S100A7 expression is localised to the so-called squamous pearls (Celis et al. 1996). As expected, S100A7 is also detectablein the urine of patients. There is a lack of persuasive data on the relationship betweenS100A7 expression and the state of malignancy of bladder carcinoma. However, the



expression of several other important genes of the 1q21 cluster is often up-regulatedwith S100A7 as a consortium. This seems to be ample justification for evaluatingthese genes as a potential noninvasive profile of markers for bladder cancer.

S100A8 AND S100A9 PROTEINS IN INFLAMMATORY DISEASES

S100A8 and S100A9 proteins, previously known by the designations MRP8 andMRP14, respectively, are CBPs of the S100 protein family (Kligman and Hilt, 1988;Kerkhoff et al. 1998). They contain two EF-hand domains, one at either end of themolecule, and they are separated by a central hinge region. The EF-hands themselvesare flanked by hydrophobic regions. Besides Ca2+, they can also bind Zn2+ ions. Thelatter might regulate some of the functional properties of these proteins.

S100A8 and A9 are regarded as myeloid-specific proteins and show an enhancedpresence in activated phagocytes. Increased plasma levels of these proteins havebeen reported in patients with inflammatory conditions, such as rheumatoid arthritis,cystic fibrosis, and chronic bronchitis.

Although localised predominantly in the cytosol, they may be secreted by acti-vated monocytes (Rammes et al. 1997). It would seem that they are translocatedfrom the cytosol to the tubulin cytoskeleton and the plasma membrane. The targetingof these molecules to this cellular compartment could be a result of phosphorylation.Guignard et al. (1996) found that translocation occurred after the proteins werephosphorylated. As an extracellular protein, several functions have been envisagedand attributed to S100A8/A9. Kerkhoff et al. (1999a) have suggested they might beinvolved in the transendothelial migration of monocytes. Among other suggestedroles are growth inhibition and cytostasis. There is some interesting evidence thatthey may regulate the inflammatory process by virtue of being able to form com-plexes with arachidonic acid and aid its transport and metabolism (Kerkhoff et al.1999b). The binding of arachidonic acid by the S100A8/A9 heterodimeric complexhas been found to be dependent on Zn2+ binding. Whereas Ca2+ binding seems tobe conducive to the binding of arachidonic acid, Zn2+ binding appears to reversethis process.

Many of these studies are still in an incipient stage and much further work isrequired in order to elucidate the mechanisms by which S100A8 and A9 putativelycarry out this range of function. Until then, most of the suggested mechanisms willremain in the realm of speculation.

S100A11 (S100C) AND POSSIBLE MODES OF ITS FUNCTION

S100A11 was isolated and characterised by Ohta et al. (1991). It is an 11-kDaprotein with two EF-hand domains and shows 41 and 37% sequence homology withS100A and S100B proteins. The S100A11 gene has been localised to the S100 genecluster at chromosome 1q21 (Moog-Lutz et al. 1995; Wicki et al. 1996a,b). Theprotein has been shown to bind to actin and inhibit actin-activated Mg2+-ATPase



activity of smooth muscle cells (Zhao et al. 2000). Zhao et al. (2000) have, in fact,shown that the inhibition of the myosin-ATPase enzyme is not due to the depoly-merisation of actin filaments but it is a direct effect of actin binding by S100A11.The cytoskeletal binding action is probably not unique to this protein. The associationof S100A4 with the cytoskeleton has now been amply demonstrated (Figure 28 andTable 17). Moreover, S100A4 also binds to the rod region of myosin and inhibitsactin-activated ATPase (Ford et al. 1997).

The function subserved by S100A11 is yet unclear. Apart from the obviousconsequences of Mg2+-ATPase inhibition to the functioning of the smooth muscle,it is of interest to note that S100A11 interacts also with annexins (Naka et al. 1994;Mailliard et al. 1996; Seemann et al. 1996), a property it shares with S100A10(Kaczanbourgois et al. 1996). These interactions of S100A11 and S100A10 couldbe important indicators of their cellular function. PKC-mediated regulation ofannexin function involves certain amino acid residues of the S100 proteins that havebeen implicated also in the interaction between annexins and S100A11 andS100A10. The interaction with cytoskeletal elements quite obviously implicatesthem in the regulation of the cytoskeletal machinery. Furthermore, there is a poten-tial, albeit indirect link between these S100 proteins and cell proliferation anddifferentiation, and, by analogy with S100A4 function, it might be involved in cancerinvasion too. This aspect of potential influences of S100A10 and S100A11 hasreceived almost no attention to date.

FIGURE 28 The biological processes influenced by S100 proteins. This is especially trueof S100A4, as discussed here. In the context of cancer, the major influences are on cellmotility, adhesive properties, and proliferation. The effects exerted by S100 proteins oncytoskeletal dynamics seem to be important in maintaining or modulating cell morphologyand also highly pertinent in cell proliferation and signal transduction. Overall, this illustrationfocuses on the breadth of biological effects brought about by S100 proteins and the need toregard S100 genes, such as S100A4, as normal genes, whose inappropriate expression couldlead to aberrant biological behaviour, rather than as “oncogenes” or “metastasis” genes. (FromSherbet and Lakshmi, 1997b, 1998.) Reprinted by permission of the publisher Academic Press.



S100P IN CANCER PROGRESSION

S100P was isolated from human placenta and characterised some years ago (T.Becker et al. 1992; Emoto et al. 1992). It has received much attention in recentyears. S100P consists of 95 amino acid residues and possesses two EF-hands thatbind calcium with different affinity. The high-affinity EF-hand occurs at the C-terminal and the one with low affinity occurs at the N-terminal region of the protein.The protein also binds other divalent ions such as Zn2+ at the C-terminal region andMg2+ at the N-terminal end. The binding of Ca2+ and Mg2+ has been shown to alterthe conformational state of S100P, and it has been postulated that the biologicalproperties of the protein may be altered by a Ca2+//Mg2+ switch (Gribenko andMakhatadze, 1998; Gribenko et al. 1998).

S100P AND ITS PUTATIVE FUNCTIONS

The presence of S100P in skin sensory corpuscles has been described by someinvestigators (Delvalle et al. 1995; Albuerne et al. 1998). Intense immunostainingfor S100P has been encountered in the lamellar cells of Meissner corpuscles andthe inner core cells of Pacinian corpuscles (Albuerne et al. 1998). In the avian Herbstcorpuscles also S100P is known to be associated with the inner core cells. Thesestudies have found no evidence of S100P in the central axons of the sensory cor-puscles. The protein may be putatively involved in sensory signal transduction. Boththese studies also refer to the occurrence of other S100 proteins. That S100P mightcontribute, together with S100A, to the structural integrity of the sensory apparatusis suggested by the finding that the pattern of expression of S100 proteins, includingS100P, was not altered in skin samples obtained from patients with spinal cord injury.However, there was a reduction in the number of sensory corpuscles that expressedthese proteins (Albuerne et al. 1998). Frank and Wolburg (1996) studied the eventsassociated with wound healing and tissue repair following injury to the optical nervein the rat. They have reported the appearance of reactive astrocytes staining forS100P about 6 days after the nerve injury, and these astrocytes might be involvedin the restructuring of the nerve fibres.

Some preliminary observations have been reported about the expression ofS100P in relation to the progression of carcinoma of the prostate. Androgens areactively involved in the growth and function of the prostate. Carcinomas of theprostate have notably been described as being androgen dependent at early stagesof their development, but becoming independent of this hormone during progressionof the disease. Averboukh et al. (1996) found that the androgen-responsive cell lineLNCaP-FGC, derived from prostate cancer, expresses S100P gene, but this is down-regulated within 30 hr after androgen deprivation. On the basis of these observations,they suggest that S100P might be involved in the aetiology of prostate cancer. Onewould concede that the expression of the gene might be regulated by androgen, butthis is a far cry from demonstrating its implication in the pathogenesis of prostatecancer. One could even tentatively suggest that S100P might conceivably functionas a suppressor gene. A number of true metastasis-suppressor genes, as contrastedwith tumour-suppressor genes, have been cloned.



In evaluating the potential role of S100P one should also take account of thefact that androgens regulate the expression of PSA. They regulate PSA gene tran-scription by means of the androgen-responsive elements that occur in the promoterregion of the PSA gene (Riegman et al. 1991). Probably cis-acting elements areinvolved also in the AR-mediated transcription of PSA (J.Y. Zhang et al. 1997). ThePSA-related protein, human glandular kallikrein-1, is also regulated by androgens.The gene that encodes it contains an androgen-responsive element (Murtha et al.1993).

A proportion of PSA occurs in the form of a complex with other proteins suchas α1-antichymotrypsin. The ratio of bound to free PSA is greater in carcinomasthan in BPH. Therefore, the proportion of free PSA found in the serum has beenregarded as a reasonable tumour marker (Becker and Lilja, 1997; Stenman et al.1999). Sartor et al. (1997) studied the rate of increase of PSA and have concludedthat a rapid increase in the level of PSA indicated metastatic disease, in contrastwith a moderate rise in PSA, which was associated with local recurrence. However,Bangma et al. (1995) did not find any relationship between PSA expression andprogression of the disease. Chopra et al. (1996) found PSA expression in normalprostate, BPH, and carcinomas. Some recent experimental work has shown that PSAcan inhibit endothelial cell proliferation and also inhibit the angiogenic effects ofFGF and VEGF. In the mouse model, PSA has been found to reduce metastaticdeposition of tumour cells in the lungs (Fortier et al. 1999). The apparent antian-giogenic and anti-metastatic effects of PSA have raised further doubts about itsreliability as a marker of tumour progression. However, because PSA formscomplexes with proteases other than antichymotrypsin, there is room for refinementsof its utility as a diagnostic agent.

PSA is a kallikrein-like serine protease and can conceivably bring about changesin the ECM that might be conducive to tumour cell invasion. Indirect evidence forthis comes from the observation that the in vitro invasive behaviour of LNCaP cellsis inhibited when the proteolytic activity of PSA is experimentally suppressed(Webber et al. 1995). In vivo, the invasive behaviour of prostate carcinoma correlateswith PSA concentration in serum (Bostwick et al. 1996). The PSA-related prostate-specific kallikrein is also regulated by androgen (Murtha et al. 1993), and its expres-sion has been reported to increase from benign epithelium through intraepithelialneoplasia to carcinomas (Darson et al. 1997). Therefore, PSA alone could be viewedas being responsible for the invasive behaviour and metastatic spread. However, onemust reconcile with the increasing androgen independence of the process of pro-gression of this cancer. In the midst of contradictory findings, there seems to be aconsensus view that PSA expression might in fact be maintained or even increasewith progression. However, the uncertainty about the relationship between PSA andtumour progression might yet allow one to dissociate the potential influence ontumour progression of S100P from that of PSA. Therefore, in spite of these variouscomplications arising from androgen-mediated regulation, S100P deserves to bestudied further. For example, it would be of much interest to know the state of itsexpression in BPH, and whether there are any discernible changes in expression inthe progression of disease from the state of intraepithelial neoplasia to invasiveadenocarcinoma.



Of considerable practical significance is the recent report by Bertram et al. (1998)of the strong association between S100P, and to a lesser extent S100A4, expressionand drug resistance. S100P was highly expressed in doxorubicin-resistant cells ascompared with those sensitive to the drug. The reasons for this association can onlybe speculated. At any rate, the need to exploit the practical value of this findingshould far outweigh the desire to understand the underlying mechanisms.

POTENTIAL VALUE OF S100 PROTEINS AS MARKERS OF CANCER PROGRESSION AND PROGNOSIS

The establishment of a marker for monitoring disease progression and possibly asan aid in predicting prognosis should contain two basic elements. One of these isthat the expression of the putative marker should show clear and unequivocal empir-ical correlation with disease state. The second, more important element is that theputative marker should manifestly function via a mechanism that involves one ormore fundamental processes of the life of the cell.

Whether measuring the expression of S100 genes has any role to play in theclinical management of human cancer is an area that now clearly warrants investi-gation. Early indications are that this could be useful. Serum levels of S100 proteinshave been measured in melanomas. Elevated levels of generic S100 proteins maybe related to metastatic outcome (Buer et al. 1997; Drummer et al. 1997). S100proteins were detectable in the serum of 79.3% patients with metastatic disease ascompared with 4% of stage I/II patients and 21.4% of stage III patients. Furthermore,there was a sharp decline in S100 levels in two patients in remission (Drummer etal. 1997). The detection of higher serum levels also has been reported in all stagesof cutaneous melanomas, as compared with levels found in normal subjects (Abra-ham et al. 1997). Immunohistochemical staining of cutaneous tumours has revealedintense staining for S100A6 in all primary lesions and in 64% (9/14) of metastaticlesions studied; but staining for S100A4 was found to be weak in these neoplasms(Boni et al. 1997).

S100A4 expression has been studied in human colorectal neoplasms. Normalcolonic mucosa and adenomas have been found to contain comparable levels ofS100A4, but adenocarcinomas express considerably larger amounts of S100A4mRNA. Furthermore, immunohistochemical analyses have revealed metastatictumours to be strongly S100A4 positive (Takenaga et al. 1997b).

It has been reported recently that, in human infiltrating ductal carcinomas of thebreast, S100A4 (h-mts1) expression correlates strongly with the potential to metasta-sise to axillary lymph nodes. S100A4 expression also inversely correlates withoestrogen and progesterone receptor status. These observations suggest that S100A4(h-mts1) expression might reflect the aggressiveness of breast cancers (Albertazziet al. 1998b). Albertazzi et al. (1998b) also reported the occurrence of a variant h-mts1v, a shorter transcript of S100A4 (h-msts1) in which exon 1 (1a, 1b) is splicedout (Albertazzi et al. 1998c). Their data have suggested the possibility that theexpression of this variant might also be indicative of aggressive disease, albeit notas powerful a marker as S100A4 (h-mts1) itself. Further work needs to be done on



the expression of both S100A4 (h-mts1) and h-mts1v in breast cancer as well as inother forms of cancer in order to consolidate these findings before pronouncing onthe clinical value of S100A4 expression in tumours. Nevertheless, there are firmdata concerning the ability of S100A4 to enhance the invasive and metastatic pro-pensities of experimental tumour models as well as in certain human neoplasms. Ofthe S100 family proteins, S100A4 seems most satisfactorily to fulfill both the criteriastated above. It appears to function via the modulation of the cytoskeletal machineryof the cell and by controlling the progression of the cell cycle. These observationsprovide considerable support for the thesis that S100A4 expression might be apowerful marker, from the S100 family of proteins, of cancer progression andprognosis.


245

Epilogue

The range of biological parameters influenced by calcium-binding proteins in generaland S100 proteins in particular is indeed most impressive (see Figure 28). CBPs arehighly versatile proteins, and highly significant is their mediation of several criticalevents closely aligned to and identifiable with specific compartments of the meta-static cascade (Table 21). Six compartments can be identified in tumour development,invasion, metastatic dissemination, and the development of overt metastatic lesions.In all these, CBPs and S100 proteins, in particular, will influence the cellularproperties that are highly associated with the behavioural or phenotypic event rele-vant to that specific metastatic compartment. This seems to begin at the beginning,in the initiation of neoplastic transformation and the expansive growth of the tumour.The transduction of signals originating from aetiological agents or down-streamsignals of TGFs or mitogenic stimuli often takes the calcium-signalling pathway.The flow of information is mediated by CBPs. The CBPs may be regarded as thetranslators and the harbingers of the calcium messages. They undergo conformationalchanges upon binding to Ca

2+

. A consequence of this seems to be the acquisition ofthe ability to recognise target proteins and translate the calcium signal into a bio-chemical function reflecting a phenotypic property. Also often involved in the path-way is a coordinated functioning of kinases and phosphatases, as, for instance, inthe transduction of the TCR binding by its antigen, in which calcineurin phospho-rylation plays an important part in information flow.

TABLE 21Metastatic Compartments and Relevant Cellular Properties Influenced by Calcium-Binding Proteins

Metastatic CompartmentRelevant Events of the Metastatic Cascade

A. Aetiology Neoplastic transformation

B. Cell proliferation, apoptosis Tumour development, expansive growth

C. ECM remodelling, modulation of cell adhesion, cell shape, motility, cytoskeletal dynamics

Invasion

D. Cytoskeletal dynamics, cell membrane malleability, cell motility, angiogenesis

Extravasation from vascular system

E. Heterotypic adhesion Arrest of cells at metastatic targets

F. Cell proliferation, apoptosis Development of overt metastases

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The expansive growth of tumours is a reflection of a net increase of the cellpopulation size, which is a function of total cell proliferation and apoptotic cell loss.The deregulation of the cell cycle seems to be the prime cause of this expansivephase of tumour development, and it is very appropriate that North (1991) describedcancer as a disease of the cell cycle. As we have seen, S100 proteins not only showcell cycle-related expression, but individual proteins such as S100A4 might beclosely linked with the regulation of cell cycle progression in conjunction with otherregulatory proteins such as p53, rb, and stathmin. The activity of these proteins isexquisitely coordinated to achieve effective regulation. Calcium signalling is anintegral part of the process of apoptosis, and the apoptotic pathway contains severaltarget enzymes activated by calcium. This is equally important in the developmentof metastatic deposits, but the kinetics of cell proliferation and apoptotic loss inprimary tumours and metastatic deposits are not strictly comparable. We have dis-cussed the potential effects of Ca

2+

-mediated activation of caspases and calpains inthe deregulation of cell proliferation and induction of apoptosis. The influence ofVD3 on cell differentiation with attendant inhibition of proliferation is a goodexample of the interplay of VDRE-mediated activation of gene transcription and thecontrol over this exerted by osteocalcin. The latter in turn is regulated by TGF

β

.The invasive phase of the metastatic cascade is replete with examples of CBP

activity. There is much empirical evidence that high levels of S100 family proteinsoccur in association with enhanced invasive propensity. Besides, some of theseproteins appear to take part in the remodelling of the extracellular matrix, which,as we have noted, figures prominently in cell adhesion and invasive behaviour.S100A4 and osteonectin are prime examples of ECM-modulating CBPs. Both seemto participate in its remodelling by having recourse to regulating the proteolyticactivity associated with the ECM. The regulation of the contractile machinery ofthe cell is obviously of considerable importance in cell motility. The regulatorycomponent of the actomyosin assembly, i.e., MLC, binds calcium in its function ofmyosin-ATPase regulation and generation of contractile forces. MLCs themselvesundergo reversible phosphorylation through the agency of Ca

2+

/CaM-dependentkinases and phosphatases. The phosphorylation of MLCs seems to be involved inthe invasion of vascular endothelium by activated PMN. As we have noted already,not only does calcium signalling

via

the activation of calpains and caspases severelyaffect tumour development, but we have also established the involvement of theseenzymes on the adhesive and invasive behaviour of cells.

The transmembrane glycoprotein cadherin forms a complex with

β

-catenin and

α

-catenin and links-up with the actin cytoskeleton. This complex is a completeexample of calcium signalling mediated by cadherin, which has calcium-bindingdomains in the extracellular part of the molecule, and the catenin serving an adhesionfunction as well as providing a signal transduction machinery. Furthermore, theremight be a feedback mechanism present, with cadherin negatively regulating thesignalling function of

β

-catenin. The cadherin–catenin complex, at the same time,is also essential for the physical process of formation of adherens junctions. Thecellular properties of heterotypic adhesion and deregulation of cell proliferationkinetics would apply equally also to the development of overt metastatic deposits.Nonetheless, one should recognise that the adhesive properties required of a tumour


Epilogue

247

cell trying to gain entry into the vascular system are probably totally different incharacter from those required for extravasation of the tumour cell in the target organ.On the other hand, one might speculate that the seeding of tumour cells at the targetorgan might be a reflection of the induction of apoptosis of endothelial cells ratherthan by a conventional form of diapedesis across the epithelium. Notwithstandingthe downstream pathway of calcium signalling, a retardation of the initial event ofcalcium influx into cells seems to affect profoundly the invasive behaviour of cellsas well as prolong survival times of mice bearing human tumour xenografts.

The vascularisation of the tumour and the decimation of tumour cells that havegained access to the vascular compartment are the major gaps in our understandingof CBP activity. Osteonectin has been attributed with both angiogenic and anti-angiogenic abilities. There is very little direct evidence that one can cite for eitherattribute. Some indirect evidence is available that suggests that S100A4 may not beinvolved with tumour vascularisation. However, there is also a report that claimsthat S100A4 can reduce the size and density of tumour-associated microvasculature.

It might be recalled here that activation of calcium influx brings about majorchanges in the shape of endothelial cells, which can lead to enhanced permeabilityand promote the diapedesis of leukocytes. Activation of calcium influx also inducesangiogenesis. Furthermore, certain agents that inhibit calcium signal transductionalso seem to be capable of inhibiting angiogenesis. Whether CBPs intervene in thisaspect of signal transduction is uncertain at present, but this is a distinct possibility.We know that calcium-dependent NOS is involved in the remodelling of vascularendothelia and in angiogenesis. Furthermore, the expression of endothelial NOS incarcinomas correlates with tumour grade. Hence, one of the strategies attempted tocontrol metastatic spread was aimed at inhibiting angiogenesis by blocking thecalcium-signalling pathway mediated by calcium-dependent NOS. That calciumsignalling might be implicated in the regulation of angiogenesis is, therefore, notaltogether in the realm of scientific speculation. There is much scope for intensiveinvestigation of this aspect of the function of CBPs in the metastatic spread of cancer.

It is now safe to say that the study of CBPs in general and of S100 familyproteins, such as S100A4, in particular have much to offer in the understanding ofcell behaviour in a normal cellular environment as well as their behaviour uponneoplastic transformation. This conclusion is based on the extensive evidence thatlinks CBPs, beyond reasonable doubt, with cellular properties that are essentialfeatures of tumour growth, differentiation, invasion, and metastatic dissemination.There is much to learn and much to be gained.


249

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349

Index

A

ABP,

see

Amyloid

β

-proteinACTH,

see

Adrenocorticotropic hormone

α

-actinin, 51, 103Action–myosin interaction, 117Actomyosin assembly, 112ACV,

see

AcyclovirAcyclovir (ACV), 61, 62Adenomatous polyposis coli (APC) protein,

104Adenylyl cyclase, 22Adhesion

cell–substratum, 58intracellular, 58-mediating proteins, 59, 94

Adrenal cortex, osteonection immunoreactivity in invasive tumours of, 193

Adrenocorticotropic hormone (ACTH), 24Alzheimer’s disease

calcineurin in, 136, 140calretinin and, 133caspases and neuronal loss in, 177CBD-immunoreactive neurones in, 129

Amyloidosis, gelsolin expression in, 42Amyloid precursor protein (APP), 178,

179

β

-Amyloid precursor protein (

β

APP), 141Amyloid

β

-protein (ABP), 141Androgen, 241Androgen receptors (ARs), 101Angina pectoris, 222Angiotensin signals, 76Annexin(s), 35

II, 208cell cycle-related expression of, 39function, PKC-mediated regulation of, 36in human foreskin fibroblasts, 37interaction between S100A11 and, 240

Antibodies, anti-keratin, 204Antichymotrypsin, 242Anti-keratin antibodies, 204

APC protein,

see

Adenomatous polyposis coli protein

Apoptosiscalpains in, 158caspase-mediated, 173dexamethasone-induced, 18PARP as marker of, 172

APP,

see

Amyloid precursor protein

β

APP,

see

β

-Amyloid precursor protein

Arabidopsis thaliana

, 96Arachidonic acid, binding of, 67ARs,

see

Androgen receptors


, 136Astrocytomas, expression of S100A3

studied in human, 225

Atriplex nummularia

, 146Autoimmune conditions, 53Autoimmune diseases, 84

B

BAE cells,

see

Bovine aortic endothelial cells

Barrett’s adenocarcinoma, villin expressed in, 45

Barrett’s metaplasia, 45, 216Basement membrane (BM), 183Basic fibroblast growth factor (bFGF), 76BDNF,

see

Brain-derived neurotropic factorBecker muscular dystrophy (BMD), 165Benign prostatic hyperplasia (BPH), 100bFGF,

see

Basic fibroblast growth factorBifonazole, 784,5-Bisphosphate, 10Bladder, squamous cell carcinoma of, 238BM,

see

Basement membraneBMD,

see

Becker muscular dystrophyBombesin, 19Bone

disease, metastatic, 60metabolism, osteocalcin in, 54scans, 61

Bovine aortic endothelial (BAE) cells, 189

0942 index.fm Page 349 Wednesday, October 25, 2000 6:48 PM

350


BPH,

see

Benign prostatic hyperplasiaBradykinin, 19Brain

-derived neurotropic factor (BDNF), 125osteonection immunoreactivity in

invasive tumours of, 193Breast

cancer(s), 70, 228aggressiveness of, 243ER-negative, 193ER/PgR-negative, 232ibandronate treatment of metastatic, 61prognosis, marker for predicting, 233

carcinomas, NDP kinase expression in, 231

ductal carcinoma of, 98epithelium, neoplastic transformation of,

111osteonection immunoreactivity in

invasive tumours of, 193Butyrate, 127

C

Cadherin, 246–catenin complexes, forms of, 212as marker for serous carcinoma of ovary,

134


, 89, 102, 154Caherin, 107CAI,

see

Carboxyamido-triazoleCalbindin

D-9K (CBD-9K), 125D-28K (CBD), 125immunoreactivity, 126neuroprotective function of, 129

Calbindin, structure and biology of, 125–130

calbindin expression in embryonic development and with ageing, 126–127

calbindin expression and metastatic phenotype, 129–130

calbindin in neuronal populations, 125neural cell lineage and regulation of

calbindin expression, 125–126neuroprotective function of calbindin,

129physiological function of calbindin,

127–128

Calcimedin, 31Calcineurin, in cell proliferation, cell

adhesion, and cell spreading, 135–144

calcineurin in Alzheimer’s disease, 140–141

calcineurin in cell proliferation and adhesion-related phenomena, 136–140

effects of calcineurin on cell adhesion and motility, 138–140

putative role of calcineurin in cell cycle progression, 136–138

calcineurin in immunosuppression, 141–144

molecular features of calcineurin, 135–136

Calcium/calmodulin-dependent protein kinases

(CAMPKs), 13capacitative energy of, 10

Calcium-binding proteins (CBPs), 1, 5cell proliferation, 245EF-hand, 29gelsolin, 40molecular configuration of, 65natural classification, 29–33neural, 79neuronal, 131non-EF-hand, 30–31photoreceptor-specific, 32posttranslational changes of, 66S100 family of, 200

Calcium signalling pathway, 5–28architectural aspects of signal

transduction machinery, 25–28cyclic AMP in calcium signaling, 21–25deregulation of inositol 1,4,5-

trisphosphate pathway, 18–20homeostatis of cell calcium, 5–10

deregulation of calcium homeostasis as primary event in carcinogenesis, 9–10

plasma membrane Ca

2+

-ATPase pump, 5–7

sarcoplasmic–endoplasmic reticulum Ca

2+

-ATPase pump, 7–8voltaged gated calcium channels, 8–9

inositol phosphates in calcium signal transduction, 16–18


Index

351

phospholipid signalling, 10–11protein kinase C and isoforms in signal

transduction, 14–16protein kinase C pathway, 13–14PTEN phosphatase in regulation of lipid

signalling, 11–13ryanodine and related receptors in

calcium mobilisation, 20–21Calcyclin, 64, 235Caldesmon, 121Calelectrin, 31Calmodulin (CaM) family, of calcium

binding proteins, 75–85calmodulin and physiological function,

75–79calmodulin and cell proliferation,

77–78calmodulin-mediated signal

transduction, 76–77calmodulin in neoplasia, 78–79structure and mode of action of

calmodulin, 75–76guanylate cyclase-activating proteins,

85recoverin subfamily of neural calcium

binding proteins, 79–85G-protein signalling pathway, 79–80mode of action of recoverin, 82post-translational modification of

recoverin, 82–83recoverin and cancer-associated

retinopathy, 83–85recoverin and function, 80–82

Calmyrin, 33Calnexin, 49Calpains, in normal and aberrant cell

physiology, 153–167calpain family of calcium-binding

proteins, 153–154calpains in cancer growth and

progression, 162–163calpains in cell proliferation and

apoptosis, 158–160calpains in cell spreading and migration,

160–161calpains in intergrin-mediated cell

adhesion and signal transduction, 161–162

calpains in muscular dystrophy, 165–167

association of calpains with Duchenne muscular dystrophy, 165–166

calpains and limb girdle muscular dystrophy, 166–167

calpains in myelodegenerative diseases, 163–165

involvement of calpains in development and differentiation, 157–158

molecular organisation of calpains, 154–155

regulation of physiological events by proteolytic function,155–157

Calponincalcium-dependent binding to, 209homology (CH), 96, 122myosin-ATPase inhibition by, 124

Calreticulin, 30, 35, 51Calretinin, 32, 33, 131–134

alternatively spliced isoforms, 131–132expression in cell proliferation and

differentiation, 133hormonal regulation of, 132as marker for serous carcinoma of ovary,

134possible neuroprotective property, 133as potential tumour marker, 133–134regulation of calretinin expression,

132–133Calsequestrin, 30, 53Caltropin, 200Calumenin, 149CaM,

see

Calmodulin family, of calcium binding proteins

CAMPKs,

see

Calcium/calmodulin-dependent protein kinases

cAMP response element binding protein (CREB), 23

cAMP response elements (CREs), 25Cancer,

see

also specific types-associated retinopathy (CAR), 83caveolin expression in, 28gelsolin in, 42involvement of fimbrin in, 100markers, experimental, 233prognosis, relationship between EGFr

expression and, 232progression

osteonectin and, 196potential role of thymosins in, 95relation of actinin to, 108


352


S100A4 expression as marker of, 244S100P in, 241

Capase-9, cytochrome c-mediated activation of, 170

Capping protein (CP), 97CAR,

see

Cancer-associated retinopathyCarboxyamido-triazole (CAI), 9Caspases, in apoptosis, cell migration,

proliferation, and neoplasia, 169–179

caspase-mediated apoptosis and cell growth inhibition in tumour expansion, 173–176

caspase-mediated proteolysis of fodrin, 176–177

caspases in apoptotic cell death, 169–172

caspases and neuronal loss in Alzheimer’s disease, 177–179

poly (ADP-ribose) polymerase as marker of apoptosis, 172–173

Caveolin, 28, 98CBD,

see

Calbindin D-28KCBD-9K,

see

Calbindin D-9KCBPs,

see

Calcium-binding proteinsCell–substratum adhesion, 58Central nervous system (CNS), 1Centrins, 145–147cGMP,

see

Cyclic guanine monophosphateCH,

see

Calponin homologyChemoattractants, 210Chinese hamster ovary (CHO) cell line,

161

Chlamydomonas

, 145, 146CHO cell line,

see

Chinese hamster ovary cell line

Clotrimazole, 78CNS,

see

Central nervous systemCofilin, 88Collagen, binding of osteonectin to, 191Colon cancer

metastatic, 163model, 43

Colorectal neoplasms, S100A4 expression in, 243

Contractile proteins, structure of, 87–124actin component of contractile machinery

of cell, 87–93actin isoforms, 87–88

cofilin in regulation of actin dynamics, 88–90

interaction of formin with profilin and Rhp GTPases, 92–93

profilin in regulation of actin dynamics, 90

regulation of actin dynamics, 88Rho GTPases in actin dynamics and

signal transduction, 90–92fimbrin family of actin-binding proteins,

96–110

α

-actinin, 102

α

-actinin isoforms, 103actinins in cell adhesion, motility, and

signal transduction, 104cadherin–catenin complex in signal

transduction and cell adhesion, 104–110

function of

α

-actinin, 104function of fimbrin in cytoskeletal

organisation, 97–99involvement of fimbrin in cancer,

100–101modulation of actin dynamics and

cancer cell dissemination, 101–102

molecular features of fimbrin, 96–97molecular structure of

α

-actinin, 102–103

regulation of fimbrin expression, 99–100

myosin filaments, 110–117actomyosin assembly, 112–115myosin heavy chain isoforms,

111–112myosin light chain phosphorylation

and function, 115–117role of thymosin family actin-binding

proteins in actin dynamics, 93–96effects of thymosins on cell

proliferation, 93–94expression of thymosins in embryonic

development, 94–95potential role of thymosins in cancer

progression, 95–96sequestration of actin by thymosins,

93thymosins and cell motility and

differentiation, 94


Index

353

troponins and tropomyosins in regulation of muscle cell contraction, 118–124

calponin, 122–124caltropin-mediated reversal of myosin-

ATPase inhibition by caldesmon and calponin, 124

regulatory role of caldesmon, 121–122regulatory role of troponins and

tropomyosins in muscle contraction, 118–119

tropomyosin isoforms in benign and malignant cells, 119–121

Corticosterone, 127CP,

see

Capping proteinCREB,

see

cAMP response element binding protein

CREs,

see

cAMP response elementsCrocalbin, 149Cyclic AMP, in calcium signalling, 21Cyclic guanine monophosphate (cGMP), 16Cyclosporin, 141Cytochalasin D, 18, 211Cytokinesis, 93, 217Cytosolic fatty acids, 237

D

DAG, see1,2-DiacylglycerolDarier’s disease (DD), 7DD,

see

Darier’s diseaseDemyelination process, autoimmune-

mediated, 164Dexamethasone, 18, 172

-inducible promoter, 206S100A4 transfectants exposed to, 226

1,2-Diacylglycerol (DAG), 13, 14, 32

Dictyostelium discoideum

, 22, 44, 96, 102Dimethyl sulphoxide (DMSO), 19DMD,

see

Duchenne muscular dystrophyDMSO,

see

Dimethyl sulphoxideDNA

binding of transcription factors to, 67damage, 72fragmentation, 170, 174hypermethylation, 73methylation, in cancer, 72

Drosophila melanogaster

, 42, 102, 150, 231Drug–immunophilin complex, 141

Duchenne muscular dystrophy (DMD), 165

E

EAE,

see

Experimental allergic encephalomyelitis

EC,

see

Endothelial cellEcdysone, 18ECM,

see

Extracellular matrixEDC,

see

Epidermal differentiation complex

EFABP,

see

Epidermal-type fatty acid-binding protein

EF-hand calcium binding proteins, 63–74

alternatively spliced variants of S100A4, 68–69

calcium binding and molecular configuration of calcium binding proteins, 65–68

functional significance of alternatively spliced isoforms, 69–70

molecular organisation, 63–64regulation of expression of S100 family

genes, 71–74DNA methylation in cancer, 72–73regulation of gene expression by DNA

methylation, 72regulation of S100 gene transcription

by methylation, 74transcriptional regulation of S100

genes, 71structure and organisation of S100 family

genes, 68EGF,

see

Epidermal growth factorEGFr,

see

Epidermal growth factor receptors

Embryonic stem (ES) cells, 51Endoplasmic reticulum, 5, 46Endothelial cell (EC), 27Endothelial nitric oxide synthase (eNOS),

26Enhancer element, 74eNOS,

see

Endothelial nitric oxide synthaseEnzymes, activation of target, 75Epidermal differentiation complex (EDC),

202Epidermal growth factor (EGF), 15, 224


354


Epidermal growth factor receptors (EGFr), 76–77

Epidermal-type fatty acid-binding protein (EFABP), 237

Epithelial–mesenchymal transformation, 214, 226

ERK,

see

Extracellular signal-related receptor kinases

ERs,

see

Oestrogen receptorsES cells,

see

Embryonic stem cellsExperimental allergic encephalomyelitis

(EAE), 164Extracellular matrix (ECM), 186

protein synthesis, 197remodelling, 214

Extracellular signal-related receptor kinases (ERK), 77

Ezrin, 108

F

FH,

see

Formin homologyFibroblast(s)

annexin in human foreskin, 37-specific protein 1 (FSP1), 214

Fibronectin (FN), 57, 60Fimbrins, 96, 100FK506, 141FLG,

see

ProfilaggrinFN,

see

FibronectinFodrin

calpain-specific degradation of, 164influence of cell adhesion and migration

by, 176Follicle-stimulating hormone (FSH), 25Follistatin, 183Formin homology (FH), 92FSH,

see

Follicle-stimulating hormoneFSP1,

see

Fibroblast-specific protein 1

G

GAP,

see

Guanosine triphosphatase activating proteins

Gastrin-releasing peptide (GRP), 19GDNF,

see

Glial cell-derived neurotropic factor

GDP,

see

Guanosine diphosphateGelation factor, 109Gelsolin, 30, 40, 43

in cancer, 42expression, in amyloidosis, 42

Generegulation, modes of, 71therapy, with cytotoxic drugs, 62

Genetic activation, signal transduction and, 2

Genistein, 104GFAP,

see

Glial fibrillary acidic protein

Ginkgo biloba

L., 46GI tract, osteonection immunoreactivity in

invasive tumours of, 193Glial cell-derived neurotropic factor

(GDNF), 132Glial fibrillary acidic protein (GFAP), 207Glutamate, 114, 206Glycoprotein(s)

adhesion mediating, 59transmembrane, 246

G-protein-coupled receptors, 26signalling pathway, 79

Growth hormone, 127GRP,

see

Gastrin-releasing peptideGTP,

see

Guanosine triphosphateGTPase,

see

Guanosine triphosphataseGuanosine diphosphate (GDP), 80Guanosine triphosphatase (GTPase), 80Guanosine triphosphatase activating

proteins (GAP), 80Guanosine triphosphate (GTP), 77Guanylate cyclase, 32, 81, 85

H

Hailey–Hailey disease, 8Heat shock protein (HSP), 84, 138, 216,

217Heme oxygenase (HO), 229HIV-1,

see

Human immunodeficiency virusHO,

see

Heme oxygenaseHPV,

see

Human papilloma virusHSP,

see

Heat shock proteinHuman immunodeficiency virus (HIV-1),

137Human papilloma virus (HPV), 156Human umbilical vein endothelial cells

(HUVEC), 175HUVEC,

see

Human umbilical vein endothelial cells


Index

355

Hyaluronic acid, 211

Hydra vulgaris

, 35Hyperthermia, 220, 221, 229Hypomethylation, 72

I

Ibandronate treatment, of metastatic breast cancer, 61

ICE,

see

Interleukin-1

β

-converting enzymesIF,

see

Intermediate filamentsIGF,

see

Insulin-like growth factorInflammatory diseases, S100A8 and

S100A9 proteins in, 239Inositol phosphates, in calcium signal

transduction, 16Insulin-like growth factor (IGF), 49Integrin receptor, cell surface, 210Interblastomere adhesion, 107Interleukin, 229Interleukin-1

β

-converting enzymes (ICE), 169

Intermediate filaments (IF), 87Intracellular adhesion, 58Invasion suppressor gene, 59

J

Jurkat T lymphocytes, 171

K

Kallikrein-1, 242Keratin filament aggregation, 32Keratinocyte differentiation, profilaggrin in,

202Keutel syndrome, 55Kidney, osteonectin immunoreactivity in

invasive tumours of, 193

L

LAK cells,

see

Lymphokine-activated killer cells

Laminin, cooperative functioning of in osteoblast migration, 59

Leiomyosarcomas, expression of calponin in, 124

Leischmania donovani

RNA, 47LGMD,

see

Limb girdle muscular

dystrophyLimb girdle muscular dystrophy (LGMD),

28, 165, 166, 167

Liriodendron tulipifera

L., 46LOH,

see

Loss of heterozygosityLong terminal repeats (LTR), 137Loss of heterozygosity (LOH), 12LTR,

see

Long terminal repeatsLung, osteonection immunoreactivity in

invasive tumours of, 193Lymphocyte proliferation, inhibition of, 143Lymphokine-activated killer (LAK) cells,

98Lymphoma, B-cell diffuse large cell, 175

M

Madin–Darby bovine kidney (MDBK), 127Major histocompatibility complex genes,

25Mammalian calpains, 153Matrilysin promoter, 109Matrix metalloproteinases, 10, 1875-MC,

see

5-MethylcytosineMCBK,

see

Madin–Darby bovine kidneyMechanoenzymes, 110Melanocyte-stimulating hormone (MSH),

22Melanomas, 234, 243Merlin, 108Mesotheliomas, cadherin and calretinin as

marker for, 134Metalloproteinases, tumour-associated, 195,

213Metastasis gene concept, 199, 241Metastatic dissemination, osteotropism of,

60

N

-Methyl-

D

-aspartate (NMDA), 135, 2065-Methylcytosine (5-MC), 72

N

-Methyl-

N

-nitrosourea (NMU), 236MHC,

see

Myosin heavy chainMicrotubules, 91, 217MLC,

see

Myosin light chainMLCK,

see

Myosin light chain kinaseMMTV,

see

Murine mammary tumour virusMoesin, 108Monocytes, transendothelial migration of,

239Mooren’s ulcer, 200mRNA(s)


356


expression, up-regulation of calcineurin, 143

ICE, 174tropomyosin, 119

MS,

see

Multiple sclerosisMSH,

see

Melanocyte-stimulating hormoneMultiple sclerosis (MS), 164Murine mammary tumour virus (MMTV),

219Muscular dystrophy, calpains in, 165Myelin basic protein, 163Myelodegenerative diseases, 163Myocardial infarction, 222Myosin

association with cytoskeleton, 22-ATPase enzyme, 240heavy chain (MHC), 110, 111light chain (MLC), 63, 64, 110

kinase (MLCK), 76phosphorylation, 115, 116, 117

N

Naegleria gruberi

, 145NCAM,

see

Neural cell adhesion molecules

NCBPs, see Neural calcium-binding proteins

N-Desmethyltamoxifen, 79NDP, see Nucleoside diphosphateNebulin, 114Nemaline myopathy, 115Neovascularisation, 191Nerve growth factor (NGF), 5Neural calcium-binding proteins (NCBPs),

79Neural cell adhesion molecules (NCAM),

206Neurite extension factor, 206Neurodegenerative disease, implication of

calpains in, 158Neurofibrillary tangles (NFTs), 140Neurofibromatosis type 2 (NF2), 155Neuronal calcium-binding protein, 131Neurones, calcium homeostasis regulation

in, 125Neurotransmission, calcineurin and, 136Neurotropin-3 (NT3), 126NF2, see Neurofibromatosis type 2

NFTs, see Neurofibrillary tanglesNGF, see Nerve growth factorNitric oxide

inhibition of focal adhesion by, 27synthase (NOS), 10, 27, 81

Nitrogen-activated protein kinase, 11NMDA, see N-Methyl-D-aspartateNMU, see N-Methyl-N-nitrosoureaNon-EF-hand calcium binding proteins,

30–31, 35–62annexins, 35–40

annexins in cancer growth and progression, 38–39

annexins in morphogenesis and differentiation, 39–40

biological functions, 36–38structure, 35–36

calreticulin and functional diversity, 46–53

calreticulin and calnexin as molecular chaperones, 49–50

calreticulin in cell adhesion, 50–51calreticulin in cell proliferation and

differentiation, 50calreticulin in intracellular calcium

storage, 48–49calreticulin in neoplasia, 51–52immunological implications of

calreticulin function, 52–53intracellular distribution of

calreticulin, 48phosphorylation of calreticulin, 47–48regulation of calreticulin expression,

46–47structure and molecular features of

calreticulin, 46calsequestrin and intracellular calcium

storage, 53–54gelsolin family of calcium-binding

proteins, 40–45gelsolin in cancer, 42–43galsolin in embryonic development

and morphogenesis, 41–42gelsolin expression in amyloidosis,

42gelsolin in severing and capping of

actin filaments, 40–41severin and cytoskeletal reorganisation,

44


Index 357

villin in differentiation and neoplasia, 44–45

osteocalcin in bone metabolism and osteotropism of cancer, 54–62

biology of osteocalcin, 54–55calcium-binding properties of

osteocalcin, 55osteocalcin in cell proliferation and

differentiation, 57–60osteocalcin gene structure and

function, 55–56osteotropism of metastatic

dissemination, 60–62regulation of osteocalcin by vitamin

D3, 56Non-small cell lung carcinoma (NSCLC), 198NOS, see Nitric oxide synthaseNSCLC, see Non-small cell lung

carcinomaNt3, see Neurotropin-3Nucleoside diphosphate (NDP), 230Nucleotide metabolism, 1

O

OA, see OsteoarthritisOdontoblast differentiation, 18517β-Oestradiol, 79Oestrogen receptors (ERs), 79, 101, 159OM, see OncomodulinOncogenes, overexpression of, 73Oncogenic retroviral genes, 120Oncomodulin (OM), 182Open reading frame (ORF), 68, 150ORF, see Open reading frameOrnithogalum virens, 122OSE, see Osteonectin silencer elementOsteoarthritis (OA), 196Osteoblast migration, cooperative

functioning of laminin and FN in, 59

Osteocalcin, 30, 35in bone metabolism, 54in cell proliferation and differentiation,

57functions of, 60gene transcription, 55

Osteoclasts, localisation of fimbrin in, 97

Osteonectin, 184, 247cancer progression and, 196expression, correlation between

angiogenesis and modulation of, 192

immunoreactivity, 193inhibition of cell spreading by, 190molecule, functional domains of, 185silencer element (OSE), 55

Osteonectin, in cell function and behaviour, 183–198

effects of osteonectin on angiogenesis, 191–192

functions and functional domains of osteonectin, 184

modulation of cell proliferation by osteonectin, 190–191

modulation of cellular adhesion, cell shape, and motility by osteonectin, 188–190

molecular structure of osteonectin, 183–184

osteonectin in embryonic development and differentiation, 187–188

osteonectin expression in cancer development and progression, 193–196

osteonetcin homologues and putative tumour suppressor properties, 197–198

osteonetcin involvement in other disease states, 196–197

osteonetcin in remodelling of extracellular matrix, 186–187

regulation of osteonectin expression, 184–186

Osteopontin-transfectant cells, 194OT, see OxytocinOvariectomy, expression of CBD gene

transcripts and, 128Ovary

calretinin as marker for serous carcinoma of, 134

carcinomas, nm23-H1 in epithelial, 231

osteonection immunoreactivity in invasive tumours of, 193

Oxytocin (OT), 132



P

PA, see Plasminogen activatorPaired helical filaments (PHF), 140Paracentrotus lividus, 216PARP, see Poly (ADP-ribose) polymeraseParvalbumin (PV), 64, 125, 181–182Paxillin, 162PCNA, see Proliferating cell nuclear antigenPDGF, see Platelet-derived growth factorPGE, see Prostaglandin EPgR, see Progesterone receptorsPhenylephrine, 123PHF, see Paired helical filamentsPhorbol 12-myristate 13-acetate (PMA), 99Phosphatidylinositol (PI), 10Phosphoglucomutase, 208Phosphoinositide-3 kinase (PI3K), 11Phospholipase C (PLC), 10Phospholipid signalling, 10Phototransduction, 81Physarum polycephalum, 40PI, see PhosphatidylinositolPI3K, see Phosphoinositide-3 kinasePKA, see Protein kinase APKC, see Protein kinase CPlasma membrane Ca2+-ATPase extrusion

pump (PMCA), 5Plasminogen activator (PA), 186Platelet

-derived growth factor (PDGF), 18integrin, 178

PLC, see Phospholipase CPMA, see Phorbol 12-myristate 13-acetatePMCA, see Plasma membrane Ca2+-ATPase

extrusion pumpPMN, see Polymorphonuclear leukocytesPoly (ADP-ribose) polymerase (PARP),

171, 172Polymorphonuclear leukocytes (PMN), 99,

115Profilaggrin (FLG), 199, 201

abnormal expression of, 204genomic and molecular features of, 203in keratinocyte differentiation, 202monomers, 204

Profilin, 90, 92Progesterone receptors (PgR), 101Programmed cell death, 159

Proliferating cell nuclear antigen (PCNA), 38

Proline, 114Prostaglandin, 229, 230Prostaglandin E (PGE), 25Prostate

adenocarcinoma cells, apoptosis of by calpain inhibitors, 159

cancer cell lines, protection of, 52carcinomas, Gleeson grade in, 95ductal carcinoma of, 98

Proteases, activation of by calcium signalling, 159

Proteasome, 166Protein(s)

actin-binding, 88, 92, 93, 176adenomatous polyposis coli, 104adhesion-mediating, 94amyloid precursor, 178, 179β-amyloid precursor, 141armadillo, 105CaM binding domains of target, 75capping, 97EF-hand, 67folding, 84glial fibrillary acidic, 207guanosine triphosphatase activating, 80guanylate cyclase-activating, 85heat shock, 84, 216intracellular, 36linking plasma membrane with actin

cytoskeleton, 108mobilisation, requirement for, 166myelin basic, 163myofibrillar, 165ras family, 26receptor, 22retroviral, 68Rho, 107S100, 214, 217stress-induced, 138tau, 179

Proteinases, 10Protein kinase A (PKA), 13, 99Protein kinase C (PKC), 13, 14

-mediated regulation, of annexin function, 36

pathway, 13Prothoracicotropic hormone (PTTH), 18


Index 359

Psoriasin, 200, 236PTEN

abnormalities, 12germline mutations, 12overexpression, 13

PTTH, see Prothoracicotropic hormonePV, see Parvalbumin

R

RA, see Rheumatoid arthritisRadixin, 108Rana rugosa, 46Rapamycin, 141Ras family proteins, 26RBP, see Retinol-binding proteinRCN, see RecoverinReceptor

proteins, 22tyrosine kinases (RTKs), 26

Recoverin (RCN), 79, 80cancer-associated retinopathy and, 83mode of action of, 82–rhodopsin kinase complex, 83

Repetin, 201, 203Reticulocalbin family, of EF-hand proteins,

149–151molecular features of reticulocalbin

homologues, 150putative functions of reticulocalbin and

homologues, 150–151Retinitis pigmentosa (RP), 81, 85Retinoblastoma

gene product, 17susceptibility regulatory element, 50

Retinoic acid, 60, 209Retinoid X receptor (RXR), 56Retinol-binding protein (RBP), 21Retroviral proteins, 68Reverse transcriptase polymerase chain

reaction (RT-PCR), 69Rheumatoid arthritis (RA), 84, 196, 204Rhodopsin kinase, 81Rho protein, 107Rod domain, 103RP, see Retinitis pigmentosaRTKs, see Receptor tyrosine kinasesRT-PCR, see Reverse transcriptase

polymerase chain reactionRubella virus (RV), 47

RV, see Rubella virusRXR, see Retinoid X receptorRyanodine receptor (RyR), 20, 53RyR, see Ryanodine receptor

S

S100 family genes, structure and organisation of, 68

S100 proteins, 199–244biological properties of S100A7,

236–239S100A7 in neoplastic disease, 238–239S100A7 in skin pathology, 237–238structure and molecular properties of

S100A7, 236–237cell cycle-related expression of, 217effects of S100 proteins on cell deformity

and cellular morphology, 205–222

cell adhesion and invasive potential of cancer cells, 210–212

cell cycle-related expression of S100 proteins, 217–219

postulated mechanism of cell cycle control by S100A4, 219–222

S100 proteins in cell proliferation, 214–217

S100 proteins in remodelling of extracellular matrix, 213–214

potential value of S100 proteins as markers of cancer progression and prognosis, 243–244

S100 proteins in cell differentiation, motility, and cancer invasion, 202–205

profilaggrin in keratinocyte differentiation, 202–205

trichohyalin, 205S100A isoforms, 222S100A2 as putative tumour suppressor,

223–224S100A3 expression in cell differentiation

and neoplasia, 224–225molecular features of S100A3,

224–225S100A3 expression in cell

differentiation and human gliomas, 225

S100A4 in cancer development and



progression, 225–235clinical potential of S100A4 as marker

for cancer prognosis, 230S100A4 expression and metastatic

potential of cancers, 225–230S100A4 in human breast cancer,

230–234S100A4 in other forms of human

cancer, 234–235S100A6 in cancer, 235–236S100A8 and S100A9 proteins in

inflammatory diseases, 239S100A11 and possible modes of function,

239–240S100P in cancer progression, 241–243

Saccharomycescerevisiae, 38, 90, 97, 145pombe, 136

Sarcoplasmic–endoplasmic reticulum Ca2+–ATPase pump (SERCA), 5

SCCA, see Squamous cell lung carcinoma antigen

Schizossaccharomyces pombe, 112Sciatic nerve regeneration, stimulation of by

S100B, 215SCLC, see Small cell lung carcinomaSDKs, see Sphingosine-dependent kinasesSecreted protein acidic, rich in cysteine

(SPARC), 183SERCA, see Sarcoplasmic–endoplasmic

reticulum Ca2+–ATPase pumpSerine

protease, kallikrein-like, 242/threonine kinase, 11

Severin, 30Shigella flexneri, 100, 109Signal transduction

genetic activation and, 2machinery, architectural aspects of, 25

Skinneoplasms, 7pathology, S100A7 in, 237tumours, trichohyalin expression in, 205

SLE, see Systemic lupus erythematosusSmall cell lung carcinoma (SCLC), 83Smith–Magenis syndrome (SMS), 1, 41SMS, see Smith–Magenis syndromeSPARC, see Secreted protein acidic, rich in

cysteine

Sphingosine-dependent kinases (SDKs), 47–48

Squamous cell carcinoma, bladder, 238Squamous cell lung carcinoma antigen

(SCCA), 162Stathmin

expression, up-regulation of, 221promoter, down-regulation of, 220

Steroid(s)calbindin expression and, 129hormone, 18

Systemic lupus erythematosus (SLE), 52, 144

T

Talin, 51Tau protein, 179T-cell

antigen receptor (TCR), 49, 141signal transduction cascade, in SLE, 144

TCR, see T-cell antigen receptorTesticular hormones, 101Thapsigargin, 172THH, see TrichohyalinThioridazine, 78Threonine phosphatase, 137Thrombospondin, 192Thymidine kinase (TK), 55Thymosins

effects of on cell proliferation, 93potential role of in cancer progression,

95Thyroid

carcinoma, human anaplastic, 107hormones, 57, 67

Tissue plasminogen activator (tPA), 186Titin, 114TK, see Thymidine kinaseT-lymphocytes, IP3R1-deficient, 17TNF, see Tumour necrosis factorTopoisomerase II, 150tPA, see Tissue plasminogen activatorTranscription factors, activator of, 156Transfection studies, 11Trichohyalin (THH), 201

expression, in skin tumours, 205genomic and molecular features of, 203

Triticum aestivum, 96


Index 361

Tropolyosin, down-regulation of, 42Tropomyosins, 118Troponin, 114, 118Troponin C, 63Tubulin monomers, 208Tumour

necrosis factor (TNF), 24progression, expression of gelsolin and

severin in, 44suppressor

gene, 73S100A2 a putative, 223

Tyrosine kinase inhibitors, 137

U

UMUC-2, human bladder cancer cell line, 43

Urokinase-type plasminogen activator, 10

V

Valine, 114Vascular cell adhesion molecule (VCAM),

139Vascular endothelial growth factor (VEGF),

27, 28Vasodilator-stimulated phosphoprotein

(VASP), 101Vasopressin (VP), 132VASP, see Vasodilator-stimulated

phosphoproteinVCAM, see Vascular cell adhesion

moleculeVD3, see Vitamin D3VDR, see VD3 receptor

VDRE, see VD response elementVD3 receptor (VDR), 56VD response element (VDRE), 56VEGF, see Vascular endothelial growth

factorVerapamil, 9, 10, 22, 23VGCCs, see Voltage-gated calcium

channelsVillin, in differentiation and neoplasia,

44Vinculin, 51Vitamin D3 (VD3), 56Vitamin D3 receptor, 67Voltage-gated calcium channels (VGCCs),

6, 8VP, see Vasopressin

W

WAS, see Wiskott-Aldrich syndromeWASP, see Wiskott-Aldrich syndrome

proteinWHO, see World Health OrganizationWiskott-Aldrich syndrome (WAS), 91

gene mutation, 92protein (WASP), 91

World Health Organization (WHO), 235

Y

Yeast budding process, 92

Z

Zinc finger transcription factors, 225


calcium signalling in cancer-sh erbet

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