p53 suppressor gene

MoLECUlAR BIOLOGY INTELLIGENCE UNIT

P53 SuPPRESSOR GENE

MoLECUlAR BIOLOGY INTELLIGENCE UNIT

P53 SUPPRESSOR GENE

Tapas Mukhopadhyay, Ph.D. Steven A. Maxwell, Ph.D.

Springer-Verlag

Jack A. Roth, M.D. University ofTexas

M. D. Anderson Cancer Center Houston, Texas, U.S.A.

Berlin Heidelberg GmbH RG. LANDEs CoMPANY AUSTIN

MoLEcULAR BIOLOGY lNTELLIGENCE UNIT

P53 SUPPRESSOR GENE

R.G. LANDES COMPANY Austin, Texas, U.S.A.

Submitted: December 1994 Published: March 1995

U.S. and Canada Copyright © 1995 R.G. Landes Company All rights reserved.

International Copyright © 1995 Springer-Verlag Berlin Heidelberg Originally published by Springer-Verlag Berlin Heidelberg in 1995 Softcover reprint of the hardcover 1 st edition 1995

All rights reserved.

International ISBN 978-3-662-22277-5

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the rime of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library ofCongress Cataloging-in-Publication Data

Mukhopadhyay, Tapas. p53 suppressor gene 1 Tapas Mukhopadhyay, Steven A. Maxwell, Jack A. Roth

p. cm.--(Molecular biology intelligence unit) Includes bibliographical references and index.

ISBN 978-3-662-22277-5 ISBN 978-3-662-22275-1 (eBook) DOI 10.1007/978-3-662-22275-1

1. p53 antioncogene. 1. Maxwell, Steven A. II. Roth, Jack A. III. Title. IV. Series. RC268. 44.P16M85 1995

616.99'4042--dc20 95-4045

CIP

PuBLISHER's NotE

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R.G. Landes Company

rr===================== CONTENTS ===================:::::::::;-]

1. The Role of p53 in Cancer .......................................................... 1 From Oncogene to Tumor Suppressor Gene ......................................... 1 Interactions with DNA Tumor Virus-Transforming Proteins ............... 2

2. Gene Structure .......................................................................... 13 Introduction ....................................................................................... 13 Structure of p53 Gene ......................................................................... 13

3. Wild-Type versus Mutant p53 .................................................. 19 Introduction ....................................................................................... 19 Structure and Function ofWild-Type p53 Protein .............................. 20 Mutational Inactivation of p53 ........................................................... 24 Methods of Assayingp53 Gene Mutation in Human Cancers ............. 30 Mutant p53 Proteins ........................................................................... 33 p53 Mutations in Human Cancer .......................... , ............................ 36

4. Biophysical and Biochemical Properties of the p53 Protein ...... 55 Introduction ....................................................................................... 55 Missense Mutations within a Conserved Region of p53

Alter Its Biological Activity ............................................................ 59 The Central Conserved Region is a Conformational

Domain of p53 .............................................................................. 60 The Conformation of p53 Determines Its Biological Activity .............. 61 Conclusion .......................................................................................... 64

5. Regulation and Modulation of the Function of p53 .................. 73 Introduction ....................................................................................... 73 Protein Interactions Can Regulate p53 ................................................ 73 Regulation of p53 by Phosphorylation ................................................ 79 Redox Mechanisms ............................................................................. 81 Allosteric Regulation Model ................................................................ 81 Regulation oflntracellular p53 Levels ................................................. 84 Regulation of Subcellular Localization of p53 ..................................... 86 Control Over the Potential Role of p53 in DNA Replication .............. 87 Cell Cycle Regulation of p53 Function ............................................... 89 Tissue- and Cell Type-Specific Regulation of p53 Activity .................. 90 Conclusions ........................................................................................ 93

6. Potential Clinical Significance of the p53 Tumor Suppressor Gene in Cancer Patients .......... 113 Second Primary Cancers ................................................................... 114 The Role of p53 in Premalignancy .................................................... 115 Prognostic Studies in Cancer Patients ............................................... 116 Gene Replacement Clinical Trials ..................................................... 116 Retroviral Vectors for Gene Delivery ................................................. 118 Adenoviral Vectors for Gene Delivery ............................................... 120 Clinical Applications ......................................................................... 122

Index .............................................................................................. 127

======PREFACE======

The p53 field has been growing at a rapid pace, with more than 2500 articles published since 1989. Publications onp53 will probably exceed 600 in 1994.

The recognition of multiple roles for p53 in biological processes of growth, apoptosis, differentiation and transformation awarded it the distinction of"Molecule of the Year" by the journal Science. The p53 protein is the most commonly mutated gene product identified to date in human cancers. The recent identification of alterations in p53 regulatory proteins, such as mdm-2, indicate that the p53 biochemical pathway of growth control may be a more frequent origin for human cancer than previously realized.

We reluctantly undertook the task of assembling the vast wealth of information on the p53 gene and its product. It was unfeasible to include all articles written on p53, and we selected those that would provide a source for additional information on the specific topics concerning p53 discussed in this book. Our main goal was to provide an overview of the p53 gene, its product, regulatory mechanisms, action and clinical implications that could be used by investigators interested in the field. We hope that this book will be a useful tool for those who are just beginning in the p53 field, as well as for researchers well established in the field.

We would like to thank Carol Torrence and Marie Bunker for their assistance in typing and editing this manuscript.

Author's Note: In this book the authors' use of p53 was used to denote the gene and p53 was used to denote the protein

AcKNOWLEDGMENTS This study was partially supported by grants (R29 CA45187

[S.A.M.] and ROl CA45187 [J.A.R.]) from the National Cancer Institute; by gifts to the Division of Surgery from Tenneco and Exxon for the Core Lab Facility; by the Cancer Core Center Support Grant (CA16672); and by a grant from the Mathers Foundation.

CHAPTERl

THE RoLE oF P53 IN CANCER

FROM ONCOGENE TO TUMOR SUPPRESSOR GENE

The product of the p53 tumor suppressor gene was first identified as a tumor antigen that bound to simian virus 40 (SV40) T anti

gen and adenovirus E1B oncoproteins. 1•3 The p53 protein originally was believed to have an oncogenic rather than a tumor suppressor function, since it could immortalize cells in culture4 and cooperate with the activated ras oncogene to transform cells in culture.5•6 Overexpression of p53 also enhanced the transformed phenotype of tumor cells.?

These observations led to the classification of p53 as an oncogene; however, the eDNA clones used in those studies contained missense mutations within a conserved region of p53 important for the conformation and biological activity of the protein. The wild-type form of the protein could not transform cells in culture and actually suppressed the transformation of cells by oncogenes.8•9 Missense mutations within conserved regions of p53 between amino acids 100 and 300 (out of a total of 393) convert the protein from a tumor suppressor protein into one with growth-promoting activity and the ability to cooperate with the ras, myc, or E1A oncoproteins to transform primary mouse embryo fibroblasts.

Mutational inactivation of the p53 gene is now recognized as one of the most frequent genetic aberrations in human cancer. 10•11 Moreover, the growth and transformed phenotype of cells that lack endogenous p53 are inhibited when transfected with the wild-type p53 gene. 12•14

These properties led to the reclassification of p53 as a tumor suppressor gene, or recessive oncogene. That loss of p53 function plays a role in the development of neoplasia is supported by observations that transgenic p53-deficient mice and Li-Fraumeni patients harboring germline p53 mutations are predisposed to early onset of a variety of tumors. 15•16

The frequency of p53 mutations argues for a critical role of p53 in the development of human cancers; however, the incidence is not absolute and ranges from 20-50% overall. The highest frequencies have been reported in oral mucosa squamous cell cancers (81 %), in small

2 p53 Suppressor Gene

cell lung cancers (70%) and in anaplastic undifferentiated thyroid cancer (68%).'7 Considering the potential of current methods to miss 15-20% of inactivated p53, a portion of human cancers probably arise through other mechanisms. 17 The observations that both p53 and ras mutations occur frequently and independently of each other in human cancers17-21 supports a concept of multiple mechanisms of tumorigenesis. The potential for inactivating mutations to reside in introns and the promoter of p53, as well as the possible inactivation of both upstream (regulatory) and downstream (p53-regulated genes) mediators of p53 action, needs to be investigated to determine the true incidence of defective p53 pathway(s) in human cancer.

The biological activity of the p53 protein is manifested as a suppression of cell growth. The p53 protein exerts its antiproliferative activity by blocking cells at the G 1 phase of the cell cycle. Overexpression of p53 blocks cell growth at a restriction point in the late G 1 phase.n-26 Furthermore, overexpression of wild-type p53 inhibited induction of proliferating cell nuclear antigen and blocked cells from entering S phaseY A temperature-sensitive mutant of p53 has been described that exhibits a wild-type phenotype that blocks division of cas-transformed cells during late G 1 at 32°C and expresses a mutant phenotype that is unable to restrict growth of transformed cells at 39°C.22 Wild-type p53 expression in other cell lines not only slowed growth but induced apoptosis.28 Thus, p53 exerts a tumor-suppressive or negative growth effect by acting as a cell-cycle checkpoint to block cell division before DNA replication at the late G1 phase. The protein also plays a role in programmed cell death.

INTERACTIONS WITH DNA TUMOR VIRUS-TRANSFORMING PROTEINS

The p53 protein was first identified in complexes with the transforming proteins of SV 40 and adenovirus. This section will discuss these protein interactions and their effects on p53. DNA tunior viruses target p53 for inactivation, possibly to increase the efficiency of viral replication. Elimination of p53 function would contribute to unrestricted cell division, benefiting virus replication, which is dependent on the host cell DNA replication machinery. 29 The elimination of p53 function leads to a increased potential for neoplastic transformation.

A. sv 40 LARGE T ANTIGEN Infection of primate and rodent cells in culture with SV 40 results

in their conversion to a neoplastic phenotype, as exhibited by increased growth rates, growth in soft agar, and the ability to form tumors in athymic mice. 30 The transforming function of SV40 is dependent on the synthesis of large T antigen, a major early protein of 708 amino acids. 31 ·32 A cellular protein of 53,000 Daltons forms a complex with

The Role of p53 in Cancer 3

large T antigen. 1-3 The same 53,000-Dalton protein (p53) was found to be overexpressed in embryonal carcinoma cells and chemically-transformed cells. 33 Approximately 10o/o of human cancer patients were found to have serum antibodies to p53. 34 Since p53 co precipitated with T antigen and was found to generate immune responses in isogeneic mice and to be overexpressed in cells transformed by means other than SV40 infection, investigators concluded that p53 was a cellular tumor antigen that complexed with large T antigen.

The levels of p53 in SV40-transformed cells were found to be regulated by large T antigen. Cells harboring a temperature-sensitive mutant ofT antigen were transformed at 32°C and expressed T antigen complexed to p53; they also expressed high levels of p53. 35 At the nonpermissive temperature of 39°C, however, the cells reverted to a normal phenotype and expressed a functionally inactive T antigen that was not complexed with p53. These cells also expressed much lower amounts of p53. A wide variety of other transformed cells expressed higher levels of p53 than their nontransformed counterparts.33•36•37 The mechanism of the higher level of expression of p53 in transformed cells involved post-translational modification, resulting in an increased half-life and stabilization of the protein. In normal cells, the half-life of p53 was less than 30 minutes, whereas in transformed cells p53 frequently exhibited half-lives up to several hours. 38•39

Considering the importance of p53 in control of cell growth, T antigen most likely targets p53 for inactivation in order to stimulate cell division and increase the efficiency of viral replication. T antigen binds to an internal domain on p53 that contains four regions that are highly conserved among all organisms from amphibians to mammals. 40 This conserved domain is located between amino acids 100 and 300, out of a total of 393 (Fig. 1.1). T antigen binds to two discontinuous regions within the conserved domain, between amino acids 123 and 215 and between residues 236 and 289.41.42 It is significant that this same region is the target for the majority of missense mutations found in human cancers that inactivate the transactivation and DNA-binding activities of p53, which are critical for its tumor suppressor activity. Missense mutations result in conformational changes in p53 that can be detected by specific monoclonal antibodies. 8.43-46

The binding ofT antigen stabilizes p53, resulting in its overexpression in SV40-transformed cells, as discussed earlier. T antigen apparently targets the wild-type growth-suppressor form of p53, since many mutant p53s are defective in binding to large T antigenY-50 T antigen probably does not induce a conformational change in p53 that would account for its stabilization, as in the case of missense mutations, since p53 bound to T antigen is of the PAb246-positive, or wild-type, immunological phenotypeY Since the region of p53 that binds to T antigen is also the site for specific DNA binding,52 the functional consequence is the loss of p53's DNA-binding and thus its transactivation activities. 53-55


SV40T

E18 224 354

E6

Nl~sac

v Fig. 1. 1. Interactions of viral transforming proteins with p53. Amino acids involved in protein binding domains are designated.

Consistent with the hypothesis that T antigen inhibits p53 is the finding that transformation by SV 40 does not involve mutational inactivation of p53. 51 Large T antigen might serve to inactivate p53, inducing DNA synthesis and allowing more efficient viral replication with an increased potential for cell transformation.

T antigen inhibits the tumor suppressor functions of p53, but binding of p53, in turn, affects several activities ofT antigen that are required for viral replication. SV 40 replication is dependent on cellular replication factors and T antigen. 29 The initiation of SV 40 DNA replication requires the physical interaction of DNA polymerase alpha/primase with T antigen in a preinitiation complex composed of topoisomerases and replication protein A. 56 The p53-binding site is located between amino acids 347 and 626 on T antigen57·58 (Fig. 1.1). DNA polymerase alpha can be displaced from T antigen by p53. 59 The p53 protein can also inhibit the helicase activity of large T antigen47·49 and bind to

sequences adjacent to the SV 40 origin of replication _47.4B.Go These ef-


fects of p53 on replication acttvtttes of T antigen, and the finding that p53 is associated with replication origins along with DNA polymerase alpha and replication protein A, 61 -63 suggest a role for p53 in replication of cellular DNA.

B. ADENOVIRUS ElA 0NCOPROTEIN Rodent cells are semipermissive for replication of human group C

adenoviruses and undergo transformation in response to infection by these same viruses. 64-66 Infection of growth-arrested rodent cells with adenovirus induces cellular DNA replication. 64 ·65 ·67 ·68 Cellular transformation by adenovirus has been shown to require two viral gene products, termed E1A and E1B. 69 The oncoprotein E1B, which is encoded by the type 2 and 5 transforming strains of adenovirus, binds to and stabilizes p53 by binding between amino acids 14 and 66,3•70 ·71 which encompass the acidic transactivation domain (Fig. 1.1). The binding site on E 1 B for p53 resides between amino acids 224 and 3 54.71

A strong correlation has been observed between the E1B protein's abiliry to inhibit the p53 transactivation function and its ability to transform primary cells in cooperation with E1A.72·73 E1B may inhibit p53 transactivation by sterically interfering with the association of the transactivation domain with components of the transcription machinery.

C. PAPILLOMAVIRUS £6 ONCOPROTEIN The human papillomaviruses (HPVs) infect the anogenital area and

are classified into two distinct groups based on their clinical associations.74 The first group is generally associated with benign anogenital warts that only rarely progress to cancer and are referred to as lowrisk viruses. The second group includes HPV types 16 and 18, which are associated with lesions at high risk for neoplastic progression and the majority of cervical carcinomas. The E6 and E7 genes of the virus are consistently retained and expressed in primary tissue and cell lines derived from cervical carcinomas. 75 Both viral genes have been shown to possess transforming potential in vitro76·77 and are required for the maintenance of the transformed phenotype of cervical cancer cells.78

In contrast to the stabilization and steric interference mechanisms ofT antigen and E1B oncoproteins, HPV E6 oncoprotein targets p53 for degradation via a ubiquitin-dependent protease pathway,79-81 resulting in a decreased half-life and very low levels of p53 in papillomavirusimmortalized keratinocytes. 82-84 The E6 protein preferentially targets the wild-type conformation of p53 exhibiting the PAb 1620 immunological phenorype.85 The C-terminal amino acid sequence between amino acids 106 and 115 of the E6 protein binds p53 (Fig. 1.1), whereas sequences within the 50 N-terminal amino acids direct degradation of p53. 86 The E6 protein requires association with a 100,000-Dalton protein (E6-AP) to complex with p53.87 Binding of the E6/E6-AP complex to


p53 results in ubiquitination of p53, selectively targeting it for proteolytic degradation. 88 In addition, E6 can abrogate p53's transactivation and DNA-binding activities. 55•89•90 That E6 targets p53 for inactivation is consistent with the observation that HPV-positive cervical cancers or cell lines transformed by HPV in general contain wild-type p53. 82•91

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72. Yew PR, Liu X, Berk AJ. Adenovirus E/B oncoprotein tethers a transcriptional repression domian to p53. Genes Devel 1994; 8:190-202.

73. Yew PR, Berk AJ. Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature 1992; 357:82-85.

74. zur Hausen H. Papillomaviruses and carcinomaviruses. In: Klein G, ed. Advances in viral oncology. New York: Raven Press, 1989:1.

75. Schwarz E, Freese UK, Gissmann L, et al. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature (London) 1985; 314:111-114.

76. Barbosa MS, Vass WC, Lowy DR, Schiller JT. In vitro biological activities of the E6 and E7 genes vary among papillomaviruses of different oncogenic potential. J Virol 1991; 65:292-298.

77. Bedell MA, Jones KH, Grossman SR, Laimins LA. Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells. J Viral 1989; 63:1247-1255.

78. von Knebel Doeberitz M, Rittmuller C, zur Hausen H, Durst M. Inhibition of tumorigenicity of C4-1 cervical cancer cells in nude mice by HPV18 E6-E7 antisense RNA. lnt] Cancer 1992; 51:831-834.

79. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990; 248:76-79.

80. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 pro-


motes the degradation of p53. Cell 1990; 63:1129-1136. 81. Shkedy D, Gonen H, Bercovich B, Ciechanover A. Complete reconstitu

tion of conjugation and subsequ_ent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system. FEBS Letters 1994; 348:126-130.

82. Scheffner M, Munger K, Byrne JC, Howley PM. The state of p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Nat! Acad Sci USA 1991; 88:5523-5527.

83. Hubbert NL, Sedman SA, Schiller JT. Human papilloma virus type 16 E6 increases the degradation rate of p53 in human keratinocytes. J Virol 1992; 66:6237-6241.

84. Lechner MS, Mack DH, Finicle AB, Crook T, Vousden KH, Laimins LA. Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription. EMBO J 1992; 11:3045-3052.

85. Medcalf EA, Milner J. Targeting and degradation of p53 by E6 of human papillomavirus type 16 is preferential for the 1620+ p53 conformation. Oncogene 1993; 8:2847-2851.

86. Crook T, Tidy JA, Vousden KH. Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and transactivation. Cell 1991; 67:547-556.

87. Huibregtse JM, Scheffner M, Howley PM. A cellular protein mediates association of p53 with the E6 oncoprotein of human· papilloma virus types 16 or 18. EMBO J 1991; 13:4129-4135.

88. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitnation of p53. Cell 1993; 75:495-505.

89. Hoppe-Seyler F, Butz K. Repression of endogenous p53 transactivation function in HeLa cervical carcinoma cells by human papillomavirus type 16 E6, human mdm-2, and mutant p53. J Virol 1993; 67:3111-3117.

90. Lechner MS, Laimins LA. Inhibition of p53 DNA binding by human papillomavirus E6 proteins. J Virol 1994; 68:4262-4273.

91. Crook T, Wrede D, Vousden KH. p53 point mutation in HPV-negative human cervical carcinoma cell lines. Oncogene 1991; 6:873-875.

CHAPTER2 ====

GENE STRUCTURE

INTRODUCTION

The use of DNA-mediated gene transfer has revealed that p53 acts as a tumor suppressor gene. Abnormalities of the p53 gene have

been reported in several human tumor cell lines and solid tumors. 1 Rapid progress has been made in the past few years in understanding the role of p53 in the biology of the tumor cell.2-4 Molecular isolation and characterization of this gene enabled determination of how p53 suppresses or inhibits the proliferation of tumor cell growth. Knowledge of the normal p53 DNA sequence was a critical prerequisite to identifying the mutant forms of p53 that cooperate with other oncoproteins in cellular transformation.

STRUCTURE OF P53 GENE The p53 gene has been found to be highly conserved in evolution.

It has been isolated from many different species, including Xenopus levis,5 rainbow trout, 6 chicken/ monkey,8 rat,9·10 mouse11-14 and human.15-18 In humans, p53 spans a region of 20 kb and is located in chromosome band 17p 13.1. 19 The mouse homologue is relatively smaller (12 kb) and resides on chromosome. 11 The relatively complex structural organization of the p53 gene is quite similar among different species: a considerable similarity exists between mouse and human p53, for example. In both cases, the gene is split into 11 exons separated by 10 introns. The first untranslated exon in both mouse and human is followed by an unusually large intron sequence. The much greater size of the human p53 gene is due partly to the longer intron sequences (Fig. 2.1), particularly the first intron, which is 10 kb in human and 6 kb in mouse. 16·21 The 2.5-kb intron 9 of the human is about three times longer than the 0.83-kb mouse intron. Except for the second exon, the splice junction sequences are highly conserved. The consensus sequences in splice donor and accepter sites, including the invariant dinucleotide GT and AG located at the 5' and 3' ends of the intron sequences, are conserved. In all cases, exon 1 of the p53 gene is a noncoding exon.

14

DNA

mRNA

PRO E1N ,.,

P 1 P2

Anti- Jtl'l1 ....

72 Pro/ Arg

Polymorprusm

2 l 4 s 6

p53 Suppressor Gene

8 9 10

l1 5 Ser

1 1

l89 Ser

Fig 2.1. Human p53 gene, mRNA and protein. p53 gene contains 11 exons with a first noncoding exon. The first intron is very long (70 kb), and appears to contain regulatory sequences. Two promoter regions are indicated asP 1 and P2. p53 protein contains three domains: acidic N-terminus, hydrophobic proline-rich domain and basic C-terminus. Five highly-conserved regions through Xenopus to human are indicated as "box". (From Osamutominage eta/, Critical Reviews in Oncogenesis 7992; 3:257,282. Reprinted by permission of CRC Press, Boca Raton, Florida.)

The human exons vary in length from 22 to 1,268 bp; exons 2, 4, 5, 7 and 8 code for five clusters of amino acid residues which are highly conserved during evolution. The first in-frame methionine codon in the human sequence is different from that of the mouse, but another ATG codon occurs further along in the mouse sequence; it is not clear which of the methionines is actually used during the translation process.

The 5' DNA sequences of the human, mouse and rat functional p53 genes each contain a noncoding exon. This 5' untranslated sequence displays an extensive diad symmetry at the 5' end of the p53 mRNA, 16·2 1 which is highly conserved during evolution. The experimental evidence suggests that the transcriptional start site lies at a point 5' from the diad element. 16 •21 It has been postulated that the presence of such an element at the 5' end of the p53 RNA would form a stable hairpin structure that may mediate transcriptional control for the gene. The human p53 gene appears to be controlled by two promoter elements separately regulated during cellular differentiation. 22·23 Primer extension experiments indicated that the stronger promoter lies in intron 1, about 1000 bp downstream of the first p53 exon, while the other promoter is located 100 to 250 bp upstream of the non coding

Gene Structure 15

first exon.B The experimental evidence suggests that these two promoter sequences are of an unusual nature in that the upstream sequences ofthep53 gene do not contain the TATA or CAAT box-like consensus sequences usually present in class II eukaryotic promoters. Nor are they like the housekeeping gene promoters, highly G+C rich. Therefore, the differential regulation of these two promoters seems to be important in modulating expression of the p53 gene during terminal differentiation. 22

The mature and spliced p53 mRNA is 2.2-2.5 kb in size and is expressed in a cell type-dependent manner. 24 ·25 The highest levels of p53 mRNA are found in spleen and thymus,26 ·27 but the mechanism of transcriptional regulation of the gene remains elusive. A more detailed analysis indicated the presence of a regulatory element in the p53 promoter, which shows binding sites for the neurofibromatosis 1 gene (NF 1) and a serum-inducible factor that resembles AP-1. 28 Further analysis indicated that both human and murine p53 promoters contain a conserved recognition sequence for the family of DNA-binding proteins that contain the basic helix-loop-helix (HLH).29 ·30 In the murine p53 promoter, this element is required for full promoter activity and contains the CACGTG motif that binds the USF transcription factor in a site-specific manner, representing the major DNA-binding activity observed in the nuclear extract. 31 Furthermore, a consensus sequence for binding the myel myoD protein family of transcriptional activators, which contain HLH, was also identified. 32 Therefore, the possibilities exist that during oncogenic transformation the altered level of USF or myc expression leads to the elevated expression of mutant p53. The locations of the HLH recognition sequences relative to the transcription start sites in the murine and human p53 genes are different, although they serve the same function.

Regulatory elements are also found in the intron sequences. Two guanidine nucleotides were discovered in intron 4 at positions 33 and 44; substitution by T and C resulted in a lack of specific DNA binding and reduced expression of p53, indicating that intron 4 increases the expression of p53. In the transgenic mouse model, intron 4 acts as an enhancer, and its activity is tissue-specificY Sequence-specific DNA binding of a protein in intron 4 may have some functional significance in p53 expression. A similar observation in a tissue culture system indicated that the optimum p53 expression required the simultaneous presence of introns 2 through 9. 33 Functional inactivation of the p53 gene has been reported where mutation in the splice site of intron 3 or 5 interferes with normal processing of the RNA. 34·35

The p53 protein is known to act as a transcription factor and, like other transcription factors, it has been implicated in the regulation of its own promoter. 36 Deletion analysis of the p53 promoter indicated that some sequences are critical for regulation of the protein. 36 This element is regulated by wild-type p53 but not by mutant p53. This


binding sequence is different from the known p53 binding consensus sequence,37 although no direct interaction of the p53 protein with this element has been demonstrated.

Alternative splicing of the p53 gene could be another dimension of its regulation. The presence of alternatively-spliced p53 RNA containing an additional 96 bases derived from intron 10 has been demonstrated in mouse tissues and human tissue culture. 38 This alternatively-spliced RNA accounts for 30% of the total p53 RNA in both normal epidermal and carcinoma cell lines suggesting that alternative splicing may be universal.

REFERENCES 1. Levine AJ. The tumor suppressor genes. Ann Rev Biochem 1993;

62:623-651. 2. Zambetti GP, Levine AJ. A comparison of the biological activities of wild

type and mutant p53. FASEB 1993, 7:855-865. 3. Montenarh M. Biochemical, immunological, and functional aspects of the

growth-suppressor/oncoprotein p53. Crit Rev Oncogenesis 1992; 3:233-256.

4. Tominaga 0, Hamelin R, Remvikos Y, Salmon RJ, Thomas G. p53 from basic research to clinical applications. Grit Rev Oncogenesis 1992; 3:257-282.


6. Caron de Fromentel C, Pakdel F, Chapus A, Baney C, May P, Sassi T. Rainbow trout p53:cDNA cloning and biochemical characterization. Gene 1992; 112:241-245.

7. Louis JM, McFarland VW, May P, Mora PT. The phosphoprotein p53 is down-regulated post-transcriptionally during embryogenesis in vertebrates. Biochim Biophys Acta 1988; 950:395-402.

8. Rigaudy P, Eckhart W. Nucleotide sequence of a eDNA encoding the monkey cellular phosphoprotein p53. Nucleic Acids Res 1989; 17:8375.

9. Coulier F, Imbert J, Albert J, et al. Permanent expression of p53 in FR3T3 rat cells but cell cycle-dependent association with large T antigen in simian virus 40 transformants. EMBO J 1985; 4:3413-3418.

10. Soussi T, De Fromentel CC, Breugnout C, May E. Nucleotide sequence of eDNA encoding the rat p53 nuclear oncoprotein. Nucleic Acids Res 1988; 16:11384.

11. Bienz B, Zukut-Houri R, Givol D, Oren M. Analysis of the gene coding for the murine cellular tumour antigen p53. EMBO J 1984; 3:2179-2183.

12. Jenkins JR, Rudge K, Redmond S, Wade-Evans A. Cloning and expression of full length mouse eDNA sequences encoding the transformation associated protein p53. Nucleic Acids Res 1984; 12:5609-5626.

13. Oren M, Levine A]. Molecular cloning of a eDNA specific for the murine p53 cellular tumor antigen. Proc Natl Acad Sci USA 1983; 80:56-59.

Gene Structure 17

14. Pennica C, Goedde! DV, Hayflick JS, Reich NC, Anderson CW, Levine AJ. The amino acid sequence of murine p53 determined from a eDNA clone. Virology 1984; 134:477-482.

15. Harlow E, Williamson NM, Ralston R, Helfman DM, Adams TE. Molecular cloning and in vitro expression of a eDNA clone for human cellular tumor antigen p53. Mol Cell Biol 1985; 5:1601-1610.

16. Lamb P, Crawford L. Characterization of the human p53 gene. Mol Cell Biol 1986; 6:1379-1385.

17. Matlashdwski G, Lamb P, Pim D, Peacock J, Crawford L, Benchimol S. Isolation and characterization of a human p53 eDNA clone: expression of the human p53 gene. EMBO J 1984; 3:3257-3262.

18. Zakut-Houri R, Bienz-Tadmor B, Givol D, Oren M. Human p53 cellular tumor antigen: eDNA sequence and expression in COS cells. EMBO J 1985; 4:1251-1255.

19. vanTuninen P, Dobyns WB, Rich DC, et al. Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome. Am J Hum Genet 1988; 43:587-596.

20. Biscoff JR, Friedman PN, Marshak DR, Prives C, Beach D. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc N ad Acad Sci USA 1990; 87:4766-4770.

21. Bienz-Tadmor B, Zakut-Houri R, Libresco S, Givol D, Oren M. The 5' region of the p53 gene: evolutionary conservation and evidence for a negative regulatory element. EMBO J 1984; 4:3209-3213.

22. Tuck SP, Crawford L. Overexpression of normal human p53 in established fibroblasts to lead to their tumorigenic conversion. Oncogene Res 1989; 4:81-96.

23. Reisman D, Greenberg M, Rotter V. Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1. Proc Natl Acad Sci USA 1988; 85:5146-5150.

24. Oren M, Bienz B, Givol D, Rechavi G, Zakut R. Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen. EMBO J 1983; 2:1633-1639.

25. Benchimol S, Lamb P, Crawford LV, et al. Transformation associated p53 protein is encoded by a gene on human chromosome 17. Somatic Cell Mol Genet 1985; 11:505-509.

26. Rogel A, Popliker M, Webb CG, Oren M. p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors. Mol Cell Biol 1985; 5:2851-2855.

27. Lozano G, Levine AJ. Tissue-specific expression of p53 in transgenic mice is regulated by intron sequences. Mol Carcinog 1991; 4:3-9.

28. Ginsberg D, Oren M, Yaniv M, Piette J. Protein-binding elements in the promoter region of the mouse p53 gene. Oncogene 1990; 5:1285-1290.

29. Matlashewski GJ, Tuck S, Pim D, Lamb P, Schneider J, Crawford LV. Primary structure polymorphism at amino acid residue 72 of human p53. Mol Cell Biol 1987; 7:2863-2869.

30. Murre C, McGraw PS, Vaessin H, et al. Interactions between heterolo-


gous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 1989; 58:537-544.

31. Reisman D, Rotter V. The helix-loop-helix containing transcription factor USF binds to and transactivates the promoter of the p53 tumor suppressor gene. Nuc Acids Res 1993; 21:345-350.

32. Ronen D, Rotter V, Reisman D. Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif. Proc Natl Acad Sci USA 1991; 88:4128-4132.

33. Hinds P, Finlay C, Levine AJ. Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation. J Virol 1989; 63:739-746.

34. Foti A, Bar-Eli M, Ahuja HG, Cline MJ. A splicing mutation accounts for the lack of p53 gene expression in a CML blast crisis cell line: a novel mechanism of p53 gene inactivation. Br J Hematol 1990; 76:143-145.

35. Takahashi T, D'Amico D, Chiba I, Buchhagen DL, Minna JD. Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer. J Clin Invest 1990; 86:363-369.

36. Deffie A, Wu HY, Reinke V, Lozano G. The Tumor Suppressor p53 Regulates Its Own Transcription. Mol Cell Biol 1993; 13:3415-3423.

37. Kern SE, Kinzler KW, Bruskin A, et al. Identification of p53 as a sequence-specific DNA-binding protein. Science 1991; 252:1708-1711.

38. Han KA, Kulesz-Martin MF. Alternatively spliced p53 RNA in transformed and normal cells of different tissue types. N uc Acids Res 1992; 20:1979-1981.

39. BeenkenSW, Karsenty G, Raycroft L, Lozano G. An intron binding protein is required for transformation ability of p53. Nucleic Acids Res 1991; 19:4747-4752.

CHAPTER3 ====

WILD-TYPE

VERSUS MuTANT P53

INTRODUCTION

What is now known as p53 was initially identified as a normal cellular protein bound to SV 40 large T antigen. 1•2 lmmunoprecipi

tation of large T antigen from a transformed mouse cell line coprecipitated a nuclear phosphoprotein of 53,000 molecular weight, hence called p53. The human p53 protein is composed of 393 amino acids and is located in the nucleus. p53 is present in all tissues but in such low quantities3-5 that it is difficult to detect by immunohistochemical techniques. On the other hand, the p53 protein has been detected at much higher levels in a large number of sporadic tumors and virally and chemically-transformed cell lines from mice and humans.6•7 Isolation and characterization of the p53 gene followed by early transfection studies indicated that p53 is capable of immortalizing primary rat embryonic fibroblast cells in culture. It was also found that p53 could cooperate with activated ras oncogene in cellular transformation of primary cells in culture. 8- 10

From these experiments, the p53 gene was initially reported to be a dominant transforming oncogene. Subsequent studies of various p53 clones indicated that they had different cellular transformation capabilities due to inherent differences in their DNA sequences. The first known clones with transforming capabilities are now known to have mutations in the p53 gene. 11 •12

Wild-type p53 competes with the mutant form and inhibits transformation. Moreover, wild-type p53 can block cellular transformation reduced by oncogenes such as myc, ras, or the adenoviral E1A. 12•13 The wild-type p53 gene is now strongly implicated as a tumor suppressor gene, and mutations in the gene have been found in a variety of common human malignancies.

20

STRUCTURE AND FUNCTION OF WILD-TYPE P53 PROTEIN

p53 Suppressor Gene

The primary structure of the p53 protein can be subdivided into three functional domains. A highly charged acidic N-terminal portion comprises the first 80 amino acids of mouse or human p53. This portion of the protein is expected to be in an alpha helical conformation. A highly charged basic C-terminal end contains amino acid residues 276 to 390 (mouse) or 319 to 393 (human) and forms an amphipathic helical structure. The middle region is highly hydrophobic in nature; this proline-rich domain contains amino acids 75 and 150 (mouse) or 80 and 150 (human). The amino acid residues between 319 and 323 retain the major nuclear localization signal for the protein14 in close proximity of cdc-2 kinase phosphorylation site (amino acid residue 316). A phosphoprotein, p53 is phosphorylated at serine residues by casein kinase I or by a DNA-dependent protein kinase. 15- 17 A serine residue at amino acid position 315 (corresponding to murine serine 312) of the p53 structural motif is phosphorylated by p34-cdc-2 kinase, 18-20 a known regulator of the cell cycle;21 residue 392, the penultimate serine, is phosphorylated by the casein kinase II enzyme. Because p53 is a nuclear phosphoprotein, its phosphorylation status and subcellular distribution in the normal cell vary through the. cell cycle. 19•22-24 The cdc-2-mediated phosphorylation of p53 reaches its maximum level during mitosis. 19

Moreover, p53 has been suggested to have associated kinase activity. 25

Comparisons of the amino acid sequences of all known p53 peptides from Xenopus through human showed about 56% homology, and five phylogenetically-conserved domains have been identified. In the human sequences, these highly-conserved regions span amino acids 13-19, 117-142, 171-181, 236-258 and 270-286.26-28 These regions with exceptional amino acid identity are of considerable importance, since with one exception (residues 72), all the point mutations observed so far in the wide variety of human cancers are found in these areas.

Wild-type p53 has been implicated in cell-cycle regulation,29•30 transcriptional regulation,31 -34 DNA replication35 and cell differentiation;36-38

it is, therefore, clearly involved in cellular growth control. The details regarding the structural and biochemical properties of p53 are described in a later chapter. Immunofluorescence studies using monoclonal and polyclonal antibodies indicated that p53 is preferentially located in the nucleus of transformed cells, whereas nontransformed cells exhibit cytoplasmic staining. 39 The p53 protein accumulates in cytoplasm during G 1 phase, enters into the cell nucleus at the early S phase, and remains there for short period of time only. These studies indicate that distribution of p53 is spatially regulated during normal cell cycle. 40

Subcellular fractionation revealed, however, that p53 was present in the chromatin, nuclear matrix fraction and nucleoplasmic fraction of both normal and transformed cells. 41

Wild-Type versus Mutantp53 21

Nuclear accumulation of the p53 protein has been shown to be mediated through a specific nuclear localization signal inherent in the primary structure of the protein. 40•18 Three nuclear localization signals (NLS I, II and III) that cluster around the C-terminus of the p53 protein have been identified. All three are required for efficient nuclear accumulation of the protein,42 although NLS I is the main nuclear localization signal in p53 and is highly conserved in genetically divergent species. Nuclear localization is a fundamental requirement of both wild-type and mutant p53 proteins in mediating tumor suppressor or cellular transformation activity, respectively. 14 Mutation in the nuclear location signal (amino acid positions 312 to 323) or substitution of arginine by glutamine at amino acid 306, which is very close to the nuclear signal location, prevent transportation of the p53 protein to the nucleus. 18

Other factors, however, seem to influence the subcellular distribution of the p53 protein. In SV40-transformed cell lines, both large T antigen and p53 are colocalized in the plasma membrane, while heat shock protein hsp70 and p53 complex are found in both the cytoplasm and in the nucleus. In nontransformed cells, the p53 protein has also been reported to be associated with the plasma membrane for a while during mitosis. 43 A striking similarity exists between the SV40 large T antigen and the human p53 nuclear localization signal motifs. 18

In a temperature-sensitive mutant cell line, p53 was located in the nucleus when the cells were growth arrested at 32°C but not at 37°C. In some human breast cancers with wild-type p53, the protein is present in the cytoplasm, which prevents it from acting as a tumor suppressor. It is likely, therefore, that p53-mediated growth control requires transactivation of other genes, for which p53 must be present in the nucleus. It has been demonstrated in situ by immunocytochemical techniques combined with electron microscope autoradiography that newlysynthesized RNP particles contained p53 protein, suggesting that p53 is involved in transcriptional regulation. Only p53 present in the nucleus can act as a negative regulator of cell proliferation. 14.44

Two hypotheses have been put forward to explain the mechanism of action of p53 in regulation of cell growth. 28•45 One proposes that p53 can interfere with the initiation of DNA replication. Wild-type murine p53 blocks the interaction of DNA polymerase a with SV40 large T antigen and prevents SV40 DNA replication;46 .47 it is possible that p53 binds to an analogous cellular replication protein to prevent the cells entering S phase. The other proposes that p53 regulates cell growth by acting directly or indirectly as a transactivator of transcription. Thus, p53 induces genes that inhibit growth or represses genes that stimulate growth. When p53 is expressed as a fusion protein using the yeast Gal-4 system, it can transactivate the reporter gene,31.32

indicating that it is able to interact with other proteins which are involved in transcriptional regulation. The mutant p53 failed to do so.


Initial interpretations of the results of transfection experiments that used p53 eDNA in cellular transformation of rat embryo fibroblast cells were erroneous, since the gene used was a mutant form. Later studies indicated that wild-type p53 confers tumor-suppressing activity and could even block cellular transformation induced by oncogenes. 13

Wild-type p53 has transformation suppressing activity in the ras complementation assay and can suppress the growth of transformed cells in culture and the formation of tumors in animals.48 The growth-inhibitory effect of the wild-type p53 has been demonstrated by gene transfer studies in glioblastoma and osteosarcoma cell lines49•50 in which cells were blocked at the GO/G 1 phase. Human colorectal cancer cell lines transfected with wild-type p53 showed lower colony-forming ability than those transfected with mutant p53, 51 indicating that overexpression of wild-type p53 could block neoplastic cell growth. It is now clear that wild-type p53 acts as a tumor suppressor.

It has been suggested that p53 is involved in transforming growth factorJ3-mediated signal transduction. It has been shown that TGFJ3 inhibits cell growth at G 1 phase of the cell cycle and that cells transfected with viral oncogenes that complex p53 are less responsive to inhibitory growth factor. 52 Thus, one mechanism of action of p53 may be the coupling of inhibitory signal transduction to the regulation of DNA replication.

A number of p53 monoclonal antibodies have been developed. 53-60

These antibodies differ greatly in recognition of different p53 protein conformations or different species-specific p53 proteins. Above all, these antibodies are useful tools in furthering our understanding of the molecular basis of p53 action. Except for the species-specific type, these antibodies fall into main two groups. One group reacts with the tumor suppressor form of p53, that is, wild-type p53, and the other with the mutant forms of p53. Monoclonal antibodies P Ab246, P Ab607 and PAb1620, for example, recognize the wild-type form of the p53, whereas PAb240 reacts mainly with mouse and human mutant forms of p53.61 The epitopes recognized by the former three monoclonal antibodies are abolished by protein denaturation, indicating that they are dependent ori conformation. 59•62 A number of antibodies have been developed against different epitopes of p53 which are subjected to change based on the protein conformation. Furthermore, it has been suggested that p53 may exist in both wild-type and mutant forms in a cell, changing with the physiology of the cell or during stages of the cell cycle. Monoclonal antibodies for example, PAb246, PAb1620, PAb240 and PAB607 react with the epitopes of the p53 protein that form in response to conformational change. In temperature-sensitive mutants, the p53 protein can adopt either wild-type or mutant conformation upon temperature shift, distinguished by antibody reactivates. Clearly, the p53 protein is a highly dynamic structure, and its interactions with the monoclonal antibodies may be confusing. Since p53 is believed to be a tumor-suppressor


gene, the wild-type p53 protein is considered to function as a negative growth regulator. In nontumorigenic fibroblast cells, however, p53 has been shown to act as an initiator of cell proliferation. 10•30•63·65 This apparent paradox in p53 function can be explained by postulating that p53 has dual functions in normal regulation of cell growth, each function being associated with a different conformation. Thus, cell growth stimulation or repression could be controlled by the transient alteration of p53 protein conformation:66 in wild-type conformation it acts as a negative growth regulator, whereas in mutant form it can stimulate growth.

The p53 gene has been implicated in the early embryonic development of mice. The protein has been detected in embryonal carcinoma cells and in 10- to 14-day-old mouse embryos, but not in 16-day-old embryos,2 •67•68 and expression of p53 mRNA declined strongly after differentiation. The steady state level of p53 mRNA expression was significantly reduced during the process of cellular differentiation,69•70

although the relative rate of p53 transcription remained unchanged during mouse embryonic development. Nuclear run-on transcription assays at various time points during differentiation of F9 cells showed that the rate of p53 mRNA transcription did not change and that the reduced amount of p53 mRNA after differentiation was due to a posttranscriptional regulation process during differentiation. Downregulation of p53 is a late event during differentiation and is associated with alteration of the cell cycle and changes in cell morphology. 71 An increased postmitotic expression of the p53 gene during mouse embryogenesis72 suggests that preregulation of p53 expression may be necessary for the inhibition of cell cycle progression and to induce differentiation. In tissue culture, during chemically-induced differentiation of the murine erythroleukemia (MEL) cell line, the expression of the p53 protein decreased to a very low level and remained low during differentiation.?3-78 Expression of an antisense p53 RNA species has been described during induction of differentiation, suggesting that antisense p53 RNA has a role in maturation of MEL cells.79 Similar reductions of p53 mRNA and protein levels were found in retinoic acid-induced differentiation of human neuroblastoma cells associated with characteristic features of neuronal cell maturation.80 Expression of p53 is higher in transformed and in undifferentiated cells than in differentiated cells, and level of expression correlates with the state of differentiation. 83•84

Intracellular p53 levels increase when the cells are exposed to DNAdamaging agents like ultraviolet or gamma irradiation.83•84 These increases are due to posttranslational stabilization of p53 after DNA damage. Induction and overexpression of wild-type p53 protein block the cell cycle; pausing) at the G 1 phase allows more time for DNA repair before the cells enter S phase. On the other hand, cells with mutated p53 protein proceed to the S phase with damaged DNA, which leads


to accumulation of mutations, increased genetic instability and even cell death. It could also result in selection of cell populations with mutations in a number of vital genes that have the properties of cancerous cells. Based on these observations, it has been proposed that wild-type p53 acts as a molecular policeman, monitoring the integrity of the genomic DNA. In case of excessive damage to the DNA, p53 switches off the cell cycle to allow extra time for DNA repair; if the cell is unable to repair its DNA, p53 may trigger suicide by apoptosis, or programmed cell death. 85 Mutant p53 lacks this capability.

MUTATIONAL INACTIVATION OF P53 Functional inactivation of the p53 gene is an almost universal step

in the development of human cancerY (Table 3.1) A number of mechanisms can lead to this inactivation, including missense mutation, deletion, insertion, rearrangement or interaction with viral or cellular proteins.2·86-93 Mutation or protein interactions often alters the biochemical and biophysical properties of the wild-type protein. The most common genetic alteration found in the p53 gene is point mutation. The p53 gene is the most highly mutated gene thus far identified in human malignancies.27·94 Many mutant p53 genes have been isolated and sequenced. Almost all the mutations cluster in four major domains spread over exons 5 through 8 which are conserved during evolution.95

Mutation often generates a protein with a longer half-life.4·53 The half-life of wild-type p53 is about 20 minutes,22 whereas all of the missense mutant p53 proteins have half-lives of hours.96·97 Mutated p53 accumulates in the cell and is easily detected by immunocytochemical techniques, whereas wild-type p53 is barely detectable (Table 3.2).

This offers a simple detection system for some mutant p53s that avoids the difficult methods of molecular biology; however, not all mutants are distinguishable from wild-type protein by reactivity with antibodies or by overexpression. A number of mechanisms may be responsible for the extended half-life of mutant p53, including cellular locality and interactions with other viral or cellular proteins. Interaction of p53 with viral and cellular proteins is discussed in chapters 1 and 5.

In nontransformed cells, the rapid degradation of the p53 protein occurs via a nonlysosomal, ATP-dependent proteolytic pathway. 145·146 A change in the normal proteolytic pathway for destruction of the p53 protein also could affect the half-life of the protein. This has not been shown directly, but ubiquitination is an important mechanism for the destruction of the p53 protein; it can be postulated that changes in this pathway can also alter the half-life of the p53 protein without accumulation of any mutation. 146 High levels of wild-type p53 have been reported in some breast cancer patients in whom the p53 gene was found inactivated without mutation. 147 The mechanism that leads to this elevation of p53 involves either sequestration of the protein or


Table 3.1. Frequency of p53 mutationa in human malignancies

Tumor Type

Colorectal cancer

Small cell lung cancer

Bladder cancer Prostate cancer Burkitt's lymphoma

Hepatocellular cancer

T-cellleukemia

Giloblastoma Non-small cell lung cancer Acute lymphoblastic leukemia Esophageal squamous cell cancer

Gastric cancer

Ovarian cancer

Acute myelogenous leukemia Neurofibrosarcoma in

von Recklinghausen neurofibromatosis

B-cell chronic lymphocytic leukemia Breast cancer

Soft tissue sarcomas Uterine cancer Oligodendroglioma Medulloblastoma Brain tumor Colonic adenomatous

polyp of familial polyposis coli

Frequency (%)

71 (5/7) 70 (23/33) 64 (16/25} 57 (8/14} 73 (11/15) 60 (6/1 0} 61 (11/18} 57 (4/7} 83 (1 0/12} 48 (26/54} so (5/1 0} so (8/16} 16(7/43} so (5/1 0} 17 (2/12} 45 (5/11) 45 (23/51) 56 (5/9) 29 (2/7) so (5/1 0) 44 (15/34} 39 (7/18} 58 (7 /12} 38 (9/24} 36 (11/30} 29 (9/13) 33 (2/6) 11 (5/46} 29 (2/7)

15 (6/40} 46 (11/26} 36 (4/11) 34 (11/32} 17 (1 0/59} 15 (2/13} 13 (8/60}b 14(6/43} 13 (3/24} 12 (2/17} 11 (2/19} 10(4/41) 7 (3/45)

a Point mutation, small insertion, small deletion b Analysis of exons '5 and 6 only.

Reference

Rodriguez NR, et al.98 Baker SJ, et al.99 Shaw P, et al. 100 lshioka C, et al. 101 Takahashi T, et al. 102 Hensel CH, et al. 103 Sidransky D, et al.104 Isaacs WB, et ai.10S

Farrell PJ, et al. 106 Gaidano G, et al.107 Bressac B, et al.108 Hsu IC, et al. 109 Murakami Y, et al.110 Cheng), etal.111 Nagai H, et al.112 Chung R, et al. 113

Chiba I, et al.114 Gaidano G, et al. 107 Sugimoto K, et al.115 Bennett WP, et al.116 Hollestein MC, et al. 117

Hollestein MC, et a1.11a Matozaki T, et al.119

Tamura G, et al. 120 Mazars R, et al.121

Okamoto A, et al.122

Slingerland JM, et al. 123

Feanux R, et al.2 37 Menon AG, et al.125

Gaidano G, et al.107 Osborne RJ, et al.126

Kovach JS, et al.124 Borresen AL, et a1. 127

Runnebaumn et al. 128

Chen LC, et al.129

Prosser j, et al. 130 Stratton MR, et al.131 Okamoto A, et al. 132

Ohgaki M, et al.133 Ohgaki M, et al.133 Mashiyama S, et al.134 Shirasawa S, et al. 135

From Osamutominage et al, Critical Reviews in Oncogenesis 1992; 3:257,282. Reprinted by permission of CRC Press, Boca Raton, Florida.


Table 3.2. Phenotypic variation in p53 mutants

Reactivity to Binding monoclonal ability Transforming antibody

Codon mutation Half·life to HSP ability 246 1620 240 Reference

Wild-type 6-20 min 0 0 + + 0 Hinds et al.11

Finlay et al.96

Struzbecher et al.137 Gannon et al. 138

Common mutant 4- 12 h + + 0 0 + Reihsaus et al.139

Kraiss et al. 140

Milner & Medcalf.141

Mouse-specific mutants 135 Ala~Val 37.5°C 3 h + ++ 0 0 + Michalovitz et al.142

32.5°C 1 h 0 0 + + 0 Ginsberg et al. 143

270 Arg~Cys 6-8 h 0 + I I I Halevy 0 et al. 144

Human-specific mutants 247 Ans~lle 37°C I I I 0 + Milner & Medcalf.141

30°CI I I + 0 175 Arg~His 3-6 h + +++ I I Hinds PW et al.97

175 Arg~His 1 h 0 0 I I Hinds PW et al.97

& 328 frame shift 273 Arg~His 7h 0 ++ I I Hinds PW et al.97

281 Asp~Giy 1.3h 0 + I I Hinds PW et al.97

315 Ser~Aia I 0 I I I Struzbecher et al.2o

Note: +, ++, +++; Relative intensity: 0: no ability or nonreactive;/: not determined;- : nonreactive owing to species specificity of monoclonal antibody. Mouse p53 mutant (135, Ala~ Val) and human p53 mutant (247, Asn~lle) are temperature sensitive. From Osamutominage et al, Critical Reviews in Oncogenesis 1992; 3:257,282. Reprinted by permission of CRC Press, Boca Raton, Florida.

defects in the p53 turn-over pathway. What is distinctive in tumors is not the expression of mutant p53, but its overexpression.

Cell lines initially derived from primary mouse fi.broblasts 148 were passaged and propagated on a 3T3 schedule to establish nontransformed cell lines. All the established cell lines examined, however, had p53 mutations. The mutation occurred during th~ selection or cloning of these cell lines. It is apparent, therefore, that the mutant p53 allele promotes immortality of cell lines in culture, 10•13•149 although several factors interplay in this process, such as tissue type, species and passage schedule.

The phenotypes resulting from p53 mutation have three distinct properties. First, most of the p53 mutants so far identified had lost


their tumor suppressor function. Adequate expression of the wild-type p53 protein in the transformed cells in culture could block the cells in G 1 phase of the cell cycle. 50·142·150·151 Second, both human and mouse missense mutations elicit gain of function. Mutant p53 introduced into a cell line with no endogenous p53 enhanced tumor growth. 48·152 When human p53 eDNA with missense mutations in major hot spots was introduced into p53-negative cell lines, the transformed cells expressed high levels of mutant p53 protein and, when injected into nude mice, induced tumorigenicity. 152 The mutated protein exhibits itself either as a dominant loss of function or a transdominant function to a wildtype protein. 13 When the cells with endogenous wild-type p53 were transfected with mutated p53, the wild-type p53 function was overridden by the mutant p53. Mutant and wild-type proteins in these cells oligomerize13·154 and form a faulty protein complex that inactivates the wild-type function,, displaying a true gain of function mutation, and enhancement of ceil growth. High stability of the mutated p53 proteins over the wild-type is an added advantage for the mutated protein to act in a transdominant fashion. The ratio of the wild-type versus the mutant p53 may be a critical issue in determination of the phenotype of the cell and its growth characteristics.

A significant number of human tumors display deletion of a small region of chromosome 17p that includes the p53 gene (Table 3.3). The high incidence of allelic loss at the 17p locus in human colon cancer has been documented, 155 leading investigators to continue their studies of the nature of the p53 gene in those primary tumors and cell lines. In colon cancer, one allele of the p53 gene is frequently deleted and the remaining allele contains a mutation.93·156 Deletion mapping showed that the common region of 17p deletion was within bands 17p12 to 17pl3.3;156 because the p53 gene is localized at 17p13.1, therefore, loss of the normal p53 gene is causally related to 17p deletion.

It has been reported that in breast cancers the more distal 17p13.3 portion is lost with much higher frequency, 58%, than to 17pl3.1, 27%.157.158 In this case the loss of heterozygosity at 17pl3.3 is associated with over-expression of p53 RNA suggesting that there are at least two loci on chromosome 17p, one at p53 and one at YNZ-22. There is a possibility that YNZ-22 regulates the p53 locus. 159 In addition, there is an association between loss of heterozygosity at 17p and erb-2 amplification. 160 Loss of heterozygosity at this second locus has been related to increased proliferation and aneuploidy in breast cancer.129 The point mutations in the remaining p53 allele are usually missense, and they are often sufficient to inactivate the tumor suppressor function of the p53 protein. In osteoblasts, allelic deletion may occur after p53, mutation in the genome.

The molecular mechanism underlying the loss of heterozygosity is unknown, but the present view is as follows. At some point during


Table 3.3. Frequency of the allele loss on chromosome 17p in various types of human tumors

Ave freq Number of cases Tumor Type (%) lost/informative Reference

Small cell lung cancer 100 16/16 (100%) Morin N, et al.165

5/5 (100%) Yokato j, et al.'"" Adenocortical cancer 100 6/6 Yano T, et al.167

Barrett's adenocarcinoma 92 12/13 Blount PL, et al.1 &a

Squamous cell lung cancer 89 8/9 Weston A, et al.1&9

Neurofibrosarcoma in 83 5/6 Menon A, et al.125

von Recklinghausen neurofibromatosis

Gastric cancer 68 13/19 (68%) Sana T, et al.170

8/12 (67%) Matozaki T, et al. 11 9

Colorectal cancer 67 22/33 (67%) Baker SJ, et al. 99

45/60 (75%) Vogelstein B, et aiY1

71/113 (63%) Delattre 0, et al.172

Osteosarcoma 65 28/37 (76%) Toguchida J, et al.m 4/12 (33%) Mulligan LM, et al. 174

Breast cancer 60 30/53 (58%) Thompson AM, et ai.1S9

51/74 (69%) Varley JM, et al.175

23/38 (61%) Mackay j, et al. 176 41/72 (57%) Devilee P, et al. 177

33/59 (56%) Sato T, et al.160 Ovarian cancer 58 16/20 (80%) Okamoto A, et al. 122

11/16 (69%) Eccles DM, et al. 178

9/14 (64%) Lee JH, et al. 179 13/28 (46%) Sato T, et al. 180

4/13 (31%) Russel SEH, et al. 181 Bladder cancer 57 20/41 (49%) Olumi AF, et al. 182

10/15 (67%) Sidransky D, et al.104

8/11 (73%) Oka T, et al. 183 Hepatocellular cancer 56 6/10 (60%) Slagle BL, et al. 184

14/26 (54%) Fujimori M, et al. 185

3/5 (60%) Bressac B, et al. 108 Esophageal 54 10/22 (45%) Wagata T, et al.186

squamous cell cancer 11/17 (65%) Meltzer SJ, et al.187 Renal cell cancer 53 15/24 (63%) Tsai YC, etal.188

6/10 (60%) Oka K, et al. 183 3/11 (27%) Bergerheim U, et ai,1B9

Medulloblastoma 45 5/11 Cogen PH, et al. 190 Astrocytoma 41 8/21 (38%) James CD, et al. 191

5/10 (50%) EI-Azouzi M, et al. 192 14/35 (40%) Fults D, et al. 193

Esophageal adenocarcinoma 30 3/10 Meltzek Sj, et al. 187 Rhabdomyosarcoma 23 7/31 Mulligan LM, et al. 174 Lymphoma 22 6/27 Cabanillas F, et al. 194 Blast crisis of chronic myelocytic 22 6/27 Marshal R, et al. 195

leukemia Non-small cell lung cancer 22 8/39 (21%) Chiba I, et al. 114

3/12 (25%) Yokato J, et al. 166 Melanoma 19 4/21 Drachopoli NC, et a1.196 Lung adenocarcinoma 18 2/11 Weston A, et al. 169 Colorectal adenoma 11 7/66 Baker SJ, et al.99

From Osamutominage et al, Critical Reviews in Oncogenesis 1992; 3:257,282. Reprinted by permission of CRC Press, Boca Raton, Florida.


tumorigenesis, proliferation of the potentially transformed cells is limited by the growth suppressor function of the wild-type p53 protein. At that point, p53 mutation provides a selective growth advantage to the cells. The mutant p53 protein at that stage exerts its "dominantnegative" effect by inactivating the wild-type p53 by oligomerization. The mutant p53 could augment the transformation process further when deletion of chromosome 17 completely eliminates the residual wildtype p53 activity. 94

Loss of heterozygosity is common in several tumor types, but it is not a universal phenomenon: for example, there is no correlation between loss of heterozygosity for chromosome 17p and p53 mutation in breast cancer. 129 A large variety of human cancers display p53 mutation where both alleles are retained. Selective elimination of the wildtype allele could, in fact, depend on the genetic background of the cell type; in some circumstances complete loss of the wild-type p53 is necessary for full activity of the mutant p53 function, but in others, expression of the mutant p53, along with wild-type p53, is sufficient for inducing the transforming function. In some instances, both p53 alleles are deleted, particularly in cell lines. The mechanism of mutant p53-mediated cell transformation is not clear, but the transformation markedly depends on the genetic set-up of the cell and distribution of cells in the macro and micro cell environments.

Individuals with the Li-Fraumeni syndrome, who have a family history of cancer, often inherit a mutant p53 allele in their genome. 161•163

Individuals with this syndrome are generally normal but have only one wild-type p53 allele and one mutant allele. This autosomal inheritance pattern is consistent with the disease; affected individuals acquire cancers at a young age (25-35 years) and develop multiple independent cancers during their life time. Germ line mutations in the p53 gene of these patients cluster around several codons between codon 245 and 258; they are not widely distributed over the reading frame like most of those responsible for sporadic cancers, possibly indicating that these mutant proteins have some unique properties or gain of function not commonly observed. The biological and biochemical properties of the mutant protein from tissue of patients with Li-Fraumeni syndrome vary considerably more than those of the same mutant from patients with most of the sporadic tumors.

Codon 248 of human p53 has been reported to be a major hot spot in Li-Fraumeni syndrome. In lung cancer cell lines, reduction of the expression of the codon 248 mutant protein by antisense RNA stimulated the growth of the cells in culture. The antisense clones formed tumors in nude mice, while the parental cell lines were nontumorigenic, indicating that p53 with a codon 248 mutation gene still retains weak tumor suppressqr function. 238

It has also been reported that mutant p53 proteins in the Li-Fraumeni group of patients are less stable and are expressed at lower levels than


the same mutant proteins expressed by sporadic tumors. The presence of these germ line p53 missense mutations does not interfere with the normal development of affected persons and is consistent with the transgenic mouse model system, where p53 knockout mice develop normally. Heterozygous mutant p53 imposes its dominant negative on the cells and could have a predisposing effect on carcinogenesis. In the transgenic mouse model, the animals having the heterozygous condition, that is, one mutant allele and one wild-type allele, show a higher incidence of cancer development. 197 It seems that the mutant p53 allele exerts its dominant negative effect on the cells of Li-Fraumeni patients and inactivates the function of the wild-type p53 alleles. A subsequent deletion of the chromosome 17 and/or accumulation of several additional mutations in the tumor-suppressor gene are required to completely escape growth control. In the murine system, p53 knockout experiments indicated that mice with homozygous p53 deletion appear normal but are prone to the spontaneous development of a variety of cancers at an early age, 6 to 8 months. 198 Thus, elimination of the p53 gene is not sufficient for cellular transformation in vivo. It is generally accepted that several genetic lesions are required to produce a cancer; this assumption is based on the exponential frequency incidence rates of adult cancers. In Li-Fraumeni patients, acquisition of p53 mutations through the germ line may be an early event, but accumulation of other genetic lesions over a period of 10 to 30 years may be required for the onset of cancer. The inherited p53 mutations in Li-Fraumeni patients probably act as weak tumor initiators.

METHODS OF ASSAYING P53 GENE MUTATION IN HUMAN CANCERS

Several methods have been used to detect p53 mutations in human cancer. Both immunological and molecular biology techniques have been successfully employed in identification and characterization of mutations. Analysis of p53 mutations in solid tumors as well as other type of tissues was greatly simplified with the advent of polymerase chain reaction (PCR), a powerful technique that allows amplification of specific regions of chromosomal DNA, 199 even from a single cell, by using Taq DNA polymerase. This technique has been used extensively in detecting and analyzing the p53 mutations in human cancers, and virtually all current methods for rapid detection of mutations use PCR products as starting materials.

Although RNase protection and chemical cleavage of mismatch heteroduplexes between the target and probe sequence are also wellestablished methods, these techniques involve several steps and require a lot of skill. By contrast, PCR-based techniques provide much faster and simpler detection with superior quality. A number of modified techniques have been successfully used for detection of p53 mutations, including allele-specific oligonucleotide hybridization (ASO), and restriction


fragment-length polymorphism (RFLP) analysis of the PCR products, both of which are nonisotopic methods. Single-strand conformation polymorphism analysis of PCR products (PCR-SSCP) is a sensitive method for detection of mutations when the exact location of the mutation is unknown. This method has been successfully employed both with and without isotopes.

Unlike the ras family of oncogenes, in which mutations occur at a few major hot spots, mutations in p53 occur in multiple sites throughout the open reading frame. Fortunately, most mutations are limited to the highly conserved region spanning exons 5 to 8. The mutation is usually identified by sequencing PCR-amplified DNA. Initial screening of the PCR-amplified DNA by SSCP for the presence of a mutation is very useful, however. The SSCP technique seems to be very sensitive: it can pick up 1 Oo/o of mutant sequence among wild-type eDNA. A powerful method for qualitatively analyzing DNA, PCRSSCP is based on the observation that the electrophoretic mobility of a DNA molecule through a neutral polyacrylamide gel can be altered by altering the size and shape of the DNA molecule. Single-strand DNA with a base substitution, for example, has a different folded structure than the wild-type sequence, giving it a different mobility in a polyacrylamide gel. Since its introduction, the PCR-SSCP technique has become widely used for detecting polymorphism in human genes.200

It is particularly useful in detection of mutations like those in the p53 gene, that is, where mutations are spread over a wide area of the open reading frame. The selection of PCR primer sequences is made such that each exon is separately amplified from the genomic DNA or eDNA by using specific primers that correspond either to th~ exon sequence or to flanking 3' and 5' intron sequences. In some cases, the nested PCR method is also used to amplify more than one exon contained between intron sequences. A good selection of p53 primer sequences and the PCR amplification conditions, followed by the sequencing technique, have been described by the Harris group (Table 3.4).201 The mixture of normal and malignant cells in most human tumors is a potential problem affecting the amplification of the target gene of interest. Identification of the tumor-bearing areas of the frozen or archival tissue sections under light microscope and subsequent scraping off the tumor tissue for the DNA extraction usually eliminate the problem.201 Touch preparations are useful for obtaining relatively pure sources of cancer cells for PCR amplification. 124 The PCR method has been employed to detect p53 mutations from cells recovered from urine and stool samples of patients.

The presence of mutations detected by PCR-SSCP is ultimately confirmed by DNA sequencing. Identification of specific base substitutions provides additional information regarding the molecular epidemiology of cancer. Although DNA sequencing is a routine procedure in the laboratory these days, the advent of the PCR technology encouraged the development of a method for direct sequencing of the


Table 3.4. Primer sequences selected for the analysis of human p53 gene mutations

Exon 1: 5'-GTG ATA AGG GTT GTG AAG GA-3' left external PCR primer 5'-AGC TGA AAA TAC ACG GAG CC-3' right external PCR primer 5'-ATT AAA TAA GAT GGT GTG AT-3' left internal Seq. primer 5'-TCA CAG CTC TGG CTT GCA GA-3' left middle internal Seq. primer 5'-TCA GAG AGG ACT CAT CAA GT-3' right internal Seq. primer 5' -TGC AGA GTC AGG ATT CTC GC-3' right middle internal Seq.primer

Exon 2: 5'-CCA GGT GAC CCA GGG TTG GA-3' left external PCR primer 5'-AGC ATC AAA TCA TCC ATT GC-3' right external PCR primer 5'-TCT CAT GCT GGA TCC CCA CT-3' left internal Seq. primer 5'-GGC CTG CCC TTC CAA TGG AT-3' right internal Seq. primer

Exon 3: 5'-CCA GGT GAC CCA GGG TTG GA-3' left external PCR primer 5'-AGC ATC AAA TCA TCG ATT GC-3' right external PCR primer 5'-CAG AGA CCT GTG GGA AGC GA-3' left internal Seq. primer 5'-AGT CAG AGG ACC AGG TCC TC-3' right internal Seq. primer

Exon 4: 5'-TGA GGA CCT GGT CCT CTG AC-3' left external PCR primer 5'-AGA GGA ATC CCA AAG TTC CA-3' right external PCR primer 5'-TGC TCT TTT CAC CCA TCT AC-3' left internal Seq. primer 5'-ATA CGG CCA GGC ATT GAA GT-3' right internal Seq. primer

Exon 5: 5'-TGT TCA CTT GTG CCC TGA CT-3' left external PCR primer 5'-AGC AAT CAG TGA GGA ATC AG-3' right external PCR primer 5'-TTC AAC TCT GTC TCC TTC CT-3' left internal Seq. primer 5'-CAG CCC TGT CGT CTC TCC AG-3' right internal Seq. primer

Exon 6: 5'-TGG TTG CCC AGG GTC CCC AG-3' left external PCR primer 5'-TGG AGG GCC ACT GAC AAC CA-3' right external PCR primer 5'-GCC TCT GAT TCC TCA CTG AT-3' left internal Seq. primer 5'-TTA ACC CCT CCT CCC AGA GA-3' right internal Seq. primer

Exon 7: 5'-CTT GCC ACA GGT CTC CCC AA-3' left external PCR/Seq. primer 5'-AGG GGT CAG CGG CAA GCA GA-3' right external PCR primar 5'-AGG CGC ACT GGC CTC ATC TT-3' left internal Seq. primer 5'-TGT GCA GGG TGG CAA GTG GC-3' right internal Seq. primer

Exon 8: 5'-TTG GGA GTA GAT GGA GCC T-3' left external PCR primer 5'-AGT GTT AGA CTG GAA ACT TT-3' right external PCR primer 5'-TTC CTT ACT GCC TCT TGC TT-3' left internal Seq. primer 5'-AGG CAT AAC TGC ACC CTT GG-3' right internal Seq. primer

Exon 9: 5'-TTG GGA GTA GAT GGA GCC T-3' left external PCR primer 5'-AGT GTT AGA CTG GAA ACT TT-3' right external PCR primer 5'-CCA AGG GTG CAG TTA TGC CT-3' left internal Seq. primer 5'-ACT TGA TAA GAG GTC CCA AG-3' right internal Seq. primer

Exon 10: 5'-TCT ACT AAA TCG ATG TTG CT-3' left external PCR primer 5'-GGA TGA GAA TGG AAT CCT AT-3' right external PCR primer 5'-CAA TTG TAA CTT GAA CCA TC-3' left internal Seq. primer 5'-CTT TCC AAC CTA GGA AGG CA-3' right internal Seq. primer

Exon 11: 5'-AGA CCC TCT CAC TCA TGT GA-3' left external PCR primer 5'-TGA CGC ACA CCT ATT GCA AG-3' right external PCR primer 5'-ATC TCT CCT CCC TGC TTC TG-3' left internal Seq. primer

5'-AGG CTG TCA GTG GGG AAC AA-3' right internal Seq. primer


double-stranded PCR-amplified DNA. This method offers several advantages: it is simple, easy to perform, involves no additional steps and consumes less time. A single allele mutation or DNA polymorphism can be detected easily. Sequencing can be performed at 74°C, which eliminates any secondary structure of the DNA and, finally, sequencing can be carried out with RNA after eDNA synthesis. A good sequencing ladder is dependent on the size and the internal sequence of the amplified DNA. Some automated DNA sequencing systems use a direct PCR sequencing procedure.

Immunocytochemical analysis has been used successfully with monoclonal and polyclonal antibodies for detection of both wild-type and mutant p53 proteins in a wide variety of tumor tissues and cells in culture. Some antibodies can detect both wild-type and mutant forms of p53 (for example, PAb1801), but others (for example, PAb240) react specifically with the mutant protein. 61 A panel of p53 antibodies has been developed that react with the different domains of p53, varying in their protein conformation depending on whether the protein is wild-type or a mutant. Some antibodies cross-react with other cellular proteins, which makes cytoplasmic location of the p53 protein difficult. For example PAb421, also reacts with the cytokeratin protein of the cell. In normal cells or tissues, the level of wild-type p53 is very low, and it is hard to detect by immunocytochemical methods. However, attempts to detect mutant proteins by using antibodies that recognize mutant p53 are not always successful because the highly dynamic p53 protein can exist in either conformation. Often during tissue preparation and fixation, the p53 protein gets denatured, and the antibodies which are supposed to react with the mutant conformation of p53 lose their reactivity. In these cases, however, mutant p53 in many cases could easily be detected. The protein, with its longer half-life, accumulates in the cell and displays differential staining with antibodies that react with only one of the two forms. Detection of p53 protein in situ by immunocytochemical methods is straightforward and provides important information regarding the relative degree of expression of the mutated protein, the heterogeneity of expression, and the coexpression of other tumor markers in association with the stage and type of tumor development. Immunoprecipitation of the p53 protein by antibodies is an established method for detection of the mutant form. It is important to note that some antibodies that react with mutant p53 because of its conformation often loses their reactivity as result of denaturation of the native protein.61

MUTANT P53 PROTEINS A large number of p53 mutations are now known that are func

tionally not identical. Different domains of this protein interact with different proteins; mutations in different parts of the gene affect the wild-type function, however, often by showing gain of function. Large


numbers of mutant phenotypes have been discovered, but only a few are well characterized. Experimental results indicate that p53 mutation often imposes a negative transdominant effect on the cells. The majority of the naturally occurring p53 mutations may not be strictly dominant in vivo, since loss of heterozygosity is very common in tumors. Early experiments showing the dominant negative effect of mutant p53 assessed this effect mainly on the transforming ability of the primary cultured cells. The mutated p53 gene has been found to cooperate with the activated ras oncogene and promote transformation of rat primary culture cells. Transfection of mutated p53 in the established cell line, which carries wild-type p53, also induced cell growth and tumorigenesis, indicating that mutant p53 can inactivate the tumor suppressor function of the wild-type p53 and exerts its dominant negative activity.

Dominant negative mutation involves inhibition of the function of a wild-type gene product by a mutant allele of the same gene. Two models have been proposed which could explain the inhibitory effect. First, the mutant protein could compete with the wild-type for the target molecule and thus inhibit the wild-type function. As an alternative, the mutant protein could form protein complexes with the wildtype to produce an inactive oligomeric complex.202 That wild-type and mutant p53 proteins differ in their binding affinity for several target proteins62·96•137·203 does not favor the first model. The second model is more applicable, because p53 is known to function as a multimer complex.204·205 There may be a common mechanism underlying the inactivation of wild-type function by oligomerization with the mutant protein, but mutant proteins differ in their transforming capabilities that can not always be explained by their ability to bind to the wild-type p53. In mice, the p53cys270 mutation has a low transforming ability, although it can bind to the wild-type protein efficiently. 144

Mutant proteins adopt a characteristic structural conformation which lacks the tumor suppressor function. A new mechanism of dominant negative function of mutant p53 has, therefore, been proposed whereby activated mutant p53 can influence the conformation of cotranslated wild-type p53 and drive the latter into the mutant phenotypic form. The wild-type conformation of the p53 protein is directly associated with ability to suppress cell proliferation. 141 Thus, by turning the wildtype p53 conformation into a mutant conformation, mutant p53 can inactivate the tumor suppressor function; such an effect may contribute a major dominant negative effect to tumor progression. A number of mutations with dominant negative activity have been reported, including murine p53vall35, a temperature-sensitive mutant, and human p53ser151, p53ile247, p53pro273 and p53leu273. A great majority of the temperature-sensitive p53 protein at 32°C exists in wild-type form and reacts with PAb246 but not with PAb240, whereas at 39°C most of the p53 protein adopts mutant conformation and reacts with


the PAb240 mutant-specific antibody. At 37°C, however, the p53 protein existed in both wild-type and mutant forms in the transformed cell and complexed with hsp70 in the cytoplasm206 during the S phase.

The wild-type p53 protein migrates into the nucleus at the beginning of the S phase. It appears to be sequestered in the cytoplasm during G 1, when it could act in the nucleus to block entry into the S phase. As such, mutant p53 acts dominantly to sequester the wildtype p53 protein in a place where it can not function. 15L206

Five different mutant alleles of human p53, p53ser151, p53ile247, p53trp248, p53pro273, and p53leu273, have been tested for their effect on cotranslated wild-type human p53. Four of them transformed the wild-type protein into the mutant phenotype; however, p53trp248 failed to drive cotranslated wild-type p53 into the mutant conformation. When translated alone, however, p53trp248 adopted mutant conformation, showing reactivity with PAb240 antibody. In this situation, both wild-type and mutant proteins coexisted in almost equal proportion as two physically distinct populations of p53 proteins.

The apparent inability of p53trp248 to drive the wild-type p53 to mutant conformation is of some interest, since this is one of the major hot spots of mutation detected in the germ line of patients with the Li-Fraumeni syndrome. This mutation appears to be weak in its ability to exert its dominant negative effect in that individuals with Li-Fraumeni syndrome carry a mutation in one allele of p53 while the other is wild-type, suggesting that this mutation might be recessive to the wild-type. 141 Germ line p53 mutations in fibroblasts derived from both affected and nonsymptomatic individuals, have been described.207•208

Individuals with the Li-F raumeni syndrome can develop a variety of soft-tissue cancers and often develop breast cancer at an early age, suggesting that wild-type p53 can function as a suppressor of cell growth in human breast cancer cells. To test this hypothesis, both wild-type and mutant p53 eDNA was introduced into the MDA-MB 468, and T47D breast cancer cell lines, which carry p53his273 and p53phe194 mutations. Following transfection, the continued expression of the exogenous wild-type p53 was incompatible with the growth of both cell lines. The data showed that the wild-type p53 gene functioned as a suppressor of cell growth in these cell lines. 209 The human mutant p53his273 gene used for cotransfection assay in a human osteosarcoma cell line also appeared to be recessive to the wild-type.210 An in vitro study has shown that wild-type p53 introduced into prostate cancer cell lines containing mutant p53 inhibited their growth. 105 Of the two cell lines used, one had a codon 126 mutation (TAC to TAG) and the other a codon 138 mutation (deleted a C, frame shift); both mutations appeared to be recessive to wild-type p53.

H358 and H23 are two lung cancer cell lines which carry a homozygous p53 deletion and a p53ile246 missense mutation, respectively. Introduction of a wild-type p53 eDNA to these cell lines greatly sup-


pressed tumor cell growth. 211 Similar studies have shown that in two colorectal cancer cell lines, introduction of wild-type p53 suppressed growth. These two cell lines, SW837 and SW480, both contain mutated p53. Cell line SW837 contains an arginine to tryptophan mutation in codon 248, whereas SW480 contains two mutations, arginine to histidine at codon 273 and proline to serine at codon 309. The substitutions at codons 248 and 273 are similar to those commonly found in sporadic tumors and occur within two of the four mutational hot spots. Cells transfected with wild-type p53 had their growth suppressed and formed fewer colonies than those transfected with mutated p53, suggesting that under these conditions these mutants are recessive to wild-type p53. Theoretically, the recessive p53 mutants would require the loss of wild-type function or allelic deletion in order to

express their phenotypic characteristics. Loss of heterozygosity of chromosome 17p or allelic deletion has

been observed in close association with p53 mutation in human lung,93·212 colon,93·99·156 breast93·159·175 and bladder carcinomas, 104 and brain tumors. 93 In colon cancer, both of these alterations are observed frequently, suggesting their involvement in tumor cell progression. A large number of studies from human cancer have documented a variety of p53 mutations. Results from all of these studies indicated that p53 mutations can be divided into two major groups. One group was recessive to wild-type p53 according to in vitro analysis by DNA-mediated gene transfer or by cotranslation studies. The other group of mutations is dominant to wild-type p53 activity. Mutations such as p53his273, p53his+ser309, p53phe194 and p53trp248 display recessive behavior,5L14L209·210 whereas mutations such as p53serl 51, p53pro273, and p53leu273, p53ile247 appear to be dominant to wild-type p53. 141 In these studies, all tumors containing the dominant p53 mutations retained both alleles, ll3,tl 4·121 whereas most of the tumors bearing the recessive type of p53 mutations showed loss of heterozygosity for chromosome 17p.93,9B,99,t03,tt4,t27,t34,2t4

Although it has been suggested that the p53 mutations occur prior to the allelic loss, there are no direct experimental data supporting this hypothesis. The p53 spectrum of mutations varies widely among human cancers. More studies are needed to assess the role of gain of function induced by mutations in interfering with the function of wild-type p53. In some cell lines in which the p53 gene has been deleted, transfection with mutant p53 changed the cell morphology and growth rate.210

The expression of different p53 mutants in different tissue and cell types may be dependent upon or influenced by the tissue origin of the tumor.

P53 MUTATIONS IN HUMAN CANCER Exogenous and endogenous mutagens are known to produce base

substitutions in some preferred sites of the p53 gene. Moreover, the patterns of mutation generated by different carcinogenic agents vary


Fig. 3.1. Distribution of p53 mutations in human tumors. These mutations were observed in cell lines or tumors from a variety of different tissue origin (brain, lung, breast, esophagus, stomach, colon, liver, bladder, ovary, bone, soft tissues, blood .. .). Conserved regions through evolution are indicated as "box". Vertical bars above the horizontal line represent the number of single base substitution identified at each codon. Vertical bars below the line represent the number of deletion mutations identified at each codon. Prominent "hot spots" of mutations are indicated at codons 175, 21 3, 242, 248, 249, 2 73 and 282. Nine mutations that have been found in introns are not shown. Data compiled from references 93,99, 101,104, 105, 110, 735, 156, 162, 175,2 72,219,229-237. Most authors have examined the mutations in exons 5 through 8 or 9. In references 93, 102, 106, 110, 132, 134, 156,201,2 12 complete p53 sequences were examined. (From Osamutominage eta/, Critical Reviews in Oncogenesis 1992; 3:257,282. Reprinted by permission of CRC Press, Boca Raton, Florida.)


according to the chemical nature of the compound. Different p53 mutations described in different types of cancer may reflect these predisposing factors. The location of the mutation in the p53 gene is critical, since certain regions of the p53 protein interacts with other factors important for its biological activity. The pattern of p53 mutation for a certain type of cancer may provide useful evidence of a certain putative carcinogen. Some of the mutations may cause the cells to acquire gain of function and may be specifically selected by a particular type of cancer.215 From the epidemiological point of view, this information is very important.

The p53 gene mutations in human cancer are spread over a large area of the open reading frame, but their locations are not random. In a survey of p53 missense mutations from 191 human cancers, all were localized between amino acid residues 120 and 290, a region which encompasses the highly evolutionary conserved exons 5 through 8.44 A comprehensive review of mutations in human cancers analyzed the mutational profile in 280 cases of base substitutions.27 Ninety-eight percent of all these mutations fell within 600 bp of the p53 eDNA, spanning codons 110 through 307 of the p53 gene. A great majority of the p53 mutations in human tumors are somatically acquired (>98%) and few germline mutations have been described in the Li-Fraumeni syndrome.

In sarcomas, p53 gene abnormalities are frequently associated with gene rearrangement, deletion, or insertions, but point mutations are not common (Fig. 3.1). Missense mutations have been reported in carcinoma of lung,93,103,114,212,216,217 colon,93.98,99 breast,44,130 brain,93 bone,174,218

bladder, 104 esophagus, 27·118·219 stomach, 120 prostate, 105 ovary,220 liver108·109 anus,221 lymphoid system. 107·11 1.222 Although a large number of p53 mutations have been described, there are a few which occur at much higher frequencies than the others, including those at codons 175, 248, 249, 273 and 282. Mutations in these amino acid residues account for 40% of the total missense mutations documented in human primary cancers. The high frequency of each of these hot spot mutations occur with respect to the tissue origin of the cancer. For example, the codon 175 mutation is quite frequent in the colorectal carcinomas (about 32%) and has less penetrance in lymphoma or esophageal carcinomas, whereas incidence of codon 249 mutation is very common in liver cancers in patients from southern China or southern Africa. The p53 codon 249 mutation occurs in hepatocellular carcinomas with a high frequency (about 53% of the cases observed), in the areas with high incidences of hepatitis B virus and aflatoxin. 108-110 These data suggest the tissue-specific distribution of hot spot mutations in human cancers.

Various mutagens or carcinogens may have specific sites of action. For example, aflatoxin B1 could induce mutation in liver, whereas lung would be the preferred site for mutations induced by benzo(a)pyrene


from cigarette smoking. There is evidence of such tissue-specific mutagenesis. In lung cancers, mutations in the p53 gene are involved in both transversions and transitions. In non-small cell lung cancer, G:C to T:A transversions are the most frequent substitutions, and in each of these cases a guanine residue was located in the nontranscribed strand. A similar situation exists in breast cancer. Such a strand bias is not apparent in p53 transitions at guanine residues in lung and breast cancers. In lung cancers, p53 transversions are more frequent than transitions; and they involve at least 16 different codons, and G to T transversions usually result from the DNA adducts typically produced by carcinogens in cigarette smoke (e.g., benz{a)anthracene). By contrast, p53 mutations in colorectal cancer are almost always transitions (79%). Most of these mutations occur at the CpG dinucleotide27•93•98•99•156 and more than half are at codons 175, 248 or 273. In breast cancer, the situation is different. The p53 missense mutations are found in 50% of breast carcinomas with or without a second wild-type allele. 223 In those breast cancers that express one p53 allele, 60% express a mutated p53. In another study, 22% of 27 breast cancers showed a wild-type p53 sequence, with cytoplasmic staining showing inactive p53 without mutation. 147 Only four of the tumors had transitions of G:C to A:T at CpG sites (13%). G:T transversions are more common in breast cancer than in colon cancer. Several breast cancer cell lines were analyzed for p53 mutation, and a range of mutations was found. Codons 175, 248 and 273 were also hot spots in breast cancer, but these mutations were not as common as in other kinds of tumors.

Tissue-specific methylation of CpG residues in the p53 gene appears to be an important contributing factor to these mutations. It is known that methylated cytosine residues in a CpG dinucleotide have a much higher rate of mutation than nonmethylated cytosine. Changes from the CpG dinucleotide to TpG have been reported.

The nature of mutagenic substances and their exposure to different target organs may differ both quantitatively and qualitatively. The aflatoxin known as a potent liver carcinogen in Africa and China induces G to T transversions in mutagenesis experiments in vitro. 164,224•225

These data agree vety well with an analysis of p53 mutations in tumors obtained from those high risk areas; all had G to T mutations in codon 249. It is noteworthy that this mutation can be ascribed to aflatoxin, which can selectively change the DNA base sequence at codon 249. When normal liver samples from the United States, Thailand and Qidong (where aflatoxin B 1 exposures were negligible, low and high) were analyzed for p53 mutations, the frequency of AGG to AGT mutations at codon 249 paralleled the level of aflatoxin exposure. 226 It is difficult, however, to explain the incidence of hot spot mutations by this mechanism alone, since no correlation was found between the liver cancers of other countries and the presence of codon 249 mutations. The particular pathobiological effect of p53ser249 mutation in the


hepatocytes is not clear, but selection of this mutation may confer some gain of function. 215 This mutation is rare, however, in other types of cancers like lung, breast or esophagus. The possibility exists that this mutant protein may interact with hepatitis B viral protein during the process of carcinogenesis. 227

Many inactivating mutations present in sporadic tumors have been detected in individuals with Li-Fraumeni syndrome, that is, from families with a cancer history. Affected individuals often carry a missense or nonsense p53 mutation in association with a wild-type allele.207·208 LiFraumeni syndrome is associated with familial breast cancer, adrenal carcinomas and leukemia. Germ line mutations in the p53 gene have been demonstrated in codons 245, 248, 252 and 258 in these tumors/07·208 however, the biological properties of some of the mutant proteins in Li-Fraumeni patients are little different from those described in sporadic cancers.228

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============= CHAPTER 4 ==========

BIOPHYSICAL AND BIOCHEMICAL PROPERTIES

OF THE P53 PROTEIN

INTRODUCTION

The biochemical mechanism of p53 in the control of cell growth is not completely understood. Considerable evidence implicates regu

lation of gene transcription as a mechanism of p53 action in controlling cell growth. The protein does resemble a transcription factor 1-5 in that it has an acidic domain that can transactivate reporter genes6-12

and a basic carboxyl terminal domain that can bind nonspecifically to DNA13 (Fig. 4.1).

The p53 protein exists in solution primarily as an elongated tetramer that can bind specifically to two DNA binding motifs through an internal hinge region located between amino acids 115 and 295 of the protein. 14- 18 (Fig. 4.1) A binding motif within the ribosomal gene cluster19 and a consensus binding site20 confer p53-dependent responsiveness to promoters containing these binding motifs linked to heterologous reporter genes. 19-21 The three-dimensional structure of the core DNA-binding domain of p53 (amino acids 102 to 292) bound to DNA has been resolved. 22 Motifs common to other DNA-binding proteins, such as helix-turn-helix or helix-loop-helix or TFIIA zinc finger motifs, are not found in the p53 DNA-binding domain. The core DNAbinding domain, instead, consists of a beta sandwich that serves as a scaffold for two large loops and a loop-sheet-helix motif. A tetrahedrally-coordinated zinc atom holds together the two loops. The zinc loops and loop-sheet-helix motifs are the target for the majority of mutations identified in human tumors, supporting the hypothesis that DNA binding is crucial for the biological activity of p53.

The p53 protein can interact with other transcription factors at gene promoters to affect transcription as a repressor or activator. 23-27 It


Fig. 4. 1. Biochemical topology of the p53 protein. A transactivation domain has been mapped between amino-terminal amino acids 20-42 and the domain involved in nonspecific DNA-binding and oligomerization of the protein is positioned within the carboxy-terminal 100 amino acids (see text for references). The sequence-specific DNA binding activity is located within the conformational domain between amino acids 100 and 300. Missense mutations observed frequently in human tumors occur within the conformational domain. The site for binding of the adenovirus E 18 protein has been identified by amino acids 14-66 and two sites involved in complex formation with SV40 large Tantigen are located between 123-215 and 236-289. A site for binding of heat-shock proteins (hsp70) is located within the carboxy/-termina/1 00 amino acids. Phosphorylation sites for cdc2 (residue 315 of human and 312 of mouse) and casein II (residue 392 of human and 389 of mouse) kinases have been identified at the carboxy terminus. Phosphorylation sites for DNA-activated (serine 15 and 37 of human, and series 7 and 18 of mouse) and a casein /-like (serine 7 of mouse) kinases are located at amino terminal residues. Two binding sites exist for replication protein (RPA) including both the 100 amino-terminal residues and the 100 carboxy-terminal residues. The TATA box-binding protein (TBP) in the holo-TFIID complex binds to amino acids 20 to 57 and the murine double minute (mdm2) protein binding site has been localized to the extreme 44 aminoterminal residues. Amino acids 88-109 and 273-217 comprise the epitopes for the conformational-sensitive antibodies PAb 1620/PAb246 and PAb240, respectively. The predominant nuclear localization signal (NLS) motif is present at amino acids 316-325 as well as two putative minor translocation sites situated further downstream.

represses promoters regulating the interleukin-6,27•28 c-fos, c-jun,Z9 multidrug resistance,30 P-glycoprotein31 and bcl-232 genes, whereas it stimulates transcription of the mdm2 protooncogene,33•34 the gadd45 gene,35 the wafllcipl gene36·37 and the retinoblastoma tumor suppressor gene.38 In addition, the p53 protein can positively regulate its own promoter. 39

Promoters of genes that are repressed by p53 lack p53-binding sites. In those cases, p53 most likely interacts with other transcription factors to effect the transcription machinery (see below). The transcriptional activation and repression domains overlap at the N-terminus.40

The positively-regulated promoters of the mdm2, gadd45, wafl! cipl, and retinoblastoma genes each contain a p53-binding site. Transactivation of the mdm2 gene by p53 activates expression of the mdm2 protein, which binds to an amino terminal domain of p53 and inactivates its transactivation function, 4 l.42 thus providing for an

Structural and Biochemical Properties of the p53 Protein 57

autoregulatory feedback loop. The gadd45 gene may play a role in DNA repair, since it is induced by the activation and increased expression of p53 that is a response to DNA-damaging agents such as mitomycin C, 43 ultraviolet light44•45 and gamma irradiation.46 The wajl lcipl or p21 gene product exerts a growth suppressor activity on tumor cells by inhibiting cyclin-dependent kinase pathways, in particular the cyclin-dependent kinase 4, thereby blocking cell division. 36.47

The ability of p53 to stimulate expression of the retinoblastoma gene38

suggests the role of both tumor-suppressor genes to act synergistically in suppression of growth.

The correlation between transcription activation/DNA-binding and growth suppression is not absolute as evident from the biochemical and biological activities of two p53 mutants. The temperature-sensitive p53ala143 mutant exhibited a higher transcriptional activation than the wild-type protein at the permissive temperature, but was less effective than the wild-type protein in inhibiting proliferation and rasinduced focus formation. 48 This phenomenon has also been observed for the his273 mutant which activated p53CON-mediated transcription but failed to inhibit cellular proliferation. 24.49 These observations suggest that p53 may regulate cell growth through mechanisms other than DNA binding and transcriptional activation. An alternative mechanism might be through the interaction of p53 with and sequestration of cellular proteins, preventing their normal inhibitory effect on cell proliferation.

Work in the SV40 DNA tumor virus system suggests that p53 has a role in control of DNA replication. SV40 replication is dependent on cellular replication factors and the virus-encoded large T antigen protein. 50 The initiation of SV40 DNA replication requires the physical interaction of DNA polymerase alpha/primase with T antigen in a preinitiation complex composed of topoisomerases and replication protein A. 51 DNA polymerase alpha can be displaced from SV40 T antigen by p53. 52 The p53 protein can also inhibit the helicase activity of large T antigen, 53-55 and bind to sequences adjacent to the SV 40 origin of replication. 5354·56 The biological effect of these p53 interactions is the inhibition of viral replication. A role for p53 in replication of cell DNA is suggested by the interaction of p53 with replication origins along with DNA polymerase alpha and with replication protein A. 57-59 Replication of human immunodeficiency virus type 1 is inhibited by wild-type p53 but enhanced by mutant p53.60 Involvement of p53 in both transcription and replication would not be a unique situation, since other transcription factors have been detected as components of DNA replication origins in eukaryotic cells. 61

Increased expression of p53 in cells exposed to DNA-damaging agents has generated a proposal that the protein might be involved in facilitating DNA repair44 .46·62 ·63 by acting as a cell-cycle check point determinant to block cell division at G 1• Cells deleted of wild-type p53 lack


G 1 cell-cycle control and have increased potential for gene amplification when challenged with the uridine biosynthesis inhibitor N-(phosphonacetyl)-L-aspartate. 64•65 Ultraviolet44•45 or gamma46·63 irradiation of cells rapidly increases the levels of wild-type p53, initiating a G 1 or G 2 block in the cell cycle. The increase in p53 induced by ultraviolet-B irradiation is the result of an increase in protein stability. 66 Cells expressing mutant p53 do not undergo radiation-induced G 1 arrest, but instead continue to proceed through S phase and undergo arrest at G 2•46 ·63

Consistent with a role for p53 in DNA replication/repair, p53 has recently been shown to bind ATP and exhibit an intrinsic ATP-stimulated DNA strand reassociation activity. 67

The physiological role of p53 is undetermined, since transgenic mice lacking p53 alleles apparently experience normal development. 68

These p53-deficient mice are predisposed to neoplasms, however, consistent with a role for p53 in tumor suppression. That patients with the Li-Fraumeni syndrome of familial cancer, who harbor inherited mutations in p53, are more likely to exhibit early onset of a variety of tumors69•72 is also consistent with this hypothesis. Although the p53 protein is dispensable for mouse development, it does play a role in cell proliferation, as discussed above. Furthermore, embryo fibroblasts from p53-deficient mice divide faster than normal embryo fibroblasts, achieve higher confluent densities and exhibit a higher fraction of division-competent cells under conditions of low cell density.73 These p53-deficient embryo fibroblasts also display a lower G0/G 1 population than normal cells and do not enter a senescent phase characteristic of p53 heterozygotic (+/-) and normal cells.

Other studies have shown that some cells require wild-type p53 in their commitment to apoptosis.74-76 The p53-dependent apoptotic response was found to be independent of the induction of RNA or protein synthesis, suggesting that p53 represses genes necessary for cell survival or is a component of the enzymatic machinery for apoptotic cleavage or repair of DNA.77 Observations of p53 associated with centrosomes and microtubules suggest that the protein might control cell division by interacting with components of these structures.78•79

A hypothesis has been proposed from the studies described above that p53 acts to regulate the cell cycle and DNA replication by transcriptional control of genes whose products are involved in aspects of G 1 control. A cell cycle check-point determinant at G 1 would allow DNA repair mechanisms to proceed with fidelity. Association with viral origins of DNA replication and the effect on replication activities of T antigen suggest a role for p53 in DNA repair/synthesis processes. A loss of p53's growth suppressor and DNA-repair functions results in unregulated cell division and lack of fidelity of DNA repair, which ultimately leads to mutations and aneuploidy, increasing the risk for neoplasia. Cells carrying normal p53 either are committed to G 1 arrest and DNA repair or undergo programmed cell death. Cells lacking functional


p53 do not undergo cell-cycle arrest and continue to divide, bypassing apoptosis;46 a result is accumulation of genetic damage. Since p53 is dispensable for control over normal development and cell differentiation,8 other factors apparently substitute for p53 in growth control and DNA repair during the course of development. However, the redundant mechanisms that replace p53 do not function as efficiently as p53, since fibroblasts from p53-deficient mice grow more rapidly than normal cells from p53-positive mice. The finding that the Wafl cipl gene can be induced by a p53-independent pathway80 underscores the potential for redundancies in the p53 biochemical pathway. The absence of p53 function may lead to accumulation of damaged DNA, increasing the incidence and progression of neoplasms. In the case of heterozygous Li-Fraumeni patients who harbor one mutant p53 allele,7° the biochemical activities of the wild-type protein are inhibited in a transdominant fashion by complexing of mutant proteins with wildtype proteins. 81

To better understand the biochemical events and mechanisms involved in the p53 pathway of cell control, it will be necessary to identify cellular genes regulated by p53 to determine how cell growth is controlled by the protein at G 1• As in all biochemical pathways, mechanisms must exist to promote or restrict the action of p53. It is conceivable that p53 controls gene expression in a tissue or cell type-specific manner. Different tissue and cell types have different requirements for gene expression events that control growth. As suggested by studies showing that p53 acts as a cell-cycle determinant, a cell-cycle-dependent mechanism might be in operation to restrict or promote p53 function at different stages of the cell cycle.

A comparative analysis of mutated and wild-type p53 proteins has provided insight into the normal regulation of the protein. This chapter will further discuss the effect of missense mutations on the biochemistry of p53.

MISSENSE MUTATIONS WITHIN A CONSERVED REGION OF P53 ALTER ITS BIOLOGICAL ACTIVITY

Almost all missense mutations identified in p53 in human cancers cluster within a domain of the protein located between amino acids 100 and 300 (of a total sequence of 393) (Fig. 4.1). 82 Within this domain, the mutations frequently are found in four evolutionally-conserved regions located between residues 117 and 142, 171 and 181, 234 and 258, and 270 and 286.4·82-85 A high degree of conservation of these regions among mammals, amphibians, birds and fish suggests that they are important to the function of p53.

Although the majority of mutations identified in human cancers cluster within the central conserved domain, others do occur in other regions of p53 and these may be more prevalent in human cancer than currently realized.86 A recent reinvestigation of 560 mutations, identified


only in those studies where the complete coding region of p53 was sequenced, 87% were in exons 5-8 and 8% and 4% were in exons 4 and 10, respectively. 86 In many of the latter, the mutation generated a stop codon or resulted in a frameshift and therefore could possibly be missed by immunohistochemical staining techniques. The number of mutations reported thus far is likely an underestimation of the frequency of inactivated p53 in human cancer.

Missense mutations within the conserved domain can exert dramatic effects on the biological properties of p53. In contrast to normal p53, mutants are unable to suppress the transformation of primary rodent embryo fibroblasts promoted by the adenovirus EIA and Harvey ras oncogenes.87•88 Certain mutations actually generate a p53 protein that can cooperate with the ras oncogene in transforming primary embryo fibroblasts,88"90 as well as enhance the transformed phenotype and growth of tumor cell lines that were devoid of endogenous p53 alleles before transfection.91 "94 Mutation within the conserved domain can somehow convert p53 from a tumor suppressor protein to one that can cooperate with oncogenes to promote cell growth and tumorigenicity. One proposed explanation for this anomaly is that missense mutation of the conserved domain allows for a "gain of function" by p53. Acquisition of growth-promoting activity might be mediated by the specific interaction of mutant p53 with a factor to influence gene regulation. For instance, even though mutant p53 is defective in binding DNA 19-21 •95 and in transactivation,6•7•9•10•19•21 it has been shown to stimulate the activity of some promoters, whereas wild-type p53 represses transcription of these same promoters.27·3°·3L93•96 Mutant p53 might be able to promote growth by an indirect protein-protein-mediated binding to promoters of one or more growth-regulatory genes.

A mutation does not always completely eliminate the tumor suppressor function of p53, as demonstrated by an experiment in which a non-small cell lung carcinoma cell line (H322j) harboring a homozygous p53 mutation at codon 248 was transfected with a p53 antisense expression vector. 97 H322j cells expressing antisense p53 expressed 90% less mutant p53 and proliferated at a faster rate in culture than the parental cells. In addition, H322j cells expressing p53 antisense were able to form tumors in athymic mice, while those expressing mutant p53 were not. Thus, this mutant p53 protein retains some growth-suppressive function. In another instance, a human p53 leu175 mutant murine p53 leu172 exhibited pseudo-wild-type properties,98•99 in contrast to the dominant negative p53his175 mutant. 100 The following two sections will further discuss the effect of mutations within the conserved domain, as well as the effects of protein binding and modifications on conformation, which determines the biological activity of p53.


THE CENTRAL CONSERVED REGION IS A CONFORMATIONAL DOMAIN OF P53

Several lines of evidence indicate that missense mutations within the conserved domain can alter the conformation of p53. First, in most cases the mutation generates a p53 protein that is more stable than its normal counterpart and exhibits an extended half-life (hours rather than minutes). 10 L102 Whereas normal p53 is expressed at levels not easily detected by immunoblotting and immunocytochemical techniques, mutants are usually overexpressed and easily detected. Second, members of the heat-shock protein family bind selectively to mutant p53 proteins. 103-106 Third, monoclonal antibodies can distinguish the distinct immunological configurations of normal and mutant p53. Generally, mutated murine p53 fails to react with the monoclonal antibody PAb246, which recognizes only normal murine p53.88•104 A similar situation exists for human p53 in that monoclonal antibody PAb1620 frequently reacts only with wild-type p53, 107•108 whereas mutant forms of the protein in many cases are specifically recognized by monoclonal antibody PAb240. 109 The epitopes for PAb246 and PAb1620 are topologically overlapping between amino acids 88 and 109 within the conserved domain, and both are sensitive to protein denaturation and conformational changes. 107•108•110 The PAb240 epitope is also located within the conformational domain at amino acids 213 to 217111 (Fig. 4.1). The conserved DNA-binding region between amino acids 100 and 300 can also be referred to as a "conformational" domain (Fig. 4.1) because of the observed effects of mutations in this area on the immunologic configuration of the protein. However, some mutations within the conformational domain allow p53 to retain its native structure. These mutations are in a class that effect those residues that directly contact the DNA and thus interfere with p53 DNA-binding activity without effecting overall structure. 112

THE CONFORMATION OF P53 DETERMINES ITS BIOLOGICAL ACTIVITY

The alterations in conformation generated by mutation and their subsequent effects on the biological activity of p53 might reflect a mechanism of control of the function of normal p53. Several studies have demonstrated that different conformations of normal p53 do exist under different conditions of cell growth. For instance, in murine lymphocytes the normal p53 protein was found to exist as two immunologically distinct species that were reciprocally expressed in quiescent and mitogenically stimulated cells. 113 The p53 protein restricted to quiescent cells was defined by expression of the epitopes for the monoclonal antibodies PAb248 and RA3.2C2, whereas the species expressed


in growth-stimulated cells was characterized by the epitopes for PAb421 and PAb122. Furthermore, addition of fresh medium to SV40-transformed cells (SV3T3) resulted in the loss of the PAb248 epitope and retention of the PAb421 epitope.u4 Wild-type p53 has been observed to undergo conversion from a PAb246-reactive (wild-type) form to a PAb246-negative (mutant) configuration in 3T3 cells within one hour of addition of fresh medium.ll4 Different conformational forms of p53 have been observed at different stages of keratinocyte differentiation. 11 5

Other examples indicate that the conformation of wild-type p53 is sensitive to growth conditions and the cell type. A conformational shift in p53 from a wild-type (PAb246-reactive) to a mutant-like conformation (PAb246-negative) was detected on stimulation of T lymphocytes with concanavalin A.ll 6 Expression of normal human p53 was elevated and reactive with the mutant-specific antibody PAb240 in growth-stimulated human lymphocytes and marrow blast cell populations.117·118 One report described 37 primary acute myeloid leukemias exhibiting elevated p53 reactive with PAb240, of which only three expressed mutant p53 genes. 117 In normal human breast epithelial cells, wild-type p53 exhibited a prolonged half-life and reactivity with PAb240. 119 Normal p53 can thus assume a mutant-like conformation in some cell types under certain conditions of growth and differentiation states. It has been proposed that the alteration of p53 conformation, rather than acquisition of point mutations, is the mechanism underlying the increased proliferation of myeloid cells in most acute myelogenous leukemia patients. 117

Milner and Watsonll4 proposed that one conformational form (wildtype) might allow p53 to carry out its growth-inhibitory function, whereas the other form (mutant-like) reflects the protein in a growth-promoting mode. The following observations do indicate that p53 has the potential to promote growth. Transfection of tumor cells devoid of p53 with mutant p53 enhances their growth and their tumorigenic phenotype.9L93·94 In addition, mutated p53 cooperates with the ras oncogene to promote cell growth and tumorigenicity90·120 and enhances the transformation ability of SV40 large T antigen. 121 Microinjection of a monoclonal antibody directed against the carboxyl terminus of p53 (PAb 122) into the nucleus of mouse fibroblasts released from G 1 arrest prevented progression into the S phase. 122·123 Moreover, reduction of p53 expression by antisense p53 inhibited growth of NIH/3T3 and methylcholanthrene-transformed mouse fibroblasts at the G1/S phase of the cell cycle. 124 Milner and Watson further postulated that missense mutations locking p53 into growth-promoting conformations are selected during tumor initiation or progression. Normal p53 may oscillate dynamically between growth-promoting and growth-suppressing conformational modes, which are determined by the state of cell growth. However, a recent investigation found no differences in p53 conformation between resting and stimulated lymphocytes in the absence or


presence of serum, respectively. 125 The inclusion of dithiothreitol in the cell lysis buffers used in this latter study might have compromised detection of mutant-like p53 molecules, because dithiothreitol has been found to convert and stabilize p53 to the wild-type conformation 126

(see below). Conformational differences might be responsible for the differences

in biological activity observed for different p53 mutants. For instance, the p53 mutated at residue 175 is about three times more efficient than that mutated at residue 273, and over five times more efficient than that mutated at amino acid 281, in cooperating with the ras oncogene in transforming primary embryo fibroblasts. 120•127•128 None of these p53 mutant alleles is able to suppress transformation; the one exception is the weakly transforming p53his273 mutant that exhibits partial tumor suppressor activity. 129 Differences in conformation among mutants of p53 are indicated by the variations in binding to the constitutively expressed member of the heat-shock protein family, hsc70. In correlation with transforming potential, the potent transforming mutants p53ala143, p53his175 and p53tyr275 bind to hsc70 (as determined in coimmunoprecipitation assays), whereas p53 proteins containing p53ser135 or p53his273 mutations, exhibiting intermediate or weak transforming capability, respectively, do not. 120•129

Different mutations in p53 can result in specific effects on DNAbinding and transcription activities of the protein. Mutations at codon 273 yielded a p53 that had the ability to bind p53 DNA binding sequences and transactivate. 49 In contrast, p53pro156, p53his175, p53leu223, p53gln248, p53trp248, and p53lys280 mutants had no transactivating activity. However, when various mutant and wild-type p53 were cotransfected with p53 responsive elements, transactivation was seen with wild-type p53 as well as with certain p53 mutants. 6•9.49•130•131

The differences in biological and biochemical activities among p53 mutants might result in differences in the biology of cancers expressing different p53 mutants.

Several studies have used monoclonal antibodies to demonstrate conformational differences that correlate with transforming potential among different p53 mutants. The strongly transforming p53ala143 and p53his175 mutants react with the mutant-specific p53 monoclonal antibody PAb240 but not with PAb1620, whereas the weakly transforming p53his273 mutant reacts with PAb 1620 but not with P Ab240. 129

A p53 protein with a mutation at either residue 132 or 247, which is reactive with the mutant-specific monoclonal antibody PAb240, exhibited a temperature-sensitive reactivity with the wild-type-specific monoclonal antibody PAb1620. 132 Conversely, ap53 protein mutated at codon 248 demonstrated temperature-independent reactivity with PAb 1620 and temperature-sensitive reactivity with PAb240. This is consistent with the apparent retention of some tumor suppressor function by codon 248 mutants as discussed previously. Other p53 mutants (p53tyr135,


p53serl51, p53pro159) reacted exclusively with PAb240 and displayed no differences in immunologic configuration at 30°C or 37°C. The type of amino acid change is important in some temperature-sensitive mutants, since a p53pro273 mutant showed no reactivity with PAb1620 at either 30°C or 37°C, while a mutant p53leu273 protein showed a temperature-dependent reactivity with PAb1620. In addition, a p53val135 mutant was reactive with PAb1620 at 30°C but not at 37°C,133 in contrast to the temperature-stable p53tyr135 mutant. 132 A p53leul75 mutant exhibited pseudo-wild-type properties as evidenced by induction of mdm-2 and repression of PCNA,99 in contrast to the p53his175 mutant, which acts as a dominant oncogene and is incapable of transactivation and DNA-binding. 16•98•100 It must be noted that, although no differences in immunologic configurations appear to exist between some mutant p53 forms, conformational differences may be present that are undetectable by the current available conformation-dependent monoclonal antibodies. Generation of other conformation-dependent monoclonal antibodies against the conformational domain will be very useful in studying the association between conformation and biological activity of mutant p53 proteins.

CONCLUSION The p53 protein is capable of transactivating promoters contain

ing a p53 responsive-element. The protein is also able to repress certain promoters that lack a p53-responsive element, suggesting the roles for other factors in controlling the regulation of gene expression by p53. An internal conserved "conformational" domain of p53 that specifically binds DNA is a target for mutations and binding of the SV 40 large T antigen, events that inactivate the p53 protein. Missense mutations produce allosteric effects that convert the protein to an inactiye form. Large T antigen probably acts sterically to interfere with p53 binding to DNA-responsive elements and other protein targets.

The p53 protein may normally oscillate between different conformational forms, either active or inactive for gene transactivation or repression. As discussed in chapter 5, the expression of these different conformations might be regulated in a cell cycle- and tissue-specific manner by protein interactions, redox mechanisms and phosphorylation.

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88. Hinds P, Finlay C, Levine AJ. Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation. J Viral 1989; 63 (2):739-746.

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90. Parada LF, Land H, Weinberg A, Wolf 0, Rotter V. Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation. Nature 1984; 312:649-651.

91. Wolf 0, Harris N, Rotter V. Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene. Cell 1984; 38:119-126.

92. Shaulsky G, Goldfinger N, Rotter V. Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins. Cancer Res 1991; 51:5232-5237.

93. Dittmer 0, Pati S, Zambetti G, et al. Gain of function mutations in p53. Nat Genet 1993; 4:42-45.

94. Ridgway PJ, Hale TK, Braithwaite AW. p53 confers a selective advantage on transfected HeLa cells. Oncogene 1993; 8:1069-1074.

95. Bargonetti J, Friedman PN, Kern SE, Vogelstein B, Prives C. Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell 1991; 65:1083-1091.

96. Deb S, Jackson CT, Subler MA, Morton OW. Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells. J Viral 1992; 66:6164-6170.

97. Mukhopadhyay T, Roth JA. A codon 248 p53 mutation retains tumor suppressor function as shown by enhancement of tumor growth by antisense p53. Cancer Res 1993; 53:4362-4366.

98. Raycraft L, Schnidt JR, Yoas K, Hao M, Lozano G. Analysis of p53 mutants for transcriptional activity. Mol Cell Bioi 1991; 11:6067-6074.

99. LiB, Greenberg N, Stephens LC, Meyn R, Medina 0, Rosen JM. Preferential overexpression of a 172leu mutant p53 in the mammary gland of transgenic mice results in altered lobuloalveolar development. Cell Growth & Differentation 1994; 5:711-721.

100. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991; 351:453-456.

101. Reich NC, Oren M, Levine AJ. Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53. Mol Cell Bioi 1983; 3:2143-2150.

102. Reich NC, Levine A. Growth regulation of a cellular tumor antigen, p53, in nontransformed cells. Nature 1984; 308:199-201.

103. Pinhasi-Kimhi 0, Michalovitz 0, Ben-Ze'ev A, Oren M. Specific interaction between the p53 cellular tumor antigen and major heat shock pro-


teins. Nature 1986; 320:182-184. 104. Sturzbecher H-W, Chumakow P, Wekh WJ, Jenkins JR. Mutant p53

proteins bind hsp 72/73 cellular heat shock-related proteins in SV40 transformed monkey cells. Oncogene 1987; 1:201-211.

105. Hinds PW, Finlay CA, Frey AB, Levine AJ. Immunological evidence for the association of p53 with a heat shock protein, hsc 70, in p53-plus-rastransformed cell lines. Mol Cell Bioi 1987; 7:2863-2869.


107. Milner J, Cook A, Sheldon M. A new anti-p53 monoclonal antibody, previously reported to be directed against the large T antigen of simian virus 40. Oncogene 1987; 1:453-455.

108. Cook A, Milner J. Evidence for allosteric variants of wild-type p53, a tumour suppressor protein. Br J Cancer 1990; 61:548-552.


110. Yewell JW, Gannon JV, Lane DP. Monoclonal antibody analysis of p53 expression in normal and transformed cells. EMBO J 1986; 59:444-452.

111. Stephen CW, Lane DP. Mutant conformation of p53: precise epitope mapping using a filamentous phage library. J Mol Bioi 1992; 225:577-583.

112. Cho YJ, Gorina S, Jeffrey PD, Pavletich NP. Crystal structure of a p53 tumor suppressor DNA complex: Understanding tumorigenic mutations. Science 1994; 265:346-355.

113. Milner J. Different forms of p53 detected by monoclonal antibodies in non-dividing and dividing lymphocytes. Nature 1984; 310:143-145.

114. Milner J, Watson JV. Addition of fresh medium induces cell cycle and conformation changes in p53, a tumour suppressor protein. Oncogene 1990; 5:1683-1690.

115. Spandau DF. Distinct conformations of p53 are observed at different stages of keratinocyte differentiation. Oncogene 1994; 9:1861-1868.

116. Wu J, Wang M, Li X, Sheng Y. Conformation changes of p53 proteins in regulation of murine T-lymphocyte proliferation. Cell Mol Bioi Res 1993; 39:27-31.

117. Zhang W, Hu G, Esley E, Hester J, Deisseroth A. Altered conformation of the p53 protein in myeloid leukemia cells and mitogen-stimulated normal blood cells. Oncogene 1992; 7:1645-1647.

118. Rivas CI, Wisniewski D, Strife A, et a!. Constitutive expression of p53 protein in enriched normal human marrow blast cell populations. Blood 1992; 79:1982-1986.

119. Delmolino L, Band H, Band V. Expression and stability of p53 protein in normal human mammary epithelial cells. Carcinogenesis 1993; 76:827-832.

120. Hinds PW, Finlay CA, Quartin RS, et a!. Mutant p53 DNA clones from


human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth & Differentation 1990; 1:571-580.

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====CHAPTERS====

REGULATION AND MoDULATION OF THE FUNCTION OF P53

INTRODUCTION

The p53 protein has a structure reminiscent of other factors involved in transcription. 1•5 As discussed previously, strong evidence that wild

type p53 functions as a regulator of transcription has come from biochemical and biological studies demonstrating that the protein can repress the interleukin-6 (IL-6) gene and stimulate the expression of the mdm2, gadd45, and waft lcipl growth-regulatory genes, whose promoters contain a p53-binding site. Proteins may bind to p53 and regulate its DNA-binding and transcription activities in a fashion similar to that observed for other transcription factors, such as those belonging to the TFII,3•6-13 the AP-1, 14-19 and the myc and max20•21 factor families.

PROTEIN INTERACTIONS CAN REGULATE P53 A premise for protein interactions controlling p53 DNA-binding

and transactivation functions was first provided by studies on the binding of SV 40 large T antigen to p53 and its effect on the biochemical activities of p53. T antigen complexes with p5Y2•23 by binding to two discontinuous regions between amino acids 123 and 215 and 236 and 289, both of which are located within the conformational domain. 24•25

The p53 protein becomes stabilized, is overexpressed in SV 40-transformed cells, and exhibits a reduced affinity for T antigen.23•25-27 T antigen apparently targets the wild-type growth-suppressor form of p53, since many mutant p53s are defective in binding to large T antigen.28-3°

Furthermore, p53 bound to T antigen is of the PAb246-positive, or wild-type, immunological configuration.31 Since the region of p53 that binds to T antigen is also the site for specific DNA binding,32-36 the functional consequence is the loss of p53's DNA-binding activity and thus its transactivation activities. 37-39 Consistent with the hypothesis that T antigen inhibits p53 is the finding that transformation by SV40 does not involve mutational inactivation of p53Y Large T antigen might


serve to inactivate p53 to induce DNA synthesis, thus allowing more efficient viral replication, with an increased potential for cell transformation.40 Although a likely mechanism forT antigen-mediated inhibition involves steric interference with DNA or cell protein targets, the possibility of a conformational change in p53 that is undetectable with the current antibodies cannot be excluded. That free, uncomplexed p53 in T antigen-transformed cells is more stable, but apparently normal by the criterion of PAb246 reactivity, suggests a change in the tertiary structure of the protein that renders it less susceptible to proteases. As alternatives, protease activity specific for p53 is reduced as a consequence of T antigen-mediated transformation or T antigen sequesters p53 from proteases (see Regulation of Intracellular p53 Levels).

Other DNA tumor virus-transforming proteins also target p53 for inactivation. The type 2 and 5 adenovirus E1B (55K) oncoproteins stabilize p53 and inactivate its transactivation function by binding to amino acids 14 to 66, 41 -43 possibly by steric interference of the transactivation domain with other proteins in a transcription initiation complex (Fig. 5.1). A strong direct correlation has been observed between the E1B (55K) protein's ability to inhibit p53 transactivation function and its ability to transform primary cells in cooperation with E1A. 44 Several amino terminal hydrophobic residues, critical for p53 transactivation activity (leu22 , trp23, pro27), are required for binding to adenovirus ElB (SSK) protein. 45 In contrast to T antigen and E1B, with their stabilization and steric interference mechanisms, papillomavirus E6 protein targets p53 for degradation via a ubiquitin-dependent protease pathway,46 resulting in a decreased half-life and very low levels of p53 in papillomavirus-immortalized keratinocytesY-49 The E6 protein requires association with a 1 00,000-Dalton protein (E6-AP) to complex with p53. 50 Binding of the E6/E6-AP complex to p53 results in ubiquitination of p53, selectively targeting it for proteolytic degradation. 51 That E6 targets p53 for inactivation is consistent with the observation that human papillomavirus-positive cervical cancers or cell lines transformed by human papillomavirus in general contain wildtype p53. 47 ·52.53 The nuclear antigen-S protein of Epstein-Barr virus has also recently been shown to form complexes with p53. 54 SV40 T antigen, adenovirus E1B, papillomavirus E6, and Epstein-Barr EBNA-5 proteins may have evolved to mimic or compete with one or more cellular proteins that normally regulate p53 function.

Evidence that a particular conformational form of p53 interacts selectively with a cellular target comes from studies demonstrating variations in affinity of mutant p53 proteins for hsc70. Generally, transforming mutant p53s, but not wild-type p53, bind one or more members of the heat-shock protein 70 family. 55-58 The constitutively expressed member of the heat-shock protein family, designated hsc70, has recently been shown to behave as a tumor suppressor by suppressing the

Regulation and Modulation of the Function of p53

Tumor Suppressor Form

(PAb 1620++)

1

Mutattons Of

Aberrant Express.on ol ~9 Proteins/Redox

Modit10rs1K11asesl Phosphatases

! X

Activation of Growth Suppressing Geoos and/or

Repression of Growth Promoting Genes

lnlermediale Form (PAb1620+1PAb240+)

75

.. X

t Muta1100s

or Abeuant E.Jcpression of Bind,ng Proloins/Rodox

Mod1liers.JKinasosl Phosphalasos GroW1h Promoling

Form (PAb240••l

1 Achvahon or Growth Promohng G&nes

anct'or Repression of Grow1h Suppress1ng Gonos

Fig 5. 1. Conformational regulation of human p53 function by protein binding, phospho· rylation, and redox modifications.

focus formation of primary rat embryo fibroblasts by mutant p53 plus ras, as well as by myc plus ras.59 As discussed earlier, a correlation between the transforming potential of various p53 mutants and their binding to hsc70 has been reported. Mutant p53val135 and p53phe 132 (PAb240-positive) stably bind hsp70 and exhibit a significantly greater activity in cooperation with the Harvey ras oncogene in transformation of rodent cells than the mutant p53cys270 (PAb240-negative), which binds hsc70 to a considerably lesser degree. 60•6 1

Other studies on the interactions of T antigen with mutant p53 proteins provide additional supporting evidence for distinct and unique conformations of p53 that exhibit specific protein-binding characteristics . It had been presumed that all mutations in p53 abolish T-antigen binding,62 until a recent reinvestigation found diversity in interaction of mutant p53s with T antigen.63 Eight of 13 point mutations in the conserved conformational domain abolished T-antigen binding, and a mutation at position 24 1 reduced the affinity of p53 forT antigen. A mutation at codon 249 retained T -antigen binding affinity similar to that of the wild-type protein. As discussed previously for temperature-sensitive p53 mutants, the type of amino acid substitution is important in determining a particular conformation and binding to T antigen. For instance, an arginine to tryptophan substitution at codon


248 abolished T-antigen binding, whereas a glutamine substitution did not. 63 As discussed in the previous chapter, normal p53 also exhibits different conformations under different conditions of cell growth, which are regulated by interactions with other proteins and modifying activities. For instance, different batches of reticulolysate yielded different immunologic configurations of p53 on in vitro translation. 64 Depending on the lysate batch, murine p53 could be translated in vitro into a form reactive (wild-type conformation) or nonreactive (mutant conformation) with monoclonal antibody PAb246. The PAb246-negative species could not bind T antigen and thus resembled a mutant-like phenotype. One explanation might be that differences in binding proteins and modifying factors existed among batches of reticulolysate, yielding variations in protein conformation.

One or more members of the heat-shock protein 70 family has been observed to play a role in the conversion of temperature-sensitive p53 from a mutant to a wild-type phenotype in vitro.65 It was suggested that p53 may stimulate the ATPase activity of one or more proteins necessary to promote the process of folding it into the wildtype configuration. Further, mutations lock p53 into a form unable to stimulate associated ATPase activity; this would be an explanation for the increased affinity of mutant proteins for hsc70. The inability of mutant p53 to activate the ATPase activity of hsc70 might prevent its dissociation from a complex with p53.

The interactions of wild-type and mutant p53 proteins with hsc70 and T antigen suggest that different mutations promote the expression of distinct conformations of p53 that associate differently with targets, each having a distinct effect on cell growth. A particular mutation might promote or lock p53 into a conformation that selectively sequesters growth-suppressing factors or expresses a higher affinity for a factor that directs it to a specific promoter of a growth-regulatory gene. A case can be made for indirect protein-mediated binding of p53 to certain promoters based on the ability of normal p53 to suppress promoters for the c-fos and c-jun genes that lack p53 DNA-binding sites.66•67

Even though defective in DNA binding, mutant p53 has been observed to enhance the activity of some promoters, suggesting that it may interact, as in the case of wild-type p53, with specific factors at promoters. Mutant p53 might then affect the activity of another transcription factor, resulting in activation of a growth-promoting gene such as c-fos or c-jun. Conceivably, both wild-type and mutant p53 proteins could have identical affinities for a factor in a transcription complex but have different effects on the activity of the factor and thus on the activity of a promoter (see below with regards to binding of p53 to TATA-binding protein, or TBP).

Indeed, a number of transcription factors have been identified that play roles in modulating the DNA-binding and transcription activities of p53. The murine double minute-2 (mdm2) gene was originally isolated

Regulation and Modulation of the Function of p53 77

after it was amplified in a spontaneously transformed BALB/c 3T3 cellline68 and was subsequently found to have tumorigenic potential.69 The product of the mouse and human mdm2 gene is a 90,000-Dalton nuclear phosphoprotein69·70 that can complex with p53 and inhibit its transactivation function.71-73 The mdm2 protein exhibits sequence similarities with other transcription and DNA-binding factors, including two putative metal-binding motifs and a highly acidic domain, suggesting the presence of a transactivation domain. 1·73 The mdm2 protein binds to the transactivation domain of p53, potentially causing steric interference of p53 with other proteins in a transcription-initiation complex and inhibiting p53's transactivation function72 (Fig. 5. 1). The amino terminal residues leu22, trp23 , leu14, and phe19 are involved both in transcription activation and binding to mdm2, suggesting that these residues interact directly with components of the transcription machinery complex.45 Mutation of these hydrophobic amino acids, while interfering with transactivation and mdm2 binding, had no effect on sequence-specific DNA-binding of p53. The p53-mdm2 complexes could not bind to a p53 DNA-binding motif/4 suggesting that a conformational change in the upstream sequence-specific DNA-binding domain had occurred or that mdm2 can also mask the conformational domain. That mdm2 has a role in inhibition of p53 in the pathogenesis of human cancer is suggested by the findings of amplification of the mdm2 gene and overexpression of the mdm2 protein in portions of bone and soft-tissue sarcomas72•75·76 and estrogen receptor-positive breast carcinomas.77 Furthermore, the mdm2 protein can overcome wild-type p53 suppression of growth of transformed cells.78

The TATA-binding protein is a component of the TFIID complex, which upon binding to the TATA sequence can initiate transcription.79 The TBP factor interacts physically with the p53 protein, suggesting that TBP is a target for p53 action at the transcriptioninitiation complex. 80-84 The ability of wild-type, but not mutant, p53 to bind to TBP has been suggested to be one mechanism that contributes to the repression by p53 of several promoters that lack p53-binding sites. 80 Another study found, however, that both wild-type and mutant p53 could bind to TBP.84 The discrepancy between these findings might have been due to the use in the binding experiments of p53 proteins from different sources. The former study isolated p53 from baculoviral and bacterial expression systems, whereas the latter purified p53 proteins from mammalian cells. Different expression systems may vary in their modifications of p53 and thus might yield variations in the conformation and the ability of mutant p53 to interact with target proteins, such as TBP. This is reflected by the finding that, although no interaction of baculovirus or bacterially expressed mutant p53 occurred with TBP in solution under nondenaturing conditions, the same mutant p53 immobilized and renatured on a membrane did bind TBP. 80


The CCAAT-binding factor (CBF) interacts and mediates wildtype p53 binding to the hsp70 gene promoter.85 The Sp1 transcription factor has also been reported to interact with p53. 86 That p53 interacts with transcription factors implicates it in interacting directly with the transcription machinery to modulate transcription at gene promoters that lack p53 DNA-binding sites. Such examples are the GC-rich box region adjacent to the SV40 origin of replication that specifically binds wild-type p53 87 and promoters of c-fos and c-jun that are repressed by p53. In contrast, promoters containing a p53 binding site such as that regulating the muscle creatine kinase (mck) gene are activated by p53. 88 Although mutant p53 has been shown to have a reduced affinity for binding to CBF and in some cases to TBF, it may have a more stable interaction with other factors, as alluded to above. As an alternative, both wild-type and mutant p53 may have the same affinity for a factor but exert different effects on its activity. This is one interpretation for the C/EBP-beta transcription factor, which positively regulates expression of the IL-6 gene, being inhibited by wild-type p53 but promoted by p53vall35 and p53phe132 mutants. 89 Furthermore, p53 proteins with mutations at co dons 175, 248, 273 or 281, which are often observed in human cancers, actually enhanced the transcriptional activity of human PCNA gene, whereas wild-type p53 repressed the activity of this promoter. 90 Such altered gene-regulation events could lead to a growth-promoting or "gain of function" activity for mutated p53.

Although no conformational changes induced by the binding of the mdm2, TBF or CBF proteins have been observed in p53, conformational changes may occur in p53 that are not detectable by the available conformation-sensitive antibodies. A mutual modulatory phenomenon between the Wilms' tumor suppressor gene (wtl) product and p53 hints of a conformational change in p53 upon binding of a transcription factor and further exemplifies and substantiates the role of interaction with transcription factors in regulating p53 activity at promoter complexes and in p53 affecting the activity of other transcription factors. The wtl transcription factor physically associates with p53 and exerts a cooperative effect on the ability of p53 to transactivate the muscle creatine kinase promoter. 91 Conversely, in the absence of p53, wtl is a potent activator of the early growth response gene 1, whereas on binding p53 it behaves as a transcriptional repressor. 91

Protein interactions at the extreme C-terminus can affect the conformation of the sequence-specific DNA-binding domain, which is located further upstream between amino acids 115 and 295.33·92 Wild-type p53 expressed in bacteria is presumably unmodified with respect to phosphorylation (see below) and only weakly binds DNA.93 DNA-binding activity could be unmasked by interaction with PAb421 (a monoclonal antibody specific for the C-terminal amino acids 370-378 of murine p53) or E. coli dnaK (a homologue of eukaryotic heat-shock proteins94),


indicating that protein interactions at the extreme C-terminus could modulate the conformation of the DNA-binding domain. 93 Some mutant p53 proteins, such as murine p53his270 or p53gln246, can be conformationally shifted by PAb421 for activation of cryptic DNAbinding activity. Three-dimensional nuclear magnetic resonance resolution of the structure of the C-terminal oligomerization domain (residues 319-360) revealed that this region may be brought in close proximity to the DNA-binding core of p53.95

Protein interactions within the conformational domain can also modulate the DNA-binding activity of wild-type p53. A bacteriallyexpressed hybrid protein containing the conformational domain of p53 (amino acids 115 to 295) fused to Staphylococcus aureus protein A exhibited specific binding to an oligonucleotide containing a p53-binding site. At least five proteins ranging in size from 35,000 to 90,000 Daltons bind to the conformational domain of p53.96 Hybrid proteins with mutant p53 sequences showed a reduced association with these proteins, possibly due to an alteration in the tertiaty structure of the conformational domain. Proteins of 42,000 and 35,000 M, that bind to the conformational domain of p53 promoted the DNA-binding activity of the conformational domain and of the full-length proteinY A potential role for the 42,000 and 35,000 M, proteins in regulating p53 DNA binding is suggested by the correlation between the affinities of mutant and wild-type conformational domains for these proteins and DNA binding. Furthermore, the expression of these p53-binding proteins is determined by the state of cell growth and varies among different non-small cell lung cancer cell types. 96 Two other proteins binding to the conformational domain of p53 appear to prevent its interaction with DNA.98 Proteins interacting with the DNAbinding, conformational domain of p53 could either regulate binding to specific DNA motifs different from those known to be regulated by p53, play a role in p53-mediated gene repression, or regulate an activity of p53 distinct from transcription and DNA-binding.

REGULATION OF P53 BY PHOSPHORYLATION A role for phosphorylation in regulation of p53 function is sug

gested by the presence of phosphorylation sites for the p34cdcl kinase and casein kinase II at amino acids 315 and 392, respectively, of the human p53 protein (amino acids 312 and 389 of the murine protein).99-102 The cdc2 103-106 and casein II107•108 kinases play important roles in regulation of cell proliferation.

Conflicting reports have emerged on the involvement of phosphorylation in the metabolic stabilization and transformation processes of p53. In one study, no significant qualitative differences in phosphorylation were found among mutant p53 in methA-transformed cells, wildtype p53 in primary BALB/c mouse embryo fibroblasts, or wild-type p53 in SV 40-transformed 3T3 cells. 109 In another study, a tempera-


ture-dependent conformational change and associated functional changes of a temperature-sensitive p53 mutant were not correlated with any discernible changes in phosphorylation, on a comparison of tryptic phosphopeptide maps. 110 However, a number of other groups have shown that phosphorylation is directly involved in regulation of the DNAbinding and antiproliferative activities of wild-type p53. The native, unmodified form of recombinant p53 expressed in bacterial systems exhibited a weak DNA-binding activity, whereas that phosphorylated by casein kinase II was activated for DNA binding. 93 Site-directed mutagenesis of a casein II kinase phosphorylation site in mouse p53 (ala389) abolished its antiproliferative capability, suggesting an important correlation between phosphorylation and the biological activity of the protein. 111 Phosphorylation modulates the conformation of the Cterminal 100 amino acids, which then propagate changes in the tertiary structure of the sequence-specific DNA-binding domain.33 The role of ser315 in the function of human p53 is unclear, since the ala31 5

p53 protein retains many of the characteristics of wild-type p53, including the ability to participate in heteroligomers, to bind to T antigen, and to localize in the nucleus and the inability to bind to heatshock proteins. 100 In addition, the corresponding murine p53ala312 mutant was still capable of inhibiting SV 40 DNA replication in vivo, like the wild-type protein. 102

Phosphorylation of sites close to the amino terminus99•112•11 3 seem to regulate the transcription activation function of p53. Phosphorylation sites have been identified at the amino terminus at serines 9, 15, 20, and 33 or 37 of primate p53114 and serines 7, 9, 18, and 37 in murine p53. 115 A casein I-like kinase from 3T3 cells phosphorylated predominantly serine 7, and to a lesser extent serines 5 and 9 of murine p53. 116 Serines 15 and 37 of human p53 and serines 7 and 18 of murine p53 have been shown to be phosphorylated by a DNA-activated protein kinase. 115•117 A potential role for phosphorylation of serine 15 in p53 function was suggested by the partial inability of a p53ala15 mutant to block cell-cycle progression at G 1 when compared with the wild-type protein, whereas mutation of serine 37 had no discernible effect. 118 In addition, phosphorylation at serine 15 was weaker in p53ile237 and p53ala143 mutants in human glioblastoma cells than in wild-type p53. 119 Furthermore, the tumor promoter okadaic acid has been shown to generate hyperphosphorylation of p53 in vitro in nuclei from rat regenerating liver and from rat fibroblasts 120 and in K-562 chronic myelogenous leukemia cells. 121 Okadaic acid inhibits protein phosphatases 1 and 2A; 122 2A dephosphorylates amino-terminal residues of p53.115· 123 Hyperphosphorylation of p53 by okadaic acid attenuated its transcriptional activation function without a discernible change in conformation. 121

In further support of a phosphorylation-regulation mechanism, changes in the phosphorylation state of wild-type p53 that resulted in


a change in immunological configuration were detected in an inducible system in which two different pools of p53 co-existed. 124 Induction of wild-type p53 resulted in G 1 arrest and in the expression of two populations: one consisting of complexes of wild-type and mutant p53 and the other composed of free wild-type protein. The free wildtype p53 protein was phosphorylated to a greater extent than mutant p53, had lost the PAb421 epitope, and could not associate with mutant p53. Phosphorylation thus may play a role in promoting different conformational forms of p53 that interact with distinct tissue-specific factors to regulate gene expression. In light of p53's many possible functional roles in growth regulation, phosphorylation may also regulate as yet unidentified activities of the protein in control of cell proliferation and differentiation. This may be evident in the observation that, although p53 was considerably more phosphorylated in actively growing breast cancer MCF-7 cells than in quiescent cells, the sequencespecific DNA-binding abilities of p53 proteins from growth-stimulated cells and of those from growth-arrested cells were much the same. 125

In another study, there was no correlation between the reduced DNAbinding properties of mutant p53 proteins and their phosphorylation levels. 126

REDOX MECHANISMS Still another possible mechanism for regulating p53 conformation

involves proteins that control the oxidation/reduction status of cysteine residues located in two putative zinc loops positioned within the conformational domain. 127•128 The presence of zinc ions is critical for stabilizing the wild-type conformation of p53, 127 possibly by interacting with the putative zinc loops within the conformational domain. The zinc chelator 1,1 0-phenanthroline converted wild-type p53 to a mutant-like conformation (PAb240-positive/PAb246-negative) that had an increased affinity for hsc70. EGTA and EDTA at much higher concentrations also promoted the mutant-like phenotype. Cysteinyl residues were implicated by the ability of chloromercuryphenylsulfonate, which can displace Zn(II) ions bound to cysteinyl residues in metalloenzymes, 129 to convert wild-type p53 to a mutant phenotype. 127

It is functionally significant that oxidation of cysteinyl residues disrupted wild-type conformation and inhibited DNA binding. 128 Furthermore, the putative zinc loops located within conserved regions 2, 3, 4 and 5 are also targeted by SV40 large T antigen, which can inhibit DNA binding of p53.

ALLOSTERIC REGULATION MODEL We propose a model for regulation of the biochemical functions

of human p53 that involves protein binding, phosphorylation and redox-mediated control over the conformation of the protein (Fig. 5.1). The model is based on the characteristics of mutant p53 proteins and


the conformational changes observed in wild-type p53 under different states of cell proliferation. As indicated by the differences among various p53 mutants in binding to hsc70 and T antigen, and in monoclonal antibody reactivities, a particular mutation will promote a specific conformation that will then determine the association of p53 with other proteins and, ultimately, its biological activity. A particular conformation exhibited by a mutation represents one of many different states of normal p53 whose manifestation depends on conditions of cell growth (Fig. 5.1). For instance, a number of mutant p53s that transform in cooperation with the ras oncogene exhibit reactivity with the mutantspecific PAb240 antibody, bind to hsc70, and are defective in binding to DNA. In contrast, a weakly transforming p53his273 protein lacks expression of the PAb240 epitope, association with hsc70, and is capable of binding p53 DNA motifs in most instancesY·130 In some instances, a p53his273 mutation was negative for DNA-binding or yielded an aberrantly-migrating DNA-protein complex in electrophoretic mobility shift assays. This may be a reflection of the host cell environment and cell type-specific ;53-binding proteins. The p53his273 protein might be trapped or locked into a state intermediate between growth-suppressing (wild-type) and growth-promoting (mutant) forms. The p53his175 protein that exhibits a gain of function is locked into the extreme growth- or transformation-promoting conformation (binds hsc70 tightly and expresses the PAb240 epitope) (Fig. 5.1). Further supporting the model, as discussed previously, are findings that normal murine p53 exhibits the mutant-specific conformation (PAb246-negative/PAb240-positive) and that normal human p53 exhibits the mutant-specific PAb240 epitope in certain cell types under specific growth conditions. The wild-type form of p53 has been shown to activate the human epidermal growth factor receptor promoter, 131 supporting the hypothesis that p53 under certain conditions and in certain cell types may contribute positively to cell proliferation. Furthermore, metabolic stabilization of p53 is strongly correlated with the transformation efficiency of SV40. 132

Generally, promoters that contain a p53 DNA-binding site are activated by p53, whereas those promoters for genes such as c-fos and c-jun that lack a binding site are suppressed. 133 The specific effects of p53 on promoters lacking a binding site implicate protein-mediated indirect mechanisms in promoter control. Other proteins potentially bind to p53 to allow it to interact with and suppress promoters lacking p53-binding sites. Certain mutations in the "conformational" domain would provide a growth advantage by locking p53 into a form that is unable to interact with a transcription factor, leading to a failure to suppress the promoter of a growth-promoting gene such as cfos or c-jun. As an alternative, mutant p53 could have a selective or increased affinity for a factor in a complex at the promoter of a growthregulatory gene. This possibility is suggested by the ability of mutant


p53 to increase the activity of the promoter function of the human PCNA90 and IL-689 genes, whereas wild-type protein repressed the promoter activity. The mechanism of activation by transforming mutants does not seem to be mediated by direct DNA binding. 131 They could interact selectively with one or more members of the general transcription factors to activate transcription. The potential for increased affinity of mutant p53 for a factor is suggested by the increased binding of hsc70 to some mutant p53s. Modulation of the factor by mutant p53 would lead either to activation of a growth-promoting gene or to suppression of a growth-inhibitory gene. A combination of these two mechanisms could exist whereby both wild-type and mutant p53 proteins have similar interactions with a transcription complex but have different modulatory effects resulting in different outcomes on the expression of a growth-regulatory gene, as in the examples of the PCNA and IL-6 genes. Only substoichiometric binding of a protein might be required to affect the conformation and activity of a large pool of p53, since mutant forms can drive the wild-type protein into a mutant conformation. 134 Mutant forms of p53 can inhibit in a transdominant manner the transactivation function of wild-type p5Jl34•135 through the oligomerization of mutant and wild-type proteins at the C-terminus. 136

Mini-p53 proteins containing only the oligomerization domain were able to inhibit wild-type p53 and to transform cells, indicating that oligomerization has importance in transdominance and transformation.

Another possible mechanism for p53 indirectly influencing gene expression involves squelching of specific factors. Wild-type and mutant p53 may selectively sequester factors required for the expression of growth-control genes. A scenario could be envisioned whereby wildtype p53 squelches the TBP factor, preventing it from binding to and activating a promoter of a growth-control gene. The effect would prevent or repress that promoter's activity; this is a possible explanation for the ability of wild-type p53 to repress promoters that lack p53-binding sites. If the same line of thinking is applied, a growth-promoting mechanism for mutant p53 might involve the selective squelching of a factor required for expression of a growth-suppressing gene.

Control over normal p53 thus involves regulation of its conformation by binding proteins, kinases/phosphatases and redox modifier enzymes. These modulators of p53 conformation could vary in a celland tissue-type fashion, as well as by the state of cell growth or differentiation. Distinct conformations of p53 have been observed at different stages of keratinocyte differentiation. 137 Each distinct conformation of p53 would have specific affinities for other factors, which target or promote interactions of p53 with initiation complexes at various gene promoters and replication origins. The proposed regulatory model also predicts that p53 plays both negative and positive roles in growth regulation and that forms of p53 exist that are intermediate between negative and positive growth conformations. Some mutations may al-


low retention of residual growth-suppressive function, as discussed earlier for a p53lys248 mutant, by inducing conformational states intermediate between tumor suppressor and growth-promoting forms. This also may be reflected in the temperature sensitivity of several mutants in their reactions with wild-type and mutant-specific antibodies as discussed earlier.

REGULATION OF INTRACELLULAR P53 LEVELS The p53 protein has been noted to exist as higher ordered struc

tures with itself3°·87 •134•138- 144 and with other proteins, including the heat-shock proteins55-57 and large T antigen. 22•23 Homo-oligomerization occurs through a carboxy-terminal basic alpha-helical region of p53. 14 1.142

The dominant negative phenomenon induced by mutant p53 occurs by oligomerization of this domain with wild-type proteins. 136 Formation of T antigen/p53 complexes appeared to be determined by the intracellular concentration of p53. 138 Formation of p53 heteroligomeric complexes with other cellular proteins functioning in DNA-binding and transactivation activities might also be dependent on the expression level of p53.

One level of control over p53 concentration could be through the expression of stabilizing proteins interacting with domains that regulate p53 conformation. Conformational changes would modulate susceptibility of p53 to degradation by a nonlysosomal ATP-dependent proteolytic pathway. 145•146 An unrelated example of the role of protein dosage in regulating a transcription factor complex is the myc transcription factor, whose intracellular concentrations are critical in determining its transcriptional activity. DNA binding by myc requires dimerization with the max protein through a carboxy-terminal basic helix-loop-helix-leucine zipper domain of myc. 147-149 Transcriptional activation is then mediated by the amino-terminal sequence of myc. 150

The active myc/max heteroligomers formed compete with the repressive max and the overly active myc homoligomers. An alteration in the level of p53 expression, as in the example of myc/max, might lead to repression or overactivation of gene expression. Monomeric p53, even though unable to bind the p53 consensus DNA site (p53CON), was capable of transactivation and growth-suppressor activities. 144 Only p53 dimers and tetramers were able to bind the p53CON sequence. Increases in the expression of p53 induced by DNA-damaging agents may promote formation of higher order homo- and hetero-oligomeric complexes that could interact with promoters containing p53CON. Other potential p53 binding sites, however, such as those in the ribosomal gene cluster and in the SV 40 origin of replication, need to be investigated for binding to monomeric p53. These data encourage speculation that dimerization and higher order oligomerization, possibly regulated by intracellular concentrations of p53, have roles in regulating the sitespecific binding of p53 to different promoters and/or DNA-replica-


tion complexes. An example of the importance of gene dosage on protein function has been shown by an inducible polyomavirus large Tantigen system. 151 The replication function of large T antigen in this system required high levels of expression of large T antigen. The function of p53 may also be dependent on its intracellular concentration. Increases in p53 protein expression induced by DNA-damaging agents152·154

also are associated with increases in sequence-specific DNA-binding activity. 155·156 These increases in p53 expression are, in part, the result of an increased stability of the protein that is generated by a labile factor, because increases in the stability of p53 required protein synthesis.156 It is not known whether changes in p53 proteases or stabilizing factors are responsible for the increases in expression of the protein induced by DNA-damaging agents.

Protein interactions, kinases/phosphatases, and redox modifiers regulate the conformation of p53, which can determine its susceptibility to proteases. The interaction of the conformational domain of p53 located between amino acids 100 and 300 with large T antigen or the domain between amino acids 14 and 66 with adenovirus E 1 B can, as mentioned previously, stabilize the p53 protein. No conformational changes in p53 bound to T antigen have been detected using monoclonal antibodies, but alterations in tertiary structure may be present that are not detected with the available monoclonal antibodies. Binding of heat-shock-related protein 70 to a region in the carboxyl terminus plays a role in the ATP-dependent conversion of a temperaturesensitive p53 protein from a stable mutant to a labile wild-type conformation.65 Binding ofT antigen or E1B-55K protein may also either sequester p53 from proteases or sterically interfere with protease activity. Stabilized p53 free from a complex with T antigen has been observed, however, in SV 40-transformed cells, 157·159 indicating that factors other than association with T antigen can stabilize the protein. Maintenance of SV40 transformation seems to require both a functional large T antigen and a metabolically stabiliz-ed p53. 159 The metabolic stabilization of p53 is strongly correlated with the transformation efficiency of SV 40. 132 T -antigen may induce a cellular state that results in metabolically stabilized p53 by activating stabilizing factors or downregulating or inhibiting a p53-specific protease. The first might explain the presence of stabilized p53 uncomplexed to T antigen, in that interaction of stabilizing factors within the conformational or T antigen-binding domain of p53 would sterically compete with or prevent complexing with T antigen.

Another possible mechanism of control of intracellular p53 levels involves recruitment of p53 to interact with proteases, as demonstrated for E6-mediated degradation of p53. Oncogenic strains of HPV types 16 and 18 encode E6 oncoproteins that can bind to p53 and promote its degradation. 48 ·52·160 The mechanism probably involves recruitment of proteins involved in the ubiquitin-dependent protease pathway by


oncogenic forms of E6.52 Complex formation between E6 proteins of oncogenic papillomaviruses and p53 requires a cellular factor, designated E6-AP (E6-associated protein), which has a native molecular mass of 100,000 Daltons.so

Mechanisms may exist in normal cells to stabilize wild-type p53; they may be dependent on the cell type and state of cell growth. For example, wild-type p53 was found to be stabilized and unreactive with the mutant-specific monoclonal antibody PAb240 in undifferentiated neuroblastoma cell lines. 161 In addition, a large proportion of primary acute myeloid leukemias exhibited elevated wild-type p53 expressing the PAb240 epitope. 162 Furthermore, the p53 protein was observed to exhibit a prolonged half-life (3 to 4 hours) in early passage normal human keratinocytes48 and in normal mammary epithelial cells. 163 Perhaps cellular factors induce p53 into multiple stable conformations that have distinct functions in regulating cell growth. The expression of stabilized p53 forms with distinct functions may be dependent on the type of cell and its state of differentiation, as well as on growth conditions.

REGULATION OF SUBCELLULAR LOCALIZATION OF P53

The subcellular localization of both wild-type and mutant p53 has been observed to be dependent on growth conditions and to differ among cell types and tissues. For instance, p53 was localized in the nuclear compartment of transformed cells but was distributed along the perimeter of the nucleus or primarily in the cytoplasm of nontransformed cells. 164 In addition, serum-stimulated normal cells were found to exhibit nuclear distribution of p53, whereas quiescent cells showed predominantly a perinuclear localization. 165 In contrast to these findings, serum stimulation of MCF-7 breast cancer cells resulted in the cytoplasmic translocation of p53 from the nucleus to the cytoplasm.125 In other cases, p53 migrated from the nucleus to the plasma membrane during mitosis166 and complexes of p53 and T antigen were detected at the plasma membrane in SV 40-transformed cells. 167-169 In growth-stimulated BALB/c 3T3 cells, p53 accumulated in the cytoplasm during the G 1 phase but then migrated to the cell nucleus at the beginning of the S phase. 170 Many other observations of differences in subcellular localization of p53 have been made in a diversity of cellular systems, ranging from cytoplasmic in cells transfected with mutant p53 to nuclear predominance in SV40-transformed cells. 164,t70,t7t Induction of growth arrest by a temperature-sensitive p53 protein was correlated with a shift to a predominantly nuclear localization, 172 suggesting that targets for p53-mediated growth suppression exist in the nucleus. Although p53 has been demonstrated to be localized mostly in the nucleus in a variety of human cancers, 173 both wild-type and mutant p53 have been retained in the cytoplasm in subsets of breast174·175


and lung tumors, 176 respectively. The heterogeneity of intracellular compartmentalizaton of mutant p53 suggests that the tumorigenic potential of p53 can be mediated through both cytoplasmic and nuclear targets. 177

At least one nuclear localization signal at the carboxy terminus of p53 plays a role in nuclear transport of the protein. 178•179 Nuclear localization of p53 has been reported to be necessary for p53 to exert control over cell division,66•172 although as noted above, p53 apparently can exert an effect over cell growth from the cytoplasmic compartment.

Cellular transport proteins do regulate the accumulation of p53 in the nucleus. A cytoplasmic mutant of p53 was able to migrate into the nucleus of cells that had been treated with protein synthesis inhibitors, indicating that a protein factor is required to anchor a cytoplasmic p53 mutant in the cytoplasm. 180 Nuclear localization signalnegative p53 migrated to the nucleus by complexing with SV 40 large T antigen, implicating other protein-binding domains in nuclear transport of p53. A temperature-sensitive mutant of p53 was found to be complexed with heat-shock protein 70 in the cytoplasm at 37°C, but at 32.5°C it converted to a wild-type phenotype and migrated into the nucleus, arresting cells at late G 1 or pre-S phase. 172 Strong evidence that the intracellular environment determines the compartmentalization of p53 is provided by observation of the diverse subcellular localization of p53 proteins, with mutations in the same codon, among different glioblastomas. 177 Since the nuclear localization signal is adjacent to a cdc2 kinase motif, phosphorylation could play a role in transport and cellular localization of p53; 179 differences in the expression of cdk kinases/phosphatases may result in cell-type variations in p53 compartmentalization. However, an ala315 human p53 protein localized to the nucleus as a wild-type protein. 100 Since transfections in this study were performed on COS cells, the ala315 mutant protein could have migrated to the nucleus in a complex with T antigen. Since conformational phenotype has also been reported as a determinant in subcellular compartmentalization, 124•181 kinases or redox-modifying enzymes might regulate p53 transport. Transport of p53 might proceed along microtubules, since complexes of T antigen and p53 associate with tubulin in vitro and with microtubules in both the cytoplasm and nucleus in vivo. 182

CONTROL OVER THE POTENTIAL ROLE OF P53 IN DNA REPLICATION

Several lines of evidence implicate p53 in some aspect of control of DNA replication or initiation of DNA synthesis, possibly regulation of the assembly or function of DNA replication complexes in the cell. Wild-type p53 promotes dissociation of DNA polymerase alpha from SV 40 large T antigen. 180 Human wild-type p53, but not


mutant forms, inhibits the DNA-unwinding activity of large T antigen, inhibiting SV40 DNA replication.28•30•183 Furthermore, p53 has been observed to co-localize with various proteins involved in DNA replication (DNA ligase 1, DNA polymerase alpha, PCNA and singlestranded binding proteins) at sites of viral replication in herpesinfected cells184 and to bind to sequences adjacent to the SV40 origin of replication. 87

The single-stranded DNA-binding protein complex RPA has been shown to bind to p53. 185•186 RPA is the first cellular factor recruited to the initiation complex, an essential first step in DNA replication involving unwinding of the DNA at the origin of replication. 187 Although compelling, the significance of the interaction of p53 with RP A is unclear, since both wild-type and mutant p53 could bind to the protein and inhibit its DNA-binding activity. 185•186 Two regions located at the N- and C-terminal sections of p53 were implicated in binding RP A. 186

Although both wild-type and mutant p53 bind to RPA, each may have a different effect on the activity of RPA in complexes with other factors in a replication complex.

Association of p53 with the nuclear matrix attachment region (MAR) DNA is compatible with the protein having a role in regulation of DNA replication. As in the case of RP A, both wild-type and mutant forms of p53 could associate with MAR DNA; 188 again, wild-type and mutant p53 could exert different effects on a replication origin, possibly inhibiting activity in the case of wild-type p53 and stimulating activity in the instance of mutant (growth-promoting) p53. The mutant that could bind MAR DNA in a manner similar to that of wildtype protein was defective in binding to the GGGCGG consensus sequence on SV 40 DNA.B? Thus, mutations do not affect DNA binding overall but selectively interfere with binding to particular DNA elements.

The nuclear matrix is involved in binding and transport of complex DNA or RNA molecules 189 and is associated with complex ribonucleoprotein particles. 190 The nuclear matrix is believed to allow for organization of DNA loops, which early in S phase are representative of DNA encoding cell- and tissue-specific genes. 191 The MAR DNA elements attach the base of the DNA loops to the nuclear matrix, where replication and transcription are initiated. 192•194 The association of p53 with MAR DNA elements may allow it to mediate control over both replication and transcription processes in response to growth conditions and differentiation states.

SV40 large T-antigen may mimic a cellular homologue that normally interacts with p53 at replication complexes. The result of competing with and blocking the binding of replication proteins to an initiation complex would be a block of cell growth at the G 1/pre-S phase of the cell cycle. Mutant p53 interacts weakly or not at all with T antigen and thus cannot compete as efficiently as wild-type p53 with DNA polymerase alpha; as mentioned earlier, cells expressing mutant


p53 are actually stimulated to grow, a result consistent with unregulated DNA replication. Some studies have suggested that p53 functions as an "SOS" protein by facilitating DNA repair in cells exposed to DNA-damaging agents. 152· 154•195 The p53 protein may be involved indirectly in promoting fidelity of DNA replication by regulating expression of genes that keep the cell cycle under G 1 control, thus allowing DNA-repair mechanisms to perform adequately. Consistent with the proposed model for transcriptional control, regulation of p53 in DNA replication might involve proteins interacting with "conformational" domains of p53 to modulate binding to a target at a replication initiation complex.

CELL CYCLE REGULATION OF P53 FUNCTION As cells progress from a resting state into the cell cycle, p53 pro

tein levels rise, peaking in late G 1 just prior to the S phase. 196 Modifying proteins might control p53 activity during progression through the cell cycle. A "feedback" mechanism regulating p53 would be necessary to allow cell division to progress from G 1 into the S phase. Levels of mdm2 protein rise at about the same time as levels of p53 in resting cells stimulated to progress from G0 to G 1.7° Because the mdm2 gene has a p53-responsive element in its first intron197 and the mdm2 protein binds to p53 to inactivate its function, 71 •72 the increases in mdm2 levels provide for a negative autoregulatory mechanism to downregulate p53 to allow cell-cycle progression into S phase. Although inactive for transactivation of promoters containing a p53-binding site, it remains to be determined whether mdm2/p53 complexes have altered specificity for promoters regulating cell-cycle control genes or exhibit other functions involved in cell-cycle progression.

Detection of alternative conformational forms of p53 in quiescent and growth-stimulated cells suggests the possibility of cell cycle-regulated allosteric control over p53 function. 18 U 98 Cyclic control over a growth-regulatory protein is well documented in the case of cell cycledependent expression of cyclins, which modulate the kinase activity of the cdc2 protein. 199 Since p53 is phosphorylated by the cdc2 kinase (at serine 312 of mouse and serine 315 of human) 100•179•200 and by the mitogen- and growth factor-stimulated casein kinase II (serine 389 in mouse, serine 392 in human),201 •202 its conformation and thus its interaction with target proteins might be influenced by phosphorylation under different states of cell growth. Phosphorylation differences resulting in conformational changes between wild-type and mutant p53 may exist at certain points in the cell cycle to influence association with other proteins or DNA. As a model for cell-cycle control of p53 function, protein conformation modulators and kinases or phosphatases would be activated to drive the protein into a growth-suppressive phenotype prior to or at the onset of the G 1 stage of cell division. On progression through late G 1 to pre-S, competing p53-modulating pro-


teins would convert the protein into a growth-promoting mode, allowing release of the cell-cycle block and progression into S phase. Mutations within the conformational domain would block interaction with the cellular modulators or lock p53 in a state normally promoted by a factor to drive the cell into S phase.

TISSUE- AND CELL TYPE-SPECIFIC REGULATION OF P53 ACTIVITY

Regulatory factors binding to p53 may be differentially expressed among different tissues and cell types. Variations in the amount of p53 expressing the PAb246 epitope have been observed among different cell lines and even clones of one cell type.58 This phenomenon may be the result of differences in expression of proteins interacting with the conformational domain of p53. These proteins may not only regulate and modulate p53 activity, but also direct and target its interaction with specific transcription-initiation complexes of growth-controlling genes. As discussed above, these regulatory factors may be other transcription factors. Transcription factors are differentially expressed in various types of tissues. For example, transcription factors tcf-1 and ets-1 are expressed only in lymphocytes,203•205 krfl is specific to keratinocytes,204 and hnf3, c/ebp, and lfb1 are restricted to the liver. In addition to tissue-specific transcription factors, cell-type effects have also been observed. In the case of the c-fos promoter, multiple regulatory sites have been identified that exert cell type-specific control of basal activity.2

Differences in expression of factors interacting with p53 may be responsible for cell-type variations in ability to support transactivation of a reporter gene by the protein.206 For instance, Gal4-wild-type p53 showed a three-fold higher transactivation of the CAT gene in nonsmall-cell lung cancer cell line H1299 than in H358 cells. The effect was not due to differences in endogenous p53, since both cell lines have deleted p53 genes. A cell-type variation was also observed in the ability of temperature-sensitive p53 mutants to transactivate at the temperature permissive for the wild-type phenotype, suggesting differences in the interaction of various p53 mutants with cellular factors. Celltype differences were observed for the ability of wild-type p53 to modulate the MCK promoter. Wild-type p53 activated the MCK promoter in CV1 cells but repressed it in HeLa cells. In contrast, wild-type p53 repressed the IL-6 promoter in both CV1 and HeLa cells. 207 In the same study, the temperature-sensitive p53val135 mutant exhibiting the wild-type phenotype at 32.5°C failed to activate the MCK promoter and activated rather than repressed the IL-6 promoter in CV1 cells. In HeLa cells, the p53val135 mutant activated both promoters. These results underscore the fact that although the p53val135 mutant exhibits a wild-type-like conformation at 32.5°C, it can exhibit an activity that is different from that of a wild-type protein in response to certain intracellular microenvironments.


The intracellular environment, rather than missense mutation, may be the prevailing determinant of the stability of normal and mutant p53 proteins. This might explain differences in expression of the same mutant p53 protein among different cell types. The importance of cellular environment was illustrated by an analysis of p53 protein levels in normal and tumor tissue of a patient with a constitutional p53cys273 mutant. 208 All tumors from this patient exhibited high levels of mutant p53 expression by immunohistochemical analysis. Only scattered staining was detected in nuclei of morphologically normal epidermal keratinocytes; however, normal lymphocytes exhibited no staining. In another instance, wild-type p53 was found to be stabilized (half-life of approximately 3 hours) and to express the PAb240 epitope in normal mammary epithelial cells, but it exhibited a half-life of only about 30 minutes in normal mammary fibroblasts. 163 Furthermore, the levels of expression of an exogenous temperature-sensitive murine mutant p53 protein in human mammary cancer cell lines was dependent on the expression level of the endogenous protein.209 The temperature-sensitive mouse p53 protein was uniformly expressed at high levels in T47D cells, which express high amounts of an endogenous mutant p53 protein. In contrast, the same temperature-sensitive mouse protein was expressed at low levels in MCF7 cells, which express endogenous wildtype protein. It was concluded that the mutant p53 protein is stable only in a cellular environment where the endogenous p53 gene product is stable. 209 Distinct conformations of p53 have been observed at different stages of keratinocyte differentiation. 137 Perhaps the state of cell growth and differentiation, which depend on the cell type, determines the expression of factors that modulate the conformation of p53 or the expression of p53-specific protease activity. Large T antigen can induce or activate a protein kinase that phosphorylates p53,210 which could result in metabolic stabilization of p53 observed in T antigentransformed cells in the absence of a complex of p53 with T antigen (see below). Because different cell types may, at different stages of differentiation and states of growth, express qualitative and quantitative differences in kinases, redox modifiers and binding proteins that influence the stability of p53, it is reasonable to assume that the stable forms of p53 perform distinct functions within each cell type or tissue. That mutant forms of p53 exert a tissue-specific effect on gene transcription is suggested by transgenic mice expressing both wild-type and mutant alleles of p53, which exhibit higher incidences of a spectrum of malignancies restricted to the lung, bone and lymphoid systems. 211

Conflicting reports on the involvement of the T antigen/p53 complex in the immortalization and transformation capabilities of SV 40 are probably the result of cell-type variations in the intracellular environment. Although in many cases complexing of p53 is closely linked with the transforming capability ofT antigen,31 •212•21 5 other reports have shown that transformation is independent of binding of T antigen to


p53. 157-159 Instead, T antigen-independent metabolic stabilization of p53 cooperated with T antigen to maintain the transformed phenotype. Complexing of p53 with T antigen has been observed in nontransformed SV40-infected cells,216 suggesting that a complex of p53 and T antigen is necessary but not sufficient for transformation. This is also suggested by the increased stability of p53 in these cells. In contrast to reports of the inhibition of p53 transactivation by T antigen,38·39 one study found that the DNA-binding and transcriptional-activation activities of endogenous p53 were not altered or disrupted in SV40-transformed COS-1 and adenovirus-transformed 293 cells.217 DNA!p53 complexes did not contain T antigen or E 1 B protein, indicating that free wild-type p53 capable of binding DNA and transactivation was present in these cells. One explanation for these variations in p53 activity observed between transiently transfected p53-negative cells and stably-transformed cells is that differences exist in the expression of modification factors among the cells lines that influence p53 conformation. Such factors may influence not only association of p53 with T antigen, but also the DNA-binding and transactivation functions of p53.

Interaction of p53 with cell-specific factors may explain why certain mutations are found more frequently in certain types of cancers. While mutation at position 273 occurs frequently in lung cancer, it has not been found in liver tumors. 218 Mutation at position 249 is found in one-half of liver cancers but rarely in lung or colon cancer.218·219 Mutation at amino acid 175 occurs with a higher frequency in colon carcinoma than in other cancer types and has not been observed in liver or lung carcinomas.220-223 One possible explanation for these mutational hotspots in certain tumors is exposure to etiologically-defined carcinogens, particularly in the cancers of the lung219 and liver. 224-226 However, another level of selection for the preponderance of mutational hot-spots must be involved, since the carcinogen benzo(a)pyrene targeted 87% of guanine residues on exons 5 to 8 in the conserved domain of p53, but only 12% of these residues were mutated in lung cancers. 219 In the case of the role of the mycotoxin aflatoxin B1 mutagenesis in hepatocellular carcinoma, the highest mutability rate was found in the G to T transversion in the third base of codon 249, resulting in the substitution of arginine by serine.227 The mutation frequency of other adjacent bases in codons 247, 248 and 250, however, was also found to be only one-half-to two-fold lower. These results indicate that the almost complete prevalence of the ser249 mutation in hepatocarcinoma is the result of selection for a particular advantage of tumor development over other mutations induced by aflatoxin B1 in codons 247, 248 and 250. 227 Perhaps certain mutations disrupt interaction with cell-specific factors. As an example of this model, a regulatory protein interacting in or around the region of amino acid 249 to restrict p53 in a growth-suppressive phenotype might be expressed in the liver but not in the colon. As another example, a regulatory


protein or target interacting in the vicinity of amino acid 175, or one unable to interact with this conformational form of p53, is expressed in the colon but not in liver or lung tissue.

Expression of differentiation-specific factors in a particular cell type may explain why, in addition to the hot-spot mutations, other mutations are selected in tumor development. 221 Cell type-specific factors may be expressed differentially or temporally during progression of cellular differentiation. The selection pressure for a p53 mutation may depend not only on the tissue or cell type, but also on the stage of cellular differentiation. Tissue- and cell-specific p53-binding proteins could direct p53 to different genes required for growth of a particular tissue. These specific proteins might be expressed differently under different growth conditions, allowing an additional level of control restricted to each type of tissue or cell. Mutations would perturb interaction with the cell- and differentiation-specific factors required for negative growth control by p53.

Differential binding of cellular proteins between mutant and wildtype p53 (i.e., as in the cases of large T antigen and hsp70) may result in differences in cellular compartmentalization that vary from cell to cell and depend on the conditions of cell growth. This is exemplified in the case of a temperature-sensitive p53 mutant that showed a temperature-dependent nuclear localization in rat embryo fibroblasts 171 •172

and a temperature-independent nuclear localization in T47D breast cancer cells. 228 Cell and tissue type-specific, as well as differentiationspecific, expression of p53-binding proteins, kinases/phosphatases, and redox modifiers thus regulate p53 conformation, which determines subcellular compartmentalization and antiproliferative or growth-promoting activity. In addition, cell type-specific and differentiation-specific variations in expression of p53 modifiers and binding proteins regulate the targeting of p53 for promoters of growth-control genes. The higher incidence of lung, bone, and lymphoid tumors in heterogenic p53 mice expressing both wild-type and mutant alleles of p53 suggests a tissue-specific selection of altered gene expression induced by complexing and inactivation of wild-type p53.

CONCLUSIONS Low levels of normal p53 are necessary to control cell growth so

that DNA replication can proceed with integrity. Although p53 is dispensable for cell growth and development, as exemplified by mice in which p53 is homozygously deleted, long-term p53 deficiency can lead to increased susceptibility to tumor development. The p53 protein does play a role in control of normal cell growth, as indicated by the intimate association of binding of the protein by DNA tumor virus proteins and transformation and by the demonstration in in vitro experimental systems of its involvement in both positive and negative regulation of cell division. In the case of p53-null transgenic mice, other factors


may be able to substitute for p53 but with less efficiency. Over a period of growth and development, this lower efficiency of gene expression by p53 substitution factors might lead to cumulative mutations in cell genomic DNA, increasing susceptibility to tumor development. In support for a redundancy in the p53-biochemical pathway is the finding of a ;53-independent induction pathway for the wafl!cipl gene.229

The normal molecular function of p53 has not yet been clearly defined; however, substantial evidence continues to accumulate implicating it in control of gene expression and efficacy of DNA replication. In situations of stress, p53 is expressed at high levels to halt cell division at Gl> allowing for DNA repair. The mechanism of DNA repair may involve transcriptional activation of genes involved in G 1

cell-growth arrest or suppression of the proteins involved in replication. A factor having effects on both transcription and DNA replication is not a unique phenomenon, since transcription factors have been noted not only to activate transcription but also to affect DNA replication.230 The wild-type p53 protein is induced to high levels by serum- or mitogen-induced growth stimulation, 196•23 1.232 suggesting that it also plays a role in growth promotion.

To understand the mechanism of action of p53 in controlling cell division, it will be necessary not only to identify the genes controlled by p53, but also to identify and characterize the putative proteins that regulate the biochemical functions of the protein. The "conformational" domain may have an important regulatory role in the transcriptional and DNA-binding activities of p53. Proteins binding to this region may mediate control over p53 function by altering its conformation. There may be large numbers of p53-binding proteins, and they may be differentially expressed in various tissues and cell types. Furthermore, expression of such proteins may be dependent on the state of cell growth. The model for regulation of normal p53 function, illustrated in Figure 5.2, proposes that modifying enzymes (kinases/phosphatases), redox enzymes, or other transcription factors interact with one or more "conformational" domains of p53 and influence its DNAbinding and transcription-activating activities by modulating conformation. Proteins interacting with p53 may control its level of expression in a posttranslational manner, thus regulating formation of oligomers active in DNA binding and transcription. Proteins interacting with conformational domains might direct p53 gene expression by altering the tertiary structure of p53 to restrict or promote components of the transcription-initiation complex.

It has been shown that mutant p53 can oligomerize with wildtype p53 142 and inhibit the ability of wild-type p53 to bind DNA and transactivate a reporter gene. 135 A mechanism for this inactivation of wild-type p53 that is consistent with the model is that mutant p53 assumes a conformation that cannot bind DNA and thus destabilizes the tetrameric complex. Cellular proteins may bind to p53 to have the

Regulation and Modulation of the Function of p53

Growth Suppressive Form

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Rele:n.e or Growth· Suppressive Factors

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Ctll Cycle • nd T+SSut ·SI)KIIoC EJ.PtOUIOI\ ol Conlormi!IOt\tll Mocl rhtrs ISt.abthZet"S. R.c:IOx Enzyme s. 1( 1~US. PhO$ph31UtS) 0

Growth Promoling Form

0 Release or Growth-Siimulatlng Factor a

Oligomerization ilnd Conversion of Wild· Type pSJ into

Mutant ConfOJmation

Stabilization of pSJ Hall-lil• ;md Overe•prusion

"F a e

A Tfanscr .p t ~<>n

~ .. 3'

5'

ActivaUon of GrOW1h Supptessor Gene Repreuion ol Growth Promoting Gen.

Activation ol Grow1h PrOf"''ttiing GeM Aeprenion of Growth Suppressor Gene

! Uncontrolled Cell Di.,ision

Nuclear Membrane ~C=o=n=• =M=ul=ol=;o=n=s=an=d=D=e=le=H=on=s===i ~

95

Fig. 5.2. Model for posttranslational regulation of p53 . Proteins and modifying enzymes regulate conformation of p53 to determine whether it exerts a negative or positive effect on cell growth. The expression of p53·conformation modulators is tissue·specific and is dependent on the state of cell growth and differentiation. Conversion to the growth· suppressor form might result in the "squelching" or sequestering of growth·stimulating factors andj or release of growth·suppressive factors. Expression of the growth-promoting form would result in release of growth·stimulating factors, conversion of wifd·type or growth·suppressor forms into mutant conformation, and elevation of p53 levels. Each conformational form regulates the activity of different transcription·initiation complexes at promoters of genes involved in growth control. Uncontrolled cell growth could result from mutation of p53 or aberrant expression of proteins that regulate conversion of the protein into a growth·suppressive configuration. Levels of nuclear·focalized p53 are regulated by nuclear transport proteins; defective expression of these proteins could result in the sequestering of p53 in the cytoplasm, thus eliminating p53 control of gene expression required for tumor suppression. However, since normal p53 has been observed at the plasma membrane of non transformed cells during mitosis, targets in cell signaling pathways involved in growth may exist at this intracellular location.


same effect on normal p53, which would provide for a feedback control mechanism. Such proteins may be expressed at substoichometric amounts to control p53 activity. Mutations in p53 would be selected to sequester the protein in a growth-promoting conformation that would be unable to activate a growth-suppressor gene or might constitutively activate expression of a gene product involved in stimulating growth. A selective growth advantage for mutant p53 has been observed in glioma cell lines where single cells possessing a p53 mutation expanded to overtake the cell culture.233

Amplification of mdm268 in sarcomas that express normal p5372

suggests that mechanisms other than deletion and mutation may exist to inactivate p53 function. In light of this possibility, defects in expression of regulatory proteins interacting with the "conformational" domains of p53 may be involved in those tumors expressing normal p53. A defect in proteins regulating p53 intracellular levels (stabilizers, proteases), nuclear localization (transport proteins), and interaction with gene promoters and replication origins (transcription factors) could result in a breakdown of growth control (Fig. 5.2). The potential for defects in transport proteins is suggested by the finding of a substantial amount of p53 in the cytoplasm of a portion of small-cell lung carcinomas176

and the cytoplasmic accumulation of p53 as an independent prognostic indicator for colorectal adenocarcinomas. 234 Aberrant control of the p53 pathway of cell regulation may be more predominant in human cancer than is now realized.

Abnormal DNA binding is a more consistent defect associated with in vivo-derived p53 mutants; 87•126 however, differences in interactions of mutant p53s with cellular factors could very well result in the variations in biological activities observed among different p53 mutants. 60•61 Although correlations between mutations and protein interactions are more complex than DNA binding for p53, the realization that certain p53 mutations with different transforming activities can be classified according to affinities for large T antigen and hsp70, and possibly transcription factors, suggests their potential prognostic importance for human cancer. Expression of mutant p53 has been correlated with more aggressive disease and a poorer prognosis in lung cancer patients.235 Different mutant p53 forms classified according to binding to cellular factors (as in the case of hsp70) may be associated with variations in tumor aggressiveness and responsiveness to chemotherapy. Further analysis of the biochemistry of p53 is clearly needed to investigate the potential of this prognostic tool in therapy of human cancers. Furthermore, identification and characterization of proteins that regulate the transcription and DNA replication control functions of p53 will enhance our knowledge of cell growth control mechanisms and provide for novel approaches for the treatment and control of cancer.

The focus of this thesis has been regulation of p53 by cellular factors; however, the other side of the issue is the potential for p53 to


affect or regulate the activity of other transcription factors. This possibility is exemplified by effects of p53 on the wtl factor and by the modulation of the ATPase, GTPase, DNA-binding, and helicase activities of SV40 large T antigen by wild-type p53.216•236 The p53/T antigen complex is defective in T antigen helicase and SV40 DNAreplication activity.

The conformational hypothesis (Milner, 1991) proposes that one configuration (PAb 1620-positive, PAb240-negative) of p53 allows it to carry out growth-suppressive activities, whereas another form (Pab 1620-negative, Pab240-positive) functions to promote cell proliferation. The model presented in this thesis further postulates that proteins interacting with and/or modulating phosphorylation sites on p53 regulate its conformation and thus its DNA-binding and transcription factor activities. Increased phosphorylation of C-terminal sites by casein kinase II and association with other factors would activate p53 for binding to its currently known consensus binding site. Inactivation of p53 binding to its consensus binding site would occur by phosphatase activity at the C-terminus or by protein binding, oxidation or mutation converting it to a growth-promoting form. The growth-promoting form of p53 could mediate, through protein interaction, the activation of growthstimulating genes and/or repression of growth-suppressing genes. The expression of particular forms of p53 would be dependent on the presence of p53-binding proteins and modifying enzymes, which could be differentially expressed in different cell and tissue types. Mutations observed in cancers are selected for their ability to lock or promote the growth-promoting form of p53. Some mutations might allow for leaky or intermediate conformations (PAb 1620+/PAb240+) as suggested by p53 mutants that are temperatu-resensitive for conformations and biological activity. An intermediate conformational state is exhibited by the p53his273 mutant species, which exhibits a pseudo-wild-type phenotype and partial tumor suppressor activity.

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132. Tiemann F, Deppert W. Stablization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences. J Virol 1994; 68:2869-2878.

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175. Moll UM, Riou G, Levine AJ. Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Nat! Acad Sci USA 1992; 89:7262-7266.

176. Iggo R, Gatter K, Bartek J, Lane 0, Harris AL. Increased expression of mutant forms of p53 oncogene in primary lung cancer. The Lancet 1990; 335:675-679.

177. Ali IU, Schweitzer JB, Ikejiri B, Saxena A, Robenson JL, Oldfield EH. Heterogeneity of subcellular localization of p53 protein in human glioblastomas. Cancer Res 1994; 54:1-5.

178. Shaulsky G, Goldfinger N, Ben-Ze'ev A, Rotter V. Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol Cell Bioi 1990; 10 (12):6565-6577.

179. Addison C, Jenkins JR, Sturzbecher HW. The p53 nuclear localization signal is structurally linked to a p34cdc2 kinase motif. Oncogene 1990; 5:423-426.

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180. Gannon JV, Lane DP. Interactions between SV 40 T antigen and DNA polymerase. New Bioi 1990; 2:84-92.

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182. Maxwell SA, Ames SK, Sawai ET, Decker GL, Cook RG, Butel JS. Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells. Cell Growth Differ 1991; 2:115-127.

183. Braithwaite AW, Sturzbevcher HW, Addison C, Palmer C, Rudge K, Jenkins JR. Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature 1987; 329:458-460.

184. Wilcock D, Lane DP. Localization of p53, retinoblastoma, and host replication proteins at sites of viral replication in herpes-infected cells. N ature 1991; 349:429.


186. Dutta A, Ruppert JM, Aster JC, Winchester E. Inhibition of DNA replication factor RPA by p53. Nature (London) 1993; 365:79-82.

187. Borowiec JA, Dean FB, Bullock PA, Hurwitz J. Binding and unwinding-How T antigen engages the SV40 origin of DNA replication. Cell 1991; 60:181-184.

188. Weibker SN, Muller BF, Homfeld A, Deppert W. Specific and complex interactions of murine p53 with DNA. Oncogene 1992; 7:1921-1932.

189. Getzenberg RH, Piente KJ, Ward WS, Coffey DS. Nuclear structure and the three-dimensional organization of DNA. J Cell Biochem 1991; 47:289-299.

190. Caron de Fromentel C, Viron A, Puvion E, May P. SV40 large T antigen and transformation-related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells. Exp Cell Res 1986; 164:35-48.

191. Herbomel P. From gene to chromosome: organizational levels defined by the interplay of transcription and replication in vetebrates. New Bioi 1990; 2:937-945.

192. Gasser SM, Laemmli UK. A glimpse at chromosomal order. Trends Genet 1987; 3:16-22.

193. Amati B, Gasser SM. Drosophila scaffold-attached regions bind nuclear scaffolds and can function as ACS elements in both budding and fission yeasts. Mol Cell Bioi 1990; 10:5442-5454.

194. Brun C, Dang Q, Miassod R. Studies of an 800-kilobase DNA stretch of the Drosophila X chromosome: comapping of a subclass of scaffold-attached regions with sequences able to replicate autonomously in Saccharomyces cerevisiae. Mol Cell Bioi 1990; 10:5453-5463.

195. Lane DP. p53: guardian of the genome. Nature 1992; 358:15-16. 196. Reich NC, Levine A. Growth regulation of a cellular tumor antigen, p53,

in nontransformed cells. Nature 1984; 308:199-201.


197. Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm 2 autoregulatory feedback loop. Genes & Dev 1993; 7:1126-1132.

198. Kastan MB, Radin AI, Kuerbitz SJ, et al. Levels of p53 protein increase with maturation in human hematopoietic cells. Cancer Res 1991; 51:4279.

199. Nurse P. Universal control mechanism regulating onset of M phase. Nature (London) 1990; 344:503-508.

200. Biscoff JR, Friedman PN, Marshak DR, Prives C, Beach D. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc Natl Acad Sci USA 1990; 87:4766-4770.

201. Meek OW, Simon S, Kikkawa U, Eckhart W. The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II. EMBO J 1990; 9:3253-3260.

202. Hermann CPE, Kraiss S, Montenarh M. Association of casein kinase II with immunopurified p53. Oncogene 1991; 6:877-844.

203. van de Weetering M, Oosterwegel M, Doojies D, Clevers H. Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J 1991; 10:123-132.

204. Mack DH, Laimins LA. A keratinocyte-specific transcription factor, KRF-1, interacts with API to activate expression of human papillomavirus 18 in squamous epithelial cells. Proc Natl Acad Sci USA 1991; 88:9102-9106.

205. Drewes T, Klein-Hitpass L, Ryffel GU. Liver-specific transcription factors of the HNF3-, C/EBP-, and LFB1-families interact with the A-activator binding site. Nucleic Acids Res 1991; 19:6383-6389.

206. Unger T, Nau M, Segal S, Minna JD. p53: A transdominant regulator of transcription whose function is ablated by mutations occurring in human cancer. EMBO J 1992; 11:1383-1390.

207. Sehgal PB, Margulies L. Cell-type- and promoter-dependent vs. phenotype of p53 vall35. Oncogene 1993; 8:3417-3419.

208. Eeles RA, Warren W, Knee G, et al. Constitutional mutation in exon 8 of the p53 gene in a patient with multiple primary tumors: molecular and immunohistochemical findings. Oncogene 1993; 8:1269-1276.

209. Vojtesek B, Lane DP. Regulation of p53 protein expression in human breast cancer cell lines. J Cell Science 1993; 105:607-612.

210. Schiedtmann KH, Haber A. Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53. J Virol 1990; 64:672-679.

211. Lavigueur A, Maltby V, Mock D, Rossant J, Pawson T, Bernstein A. High incidence of lung, bone and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 ocogene. Mol Cell Bioi 1989; 9:3982-3991.

212. Dobbelstein M, Arthur AK, Dehde S, van Zee K, Dickmanns A, Fanning E. lntracistronic complementation reveals a new function of SV40 T antigen that cooperates with RB and p53 binding to stimulate DNA synthesis in quiescent cells. Oncogene 1992; 7:837-847.

213. Zhu J, Rice PW, Gorsch L, Abate M, Cole CN. Transformation of a continuous rat embryo fibroblast cell line requires three separate domains


of simian virus 40 large T antigen. J Virol 1992; 66:2780-2791. 214. Efrat S, Baekkeskov S, Lane D, Hanahan D. Coordinate expression of

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215. Schmieg Fl, Simmons DT. p53 mutants with changes in conserved region II: three classes with differing antibody reactivity, SV 40 T antigen binding and ability to inhibit transformation. Oncogene 1993; 8:2043-2050.

216. Tack LC, Wright JH, Deb SP, Tegtmeyer P. The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen. J Viral 1989; 63:1310-1317.

217. Chumakov AM, Miller CW, Chen DL, Koeffler HP. Analysis of p53 transactivation through high-affinity binding sites. Oncogene 1993; 8:3005-3011.

218. Chiba I, Takahashi T, Nau MM, et al. Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene 1990; 5:1603-1610.

219. Puisieux A, Lim S, Groopman J, Ozturk M. Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res 1991; 51:6185-6189.

220. Nigro JM, Baker SJ, Preisinger AC, et al. Mutations in the p53 gene occur in diverse human tumor types. Nature 1989; 342:705-708.


222. Prosser J, Thompson AM, Cranston G, Evans HJ. Evidence that p53 behaves as a tumour suppressor gene in sporadic breast tumours. Oncogene 1990; 5 (10):1573-1580.

223. Gaidano G, Ballerini P, Gong JZ, et al. p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc Natl Acad Sci USA 1991; 88:5413-5417.

224. Bressac B, Kew M, Wands J, Ozturk M. Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature 1991; 350:429-431.

225. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC. Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 1991; 350:429.

226. Aguilar F, Harris CC, Sun T, Hollstein M, Cerutti P. Geographic variation of p53 mutational profile in nonmalignant human liver. Science 1994; 264:1317-1319.

227. Aguilar F, Aguilar F, Hussain SP, Cerutti P. Aflatoxin B1 induces the transversion of G to T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc Natl Acad Sci USA 1993; 90:8586-8590.

228. Vojtesek B, Lane DP. Regulation of p53 protein expression in human breast cancer cell lines. J Cell Sci 1993; 105:607-612.

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====CHAPTER6 ====

PoTENTIAL CLINICAL SIGNIFICANCE OF THE P53 TUMOR SUPPRESSOR GENE

IN CANCER PATIENTS

Mutations of p53 are common in a wide spectrum of tumors.' They occur commonly in both non-small cell lung cancer (NSCLC)

and small cell lung cancer cell lung cancer (SCLC), as well as breast, colon, head and neck, esophageal, hepatocellular and brain cancers. 2•3

The precise role of these mutations in oncogenesis and the mechanisms involved are subjects of active investigation. The type of mutation found in many cancers suggests the mutations are caused by the interaction of DNA with a specific carcinogen.4 For example, mutations in p53 positively correlate with lifetime cigarette consumption.5

Radon exposure, which increases lung cancer risk, is also associated with p53 mutations, although the types of mutations differ from those seen in tobacco-associated lung cancer.6 Insertion of a wild-type p53 into lung cancer and colon cancer cell lines with a mutant or deleted p53 can suppress their growth even though the cells have multiple genetic lesions.?-9 The p53 protein may be overexpressed in lung cancer cells, although this is not always associated with the presence of a mutant p53 gene. However, overexpression of the p53 protein has correlated positively with a poor prognosis for some cancers. 10•11 Deletions in 17p, p53 mutations, and 3p deletions have been detected in preneoplastic lesions of the lung such as severe dysplasia. 12 Mutations in p53 and the ras oncogene appear to be independent events. 13 Mutations in the p53 gene may have significance for several aspects of the clinical practice of oncology including cancer diagnosis, prognosis and staging, prevention and treatment, and these will be discussed in this chapter.


SECOND PRIMARY CANCERS Patients who have had a primary epithelial cancer of the upper

aerodigestive tract (head and neck, esophagus, or lungs) have a higherthan-average risk of developing a simultaneous or subsequent second primary cancer. It has been hypothesized that the epithelial surface of the upper aerodigestive tract shares a common carcinogen exposure and thus an increased risk of cancer development. Many researchers believe that epithelial cancers can arise independently as separate primary cancers following prolonged carcinogen exposure, an effect called field cancerization. Such field effects have also been proposed for breast and colorectal cancers. This hypothesis is testable in that it predicts that, if a common carcinogen is involved, second primary cancers would arise from similar but independent events.

A difference in p53 mutations between the primary cancer and second primary cancers provide evidence of independent origin for these multifocal tumors.

A molecular marker that can distinguish cancers of independent origin must have several characteristics. The marker should be associated with the development of the cancer. Alterations in the marker such as mutations should occur early in the development of the cancer. Finally, these mutations should be clonally preserved. The p53 gene fulfills these criteria for the three most common primary and second primary cancers of the aerodigestive tract: those of the head and neck, lung and esophagus. Many studies have shown that inactivation of p53 by mutation or deletion results in cell transformation. Mutations in the p53 gene occur early in the development of upper aerodigestive tract cancers and are detected frequently in fresh tumor specimens.6•14-16 They are found in premalignant lesions from aerodigestive tract cancers and have frequently been detected in early stage cancers of the head and neck, as well as of the lung and esophagus. The incidence of these mutations does not increase with advancing stage of disease, as would be expected if p53 mutations were associated with cancer progression. Moreover, these mutations show clonal fidelity for recurrent cancers and metastases. The tumors of 31 patients with primary cancers of the head and neck and associated second primary cancers were studied by single-strand conformation polymorphism analysis (SSCP) and DNA sequencing. 17 The overall frequencies of p53 mutations among primary tumors and second primary cancers were 42% (13/31) and 37% (13/35), respectively. Mutations were found in 19 of 52 head and neck squamous cell carcinomas (36%), in four of seven squamous cell carcinomas of the lung (57%), in two of five adenocarcinomas of the lung, and in one of two squamous cell carcinomas of the esophagus. Twelve samples had p53 mutations on exon 5, nine on exon 7, and 11 on exon 8. Six samples had mutations in more than one exon. Twenty-one of 31 patients (68%) had p53 mutations in one or more specimens. In all 21 cases the genetic lesions were discordant such

Potential Clinical Significance of the p53 Tumor Suppressor Gene in Cancer Patients 115

that the presence or location of the mutations were different from those of the second and third primary cancers. In the five patients with p53 mutations in both the initial primary cancer and second primary cancer, the mutations occurred in different regions of the p53 gene. In the other 16 patients, a p53 mutation was found in one primary tumor but not in the other. In 8 of these 16 cases, a mutation was found in the first primary but not the subsequent primary cancer, and in the other eight cases, a p53 mutation was not detected in the initial primary but was detected in subsequent primary cancers.

These discordances for p53 mutations suggest that these cancers arise as independent events. These observations provide the first indication of a molecular basis for field cancerization effects in cancers of the upper aerodigestive tract.

THE ROLE OF P53 IN PREMALIGNANCY A number of common cancers are preceded in their development

by histologically well-characterized premalignant lesions. Adenocarcinoma of the esophagus is one such cancer and is notable for its increasing incidence. 18 It is frequently associated with replacement of the squamous epithelial lining of the lower esophagus with columnar epithelium, a condition commonly called Barrett's esophagus. Patients with Barrett's esophagus have a 50-fold increase in adenocarcinoma incidence. Although Barrett's esophagus is common, occurring, for example, in 10% of patients with esophageal reflux, only a small percentage of individuals with the condition will develop cancer. Because markers such as dysplasia are not reliable for predicting development of malignancy, it would be helpful to identifY markers that predict which patients with Barrett's esophagus will develop esophageal cancer. If such prediction were possible, esophagectomy could be done before the development of invasive cancer in high-risk patients, and it would be curative. Once invasive esophageal cancer develops, fewer than 10% of patients are cured.

Specimens of Barrett's epithelium were analyzed for p53 mutations by Casson and coworkers. 14 Unlike the ras family of oncogenes in which mutations occur in two "hot spots," mutations of p53 occur in many sites throughout the open reading frame. Fortunately, most mutations are limited to a region highly conserved among species that spans exons 5 to 8. 1 Because it is impractical when handling large numbers of samples to directly sequence the polymerase chain reaction (PCR) products to identifY mutations, one can screen the conserved region of p53 for mutations by using single-strand conformation polymorphism (SSCP) analysis. The combination of PCR and SSCP is a powerful approach for qualitative analysis of the DNA. The method is based on the observation that the electrophoretic mobility of a DNA molecule through a neutral polyacrylamide gel can be altered by altering the size or shape of the DNA molecule. Under nondenaturating conditions, single-stranded


DNA has a folded structure that is determined by intramolecular interaction related to its base sequence. A mutated single-stranded DNA has a different folded structure than the wild-type sequence and has different mobility in a polyacrylamide gel. Since the introduction of this technique for detection of polymorphism in the human gene, it has become widely used. This property of the DNA molecule has been utilized to identify the mutations in a variety of genetic abnormalities.

Mutations in the p53 gene were identified in Barrett's epithelium associated with adenocarcinoma. The mutations were C:A ~ T:G transitions, and they resulted in amino acid substitutions in the p53 protein. In an ongoing prospective study, mutations were found in patients with Barrett's metaplasia and a coexisting adenocarcinoma, whereas they were not detected in specimens from Barrett's metaplasia with no evidence of cancer. Mutations in the p53 gene appear to be independent of the development of dysplasia. Barrett's epithelium is a multiclonal proliferative process. 19 Our data are consistent with this and suggest that in some cases the p53 mutation leads to a selective growth advantage, resulting in tumor formation. In other cases the presence of p53 mutations indicates genetic instability in the epithelium, with a nonmutant clone eventually forming the tumor. Thus, p53 mutations in Barrett's epithelium may be an independent marker for the risk of developing esophageal cancer.

The p53 protein is strongly expressed in premalignant lesions such as hyperplasia and dysplasia associated with head and neck and lung carcinogenesis. 20•21 The incidence of expression of this protein increased with increasing severity of the premalignant lesion. Mutations in p53 are present in dysplasia associated with invasive cancer.6 Thus, it is likely that p53 is altered prior to the development of tumors in many cases.

PROGNOSTIC STUDIES IN CANCER PATIENTS The value of p53 as a prognostic marker for cancers is an area of

active investigation. The results of studies have been somewhat conflicting. One study found that increasing levels of p53 protein detected by immunohistochemical analysis identified a subset of patients with Stage I or II lung adenocarcinoma or squamous carcinoma who had a worse prognosis than other patients with this stage of disease. 10 Another study showed that p53 mutations were an independent unfavorable prognostic factor in patients with non-small cell lung cancer. 11

Another study, however, found no relationship between p53 protein expression and direction of survival in patients with squamous carcinoma of the lung.22 In breast cancer patients no relationship was found between p53 expression and clinical stage. 23

GENE REPLACEMENT CLINICAL TRIALS The advances in our understanding of the molecular genetics of

cancer present an opportunity to develop prevention and treatment


strategies based on the reversal of specific genetic lesions. Clinical trials have recently begun to assess the efficacy of recombinant DNA constructs in the treatment of advanced cancer. Current trials are using these constructs to augment existing therapeutic strategies. These include immunotherapy, with the adoptive transfer of lymphocytes genetically altered to enhance the anti-tumor immune response, and transfer of the multiple drug resistance gene (MDR) to marrow stem cells for chemoprotection. The advances in our understanding of the molecular genetics of cancer present an opportunity to develop prevention and treatment strategies based on the reversal of specific genetic lesions in the cancer cell.

The gene families implicated in carcinogenesis include dominant oncogenes and tumor suppressor genes.24•25 Proto-oncogenes {normal homologues of oncogenes) participate in critical cell functions, including signal transduction and transcription. Only a single mutant allele is required to convert a proto-oncogene to an oncogene inducing malignant transformation. Primary modifications in the dominant oncogenes that confer gain of transforming function include point mutations, amplification, translocations and rearrangements.

A second more recently described gene family is the tumor suppressor genes. Tumor suppressor genes appear to require homozygous loss of function by mutation, deletion or a combination of these. Some tumor suppressor genes appear to play a role in the governance of proliferation by regulation of transcription. A dynamic interplay exists within the cell between dominant oncogenes, which promote cell proliferation, and tumor genes suppressor which constrain cell proliferation. It is possible that modification of the expression of dominant and tumor suppressor oncogenes may influence certain characteristics of cells that contribute to the malignant phenotype. Our increasing knowledge of the mechanisms involved in oncogenemediated transformation has lead to progress in developing therapeutic strategies that may alter or replace abnormal transformed genes in cancer cells.

Many different genetic abnormalities are found in cancer cell lines and fresh tumor samples. 26-29 This is evident at the chromosomal level where multiple chromosomal abnormalities have been identified. Moreover, increasing numbers of oncogenes and tumor suppressor genes have been identified. It was once thought that gene replacement cancer therapy would not be possible because of the difficulties associated with correcting multiple genetic abnormalities in one cell. However, several studies have shown that correction of a single genetic defect, such as eliminating expression of a dominant oncogene or adding a normal copy of a tumor suppressor gene to a cell with deleted or mutated copies, reduced or eliminated critical characteristics of the malignant phenotype such as tumorigenicity or anchorage-independent growth.7·30-32


RETROVIRAL VECTORS FOR GENE DELIVERY A major obstacle to direct correction of genetic lesions in cancer

cells is the difficulty of efficiently delivering genetic constructs to the cells. Retroviruses have been extensively studied as delivery vehicles in gene transfer protocols. 33 Retroviral vectors have been created that lack genes essential for replication. The replication-defective vectors are capable of infecting cells and integrating as a provirus which will then express recombinant genes.

Because gene constructs transduced by retroviruses are integrated preferentially in dividing cells, this technique gives proliferating cancer cells a selective advantage for expressing the gene construct. Retroviruses and cells modified by retroviral transduction have little acute toxicity, making multiple treatments with high-titer preparations feasible.

The p53 gene presents a logical choice for a gene replacement strategy for cancer. The p53 gene is 'the most commonly mutated gene yet identified in human cancers. 1 Missense mutations are common in this gene and in many cases will functionally impair the p53 gene product.34·35 The mechanism of p53 transformation may vary, depending on the type of p53 mutation. The p53 gene encodes a 373-amino-acid phosphoprotein that can form complexes with host proteins such as large-T antigen and ElB. The gene appears multifunctional, with major domains that can transactivate, bind proteins, bind sequence-specific DNA and oligomerize with p53. Abnormalities in of one or more of these functions could contribute to abrogation of the tumor suppressor function of p53. Fa~!ure of the mutant p53 to activate transcription of molecules essential for regulating the cell cycle and DNA repair or the untimely expression of molecules transcriptionally enhanced by the mutant p53 may make the cell more susceptible to genetic instability. Certain mutations also have a dominant transforming capability. The wild-type p53 gene may suppress genes that contribute to uncontrolled cell growth and proliferation or activate genes that suppress uncontrolled cell growth. Thus, the absence of or inactivation of wild-type p53 may contribute to transformation. Some studies indicate, however, that the mutant p53 must be present for full expression of the transforming potential of the gene.

To assess the role of the p53 gene in the development of human cancer, wild-type p53 eDNA in either sense or antisense orientation was introduced into human non-small cell lung cancer celllines.36 The cell line H226b has a wild-type p53 gene, whereas H322a has a codon 248 mutation. H226b cells transfected with the p53 sense gene construct grew more slowly than the parent cells. We were unable to recover any H322a sense-transfected clones. Transfection with antisense p53 also reduced colony formation, although some clones transfected with antisense p53 showed increased proliferation. Elevated levels of antisense p53 RNA in transfected cells reduced the levels of wild-type


and mutated p53 proteins. Although parental H322a and H226b cells formed tumors in nu/nu mice after a long latency period, their antisense transfectants, with reduced levels of p53 proteins, formed large tumors in 15 days. Functional inactivation of mutated and wild-type p53 by antisense RNA provides a direct experimental demonstration of p53 tumor suppressor function and suggests that at least some p53 mutations also have residual cell growth and tumor suppressor functions which may be dose dependent. Although, mutations in the p53 tumor suppressor gene are common in human lung cancers, the wildtype form of p53 is dominant over the mutant, and thus restoration of wild-type p53 function in lung cancer cells may suppress their growth as tumors. For lung cancer, therefore, introduction of a normal p53 gene into cancer cells would be preferable· to using an antisense construct to inactivate mutated p53.

A retroviral vector-mediated system was established to allow efficient transduction of the wild-type p53 gene into human lung cancer cell lines H358a (deleted p53) and H322a (mutant p53). 9 These LNSX/ p53 constructs incorporating p53 eDNA driven by a ~-actin promoter, mediated stable integration of p53. p53 mRNA and protein were detected in these cell lines six months after transduction by northern and western blot analyses. Restoration of the wild-type p53 gene suppressed growth in the two transduced cell lines but had no effect in another transduced tumor cell line, H460a, which has an endogenous wild-type p53 gene. A high transduction efficiency (90%) was obtained after five cycles of transduction in vitro. Mixing experiments showed that transduced cells could reduce the growth rate of nontransduced cells; this reduction may have been mediated by factors shed into the supernatant of the transduced cell cultures.

A critical issue with respect to the use of viral vectors to deliver genes to tumors is the ability of these vectors to penetrate three-dimensional tumor matrices. Multicellular tumor spheroids (MTS) represent a three-dimensional culture model in which the tissue approaches the degree of structural and functional differentiation of primary and metastatic tumors. Cells grow in a spherical configuration in suspension culture. The effects of retrovirus-mediated transduction of wildtype p53 (wt-p53) were studied on MTS of human non-small cell lung cancer cell lines H322a, which has a homozygous mutated p53 (mutp53) gene (codon 248), and WT226b which has endogenous wt-p53Y The growth of WT226b, spheroids was not affected by exogenous wtp53, but the growth of H322a spheroids was significantly inhibited by the addition of wt-p53 viral stocks, whereas the vector alone or the mut-p53 vector had no effect. Transduction of cells by the wt-p53 vector in H322a spheroids was demonstrated by using in situ polymerase chain reaction/hybridization with the neomycin-resistant gene neo probe. Apoptotic changes were observed in H322a spheroids treated with the wt-p53 virus. These results suggest that retroviral vectors can


penetrate three-dimensional tumor masses and induce potentially therapeutic effects.

We investigated the therapeutic efficacy of direct administration of a retroviral expression vector (LNp53B) in an orthotopic human lung cancer model. Irradiated (350 cGy) nulnu mice were intratracheally inoculated with 2 x 106 H226Br cells (codon 254 mutation) and treated beginning 3 days later with an intratracheal instillation of LNp53B retroviral supernatant for 3 days. Infection with LNp53 inhibited proliferation of H226Br cells in vitro. Thirty days after tumor cell inoculation, 63-80% of the control mice showed macroscopic tumors of the right mainstem bronchus. LNp53B suppressed H226Br tumor formation in 62-100% of mice, and the effect was dose dependent. These results suggest that direct administration of a retroviral vector expressing wt-p53 may inhibit local growth in vivo of human lung cancer cells with abnormal p53 expression. We conclude that development of gene replacement treatment strategies based on the type of mutations found in target cancers is warranted and may lead to the development of new adjunctive therapies and gene-specific prevention strategies for lung cancer.

Replication-defective retroviruses have some theoretical and practical limitations as gene delivery vehicles for tumors in vivo. Retroviruses may undergo rearrangement in packaging cell lines and become able to replicate. Although the integration of retroviral vectors into the host genome is advantageous for long-term gene expression, the site of integration is unpredictable and could theoretically cause activation of proto-oncogenes. Moreover, current production techniques for retroviral vectors are laborious and the resultant titers are limited. Adenoviral vectors, an alternative, can transduce both dividing and nondividing cells and may have tropism for lung epithelium. Adenoviral vectors can be produced in large amounts. Although their integration into the host genome is rare, transient expression is not necessarily a disadvantage when dealing with cancer.

ADENOVIRAL VECTORS FOR GENE DELIVERY We developed an adenoviral vector for delivery of wild-type p53.

The p53 expression cassette, which contains human cytomegalovirus promoter, wild-type p53 eDNA, and SV40 early polyadenylation signal, was inserted between the Xba I and Cla I sites of plasmid pXCJL.1 (a gift from Dr. F.L. Graham). The p53 shuttle vector (pEC53) and the recombinant plasmid p]M1738 were cotransfected into 293 cells39

by liposome-mediated transfection with DOTAP. A high level of expression of exogenous p53 was achieved in the H358 cells that were infected by Ad5CMV-p53 at a multiplicity of infection (MOI) of 30 plaque forming units (PFU)/cell. When H322 or H460 cells were infected at the same MOI, the level of expression of the exogenous p53 gene was three times higher than that of the endogenous mutated protein


in H322 and 14 times higher than that of the endogenous wild-type protein in H460 cells.

The time course of the expression of the exogenous p53 after a single infection of 10 PFU I cell was studied in H3 58 cells. The protein expression peaked at postinfection day 3, sharply decreased after day 5, and lasted for at least 15 days. This is a critical point with respect to safety of the vector. Transient p53 expression is sufficient for mediating apoptosis, but normal cells taking up the vector will express the exogenous p53 for too short a time for apoptosis to occur. Ad5CMV-p53 inhibited the proliferation of lung cancer cells with mutated or deleted p53 but only minimally affected the growth of cells expressing wild-type p53.

The efficacy of Ad5CMV-p53 in inhibiting tumorigenicity was evaluated in the mouse model of orthotopic human lung cancer. 40

H226Br which originated from a squamous lung cancer that metastasized to brain and has a point mutation (ATC to GTC) at exon 7, codon 254, of the p53 gene was used. The irradiated nude mice were inoculated with 2 x 106 H226Br cells/mouse by intratracheal instillation. Three days after inoculation, each of the mice (8-1 0 per group) were treated with 0.1 ml of Ad5CMV-p53, Ad5/RSV/GL2 (5 x 107

PFU/mouse) or vehicle phosphate-buffered saline by intratracheal instillation once a day for 2 days. After 6 weeks only 25% of the Ad5CMVp53-treated mice formed tumors, whereas in the vehicle or Ad5/RSV/ GL2 control groups, 70-80% of the treated mice formed tumors. In the mice treated with Ad5CMV-p53 that did develop tumors, the average tumor size was significantly smaller than those of the control groups. These results indicate that Ad5CMV-p53 can prevent H226Br from forming tumors in the mouse model of orthotopic human lung cancer.

Previous experiments. showed that expression of wild-type p53 in human lung cancer cells can mediate apoptosis, but not all cell lines tested showed this. H358a cells, which have a homozygous p53 deletion, showed a reduction in their rate of proliferation after transduction with wild-type p53, but the cells did not undergo apoptosis. We hypothesized that if the level of DNA damage in the cell could be elevated acutely at the time the wild-type p53 gene was expressed, apoptosis would occur. We examined whether Ad-p53 and cisplatin given in a sequential combination could induce synergistic tumor regression in vivoY Following 3-day direct intratumoral injection of Adp53, H358a tumors subcutaneously transplanted in nu/nu mice showed a modest slowing of growth; these tumors, regressed, however if cisplatin was administered intraperitoneally for 3 days. Histologic examination revealed necrosis of tumoral tissue in the area where Ad-p53 was injected in mice treated with cisplatin. In situ staining showed extensive areas of apoptosis. In contrast, tumors treated with cisplatin alone or Ad-p53 alone showed no apoptosis.


CLINICAL APPLICATIONS These studies provide a rationale for a new clinical protocol, re

cently approved by the NIH Recombinant DNA Advisory Committee, to inhibit expression of mutant K-ras p21 or replace a defective p53 gene with intratumoral injection of recombinant retrovirus expressing antisense-K-ras or normal p53, respectively. Patients with unresectable lung cancer which obstructs a bronchus and has a K-ras or p53 mutation will first undergo endoscopic resection, the remaining tumor will be directly injected with the appropriate retroviral supernatant. Toxicity, integration of the proviral DNA by tumor cells, and rate of tumor regrowth will be monitored.

A second protocol, which has also received NIH Recombinant DNA Advisory Committee approval, will be an open-label upward dose-ranging study of the Ad5CMV -p53 vector. The study will be done in two phases. It is not known what toxic effects, if any, will be caused by the adenovirus. The first phase of the study will allow assessment of the toxic effects related only to the vector. Patients will receive one intratumor or intrapleural injection of Ad5CMV-p53. The initial dose will be 106 PFU. Following completion of the first vector-only phase, a second phase will evaluate Ad5CMV-p53 and cisplatin administered concurrently. Patients treated in this phase will receive one intratumoral injection of Ad5CMV-p53 with concurrent cisplatin at 30 mg/m2, with two additional doses of cisplatin on days 2 and 3. Three patients will be entered at each dose level, with six patients entered at the maximum tolerated or maximum attainable dose (limitation imposed by production of the adenovirus). The adenovirus dose will increase in one-log10 increments for each group. The objectives of the trial are: (1) to determine the maximum tolerated dose of the wild-type p53 adenoviral vector given with and without cisplatin in patients with refractory non-small cell lung cancer; (2) to"Jdetermine the qualitative and quantitative toxicity and reversibility of toxicity of this treatment approach; and (3) to document the antitumor activity of this treatment approach.

Successful therapy and prevention interventions that reverse genetic lesions may be possible. Genetic constructs could specifically inhibit expression of mutant proteins by dominant oncogenes and could replace the function of deleted or mutated tumor suppressor genes if they could be delivered with high efficiency to tumor cells in vivo. Viral vectors have the potential for this. The aerodigestive tract is suited to this approach because high concentrations of these relatively nontoxic agents could be achieved with local installation, thus avoiding the dilutional effects of intravenous injection. Intervention to halt the progression of premalignant lesions to invasive cancer may be possible. Premalignant lesions such as Barrett's epithelium have tumor suppressor gene mutations. 14 Preventing the development of invasive cancers would clearly be preferable to treating established cancer. These agents


may also have a role in the treatment of patients with more advanced cancer.

Local recurrence or persistence of local disease is still a major management problem for many cancers such as lung, head and neck, and pancreas. lntralesional injection or adjuvant use of genetic constructs to prevent local recurrence after surgery could be considered. Limited metastatic disease could be injected with these agents percutaneously. If these agents are efficacious, their lack of toxicity may provide a sufficiently high therapeutic index such that they could be used as an adjuvant to surgery to treat patients with earlier stages of cancer or as prevention of second primary cancers for individuals with genetic abnormalities in premalignant lesions. The high titers achievable with adenoviral vectors suggest that they could be used systemically. Vector targeting by expression of receptor ligands in the viral capsid is also possible. Although much research needs to be done, the possibility of specific gene targeting with a high therapeutic index makes this an exciting and promising area for investigation.

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suppressor gene in primary lung cancer in Japan. Biochem Biophys Res Commun 1992; 183:1139-1146.

3. Damico D, Carbone D, Mitsudomi T, et al. High frequency of somatically-acquired p53 mutations in small-cell lung cancer cell lines and tumors. Oncogene 1992; 7:339-346.

4. Harris CC, Hollstein M. Clinical Implications of the p53 tumor-suppressor gene. N Eng! J Med 1993; 329:1318-1327.

5. Suzuki H, Takahashi T, Kuroishi T, Suyama M, Ariyoshi Y, Veda R. p53 Mutations of non-small cell lung cancer in Japan: association between mutations and smoking. Cancer Res 1992; 52:734-736.

6. Vahakangas KH, Samet JM, Metcalf RA, et al. Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners. Lancet 1992; 339:576-580.

7. Takahashi T, Carbone D, Nau MM, et al. Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic lesions. Cancer Res 1992; 52:2340-2343.

8. Cajot JF, Anderson MJ, Lehman TA, Shapiro H, Briggs AA, Stanbridge EJ. Growth suppression mediated by transfection of p53 in hut292DM human lung cancer cells expressing endogenous wild-type p53 protein. Cancer Res 1992; 52:6956-6960.

9. Cai DW, Mukhopadhyay T, Liu T, Fujiwara T, Roth JA. Stable expression of the wild-type p53 gene in human lung cancer cells after retrovirusmediated gene transfer. Human Gene Ther 1993; 4:617-624.

10. Quinlan DC, Davidson AG, Summers CL, Warden HE, Doshi HM.


Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Res 1992; 52:4828-4831.

11. Horio Y, Takahashi T, Kuroishi T, et al. Prognostic significance of p53 mutations and 3p deletions in primary resected non-small cell lung cancer. Cancer Res 1993; 53:1-4.

12. Sundaresan V, Ganly P, Hasleton P, et al. p53 and chromosome-3 abnormalities, characteristic of malignant lung tumors, are detectable m preinvasive lesions of the bronchus. Oncogene 1992; 7:1989-1997.

13. Mitsudomi T, Steinberg SM, Nau MM, et al. p53 gene mutations in non-small cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene 1992; 7:171-180.

14. Casson AG, Mukhopadhyay T, Cleary KR, Ro JY, Levin B, Roth JA. p53 gene mutations in Barrett's epithelium and esophageal cancer. Cancer Res 1991; 51:4495-4499.

15. Ramel S, Reid BJ, Sanchez CA, et al. Evaluation of p53 Protein Expression in Barrett's esophagus by two-parameter flow cytometry. Gastroenterology 1992; 102:1220-1228.

16. Sundaresan V, Ganly PS, Hasleton P, et al. Genetic changes in pre-invasive lesions of the respiratory tract. Lung Cancer 1991; 7 supplement:17.

17. Chung KY, Mukhopadhyay T, Kim J, et al. Discordant p53 gene mutations in primary head and neck cancers and corresponding second primary cancers of the upper aerodigestive tract. Cancer Res 1993; 53:1676-1683.

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=============================INDEX =============

Page numbers in italics denote figures (/) and tables ( t ).

A Adenovirus

E lA oncoprotein, 5 ElB oncoprotein, 4/, 5, 74, 118 gene replacement vectors, 120-121

Aflatoxin B 1, 38 AP-1, 15 Apoptosis, 58

lung cancer, 121 ASO (allele-specific oligonucleotide hybridization,

30-31

B Barrett's esophagus, 115-116, 122 Benzo(a)pyrene, 38 Bladder cancer, 36 Bone cancer, 93 Brain cancer, 36 Breast cancer, 27, 36, 39

c Cancer. See also Human tumors.

aerodigestive, 114 premalignancy, 115-116 second primaries, 114-115

Carcinogens, 113 Casein kinase II, 79, 80, 89 Casson AG, 115 CAT, 90 CCAAT-binding factor (CBF), 78 cdc2 kinase, 89 Cell cycle control, 58-59, 89-90

check point determinant, 57 c-fos, 76, 78, 82, 90 Cigarette smoke, 113 Cisplatin, 122 c-jun, 76, 78, 82 Colon cancer. See Colorectal cancer. Colorectal cancer, 27, 36, 38, 39, 92, 93, 113

D DNA polymerase alpha, 4, 5, 57, 87,88 DNA repair, 24, 89 DNA replication, 57, 58 DNA sequencing, 31, 33, 114

E EBNA-5 (Epstein-Barr nuclear

antigen- 5 ), 7 4 Embryonic development, 23 Epitopes. See Monoclonal antibodies. Epstein-Barr virus (EBV), 74 Esophageal cancer, 38, 114, 115

F Field cancerization, 114-115

G gadd45, 56, 57 Gene replacement, 116-117

adenoviral vectors, 120-121 retroviral vectors, 118-120

H Heat shock protein [constitutively

expressed} (hsc), 74, 75, 76, 82 Helix-loop-helix (HLH), 15 Human paplillomavirus (HPV), 5 Human tumors, 28t

I

loss of heterozygosity chromosome 17p, 36

p53 mutations, 36, 37/, 38-40 assay methods, 30-31, 33

primer sequences, 32t

Immunocytochemical analysis, 33 Interleukin-6 (IL-6), 83, 90 Irradiation, 22-23, 58

K K-ras, 122

L Large T antigen. See SV 40 (simian virus

40) large T antigen. Li-Fraumeni syndrome, 1, 29-30, 35, 40,

58, 59 Liver cancer, 92, 93 Lung cancer, 36, 39, 92, 93, 113, 121,

122 Lymphoid cancer, 93

128

M Matrix attachment region (MAR), 88 MCK promoter, 90 mdm2 (murine double minute-2), 56, 64, 76-77,

96 Methylation, 39 Milner J, 62 Missense mutations, 1, 3, 27, 59-60

human cancers, 38, 118 MEL (murine erythroleukemia cell line), 22 Monoclonal antibodies, 22-23

conformational changes in p53 protein, 61-63 PAb122, 62 PAb240,61,62,63,64,82,86,91,97 PAb246,61,62,84,90 PAb248, 61, 62 PAb421, 62 PAb1620, 61, 63, 64, 97

Multicellular tumor spheroids (MTS), 119 Multiple drug resistance gene, 117 Muscle creatine kinase (mck), 78 Mutagens, 38-40 myc, 84 myc/max, 84 myc/myoD protein family, 15

N Neurofibromatosis 1 gene (NF1), 15 Non-small cell lung cancer (NSCLC), 79 Nuclear matrix attachment region (MAR). See

Matrix attachment region (MAR).

p p21. See wafllciipl, 56, 57. p53 gene

structure, 13-16, 14/ p53 protein

biophysical and biochemical properties, 55-59, 56/

central conserved region, 61 conformation, 61-64, 7 5 f

cell cycle control of, 89-90, 94 gene replacement

adenoviral vectors, 120-121 clinical applications, 122-123 retroviral vectors, 118-120

intracellular levels, 84-86 mechanism of action, 21-24, 64, 93-94, 95[,

96-97 DNA replication, 57, 58,87-89, 94 embryonic development, 23 transcription activation, 55-56, 57

monoclonal antibodies, 22-23

p53Suppressor Gene

mutation, 24, 25t, 26-27, 33-36 allelic loss, 27, 28t assay methods in human cancer, 30-31, 33t,

34 conformation, 61-64, 97 loss of heterozygosity, 27, 29, 36 missense, 59-60 p53cys270, 75 p53his273, 35 p53ile246, 35 p53phe194, 35 p53trp248, 35 phenotypic variation, 26-27, 26t

premalignancy, 115-116 prognostic studies, 116 regulation by

allosteric model, 81-82, 94, 96, 97 phosphorylation, 79-81, 89, 94, 95/ protein interactions, 73-79, 7 5/ redox mechanisms, 81, 94, 95/ tissue- and cell-specific, 90-93, 95/

second primaries, 114-115 structure, 20-21

nuclear accumulation, 21 subcellular localization, 86-87

p53CON, 84 . Papillomavirus E6 oncoprotein, 5-6, 74, 85-86 Polymerase chain reaction (PCR), 30

SSCP (single-strand conformation polymorphism), 31, 115

Polyomavirus large T antigen, 85 Premalignancy, 115-116 Proto-oncogene, 117

R Redox mechanisms, 81 Replication protein A (RPA), 4, 5, 57, 88 Retinoblastoma gene, 56, 57 Retroviral vectors

gene replacement, 118-120 replication-defective, 120

RFLP (restriction fragment-length polymorphism), 31

s Sarcomas, 38 Sp1, 78 SSCP (single-strand conformation polymorphism),

31, 114 SV40 (simian virus 40) large T antigen

p53 interaction, 2-5, 4f, 73-74, 75-76, 85, 91-92, 118 DNA replication, 57, 88

Index

T TATA-binding proteinn (TBP), 77 Transcription factors, 55-56, 57, 97 Transforming growth factorb (TGFb), 22 Tumor suppressor genes, 117

u Ubiquitination, 5

w wafllcipl, 56, 57, 94 Warson JV, 62 Wilms' tumor suppressor gene (wtl), 78

129

y YNZ-22, 27

z Zinc (Zn), 81

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