Van Andel Research Institute

Scientific Report

2002

© 2002 by the Van Andel InstituteAll rights reserved

Van Andel Institute333 Bostwick Avenue, N.E.

Grand Rapids, Michigan 49503, U.S.A.

Title page photo: HGF/SF-induced scattering of MDCK cells in vitroShown are dog kidney–derived (MDCK) cells stained for DNA (using DAPI, green) and actin (phalloidin-rhodamine, red). (Phalloidin is a protein that preferentially binds to actin and can be labeled with a fluo-rescent dye, here rhodamine.) Although DAPI is normally seen as blue, here the DAPI emission is placedin the green channel so that co-localization can be seen as yellow. These cells were treated with scatterfactor (HGF/SF) to induce separation from their substrate and begin their migration/scattering. There is alarge cluster of cells in the center and random, attenuated cells with longer cellular processes in the leftcenter and upper right that are starting the scattering process.(Resau)

Contents

Director’s Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Laboratory Reports

Laboratory of Cell Structure and Signal Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Arthur S. Alberts, Ph.D.

Antibody Technology Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Brian Cao, M.D.

Mass Spectrometry and Proteomics Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Gregory S. Cavey, B.S.

Laboratory of Signal Regulation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Sara A. Courtneidge, Ph.D.

Developmental Cell Biology Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Nicholas S. Duesbery, Ph.D.

Bioinformatics Core Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Kyle Furge, Ph.D.

Laboratory of DNA and Protein Microarray Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Brian B. Haab, Ph.D.

Laboratory of Cancer Pharmacogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Han-Mo Koo, Ph.D.

Laboratory of Integrin Signaling and Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Cindy K. Miranti, Ph.D.

Analytical, Cellular, and Molecular Microscopy Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37James H. Resau, Ph.D.

Laboratory of Germline Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Pamela J. Swiatek, Ph.D.

Laboratory of Cancer Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Bin T. Teh, M.D., Ph.D.

Laboratory of Molecular Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45George F. Vande Woude, Ph.D.

Tumor Metastasis and Angiogenesis Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Craig P. Webb, Ph.D.

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Laboratory of Chromosome Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Michael Weinreich, Ph.D.

Laboratory of Cell Signaling and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Bart O. Williams, Ph.D.

Laboratory of Structural Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59H. Eric Xu, Ph.D.

Laboratory of Developmental Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Nian Zhang, Ph.D.

Daniel Nathans Memorial Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Postdoctoral Fellowship Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Student Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

VARI Seminar Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Conference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

VARI Photos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

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Director’s Introduction

I began writingthis introduction dur-ing our 4th annual sci-entific retreat, held ata lodge in the quietbeauty of upperMichigan, where all ofour scientists present-ed overviews of theirresearch and their

future plans. Such a retreat is intense butextremely valuable, because it provides a forumfor promoting collaborations and for testinghypotheses before a lively audience. I am pleasedwith the progress that the Van Andel ResearchInstitute (VARI) has made in a very short periodof time on so many fronts. Some of the highlightsof the past year are quite impressive to me, and Ihope you will find them to be also.

Each year we select a member of the scien-tific community to receive the Daniel NathansMemorial Award, choosing a scientist who hasemulated Dr. Nathans’ extraordinary contribu-tions and special character. Our most recentrecipient was Dr. Francis Collins, who receivedthe award for leading this nation’s effort in deter-mining the sequence of the human genome. Histwo lectures in Grand Rapids on October 2,2001—one to the scientific/medical communityand one to the general public—were exciting andvisionary (see page 65).

We are delighted to have recruited Eric Xu,who came to us from GlaxoSmithKline to headour Laboratory of Structural Sciences. Eric is asuperb crystallographer whose work has focusedon the crystal structures of nuclear hormonereceptors; at our Institute, he will continue study-ing the structural basis for protein/DNA interac-tions, protein/ligand interactions, and multipro-tein complexes. Our success in attracting Eric toVARI was leveraged by funding from theMichigan Life Sciences Corridor (MLSC) for theMichigan Center for Structural Biology(MCSB), which will build and operate a state-of-the-art synchrotron beamline at ArgonneNational Laboratory. This technology has revo-lutionized our ability to solve the structure ofmacromolecules, proteins, and nucleic acids, and

having access to this technology was instrumen-tal in our efforts to recruit Eric. The members ofthe MCSB (VARI, University of Michigan,Michigan State University, and Wayne StateUniversity) partnered with NorthwesternUniversity to secure the beamline.

We also were successful in recruiting GregCavey to establish our proteomics core facility.Greg comes with glowing credentials, havingbeen responsible for establishing a proteomicscore at Pharmacia in Kalamazoo, Michigan. Theproteomics core will provide us with powerfultools necessary for measuring infinitesimal quan-tities of protein that contribute to cancer. At thiswriting, we are eagerly awaiting the arrival of ourmass spectrometer, and Greg already has a back-log of samples to work with once the equipmentis in place. We are grateful to Jack and NancyBatts of Grand Rapids for endowing this labora-tory through a Charitable Remainder Trust, andalso to the Wege Foundation for their gift.

We are pleased to announce the recruitment ofDavid Nadziejka, our science editor. David has 22years of experience in science editing and writing,including eight years as the lead technical editorfor biology and biomedical science at ArgonneNational Laboratory. Since 1996, David has beenthe recipient of three of the four national awardsgiven in the field of technical communication. Hewill be a valuable asset to our investigators as theyprepare grant proposals, manuscripts, site visitreports, and other scientific documents.

We have continued to expand our archive tis-sue repository and to establish tissue acquisitionagreements with area hospitals. We have estab-lished agreements with Spectrum Health (GrandRapids), Pennock Health Services and HastingsSurgeons P.C. (Hastings), Hackley Hospital(Muskegon), Ferguson Clinic (formerlyFerguson Hospital; Grand Rapids), and HollandCommunity Hospital (Holland). We are workingon agreements with St. Mary’s Mercy MedicalCenter (Grand Rapids), and MetropolitanHospital (Grand Rapids). Jim Resau, Rick Hay,Craig Webb, Bin Teh, and Brian Haab haveworked out the methodology and conditions forcollecting, storing, and processing specimens forgene expression profiling (microarray analysis)

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George F. Vande Woude

Director’s Introduction

and proteomics analyses. These investigators,together with Kyle Furge and Bryon Campbell’steam, generated the bioinformatics software ofrelational databases needed for processing, com-paring, and annotating the clinical and molecularinformation. Collectively, we are establishing anetwork for translation of this research into clin-ical application. Our collaborations with sur-geons, oncologists, and pathologists in a commu-nity hospital setting are unique and will becomea powerful asset to Western Michigan.

We have upgraded our imaging facility withthe purchase of a multiphoton microscope. Thismicroscope has a special detector that will enableus to see fluorescently labeled cells deep withinliving tissues without introducing the damageusually associated with ultraviolet light. Thiswill enable us to not only see cells and tissues,but to follow their biology over time (as theygrow, migrate, and develop) and to quantify thechanges. We also are developing a state-of-the-art Acusome ultrasound facility for in vivo imag-ing of mouse development, as well as of tumorgrowth and metastases using molecular markersand newly developed image-contrast reagents.Bart Williams led an effort to purchase an instru-ment that allows us to quickly determine bonemineral density and body fat content in livingmice. This machine, obtained from GE Lunar,will be valuable in examining mutant mice thatare susceptible to osteoporosis and diabetes, aswell as in determining how mouse models ofhuman cancer respond to various treatments.

In June 2002, the Michigan EconomicDevelopment Corporation announced plans tofund 18 of the 111 full proposals submitted forthe FY2002 competition. These projects,designed to advance the research and commer-cialization of cutting-edge life sciences products,represent a $45 million commitment from theMLSC Fund. A multi-institutional collaborationto develop new approaches for imaging and treat-ing prostate cancer, using the molecule Met as adiagnostic and therapeutic target, was funded bythe MLSC for a three-year project. This effortinvolves three institutions within Michigan(VARI, MSU, and the Veterans Affairs HealthCare System in Ann Arbor) and two inWashington State (Fred Hutchinson CancerResearch Center and the Gerald P. MurphyCancer Foundation). VARI investigators on the

grant are myself, Rick Hay, James Resau, andBrian Cao. VARI will also receive funding forthe core services it provides as part of the CoreTechnology Alliance (CTA; comprising VARI,the University of Michigan, Michigan StateUniversity, and Wayne State University). TheCTA is important to Michigan’s research effortsin that it provides cutting-edge biomedical tech-nology services in proteomics, genomics, bioin-formatics, structural biology, and animal modelsto all scientists in the state. Given sufficienttime, this will propel Michigan into a leadershiprole in biotechnology. Pam Swiatek from ourInstitute has been a leader in helping to establishthe CTA cores and is director of the AnimalModels Consortium in Michigan.

With Drs. Brian Ross and AlnawazRehemtulla at the University of Michigan, we havereceived an In Vivo Cellular Molecular ImagingCenter (ICMIC) P50 grant for in vivo imaging ofoncogene activities and metastases. This grant wasawarded by the National Cancer Institute (NCI).VARI investigators on the grant are myself, Ilanand Galia Tsarfaty, Pam Swiatek, Bryn Eagleson,and Jim Resau. Ilan, a cancer biologist, and Galia,a radiologist, are visiting scientists from Tel AvivUniversity and the Chaim Sheba Medical Center,respectively; they have a major interest in imagingand were key to our success in obtaining this grant.We are making a significant commitment to themolecular imaging of cancer cells and tumors inanimal models as a means to create a knowledgebridge between basic science and the clinical diag-nosis and therapy of human pathological states.

Craig Webb and Brian Haab have begun acollaborative research project with Dr. AnthonySchaeffer, chairman of the Department ofUrology at Northwestern University. They willexamine, by gene expression analysis and pro-teomics, prostatic fluids and tissue specimensfrom patients with various diseases of theprostate, including prostatitis, benign prostatehyperplasia, and prostate carcinoma. They willperform gene and protein profiling on these sam-ples for different pathological states to identifyuseful clinical markers. Further, Brian Haab hasestablished a collaboration with Dr. Jose Costa,director of anatomical pathology of the YaleSchool of Medicine, to identify markers in pan-creatic cancer; the work is funded through NCI’sEarly Detection Research Network (EDRN).

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In a first for VARI and the Grand Rapids com-munity, Han-mo Koo helped develop a clinical trialfor treating pancreatic cancer, which stems fromwork we began at NCI. This clinical trial is headedby Dr. Marianne Lange, together with Drs. TimO’Rourke and Alan Campbell, through the GrandRapids Clinical Oncology Program (GRCOP). Thetrial is managed by the GRCOP under the directionof Connie Szczepanek. This trial is unique in that itis the first Phase II trial to be developed locally andis locally managed through the GRCOP as a con-sortium. The members of the consortium are BattleCreek Health System (Battle Creek), HackleyHospital (Muskegon), Holland CommunityHospital (Holland), Metropolitan Hospital (GrandRapids), Munson Medical Center (Traverse City),Spectrum Health (Grand Rapids), St. Mary’s MercyMedical Center (Grand Rapids), and MecostaCounty General Hospital (Big Rapids).

One of the most important and dramaticchanges occurring in cancer diagnosis and treat-ment is that we are moving from phenomenologicalapproaches to molecular-based medicine. This willrevolutionize the detection and treatment of cancerand, for that matter, of all pathological states,because visualized changes will be converted intoquantifiable measurements. For example, BinTeh’s laboratory—using gene microchips generat-ed at VARI by Brian Haab’s lab—has discovered,in retrospective studies, alterations in the expres-sion of specific genes in kidney cancer specimensthat correlate with poor prognosis. The identifica-tion of these genes and their correlation with theaggressiveness of the disease at the time of diagno-sis is helpful in identifying new targets for drugdevelopment that may lead to improved therapiesfor this group of patients. Using this technology,and in collaboration with urologists and patholo-gists from hospitals in Grand Rapids, the Universityof Chicago, and the University of Tokushima(Japan), Teh’s lab has molecular evidence that kid-ney cancers can be divided into five classes basedon gene expression profiles. Similarly, togetherwith members of the DeVos Children’s Hospital ofGrand Rapids, they have identified gene expressionprofiles that correlate with Wilms’ tumors, a rarechildhood kidney cancer that is refractory to treat-ment. Bin Teh’s lab has also collaborated with Drs.Lawrence Einhorn and Richard Foster from IndianaUniversity, looking into testicular cancer that isrefractory to chemotherapy.

The VIII International Workshop on MultipleEndocrine Neoplasia (MEN) was organized byBin Teh and held at VARI on June 16–18, 2002.The meeting was well attended, with over 180researchers representing 18 countries (see page85). VARI will host the Cancer Intervention2002 meeting in Grand Rapids on October 2–6,2002, at which we will explore progress on thefronts of cancer diagnosis, treatment, and pre-vention with the leading investigators in cancerintervention strategies. We are excited aboutbringing this cutting-edge science to the GrandRapids medical community.

This year we have implemented a memoran-dum of understanding with Michigan StateUniversity (MSU) that establishes a cooperativerelationship to enhance graduate recruitment, edu-cation, and research training opportunities forselected students in doctoral programs in one ormore of the biomedical and life sciences programsat MSU. Sara Courtneidge, Cindy Miranti, andMichael Weinreich have been appointed adjunctprofessors at MSU, and we anticipate graduatestudents from MSU training in VARI laboratories.

The visual documentation of science some-times produces images that are not only scientifi-cally important, but aesthetically pleasing asworks of art. One example is the photograph onthe back cover of this report, contributed by ArtAlberts, that shows the effects of a gene that neg-atively regulates cell motility. I hope you enjoythe other examples of nature’s artwork displayedthroughout this report.

In conclusion, I wish to express my apprecia-tion and gratitude to all who have helped get us thisfar in a few short years. We are especially indebtedto Jay Van Andel and his family and to all of ourbenefactors. Our community has many individualswho believe in what we are doing and have gener-ously contributed to our research through the Hopeon the Hill Foundation. We are very grateful for allthe donations from organizations, individuals, andemployees, as well as for the proceeds from fund-raising events. It is very moving and encouragingthat so many are partnering with us to build a centerof excellence for world-class scientists and world-class medical science. This support, both moral andfinancial, will enhance our ability to develop newknowledge and make new discoveries about cancerthat will lead to better diagnostic and therapeuticstrategies for treating this dreaded disease.

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Van Andel Research InstituteLaboratory Reports

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An mDia2 mutant that disrupts the actin cytoskeleton

This photo shows the effects of the expression of an altered form of the Diaphanous-related formin (DRF)mDia2, a protein that controls cytoskeletal remodeling. The mutation, an alanine substitution at methionine1041, disrupts the ability of mDia2 to regulate itself. mDia2 then assumes the “open” conformation and cannotregulate the formation of actin fibers. Red marks the actin fiber network, green marks the expressed protein.This work shows that proper autoregulation of DRF proteins is crucial for normal function. Similar mutationshave arisen in human DRF proteins that have led to inherited deafness, infertility, and defects in cell growth.(Wallar and Alberts)

Laboratory of Cell Structure and Signal Integration

Arthur S. Alberts, Ph.D.Dr. Alberts received his Ph.D. in physiology and pharmacology at the University ofCalifornia, San Diego, in 1993, where he studied with James Feramisco. From1994 to 1997, he served as a Postdoctoral Fellow in Richard Treisman’s laboratoryat the Imperial Cancer Research Fund in London, England. From 1997 through1999, he was an Assistant Research Biochemist in the laboratory of FrankMcCormick at the Cancer Research Institute, University of California, SanFrancisco. Dr. Alberts joined VARI as a Scientific Investigator in January 2000.

Laboratory Members

StaffJun Peng, M.D. Stephen Matheson, Ph.D.Brad Wallar, Ph.D.Akiko Vankirk, M.S.

StudentsNicole NeumanDare Odomosu

ormal cells base growth decisions on thesum of positive and negative inputsderived from extracellular cues. These

signals are processed by biochemical networkscomposed of thousands of interacting proteins andsmall chemicals that shuttle information from oneto the other. If the system becomes unbalanced—due to the presence of viral factors or DNA dam-age, for example—the cells will arrest and/orundergo a form of programmed cell suicide (apop-tosis) in order to protect surrounding cells or tis-sues. In some cases, the protection system is over-ridden and damaged cells continue to live. As anafflicted cell loses control and continues to divideunchecked, it may incur further mutations thatlead to tumor formation and disease.

Our lab is interested in the intracellular sig-naling networks that regulate proliferation, cellshape, and motility, as well as how cells becomehijacked by disease. Our approaches dependupon a combination of molecular and cell bio-logical techniques to define signal transductionand transformation mechanisms. In particular,we focus on the biology of the single cell and itsinstantaneous response to growth factor stimula-tion. Understanding these mechanisms is crucialin the development of anticancer treatments,because each step in a pathway may eventuallybe exploited for drug and gene therapy targets.

The Rho family of GTP-binding proteins aresignaling factors involved in cell growth respons-es, including changes in gene expression, cellshape, and motility. They act as molecular

switch proteins: they are “on” when bound to thechemical GTP and “off” after they convert GTPto GDP. This on/off cycle is regulated by gua-nine-nucleotide exchange factors, or Rho GEFs.

Rho GEFs are positive activators of the Rhoproteins, inducing the Rho proteins to bind GTP.Once GTP is bound, Rho proteins can bind totarget factors. These targets can act as Rhoeffectors and directly participate in signaling.Alternatively, the Rho proteins target other“switch” proteins that are part of a signaling net-work. The Rho proteins are from the same fam-ily of GTP-binding proteins as Ras. Ras is anoncogene mutated in many tumors. Mutant Rasprotein is locked in a GTP-bound active state, butunlike Ras, similarly active GTP-bound Rhomutants are unable to transform cells. Thoughnontransforming, the Rho proteins are requiredfor Ras transformation. The role that Rho pro-teins play in transformation is unclear, but recentwork suggests that they may indirectly regulatethe cell cycle. They do, however, have an impor-tant role in cancer metastases by regulating cellshape and mobility during the invasion of sur-rounding tissues. Oncogenic mutant Rho GEFstransform cells by essentially force-feeding GTPto Rho proteins. But unlike mutant Rho proteinslocked with GTP, Rho GEFs allow cyclingbetween GTP and GDP bound states. Thiscycling process appears to be key to their abilityto transform cells. We are investigating this inmore detail by comparing signals generated byactivated Rho proteins and the oncogenic Rho

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NResearch Projects

GEFs. On a molecular level, we are analyzingthe regulation of Rho GEFs by phosphorylation,by transcriptional regulation, and by direct bind-ing to viral and cellular proteins.

The Diaphanous-related formins (DRFs)bind to activated Rho proteins. These molecularscaffolds bridge multiple growth factor–regulatedsignaling proteins and regulators of the cytoskele-ton. One of these is Src tyrosine kinase. Src is aprotooncogene whose expression is amplified inmost breast tumors and whose function is criticalfor the growth of breast cancer cells. We havefound that the DRFs bridge Src and Rho proteinsin growth factor signaling. Our observationestablishes an important link between two paral-lel signaling networks that control proliferation inresponse to growth factor stimulation.

The DRFs are controlled by intramolecularautoinhibition, as illustrated in Figure 1. TheGTPase binding domain (GBD) associates withthe carboxy-terminal DAD (Dia-autoregulatorydomain). This interaction is disrupted by GTP-bound Rho. We are characterizing subsequentmolecular events that occur as a result of Rhobinding by posing the following questions:Where does DRF activation occur in cells fol-lowing growth factor stimulation? Is the interac-

tion regulated during the cell cycle or in directedcell migration? And, does Rho binding affectother DRF-binding partners activity or subcellu-lar targeting? We are also testing the hypothesisthat the DRF mDia2 binds to Src near its GTPasebinding domain and that this might activatedownstream signaling by disrupting autoinhibi-tion. We are addressing these questions througha variety of methods that include digital time-lapse microscopy and targeted gene disruption ofthe murine DRF family members in collabora-tion with the VARI’s Laboratory of GermlineModification headed by Pam Swiatek.

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Molecular regulation of the Diaphanous-related formins

Actinremodelingmachinery?

Rho-GDP

ON

OFF GBD FH1 FH2 DADFH3

activation andtargeting?

?

SrcWISH

profilinIRSp53

actin nucleation

Rho-GTP

Src?

Figure 1. Molecular regulation of the Diaphanous-related formins

External Collaborators

Pierre Chardin, Institut de Pharmacologie du CNRS, Valbonne, France

Phillipe Chavrier, Marie Curie Institut, Paris, France

Jeff Frost, University of Texas, Houston

Gregg Gundersen, Columbia University, New York

George Prendergast, Lankenau Institute for Medical Research, Wynnewood, Pennsylvania

Fred Wittinghofer, Max-Planck-Institut, Dortmund, Germany

Publications

Collins, Colin, Stanislav Volik, David Kowbel, David Ginzinger, Bauke Ylstra, Thomas Cloutier,Trevor Hawkins, Paul Predki, Christopher Martin, Meredith Wernick, Wen-Lin Kuo, ArthurAlberts, and Joe W. Gray. 2001. Comprehensive genome sequence analysis of a breast canceramplicon. Genome Research 11(6): 1034–1042.

Palazzo, Alexander F., Hazel L. Joseph, Ying-Jiun Chen, Denis L. Dujardin, Arthur S. Alberts, K.Kevin Pfister, Richard B. Vallee, and Gregg G. Gundersen. 2001. Cdc42, dynein, and dynactinregulate MTOC reorientation independent of Rho-regulated microtubule stabilization. CurrentBiology 11(19): 1536–1541.

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From left to right, back row: Waller, Alberts, Matheson front row: Vankirk, Peng, Odomosu, Newman

Antibody Technology Laboratory

Brian Cao, M.D.Dr. Cao obtained his M.D. from Beijing Medical University, People’s Republic ofChina, in 1986. On receiving a CDC Fellowship Award, he was a Visiting Scientistat the National Center for Infectious Diseases, Centers for Disease Control andPrevention (1991–1994). He next served as a Postdoctoral Fellow at Harvard(1994–1995) and Yale (1995–1996). From 1996 to 1999, Dr. Cao was a ScientistAssociate in charge of the Monoclonal Antibody Production Laboratory at theAdvanced BioScience Laboratories–Basic Research Program at the NationalCancer Institute–Frederick Cancer Research and Development Center, Maryland.Dr. Cao joined VARI as a Special Program Investigator in June 1999.

Laboratory Members

StaffHuiying Zhang, Ph.D.Ping Zhao, M.S.Jessica Kalbfleisch, B.S.

StudentsJennifer Edgar, B.S.Josie ClowneyPaul Veldhouse

he antibody technology facility pro-duces, purifies, and characterizes mon-oclonal and polyclonal antibodies. Our

laboratory works with investigators on a varietyof research projects, including the identificationand characterization of novel proteins; affinitypurification for structural analysis; developmentof clinical immunodiagnostic methods and kits;and engineering and humanizing monoclonalantibodies that have potential application forclinical diagnosis, prognosis, and immunothera-py in cancers and infectious diseases.

Hepatocyte growth factor/scatter factor(HGF/SF)-Met, a ligand-receptor pair, playsimportant roles in tumorigenesis, angiogenesis,and metastasis. Tumors with an autocrine loopof HGF/SF-Met are highly malignant and have apoor prognosis. We have generated a panel ofmonoclonal antibodies (mAbs) to the ligand andfound some of them with biologic neutralizingactivity when they are used in combination. Wehave characterized their antitumor effect both invitro and in vivo. A panel of mAbs raised againstthe Met receptor extracellular domain has beengenerated; two of these mAbs are under furthercharacterization for clinical imaging/diagnosticapplication. Both anti-ligand and receptor mAbshave been patented. Angiogenesis contributessignificantly to the progression of cancer and, astumors grow, they begin to produce a wider arrayof angiogenesis molecules. In collaboration with

Brian Haab’s microarray technology core facili-ty and James Resau’s cellular/molecular imagingcore facility at VARI, we are developingxenograft animal models for several human can-cers, including glioblastoma and soft-tissue sar-coma, in order to understand the correlationbetween key growth factors (VEGF, HGF/SF,EGF, etc.) and their receptors, which stimulatetumor angiogenesis and metastasis. Moreover,we seek to evaluate the effect of mAbs on thesegrowth factors or receptors, individually or incombination, for potential clinical immunothera-peutic application.

In collaboration with the University ofMichigan, we are currently using our antibodiesto HGF/SF and Met for clinical nuclear imagingdiagnostic applications. We are in the process ofcharacterizing several radiolabeled mAbs thathave high affinity to this ligand-receptor in ani-mal models bearing human tumors. In addition,we are using several combined polyclonal andmonoclonal antibodies to analyze quantitativelythe expression levels of various growth factorsand their receptors in human cancer tissues andthe levels of their normal counterparts from clin-ical specimens. These experiments may identifypotential diagnostic and prognostic indicators forthese diseases.

Anthrax is a zoonotic disease transmissiblefrom animal to man that is caused by the Gram-positive, spore-forming bacterium Bacillus

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TResearch Projects

anthracis. The three proteins of the exotoxinsecreted by the organism are protective antigen(PA), lethal factor (LF), and edema factor (EF).The PA63 fragment forms a heptameric complexon the cell surface that is capable of binding withthe 90 kDa LF protein to form lethal toxin(LeTx). It is known that macrophages are partic-ularly sensitive to LF: at low concentrations, LFstimulates TNF-α and IL-1β; at high concentra-tions, LF causes the death of macrophages andthe release of cytokines into the bloodstream.The symptoms of systemic anthrax are inducibleby injection of LeTx alone in animal models; it islikely that death is caused by cytokine-inducedshock. Because of its rarity, anthrax is not oftenincluded in differential diagnosis; in cases ofinhalational anthrax, the diagnosis is rarely madeuntil the patient is moribund. Antibiotic treat-ment should be initiated at the earliest stage ofinfection. By the time characteristic symptomsappear, the bacteria are already multiplying rap-idly in the bloodstream and have produced mas-sive amounts of toxin. Killing the bacteria can-not eliminate the toxin, and its effects result indeath of the host despite antibiotic treatment atthis point. Therefore, we hypothesize that ahigh-affinity neutralizing monoclonal antibodyto LF would be a potentially useful reagent forthe treatment of anthrax infection, in combina-tion with the use of antibiotics.

In collaboration with the laboratories ofNicholas Duesbery and Han-Mo Koo at VARI,we have developed two panels of mAbs againstprotective antigen and lethal factor. Our majorgoal of producing these mAbs is to find thosethat have properties of specifically blocking thebiological functions of LF and to further evaluatethose antibodies as potential agents for the treat-ment of anthrax infection. We have character-ized their biological neutralization properties toPA and LF by in vitro bioassays and found a par-ticular anti-LF mAb that has strong neutralizingactivity to LF: at a low molar ratio to LF, it shiftsthe effective concentration (EC50) over 500-foldin the macrophage cell line J774A.1 (Figure 1).Our results also showed that this antibody specif-ically recognizes the binding site of LF to PA andblocks their binding, preventing the formation oflethal toxin (Figure 2). We will further test this

mAb in vivo on a variety of animals models(including mouse, rat, and rabbit) to observe if itneutralizes LF in vivo and protects the animalfrom death caused by challenges with either puri-fied LeTx or live bacteria. Meanwhile, we havealso epitope-mapped this mAb using the phage-display technique, and a few synthetic small pep-tides based on the epitope mapping informationare being tested to determine if they could be LFantagonists with biological functions similar tothose of LF-neutralizing mAbs. Our overallgoals in this project are to characterize the in vivoneutralizing activities of anti-LF mAbs and/orsynthetic peptides as LF antagonists for theirsuitability as passive protection for animals,including their potential clinical application totreat anthrax infection.

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Figure 1. Macrophage cell survival

Figure 2. Gel shifting assay. Lane 1: molecular weight marker 66 kDa; lane 2: PA63 alone; lane 3:PA63 plus anti-LF mAb; lane 4: LF alone; lane 5: LFplus mAb; lane 6: PA63 plus LF; lane 7: PA63, LF,and mAb.

External Collaborators

Lonson Barr, Michigan State University, Lansing

Milton Gross, Department of Veterans Affairs Medical Center – University of Michigan MedicalCenter, Ann Arbor

Yi Ren, Royal Mary Hospital, Hong Kong University

Wei-cheng You, Beijing Institute for Cancer Research, People’s Republic of China

Dong-zheng Yu, Institute of Epidemiology and Microbiology, Chinese Academy of PreventiveMedicine, Beijing, People’s Republic of China

Publications

Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, George F. VandeWoude, and Milton Gross. 2002. Radioimmunoscintigraphy of tumors autocrine for human Metand hepatocyte growth factor/scatter factor. Molecular Imaging 1(1): 56–62.

Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-MeiWang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H.Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patients withlate-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596.

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From left to right: Zhao, Edgar, Kalbfleisch, Cao

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Immunostaining of human hepatocyte growth factor/scatter factor (HGF/SF)

Formalin-fixed S-114 cells (NIH 3T3 cells transformed with human HGF/SF and its receptor Met) are stainedwith a rabbit anti-HGF/SF polyclonal antibody with rhodamine conjugate (red) and a mouse mAb (7-2) withFITC conjugate (green). Both antibodies were produced by the Antibody Technology Lab at VARI. The blackand white is a Nomarski-DIC (differential interference contrast) image of the cellular detail. The yellowimage indicates co-localized staining of the green monoclonal and red polyclonal antibodies.(Hudson, Zhao, Resau, and Cao)

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A) Clustering of 15 Wilms tumors and clinical information; B) Three-dimensional clustering

The clustering of patients (using Pearson’s correlation) is based on global gene expression profiles consisting ofmedian polished data of 5,594 well-measured spots. The tumors approximately clustered into two main groups,with one group consisting of mostly tumors of high stage (stages III and IV) and the other consisting of mostlytumors of low stage (stages I and II). Two patients with high-stage tumors who died of cancer (Wilms 6, 15)and one patient who had recurrence (Wilms 11) were closely clustered together. One patient (Wilms 14), repre-sented by the orange circle in panel B, who had stage II disease but developed recurrence and died of cancer,was clustered with high-stage tumors.(Takahashi and Teh)

Mass Spectrometry and Proteomics Laboratory

Gregory S. Cavey, B.S.Mr. Cavey received his B.S. degree from Michigan State University in 1990. Priorto joining VARI, he was employed at Pharmacia in Kalamazoo, Michigan, for near-ly 15 years. As part of a biotechnology development unit, he was a group leader fora protein characterization core laboratory. More recently as a research scientist indiscovery research, Greg was principal in the establishment and applications of astate-of-the-art proteomics laboratory for drug discovery. He joined VARI as aSpecial Program Investigator in July 2002.

roteomics is fast becoming a majoreffort in most research institutions. Therapid development of technology is

providing powerful new tools for probing thefunction of proteins and for extracting informa-tion from the Human Genome project. Sincenearly all drug targets are proteins, there is clearincentive to apply proteomics as a complementa-ry approach to genomics research. For pro-teomics, the most important technology is thecoupling of quantitative analytical protein sepa-ration and modern mass spectrometers to identi-fy and characterize proteins with unprecedentedsensitivity and throughput. This laboratory willcollaborate with VARI investigators in applyingcurrent proteomics technology toward theirresearch goals and will build external collabora-tions to speed the development of new tools tomeet the many challenges in cancer research.

Expression proteomics and cell-mappingproteomics will be pursued, as will characteriza-tion of posttranslational modifications such asphosphorylation. For expression proteomics, wewill initially rely on two-dimensional (2D) gelelectrophoresis to display differentiallyexpressed proteins of a given disease state,genetic manipulation, or drug treatment. Thismay require implementing an array of sampleisolation, solubilization, and fractionation tech-

niques. Once the proteins are displayed on 2Dgels, quantitative data can be used to identify pro-teins of interest, followed by automated identifica-tion using mass spectrometry and database search-ing. Forthcoming will be the use of an isotope-coded affinity tagging (ICAT) approach that willallow quantitative measurement of proteins in con-trol vs. experimental samples. This approach willrequire the set-up and use of a multidimensionalliquid chromatography system for the separationof complex mixtures of proteins.

Cell-mapping proteomics will be used to iden-tify components of protein complexes under vari-ous conditions in order to help understand the reg-ulatory mechanism(s) of a given pathway. In thisapproach, a nondenatured sample is affinity-puri-fied using either antibodies, a known protein car-rying an affinity tag, or immobilized small mole-cules. Binding partners are separated by 2D orSDS polyacrylamide gel electrophoresis (PAGE)and are identified using mass spectrometry anddatabase searching.

Posttranslational modification of proteins—inparticular phosphorylation—is known to be a regu-latory event in signal transduction. The proteomicslab will work with various investigators to map phos-phorylation sites of proteins. The unique talents,research, and resources at VARI provide numerousopportunities for proteomics applications.

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PResearch Projects

Laboratory of Signal Regulation and Cancer

Sara A. Courtneidge, Ph.D.Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Researchin London. She began her career in the basic sciences in 1978 as a PostdoctoralFellow in the laboratory of J. Michael Bishop at the University of California Schoolof Medicine. She later joined her alma mater as a member of the scientific staff. In1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as GroupLeader and in 1991 was appointed Senior Scientist with tenure. She joined Sugenin 1994 as Vice President of Research, later becoming Senior Vice President ofResearch and then Chief Scientist. Dr. Courtneidge was appointed SeniorScientific Investigator and Deputy Director of the Van Andel Research Institute inJanuary 2001.

Laboratory Members

StaffEduardo Azucena, Ph.D.Hasan Korkaya, Ph.D.Darren Seals, Ph.D.Rebecca Uzarski, Ph.D.Rebecca Cruz, M.S.Daniel Salinsky, M.S.

StudentsErik Freiter, B.S.Lisa MaurerTherese Roth

ur lab wants to understand at the molec-ular level how proliferation is con-trolled in normal cells and by what

mechanisms these controls are subverted intumor cells. We largely focus on a family ofoncogenic tyrosine kinases, the Src family. Theprototype of the family, vSrc, originally discov-ered as the transforming protein of Rous sarcomavirus, is a mutated and activated version of a nor-mal cellular gene product, cSrc. The activity ofall members of the Src family is normally understrict control; however, the enzymes are fre-quently activated or overexpressed, or both, inhuman tumors. In normal cells, Src familykinases have been implicated in signaling frommany types of receptors, including receptor tyro-sine kinases, as well as integrin receptors and Gprotein-coupled receptors. Signals generated bySrc family kinases are thought to play a role incell cycle entry, cytoskeletal rearrangements, cellmigration, and cell division. In tumor cells, Srcmay play a role in growth factor–independentproliferation or in invasiveness. In addition,some evidence points to a role for Src familykinases in angiogenesis. Some of the currentprojects in the laboratory are outlined below.

Novel Src substrates

We recently described a method for identify-ing tyrosine kinase substrates by using anti-phos-

photyrosine antibodies to screen tyrosine-phos-phorylated cDNA expression libraries. Severalpotential Src substrates were identified, includingFish, which has five SH3 domains and a phox

OResearch Projects

Src-transformed cells were stained to visualizeFish (green) and F-actin (red). The podosomes arevisible as rings of intense F-actin staining. Much ofFish is also present in these podosomes.

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homology (PX) domain. Fish is tyrosine phospho-rylated in Src-transformed fibroblasts (suggestingthat it is a target of Src in vivo) and in normal cellsafter treatment with several growth factors.

We have recently found that in Src-trans-formed cells, Fish is localized to specializedregions of the plasma membrane calledinvadopodia or podosomes. These actin-richprotrusions from the plasma membrane are sitesof matrix invasion and locomotion. We have alsodetermined that the PX domain of Fish associateswith phosphatidylinositol 3-phosphate and thatthis domain targets Fish to the podosomes.Furthermore, the fifth SH3 domain of Fish medi-ates its association with members of the ADAMsfamily of membrane metalloproteases, which inSrc-transformed cells are also localized topodosomes. Our current research aims to probethe role of Fish in invasion in both Src-trans-formed cells and in human tumor cell lines.

Other novel proteins identified in the sub-strate screen have also been shown to be tyrosinephosphorylated in Src-transformed cells and arebeing characterized.

Using a Src-selective inhibitor to probe its rolein signaling pathways

The use of small molecule inhibitors to studymolecular components of cellular signal transduc-tion pathways provides a complementary means ofanalysis to techniques such as antisense, dominantnegative (interfering) mutants and constitutivelyactivated mutants. We have recently identified andcharacterized a small molecule inhibitor, SU6656,which exhibits selectivity for Src and other mem-bers of the Src family. The use of SU6656 con-firmed our previous findings that Src family kinas-es are required for both Myc induction and DNAsynthesis in response to PDGF stimulation of NIH3T3 fibroblasts. We are currently comparing bothPDGF-stimulated gene expression and tyrosinephosphorylation events in untreated and SU6656-treated cells to define which events require Srcfamily kinases. SU6656 should prove a useful toolfor further dissecting the role of Src kinases in thisand other signal transduction pathways.

The connection between Src and p53

The tumor suppressor p53 is present at lowlevels in growing cells. Many DNA tumor virus-es encode proteins that inactivate p53 by directassociation or ubiquitination-mediated degrada-tion, presumably to facilitate the entry of cellsinto cycle and therefore viral replication. Wehave recently shown that the product of one suchDNA tumor virus, the SV40 large T antigen,bypasses the requirements for several signalsemanating from growth factor receptors. In par-ticular, cells that lack p53, or in which p53 hasbeen inactivated by T antigen binding, no longerrequire Src family kinases for growth factor sig-naling. These findings suggest that Src kinasesare required to overcome the inhibitory effects ofp53. There are perhaps also implications for theuse of signal transduction inhibitors in humancancers where negative regulators such as p53are frequently mutated or absent. We are nowinvestigating in more detail the way in which Srckinases impact p53 function.

Breast cancer

Increased Src activity can be demonstrated inthe majority of breast cancers, both estrogen-dependent and estrogen-independent, yet the roleof Src in breast tumorigenesis has not been estab-lished. We have begun to characterize the role ofSrc in both EGF-stimulated and estrogen-stimu-lated signal transduction pathways in breast can-cer cell lines.

Novel members of the Src family

We are particularly interested in the humankinase Frk. This kinase has a domain structuretypical of Src family kinases and is probably reg-ulated in a similar manner. However Frk lacksthe amino-terminal myristylation sequences andinstead has a nuclear localization sequence in itsSH2 domain. Interestingly, Frk is predominant-ly expressed in epithelial cells and is overex-pressed in a high proportion of human tumorsand tumor cells lines, particularly those derivingfrom lung. We have begun an extensive charac-terization of Frk, including its substrate specifici-ty, regulation, transforming ability, and function.

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Publications

Courtneidge, S.A. 2002. The role of Src in signal transduction pathways. Biochemical SocietyTransactions 30(2): 11–17.

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From left to right, back row: Cruz, Mauer, Courtneidge middle row: Korkaya, Freiter, Seals front row: Salinsky, Azucena, Uzarski, Roth

Developmental Cell Biology Laboratory

Nicholas S. Duesbery, Ph.D.Dr. Duesbery received both his M.S. (1990) and Ph.D. (1996) degrees in zoologyfrom the University of Toronto, Canada. Before his appointment at VARI, he servedas a Postdoctoral Fellow in the laboratory of George Vande Woude in the MolecularOncology Section of the Advanced BioScience Laboratories–Basic ResearchProgram at the National Cancer Institute–Frederick Cancer Research andDevelopment Center, Maryland (1996–1999). Dr. Duesbery joined VARI as aScientific Investigator in April 1999.

Laboratory Members

StaffXudong Liang, M.D.Arun Prasad Chopra, Ph.D.Sherri Boone, B.S.

Visiting ScientistJean-François Bodart, Ph.D.

StudentsJonathon DouglasMarie GravesJeanine Myles

ur research group focuses on cellularaspects of oogenesis, meiosis, andmitosis in a variety of vertebrate model

organisms. Using biochemical and molecularapproaches, we seek to identify regulatory mech-anisms involved in egg cell formation, fertiliza-tion, and early embryonic development, as wellas to ascertain the roles of these mechanisms inhuman health and disease.

Our current understanding of meiotic matura-tion in amphibian oocytes is largely based onextensive analyses of cell cycle regulation inXenopus laevis. Immature amphibian oocytes,arrested at prophase of meiosis I, resume meiosisin response to hormonal stimulation. Theresumption of meiosis is followed by germinalvesicle breakdown (GVBD) and the outwardappearance of a white spot. Oocytes subsequent-ly arrest at metaphase II as mature oocytes oreggs in anticipation of fertilization. In 1971,Masui and Markert found that cytoplasm from anegg, when injected into oocytes, was capable ofinducing meiotic maturation. They thus conclud-ed that eggs contain an activity, which they calledmaturation promoting factor (MPF), that was suf-ficient to induce maturation. Subsequent purifi-cation showed that MPF is a complex of two sub-units: a catalytic subunit, p34cdc2, and a regulatorysubunit, cyclin B. Since that initial report, MPFhas been shown to be a universal regulator ofentry into meiotic and mitotic metaphase.

MPF in X. laevis oocytes possesses the abil-ity to activate itself; injection of MPF from eggscan induce recipient oocytes to undergo GVBD

in the presence of cycloheximide. The preexis-tence of cyclin B2 in immature oocytes mayexplain why amplification of MPF is observedeven in the absence of protein synthesis. It hasbeen proposed that X. laevis oocytes contain aprecursor of MPF, called pre-MPF, that is acti-vated by the injection of MPF. However, themechanisms of autoamplification of MPF remainpoorly understood. It has been shown that MPFcan phosphorylate and activate its positive regu-lator Cdc25 in vitro and that this mechanism mayplay a role in the autoamplification of MPF.Still, even if Cdc25 phosphorylation by p34cdc2 isrequired for the autoamplifying activity of MPF,it is not sufficient. Numerous kinases have alsobeen implicated in the MPF autocatalytic loop;among them, members of the polokinase familyhave been involved in p34cdc2 activation by phos-phorylating Cdc25 and have been characterizedin X. laevis.

Mitogen-activated protein kinase (MAPK) isactivated during meiotic maturation of X. laevisoocytes at the same time that maturation promot-ing factor is. Though it remains unclear whetherMAPK is activated before MPF, interconnec-tions exist between the MPF and MAPK path-ways. MAPK kinase (MEK1) injection or con-stitutively active thiophosphorylated-MAPKinjection into X. laevis oocytes can induceresumption of meiosis. The mechanisms bywhich MAPK activation induces meiotic matura-tion seem to involve Myt1 inhibition by p90rsk.Indeed, it has been shown that p90rsk, which isphosphorylated and activated by MAPK, is able

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OResearch Projects

to bind to Myt1, to phosphorylate it, and thus toinactivate the protein. However, GVBD canoccur even in the absence of MAPK activity, afact that leads us to conclude that MAPK activi-ty might not be required for meiosis I in X. laevisoocytes. Nevertheless, MAPK activity isrequired for reactivation of MPF and suppressionof DNA replication between meiosis I and II.

Despite the advantages of X. laevis as amodel system, its amenability to geneticapproaches is limited as a consequence of itspseudotetraploidy. The use of Xenopus tropi-calis, a diploid member of the same genus, hasbeen proposed as a way of circumventing thisproblem. In addition to its promise as a geneticmodel, X. tropicalis may also be useful as a com-parative model to complement studies in X. lae-vis. Although X. tropicalis development superfi-cially resembles that of X. laevis, it may not beassumed that this similarity holds at all levels,because these species evolutionarily diverged30–100 million years ago.

Consequently, we have compared the bio-chemical regulation of oocyte maturation in thetwo species, focusing on the regulation of MPFactivation and MAPK activation in X. tropicalis.The time required for progesterone-induced matu-ration of X. tropicalis oocytes was shorter(GVBD50 = 148.8 ± 44 min) than that of X. laevisoocytes. The maturation of X. tropicalis oocyteswas marked by the appearance of a white dot andthen the formation of a dark ring coincident,respectively, with entry into meiosis I and theonset of anaphase I. As with X. laevis, X. tropi-calis maturation required protein synthesis but nottranscription. The activity of MPF during matura-tion first peaked at 0.67 GVBD50, transientlydeclined, and remained stable thereafter. Crudelysates and cytoplasmic extracts of mature X. trop-icalis oocytes could induce immature oocytes tomature. X. tropicalis oocytes, however, appearedto lack stores of pre-MPF, because these extractscould not induce GVBD in the presence of proteinsynthesis inhibitors. MAPK activity increased inparallel with that of MPF but remained elevatedafter the first meiotic division. Whereas injectionof constitutively active MEK2 triggered GVBD,

MAPK appeared not to be required for GVBD inX. tropicalis oocytes. However, maturation in theabsence of MAPK activation was delayed, andmeiotic spindles failed to form.

Our results indicate that the biochemical reg-ulation of oocyte maturation in both of thesespecies is similar in most respects, with thenotable exception that X. tropicalis oocytes do notmature when injected with MPF in the presence ofprotein synthesis inhibitors. We are currentlyusing a comparative approach to characterize theproteins present in MPF complexes isolated fromX. laevis and X. tropicalis oocytes in order to iden-tify elements required for MPF autoamplification.

In the course of our studies, we serendipi-tously identified anthrax lethal factor (LF), a com-ponent of the toxin produced by Bacillusanthracis, as a proteolytic inhibitor of the MAPKpathway. Specifically, LF was found to removeseven amino acids from the amino terminus ofMAPK kinase 1 (MEK1), the loss of which result-ed in its inactivation. Given the importance ofMEK signaling in tumorigenesis, we assessed theeffects of anthrax lethal toxin on tumor cells. LFwas very effective in inhibiting MAPK activationin V12 H-ras–transformed NIH 3T3 cells.Treatment of transformed cells in vitro with LFcaused them to revert to a nontransformed mor-phology and also inhibited their ability to formcolonies in soft agar and to invade Matrigel, with-out markedly affecting cell proliferation. In vivo,LF inhibited the growth of ras-transformed cellsimplanted in athymic nude mice—in some casescausing tumor regression—at concentrations thatproduced no apparent animal toxicity.Unexpectedly, LF also greatly decreased tumorneovascularization. These results demonstrate thatLF potently inhibits ras-mediated tumor growthand is a novel, potential antitumor therapeutic.

Current research efforts in our lab aredesigned to characterize the protein regions neces-sary for LF-MEK interaction, to identify regionsof LF that are important for cleaving MEK, and todetermine how this cleavage results in inactivationof MEK. Such information may allow us to devel-op drugs that would interfere with this interactionand ultimately block anthrax toxin activity.

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External Collaborators

Jiahuai Han, Scripps Research Institute, San Diego, California

Stephen Leppla, National Institute of Dental and Craniofacial Research, Bethesda, Maryland

Robert Liddington, Burnham Institute, La Jolla, California

Angel Nebreda, European Molecular Biology Laboratory, Heidelberg, Germany

David Waugh, National Cancer Institute, Bethesda, Maryland

Publications

Bodart, Jean-François, Arun P. Chopra, Xudong Liang, and Nicholas S. Duesbery. 2002. Anthrax,MEK, and cancer. Cell Cycle 1(1): 10–15.

Bodart, Jean-François, Davina V. Gutierrez, James H. Resau, Bree D. Buckner, Angel R. Nebreda,and Nicholas S. Duesbery. 2002. Characterization of MPF and MAPK activities during meioticmaturation of Xenopus tropicalis oocytes. Developmental Biology 245: 348–361.

Koo, Han-Mo, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Anthrax toxins, mitogen-activated protein kinase pathway, and melanoma treatment. Directions in Science 1: 123–126.

Koo, Han-Mo, Matt VanBrocklin, Mary Jane McWilliams, Stephan H. Leppla, Nicholas S.Duesbery, and George F. Vande Woude. 2002. Apoptosis and melanogenesis in humanmelanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinasekinase. Proceedings of the National Academy of Sciences USA 99(5): 3052–3057.

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From left to right: Chopra, Bodart, Liang, Boone, Douglas, Duesbery

Bioinformatics Core Program

Kyle A. Furge, Ph.D.Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University Schoolof Medicine in 2000. Prior to obtaining his degree, he worked as a software engi-neer at YSI, Inc., where he wrote operating systems for embedded computerdevices. He did his postdoctoral work in the laboratory of George Vande Woudeand became a Bioinformatics Scientist at VARI in June 2001.

Laboratory Members

StaffEd Dere, B.S., B.Eng.

StudentJoe Crawley

s high-throughput biotechnologies suchas DNA sequencing, gene expressionmicroarray, and genotyping become

more accessible to researchers, analysis of thedata produced by these technologies becomesincreasingly difficult. A relatively new field,termed bioinformatics, has emerged to store, dis-tribute, integrate, and analyze this flood of biolog-ical data. Bioinformatics is a field that encom-passes aspects of several disciplines, includinginformation technology, computer science, statis-tics, and molecular biology/genetics. The bioin-formatics program at VARI focuses on using acomputational approach to understand how cancercells differ from normal cells at the molecularlevel. In addition, we assist in the analysis of largeand small data sets that are generated both withinVARI and as part of external collaborations.

Assembled DNA sequence information forhumans and mice has recently become available. Toallow investigators at VARI to take advantage of thisknowledge, we have downloaded the Ensembl ver-sion of the public human sequence database. Inaddition, we have several subscriptions to the Celerahuman and mouse databases. As sequence annota-tions are constantly being updated by the EuropeanBioinformatics Institute, the National Center forBiological Information, and other institutes, we col-lect the sequence information from the varioussources and summarize and distribute the results. Inaddition, we constantly monitor public genesequence databases to ensure that as more genesequences have cellular functions attributed to them,this information is available to our researchers.

We also have active areas of research in theanalysis of DNA microarray data. The DNA

microarray technology allows the measurementof expression levels for tens of thousands ofgenes in a single experiment. To help determinegene expression values that change in a signifi-cant way between two sample groups (i.e., nor-mal tissue versus tumor tissue), we have devel-oped several programs to perform specialized sta-tistical analysis on microarray data sets. Of spe-cial interest is a new technology we have devel-oped to identify tumor cell chromosomal abnor-malities from gene expression microarray data.This technique organizes genes by their genomemapping location and then scans for genomicregions that contain a disproportionate number ofgenes that show either increased or decreasedexpression (Figure 1). We have termed thisanalysis comparative genomic microarray analy-sis, or CGMA, as regional gene expression bias-es often indicate chromosomal losses or gains.We hope to develop this technology further toallow more in-depth analysis of chromosomalchanges in cancer cells and to identify candidategenes whose expression changes most in regionsof frequent cytogenetic change.

Because many types of data analysis are com-putationally intensive, we are developing an infra-structure (as part of a collaboration) that will allowmore-sophisticated computational analysis. Thisinfrastructure, called cluster or grid computing, dis-tributes a large computational workloads over manylow-cost computers. Following completion of theanalysis, a monitoring computer collects all of thedata from the smaller computers and assembles theresults. This type of computing is beneficial as arelatively small group of low-cost computers canefficiently process a large computational workload.

AResearch Projects

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External Collaborators

Greg Wolffe, Grand Valley State University,Allendale, Michigan

From left to right: Crawley, Furge, Dere

Publications

Furge, Kyle A., Ramsi Haddad, Jeremy C. Miller, J. Schoumans, Brian B. Haab, Bin T. Teh, Lonson Barr,and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon ade-nocarcinoma and associated peritoneal metastasis reveals consistent cytogenetic and transcriptionalaberrations associated with progression of multiple metastases. Applied Genomics and Proteomics.

Furge, Kyle A., David Kiewlich, Phuong Le, My Nga Vo, Michel Faure, Anthony R. Howlett,Kenneth E. Lipson, George F. Vande Woude, and Craig P. Webb. 2001. Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase.Proceedings of the National Academy of Sciences U.S.A. 98(19): 10722-10727.

Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, B.T. Teh, L. Barr, andCraig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adeno-carcinoma and associated intraperitoneal metastases reveals consistent cytogenetic and transcriptionalaberrations associated with progression of multiple metastases. Applied Genomics and Proteomics.

Rhodes, Daniel R., Jeremy C. Miller, Brian B. Haab, and Kyle A. Furge. 2002. CIT: identification ofdifferentially expressed clusters of genes from microarray data. Bioinformatics 18(1): 205–206.

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Figure 1. Identification of gene expression biaseson chromosome 8. A sliding window algorithm wasused to scan for regional gene expression biases onchromosome 8. Regions of amplification (red) or dele-tion (green) can be detected by finding regions ofexpression biases that pass a significance threshold (inthis example, the 95% confidence interval). Here, theportions of the p-arm of chromosome 8 above 95% arelost and the portions of the q-arm are gained. The 8ploss and 8q gain was confirmed by comparativegenomic hybridization.

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Serum protein profiling and marker identification using antibody microarrays

A) Scanned image of an antibody microarray, in which 48 antibodies targeting serum proteins were each spottedeight times on the array. Two serum samples—a test sample and a reference sample—were each labeled with oneof two different-colored fluorescent dyes and incubated on the array. The array was scanned for sample-specific andreference-specific fluorescence, which reveal the relative protein binding to each antibody from the test and the ref-erence samples.

B) Two-way hierarchical clustering of microarray data from 53 serum samples (horizontal axis) and antibody meas-urements from four replicate experiment sets (vertical axis). Each colored square represents one antibody measure-ment from one array. The color and intensity of each square represents the relative protein binding from the sampleversus the reference, red representing higher from the sample and green, higher from the reference. The red branchesof the dendrogram indicate serum samples from prostate cancer patients, and the blue branches indicate serum sam-ples from the controls. ELISA measurements of various serum proteins cluster tightly with the microarray measure-ments from the respective antibodies, showing the accuracy of the microarray measurements.

C) Proteins with significantly different serum levels between the prostate cancer samples and the controls. The soft-ware program CIT calculated p-values for each antibody in the data from panel B. In the cancer patients, vonWillebrand factor was higher and the other proteins were lower; all varied independently of PSA (column 3). Theseproteins, together with other markers or clinical indicators, may be useful in the clinical evaluation of prostate cancer.

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Laboratory of DNA and Protein Microarray Technology

Brian B. Haab, Ph.D.Dr. Haab obtained his Ph.D. in chemistry from the University of California atBerkeley in 1998. He then served as a Postdoctoral Fellow in the laboratory ofPatrick Brown in the Department of Biochemistry at Stanford University. Dr. Haabjoined VARI as a Special Program Investigator in May 2000.

Laboratory Members

Core facilityRamsi Haddad, Ph.D.Peterson Haak, B.S. Joshua Kwekel, B.S.Paul Norton, B.S.

Research laboratoryHeping Zhou, Ph.D.Kerri Kaledas, B.S.Mark Schotanus, B.S.

StudentDaniel Diephouse

he discovery of new disease markers isparticularly necessary for diseases diffi-cult to detect or diagnose at an early,

curable stage. For example, the differentiation ofmalignant from benign disease and the earlydetection of pancreatic cancer are extremely dif-ficult with current imaging and cytological meth-ods. An improved screening tool, such as a reli-able and specific serum assay, would both avoidunnecessary surgery and allow performance ofneeded procedures at a curative stage. The diffi-culties of high-throughput protein detection andquantification make the discovery of a new dis-ease marker challenging.

Antibody microarray analysis of fluids fromcancer patients

A new tool that is potentially well suited tomeet this challenge is the protein microarray. Themicroarray enables highly multiplexed detection ina low-volume, rapid, and sensitive assay. Arobotic arrayer prints antibodies targeting putativeserum markers and cancer-related genes on deriva-tized glass surfaces. Serum samples are incubatedon the surfaces of the arrays, and individual serumproteins bind to the surfaces through specific anti-body–antigen interactions. We are developing andvalidating a variety of methods to detect boundproteins according to the concentration range ofthe proteins. A promising method for high-sensi-tivity detection of low-abundance proteins isrolling circle amplification (RCA), which we aredeveloping and applying in a project with PaulLizardi and Jose Costa at Yale University. We alsoperform direct labeling of serum proteins with Cy3or Cy5 to detect higher-abundance proteins, andwe are developing microspot ELISA for the detec-

tion of very-low-level proteins. These detectionmethods taken together allow us to profile the widerange of protein concentrations that are present inphysiological samples.

We use these methods to acquire protein pro-files of serum and other fluid samples from can-cer patients and controls. The antibodies arechosen to target putative markers and proteinsinvolved in functions that are modulated by can-cer, such as immune system proteins, angiogene-sis proteins, acute phase reactants, growth fac-tors, cytokines, and coagulation proteins. Thepatterns of protein abundances in the serum sam-ples are compared with clinical information toachieve two goals: 1) to define sets of proteinswith potential diagnostic or prognostic informa-tion and 2) to gain insight into the relationshipbetween circulating factors and states of diseaseprogression. In a collaborative study with Bin S.Teh of the Baylor College of Medicine, we vali-dated accurate and specific detection of multipleserum proteins using the microarray assay, andwe identified five serum proteins (vonWillebrand Factor, IgM, IgG, α1-antichy-motrypsin, and villin) that statistically differenti-ated prostate cancer serum samples from controlserum samples (see page 26). Four of these pro-teins had been reported previously as associatedwith prostate cancer, and each has implicationsfor the host response to the cancer. Furtherinsight into the alterations of the secretory activ-ity of prostatic epithelial cells is being gatheredin a project with Anthony Schaeffer and JohnGrayhack to study the protein profiles of prosta-tic fluid samples. In collaboration with PaulLizardi and Jose Costa at Yale University, we are

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TResearch Projects

studying the protein alterations in the sera of pan-creatic cancer patients.

Microarray core facility

The DNA microarray is widely regarded as arevolutionary technology in biological research.Since its introduction about 10 years ago, microar-rays have grown in use and usefulness and havecontributed to many significant discoveries.VARI’s microarray core facility makes this tech-nology accessible and useful to its researchers andto external collaborators. We have acquired setsof 40,000 human cDNA clones, 15,000 mousecDNA clones, and through our collaboration withthe Core Technology Alliance of the MichiganLife Sciences Corridor, 20,000 rat cDNA clones.A high-throughput liquid-handling robot is usedto prepare these DNA sequences for microarrays,and a robotic arrayer spots the DNAs at high den-sity onto the surfaces of glass slides (about 20,000spots in 2 × 4 cm). The high-density spotting ofDNA sequences allows simultaneous hybridiza-tion assays on thousands of genes. Rigorous qual-ity control at every level of the microarray pro-duction and use has allowed us to routinely gener-ate high-quality data, and more than 1,000microarray experiments will have been performedat VARI in the year 2002.

The core facility provides training in the useand analysis of microarrays and access to the lat-est analysis tools. Support from VARI bioinfor-maticians Kyle Furge and Edward Dere, along

with a new microarray database built by theInformation Technology department at VARI,provides extensive informatics capability formicroarray users. The core is also involved intechnology and methods development. Newarrays are being produced that comprise onlysequences from verified and named genes, orsequences pertaining to particular projects.Focused gene sets allow replicate spotting ofeach sequence and better verification of the qual-ity of each sequence. Arrays made from sets of70-base oligonucleotides, which may providereduced cross-reactivity with other genes relativeto cDNA clones, are being validated and charac-terized to complement our existing cDNA arrays.In addition, we are implementing methods ofsignal amplification to allow detection of lowquantities of RNA.

Applications of DNA microarrays includethe study of the mRNA expression patterns inhuman tumor samples and the study of changesin cell-line gene expression after perturbations.The data are analyzed to identify genes that sta-tistically correlate with other genes, phenotypes,clinical parameters, disease states, or perturba-tion states. A detailed analysis of gene expres-sion programs can yield insight into the functionand interrelationship of genes and can suggeststrategies of intervention in disease. Many VARIresearchers, as well as external researchers, aremaking use of VARI’s microarray facility forsuch studies.

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External Collaborators

Phil Andrews, Samir Hanash, and Gil Omenn, University of Michigan, Ann Arbor

Jose Costa and Paul Lizardi, Yale University School of Medicine, New Haven, Connecticut

Yi Ren, University of Hong Kong

Anthony Schaeffer and John Grayhack, Northwestern University, Evanston, Illinois

Peter Schirmacher, University of Cologne, Germany

Bin S. Teh, Baylor College of Medicine, Houston, Texas

Cornelius Verweij, University of Amsterdam, The Netherlands

Tim Zacharewski, Michigan State University, Lansing

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Publications

Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, Bin T. Teh, L. Barr,and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colonadenocarcinoma and associated intraperitoneal metastases reveals consistent cytogenetic andtranscriptional aberrations associated with progression of multiple metastases. AppliedGenomics and Proteomics.

Miller, Jeremy C., E. Brian Butler, Bin S. Teh, and Brian B. Haab. 2001. The application of proteinmicroarrays to serum diagnostics: prostate cancer as a test case. Disease Markers 17(4):225–234.

Miller, Jeremy C., Heping Zhou, Joshua Kwekel, Robert Cavallo, Jocelyn Burke, E. Brian Butler,Bin S. Teh, and Brian B. Haab. In press. Antibody microarray profiling of human prostate can-cer sera: antibody screening and identification of potential biomarkers. Proteomics.

Rhodes, Daniel R., Jeremy C. Miller, Brian B. Haab, and Kyle A. Furge. 2002. CIT: identificationof differentially expressed clusters of genes from microarray data. Bioinformatics 18(1):205–206.

Robinson, William H., Carla DiGennaro, Wolfgang Hueber, Brian B. Haab, Makoto Kamachi, ErikJ. Dean, Sylvie Fournel, Derek Fong, Mark C. Genovese, Henry E. Neuman de Vegvar, GunterSteiner, David L. Hirschberg, Sylviane Muller, Ger J. Pruijn, Walther J. van Venrooij, Josef S.Smolen, Patrick O. Brown, Lawrence Steinman, and Paul J. Utz. 2002. Autoantigen microar-rays for multiplex characterization of autoantibody responses. Nature Medicine 8(3): 295–301.

From left to right, back row: Haak, Kwekel, Vreder, Diephouse, Schotanusfront row: Norton, Zhou, Kaledas, Haddad, Haab

Laboratory of Cancer Pharmacogenetics

Han-Mo Koo, Ph.D.Dr. Koo received his Ph.D. in microbiology and molecular genetics at Rutgers–TheState University of New Jersey in 1993. He then served as a Senior PostdoctoralFellow in the laboratory of George Vande Woude in the Molecular Oncology Sectionof the Advanced BioScience Laboratories–Basic Research Program at the NationalCancer Institute–Frederick Cancer Research and Development Center, Maryland.In June 1999, Dr. Koo joined VARI as a Scientific Investigator.

Laboratory Members

StaffKate Eisenmann, Ph.D.Matt VanBrocklin, M.S.Nancy Staffend, B.S.

StudentsSusan KitchenTracey Millard

dvances in our understanding of themolecular pathophysiology of humancancers open promising opportunities

for the prevention of and intervention in cancer.Our laboratory is interested in studying mecha-nisms of drug actions, identifying novel thera-peutic targets, and developing novel anticanceragents by means of molecular-targetingapproaches.

Mitogen-activated protein kinase (MAPK)signaling pathways are highly conserved amongall eukaryotes and are integral for the transduc-tion of a variety of extracellular signals.Furthermore, constitutive activation of MAPKsignaling (e.g., the Raf-MEK1/2-ERK1/2 path-way) contributes to many aspects of human can-cers; hence, the pathway has been identified as apotential therapeutic target for cancer interven-tion. Typically, cancer cells exhibit a cytostatic(growth arrest) response to the disruption ofMAPK signaling. However, we have recentlydemonstrated that interfering with the MAPKsignaling pathway evokes a cytotoxic response(apoptosis) in human melanoma cells but not innormal melanocytes: either anthrax lethal toxin(which proteolytically cleaves MAPK kinases[MEKs]) or small-molecule MEK inhibitors(such as PD90859 and U0126) triggers an apop-totic response in human melanoma cells.Normal melanocytes treated with the sameinhibitors, on the other hand, simply arrest in theG1 phase of the cell cycle. More importantly, invivo treatment with anthrax lethal toxin ofhuman melanoma xenograft tumors in athymicnude mice renders either significant or complete

tumor regression without apparent side effects.These results indicate that the MAPK signalingpathway represents a tumor-specific survival sig-naling in melanoma and that inhibition of thispathway may be a useful and potentially selec-tive strategy for treating this cancer.

Our current research focuses on molecularcharacterization of the MAPK pathway–associ-ated survival signaling in melanoma cells. Inparticular, we are investigating the phosphoryla-tion and inactivation of the pro-apoptotic proteinBad mediated by the 90 kDa ribosomal S6kinase. The molecular mechanism by which theinhibition of MAPK signaling specifically trig-gers apoptosis in human melanoma cells shouldreveal additional molecular targets useful forprevention of and intervention in melanoma, aswell as in other MAPK-associated cancers suchas pancreatic, lung, colon, and breast carcino-mas, as well as gliomas. Additionally, furthervalidation studies are ongoing to clinically devel-op the MAPK signaling pathway as a therapeutictarget for melanoma treatment.

Activating mutations in RAS oncogenes arethe most frequent gain-of-function mutationsdetected in human cancers. Besides their well-documented role in cellular transformation andtumorigenesis, we have previously shown thatthe RAS oncogenes play an important role insensitizing tumor cells to deoxycytidine ana-logues such as 1-β-D-arabinofuranosylcytosine(Ara-C) and gemcitabine, as well as to topoiso-merase (topo) II inhibitors, more prominently toetoposide. These results are supported by clini-cal findings that patients who have RAS onco-

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AResearch Projects

gene–positive acute myeloid leukemia show anincreased remission rate, longer remission dura-tion, and improved overall survival in response toa combination therapy of Ara-C plus topo IIinhibitor. To translate our results into a clinicaltrial, we have established a collaboration with theGrand Rapids Clinical Oncology Program and

the Spectrum Health Cancer Program. This sum-mer, through this collaboration, we have initiateda Phase II trial to evaluate the gemcitabine +etoposide combination treatment for patientswith locally advanced or metastatic pancreaticcarcinomas, which display RAS oncogene acti-vation in over 95% of the cases.

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External Collaborators

Thomas M. Aaberg, Jr., Associated Retinal Consultants, Grand Rapids, Michigan

Alan Campbell, Spectrum Health Cancer Program, Grand Rapids, Michigan

Marianne K. Lang, Timothy J. O’Rourke, and Connie Szczepanek, Grand Rapids Clinical OncologyProgram, Michigan

Won Kyu Lee, Kent Pathology Laboratory, Ltd., Grand Rapids, Michigan

Judith S. Sebolt-Leopold, Pfizer Global Research & Development, Ann Arbor, Michigan

Lilly Research Laboratories, a division of Eli Lilly and Company, Indianapolis, Indiana

Publications

Koo, Han-Mo, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Anthrax toxins, mitogen-activated protein kinase pathway, and melanoma treatment. Directions in Science 1: 123–126.

Koo, Han-Mo, Matt VanBrocklin, MaryJane McWilliams, Stephan H. Leppla, Nicholas S. Duesbery,and George F. Vande Woude. 2002. Apoptosis and melanogenesis in human melanoma cellsinduced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase.Proceedings of the National Academy of Sciences U.S.A. 99(5): 3052–3057.

From left to right: Kitchen, VanBrocklin, Millard, Staffend, Eisenmann, Koo

Laboratory of Integrin Signaling and Tumorigenesis

Cindy K. Miranti, Ph.D.Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982and her Ph.D. in biochemistry from Harvard Medical School in 1995. She was aPostdoctoral Fellow from 1995 to 1997 in the laboratory of Joan Brugge at ARIADPharmaceuticals, Cambridge, Massachusetts, and from 1997 to 2000 in theDepartment of Cell Biology at Harvard Medical School. Dr. Miranti joined VARI asa Scientific Investigator in January 2000; she is also an Adjunct Assistant Professorin the Department of Physiology at Michigan State University.

Laboratory Members

StaffSuganthi Chinnaswamy, Ph.D.Andrew Putnam, Ph.D.Veronique Schultz Patacsil, B.S.

StudentsHeather Bill, B.S.Andrea Pearson

ur laboratory is interested in understand-ing the mechanisms by which integrinreceptors interacting with the extracellu-

lar matrix regulate cell function in normal andtumorigenic processes. Alterations in integrinreceptors and their downstream signaling targetsare common events in tumorigenesis, leading to adisruption of normal cell function. Using tissue-culture models, biochemistry, molecular genetics,and ultimately mouse models, we are definingthe signaling pathways and molecular eventsinvolved in integrin-dependent adhesion andmigration that are important for tumorigenesis ingeneral and specifically for melanoma andprostate cancer.

Role of integrins in tumorigenesis

Integrins are a class of heterodimeric trans-membrane receptors for which there are current-ly 24 known family members: 15 alpha and 9beta subunits. Each subunit contains a shortcytoplasmic region with no known enzymaticactivity, but through protein-protein interactions,subunits are able to interact with actin-contain-ing microfilaments and important signaling mol-ecules. Thus, the engagement of the integrinreceptor by extracellular matrix componentsinduces changes in actin structures, as well as theinduction of several signal transduction path-ways. Both the loss and gain of different inte-grins contribute to tumorigenesis and metastasisin many tumor types. In addition to changes inintegrin expression, other contributors to tumori-genesis are alterations in integrin ligands, altered

regulation of integrin function, or alterations inintegrin-dependent signal transduction pathways.

How integrins activate growth factor receptors

Recent work in our laboratory has focusedon characterizing the interactions between inte-grins and receptor tyrosine kinase. Adhesion ofepithelial cells to several different extracellularmatrices induces ligand-independent activationof the epidermal growth factor receptor (EGFR)and the Met receptor. Overexpression or muta-tion of EGFR family members or the Met recep-tor are common events in many epithelialtumors. We have shown that by recruitingEGFR, integrins are able to activate a subset ofintegrin-induced signaling pathways (Figure 1).In the absence of EGFR activation, the ability ofthe cells to induce the Ras/Erk signaling pathwayand Akt is severely impaired. However, not all

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OResearch Projects

Figure 1. Integrin-induced activation of EGFR isrequired for a subset of integrin-regulated signal-ing pathways

integrin signaling pathways are dependent onEGFR (e.g., FAK, Src, and PKC).

We further have demonstrated that integrin-mediated adhesion of epithelial cells, includingprimary prostate epithelial cells, is sufficient toinduce several G1 cell cycle events, includingincreases in cyclin D1, p21, cdk4 kinase activity,and Rb phosphorylation. This is dependent onintegrin activation of EGFR, Erk, and PI-3K(Figure 2). However, adhesion alone was not

sufficient for induction of DNA synthesis, indi-cating that additional signals are required. Weare currently attempting to define what steps inG1 are blocked. Interestingly, HGF-mediatedinduction of DNA synthesis through the Metreceptor was also dependent on integrin activa-tion of EGFR. These data indicate that integrinregulation of EGFR activation is a critical medi-ator of cell cycle regulation. Integrin-mediatedregulation of EGFR may be one mechanism thattumor cells use to regulate cell growth in theabsence of exogenous growth factor. We are alsoexploring the mechanisms by which integrinsactivate EGFR and how integrins cooperate withthe Met receptor to regulate cellular signaling.

Integrin signaling in prostate cancer

The development of metastatic prostate can-cer is slow and is accompanied by the loss ofandrogen sensitivity. In normal epithelial cells,the α6 integrin is usually found in associationwith β4 integrin (α6β4) and is specifically local-ized to desmosomal junctions. However, inprostate carcinoma, β4 integrins are often lost,and there is a concomitant increase in the α6β1

and α3b1 integrins and a loss of typical epithelialstructures. We are interested in understandinghow α6β1 and α3β1 integrins contribute to late-stage prostate carcinoma and how androgen mayregulate this process.

The integrins α6β1 and α3β1 are knowninteract with an integrin-associated protein calledCD82 (or alternatively, KAI1). Loss of expres-sion of CD82 correlates with prostate metastasis,and the loss of CD82 would be predicted to alterthe function of α6β1 and α3β1 integrins. Usingprimary prostate epithelial cells, which expresshigh levels of CD82, as well as several prostatetumor cell lines that do not, we are exploring therole of CD82 in regulating α6β1- and α3β1-mediated cell adhesion, migration, and cell sig-naling. We are using molecular genetic approach-es such as mutagenesis, siRNA, and mouse mod-els to alter CD82 expression in prostate cells.

Integrin regulation of melanoma progression

The incidence of melanoma has been steadi-ly increasing in the last 10 years. If caught at anearly stage it is usually curable, but once it hasbecome invasive, metastatic melanoma is virtual-ly untreatable and progresses very rapidly.Induced expression of the αvβ3 integrin corre-lates with increased invasive capacity ofmelanomas, yet the mechanisms underlying thisshift in expression and increased invasiveness areunknown. We have initiated studies to determinehow expression of the αvβ3 integrin in normalmelanocytes alters cell function and the integrin-dependent signaling pathways involved.

The serine/threonine protein kinase family,PKC, is a family of 11 related kinases that canbe separated into three major classes: classical,novel, and atypical. This kinase family has beenimplicated in differentiation, growth regulation,cell survival, cell adhesion, cell migration, andtumorigenesis, but the exact role of each of thesekinases is largely unknown. In normalmelanocytes, PKC is required for cell growthand survival; in tumor cells, however, stimula-tion of PKC activity can result in growth arrestand cell death. In addition, PKC plays an impor-tant role in regulating cell adhesion and migra-tion. We are interested in understanding howchanges in expression of different PKC isoformscan regulate melanoma proliferation, migration,and invasion.

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Figure 2. Integrin-induced activation of EGFR,and subsequently Erk and PI-3K, is required for aentry into G1 of the cell cycle, but is not suffi-cient for entry into S phase

The adhesion of normal melanocytes to theextracellular matrix induces the formation of focaladhesion complexes and actin stress fibers (Figure3), but in a highly metastatic melanoma cell line,these structures are absent. We are exploring thebiochemical basis for this difference. We havefound that the activity level of Rac (a small GTPaserequired for regulating actin structure) is elevatedand that the inhibition of PKC blocks this activity.The levels of PKCa are elevated in these cells aswell. We are exploring the effects on cell structure,adhesion, and migration of PKCα levels.

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Figure 3. Focal adhesions (green spots) and stressfibers (red fibers) are absent in metastatic melanoma

External CollaboratorsJoan Brugge, Harvard Medical School, Boston, Massachusetts

Beatrice Knudsen, Cornell University Medical College, New York, New York

Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York

Benjamin Neel, Beth Israel Deaconess Medical Center, Harvard Institute of Medicine, Boston, Massachusetts

David Shalloway, Cornell University, Ithaca, New York

Sheila Thomas, Harvard Institute of Medicine, Boston, Massachusetts

Publications

Miranti, Cindy K. 2002. Application of cell adhesion to study signaling networks. In Methods in Cell MatrixAdhesion, J.C. Adams, ed. Methods in Cell Biology series, San Diego: Academic Press, pp. 359–383.

Miranti, Cindy K., and Joan S. Brugge. 2002. Sensing the environment: a historical perspective onintegrin signal transduction. Nature Cell Biology 4(4): E83–E90.

Woodside, Darren G., A. Obergfell, Lijun Leng, Julie L. Wilsbacher, Cindy K. Miranti, Joan S. Brugge,Sanford J. Shattil, and Mark H. Ginsberg. 2001. Activation of Syk protein tyrosine kinase throughinteraction with integrin β cytoplasmic domains. Current Biology 11(22): 1799–1804.

Left to right: Pearson, Putnam, Bill, Patacsil, Miranti

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Human breast ductal epithelium

This tissue was stained with two antibodies. In red is c-Met (polyclonal antibody c28) and in green is c-neu(monoclonal antibody OCS). c-neu stains for the tyrosine kinase receptor Her2-neu (involved in cell signalprocesses) that is amplified in breast cancer in 10–20% of primary cases. The protein c-Met is also a tyrosinekinase receptor that is activated by the ligand hepatocyte growth factor/scatter factor (HGF/SF); c-Met hasbeen shown to be a prognostic marker for human breast cancer. Co-localized foci are colored yellow, whilered and green label the Met and her2neu separately. The c-Met is evident on the lumenal and basal border; c-neu is less specific but may be increased in the lateral boundaries between cells.(Resau)

Analytical, Cellular, and Molecular Microscopy Laboratory

James H. Resau, Ph.D.Dr. Resau received his Ph.D. from the University of Maryland School of Medicine in1985. Between 1968 and 1994, he was in the U.S. Army (active duty and reserveassignments) and served in Vietnam. From 1985 until 1992, Dr. Resau was a fac-ulty member of the University of Maryland, School of Medicine, Department ofPathology, and was a tenured Associate Professor from 1990–1992. Dr. Resauthen went to the NCI to be Director of the Analytical, Cellular and MolecularMicroscopy Laboratory in the Advanced BioScience Laboratories–Basic ResearchProgram at the National Cancer Institute–Frederick Cancer Research andDevelopment Center, Maryland (1992–1999). Dr. Resau joined VARI as a SpecialProgram Investigator in June 1999.

Laboratory Members

StudentsHien DangMarie GravesLateefah GrayMaketta Hassen

Brandon LeeserMatthew MainChristine MooreJeanine Myles

StaffBree Buckner, B.S., HTL (ASCP), QIHCEric Hudson, B.S. J.C. Goolsby

ur laboratory works closely with VARIinvestigators, as well as in collaborationwith outside parties, to provide a num-

ber of microscopy needs. We have a special inter-est in the quantification of imagery. We have twoconfocal microscopes that enable us to visualizeorganelles and processes in cells and tissues suchas receptor–ligand interactions and co-localiza-tion of proteins with organelles. We have studiedthe location of two gene-targeted proteins withina cell (i.e., GFP and RFP) with a DNA marker,DAPI, in three dimensions. We have integratedlaser-capture microdissection instrumentationinto the program, as well as paraffin and frozen-section staining. We also provide histotechnolo-gy services, consultation on staining, and direc-tion for the human tissue services.

In collaboration with George Vande Woudeand Rick Hay of VARI, we have developed theTissue Collection Initiative between our Instituteand the Spectrum Health System in Grand Rapids.We have expanded the program to include theHolland, Pennock, and Hackley hospitals. Thisprogram provides for the collection and characteri-zation of fresh-frozen surgical tissues that willallow investigators to create a working repositoryfor a wide range of projects. Surgically removedhuman tumors and normal tissue will be evaluatedin institutional review board (IRB)–approved basicand translational research projects. This collectionwill at the same time provide to the physician

researcher access to research collaborations withthe intention of facilitating translation of new diag-nostic, treatment, and evaluation protocols. Weplan to generate gene expression profiles (microar-ray), establish new tumor cell lines, and developnew diagnostic and therapeutic agents through thiscollaboration. Epidemiologic evaluations will alsobe greatly improved by the coordination of clinicalinformation, diagnosis, and research results. Thegoal of this project is to develop genetic-baseddiagnostic classification of human disease. There isa Scientific Advisory Board for this project com-prising members of VARI and the Spectrum Healthpathology, surgical and medical oncology, and sur-gery departments. Tissue is collected with explicitwritten permission of the participating physiciansand patients. Protocols for the use of the materialin this archive require the approval of both theVARI and the Spectrum Health IRBs.

Directly related to this archive is the paraffin-block repository called SPIN (Shared PathologyTissue and Informatics Network), a project thatinvolves the same hospitals in west Michigan. Thisarchive stores and catalogs paraffin blocks that areolder than five years and ordinarily would bedestroyed. Currently the archive holds approxi-mately 150,000 tissue samples/paraffin blocks.They are not directly linked to any personal identi-fiers or names and there is limited demographicinformation available. Of the 150,000 samples orblocks, clinical and demographic data is available

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OResearch Projects

for about 20%. The material from future years willbe available with digital information on age, sex,and diagnosis. The current samples were collectedby the hospitals with nondigital database technolo-gy, and the information is slowly being transcribed.These samples will be used in cellular and molecu-lar protocols approved by our IRB. The samplesand demographics are identified with basic infor-mation (such as diagnosis, age, sex, etc.) in a web-based, interactive format for determination of prog-nosis, diagnosis, and therapy. In the first six monthsof operation of SPIN there have been 12 users reg-istered who have submitted 83 requests for search-es and 33 subsequent tissue requests. We have pro-vided tissue and histopathology services for 12VARI investigators and have generated over 72,000microscopic images and related image files for thecollaborations. A major part of both the tissue ini-tiative and SPIN programs will be to annotate andupdate information on each specimen.

Our own research interest is in the retrospectivereview of archival tissues from individual sampleswith known clinical outcomes. We identify andquantify the location of particular proteins andexamine the relationship between their pattern ofexpression and the prognosis of disease progres-sion. We have submitted the results from our U.S.Army Breast Cancer–funded analysis of Met andHer2neu in human breast cancer and have nearlycompleted a major study in collaboration withinvestigators in Chicago on the same problem. Theadvantage of archival material is that new markersof prognosis can be evaluated. We can measure upto four proteins simultaneously, and we are devel-oping methods to combine confocal microscopy,gene expression, and laser-capture microdissection

with traditional surgical pathology diagnostic pro-cedures to determine prognosis via an objective andquantifiable method(s). These tools—and, moreimportantly, the process—will have application tomany types of human disease.

We have recently obtained NIH funding for amajor effort in multiphoton imaging of develop-mental and carcinogenic events in GFP-expressingtransgenic mice. George Vande Woude, IlanTsarfaty, and I will evaluate the role of Met andHGF/SF in branching morphogenesis, carcinogen-esis, and therapy of cancer-related compounds.Other collaborations within VARI involve Met andHGF-SF in cells and tissues; the role of Met andHGF-SF in prostate and breast carcinogenesis; thelocation of gene-targeted proteins in rodents; evalu-ation of monoclonal antibodies as diagnosticreagents; application of human tissues to molecu-lar-based studies; and the cellular and subcellularlocalization and quantification of proteins. We alsohave ongoing collaborations with scientists in othercountries. We have in development a molecularimaging project with scientists at Tel AvivUniversity in Israel, and we have an internationalbreast cancer project to evaluate Her2/neu andMet/HGF interactions with scientists in Germany.

In addition to our research program, we have along-standing interest in science education.Together with Grand Valley State University andGrand Rapids Community College, we havereceived NIH funding as part of the Bridges to theBaccalaureate program to support the recruitmentand graduation of women and minorities into sci-ence, mathematics, and research careers. Dr. Resauis a co-investigator and site coordinator for theBridges program.

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External Collaborators

Stephan Baldus, University of Cologne, Germany

Maria Birchenall-Roberts, Francis Ruscetti, Jerrold Ward, and George Pavlakis, National CancerInstitute, Frederick, Maryland

Ruth Heilmann, University of Chicago, Illinois

Iafa Keydar and Ilan Tsarfaty, Tel Aviv University, Israel

Justin McCormick, Michigan State University, Lansing

Toshio Mura, National Cancer Institute, Bethesda, Maryland

John Sacci, University of Maryland, Baltimore

Duane Smoot, Howard University, Washington, D.C.

Publications

Albright, Craig D., Philip M. Grimley, Raymond T. Jones, and James H. Resau. 2002. Differentialeffects of TPA and retinoic acid on cell-cell communication in human bronchial epithelial cells.Experimental and Molecular Pathology 72(1): 62–67.

Kino, Tomoshige, Roland H. Stauber, James H. Resau, George N. Pavlakis, and George P. Chrousos.2001. Pathologic human GR mutant has a transdominant negative effect on the wild-type GR byinhibiting its translocation into the nucleus: importance of the ligand-binding domain for intra-cellular GR trafficking. Journal of Clinical Endocrinology and Metabolism 86(11): 5600–5608.

Kort, Eric, Bryon Campbell, and James H. Resau. In press. A shared pathology informatics net-work. Computer and Programs in Biomedicine.

Miura, Koichi, Kerry M. Jacques, Stacey Stauffer, Atsutaka Kubosaki, Kejin Zhu, Dianne SnowHirsch, James Resau, Yi Zheng, and Paul A. Randazzo. 2002. ARAP1: a point of convergencefor Arf and Rho signaling. Molecular Cell 9(1): 109–119.

Miura, Koichi, Shoko Miyazawa, Shuichi Furuta, Junji Mitsushita, Keiju Kamijo, Hiroshi Ishida,Toru Miki, Kazumi Suzukawa, James Resau, Terry D. Copeland, and Tohru Kamata. 2001. TheSos1-Rac1 signaling: possible involvement of a vacuolar H+-ATPase E subunit. Journal ofBiological Chemistry 276(49): 46276–46283.

Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-MeiWang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H.Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patientswith late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596.

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From left to right, back row: Hassen, Graves, Moore, Buckner, Resau, Leeser, Hudsonfront row: Myles, Goolsby

Laboratory of Germline Modification

Pamela J. Swiatek, Ph.D.Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology fromIndiana University. From 1988 to 1990, she was a Postdoctoral Fellow at theTampa Bay Research Institute. From 1990 to 1994, she was a Postdoctoral Fellowat the Roche Institute of Molecular Biology in the laboratory of Tom Gridley. From1994 to 2000, Dr. Swiatek was a Research Scientist and Director of the TransgenicCore Facility at the Wadsworth Center in Albany, New York, and an AssistantProfessor in the Department of Biomedical Sciences at the State University of NewYork at Albany. She joined VARI as a Special Program Investigator, Laboratory ofGermline Modification, in August 2000.

Laboratory Members

StaffKathy Davidson, B.S.Kelly Sisson, B.S.

StudentCassandra Van Dunk

he Germline Modification Laboratoryprovides transgenic and gene-targetingtechnology services to develop mouse

models of human disease. These well-establishedand powerful techniques are used to insert specificgenetic changes into the mouse genome in order tostudy the effect of these mutations in the complexbiological environment of a living organism.These changes can include the introduction of agene into a random site in the genome (transgen-ics), introduction of a gene into a specific site inthe genome (gene knock-in), or the inactivation ofa gene already present in the genome (gene knock-out). Since these mutations are introduced into thereproductive cells known as the germline, they canbe used to study the developmental aspects of genefunction associated with inherited genetic diseases.

Transgenic mice are produced by injectingsmall quantities of foreign DNA into a pronucle-

us of a one-cell fertilized egg. Fertilized eggscontain two pronuclei, one that is derived fromthe egg and contains the maternal genetic mate-rial and one derived from the sperm that containsthe paternal genetic material. As developmentproceeds, these two pronuclei fuse, the geneticmaterial mixes, and the cell proceeds to divideand develop into an embryo. DNA microinject-ed into a pronucleus randomly integrates into themouse genome and will theoretically be presentin every cell of the resulting organism.Expression of the transgene is controlled bygenetic elements called promoters that are genet-ically engineered into the transgenic DNA.Depending on the selection of the promoter, thetransgene can be expressed in every cell of themouse or in specific cell populations such as neu-rons, skin cells, or blood cells. Temporal expres-sion of the transgene during development can

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Bryn Eagleson, A.A.Bryn Eagleson began her career in laboratory animal services in 1981 with LittonBionetics at the National Cancer Institute’s Frederick Cancer Research andDevelopment Center (NCI-FCRDC) in Maryland. In 1983, she joined the Johnson& Johnson Biotechnology Center in San Diego, California. In 1988, she returned tothe NCI-FCRDC, where she continued to develop her skills in transgenic technolo-gy and managed the transgenic mouse colony. During this time she attendedFrederick Community College and Hood College in Frederick, Maryland. In 1999,Bryn joined VARI as the Vivarium Director and Transgenic Core Manager.

Managerial StaffJason Martin, RLATG

Technical StaffDawna Dylewski, B.S.Audra Guikema, B.S., L.V.T.Lori Ruff, B.S., RALATKristen Van Noord, B.S.,

RALAT

Vivarium StaffShawn Ballard, A.S., B.A.,

RALATBen Buckrey, B.S.Elissa Boguslawski

TResearch Projects

also be controlled by genetic engineering. Thesetransgenic mice are excellent models for studyingthe expression and function of the transgene inthe biological environment of the living mouse.

Gene-targeting mutations are introduced intothe mouse by genetic manipulation of pluripotentembryonic stem (ES) cells. ES cells, which arederived from 3.5-day-old embryos called blasto-cysts, have the potential to contribute to all tis-sues of a developing mouse. Genomic DNA con-taining the gene of interest is isolated, mutated,and inserted into ES cells. The mutated geneintegrates into the genomes of the ES cells and,by a process called homologous recombination,replaces one of the two wild-type copies of thegene in the cells. These genetically modifiedcells, containing one mutant copy of the gene,are injected into wild-type blastocysts wherethey integrate into the developing embryo. Theseembryos, containing a mixture of wild-type andmutant ES cells, develop into offspring calledchimeras. Offspring of chimeras that inherit themutated gene are called heterozygotes, becausethey possess one copy of the mutated gene. Theheterozygous mice are bred together, or inter-crossed, to produce mice that completely lack thenormal gene; these homozygous mice have twocopies of the mutant gene and are called gene-targeted or gene “knock-out” mice. A relatedtechnology, gene knock-in, employs similarmethods to insert functional genes into specificlocations in the mouse genome. Ultimately,gene-targeted mice can be observed for abnor-malities associated with the inserted geneticchange, and they provide powerful research toolsfor studying gene function in living organisms.

The Germline Modification Laboratory is afull-service lab that functions at the level of serv-ice, research, and teaching. VARI and Michigan

Life Science Corridor clients will be assisted inthe design and implementation of transgenic andgene-targeting experiments and, if necessary,trained in these techniques. New stem cell linescan be derived, and spectral karyotypic (SKY)analysis of mouse chromosomes—using high-quality, 24-color fluorescent in situ hybridizationpaints—can aid in the detection of subtle andcomplex chromosomal rearrangements in EScells. Upon production of the genetically modi-fied mice, our lab will assist in developing breed-ing schemes and provide for the complete analy-sis of the mutant mice.

The vivarium utilizes two TopazTechnologies software products, Granite andScion, for integrated management of the vivari-um finances, the mouse breeding colony, and theInstitutional Animal Care and Use Committee(IACUC) protocols and records. The efficiencyof mutant mouse production and analysis isenhanced using the Autogen 9600, a robotic,high-throughput DNA purification machine.Imaging equipment, such as the PIXImus MouseDensitometer and the Acuson Sequoia 512 ultra-sound machine, is available for noninvasiveimaging of mice. Mouse strains are archivedusing sperm cryopreservation and reconstitutedusing in vitro fertilization techniques. Additionalservices provided by the vivarium technical staffinclude an extensive xenograft model develop-ment and analysis service, rederivation, surgery,dissection, necropsy, breeding, and health-statusmonitoring. In summary, the goal of thegermline modification laboratory is to develop,provide, and support high-quality mouse model-ing technology services for the Van AndelResearch Institute investigators, Michigan LifeScience Corridor collaborators, and the greaterresearch community.

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External Collaborators

Narayanan Parameswaran, Bill Smith, and Bill Spielman, Michigan State University, Lansing

Douglas Ashley Monk, Michigan State University, Lansing

Gary Litman, University of South Florida, Tampa

Dan Rosen, Wadsworth Center, New York State Department of Health, Albany

Publications

Su, Ting, Qing-Yu Zhang, Jianhua Zhang, Pamela J. Swiatek, and Xinxin Ding. 2002. Expression ofthe rat CYP2A3 gene in transgenic mice. Drug Metabolism and Disposition 30(5): 548–552.

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From left to right: Dylewski, Ruff, Eagleson, Martin, Boguslawski, Buckrey, Guikema, Van Noord, Ballard

From left to right: Swiatek, Van Dunk, Sisson, Davidson

Laboratory of Cancer Genetics

Bin T. Teh, M.D., Ph.D.Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, andhis Ph.D. from the Karolinska Institute, Sweden, in 1997. Before joining the VanAndel Research Institute, he was an Associate Professor of medical genetics at theKarolinska Institute. Dr. Teh joined VARI as a Senior Scientific Investigator inJanuary 2000.

Laboratory Members

StaffMiles Chao-Nan Qian, M.D.,

Ph.D.Libing Song, M.D.. Ph.D.Jun Sugimura, M.D., Ph.D.Jindong Chen, Ph.D.Sok Kean Khoo, Ph.D.David Petillo, Ph.D.Chun Zhang, Ph.D.Eric Kort, M.S.Olga Motorna, B.S.Jason Yuhas, B.S.

StudentsKatherine KahnoskiTodd LaveryCasey MaduraGrace MiguelRadoslav NickolovSarah Scolon

Visiting ScientistsCharlotta Lindvall, M.D.,

Ph.D.Joe Chien-Chung Chou,

M.D.Carola Haven, M.D.Kanthimathi M.S., Ph.D.Vivve Howell, M.S.Jacqueline Schoumans,

M.S.

ancer formation is a multistep processthat results from genetic instability inthe cells. At the molecular level it is

characterized by multiple alterations in genesthat play key regulatory roles in various cellularfunctions. Our laboratory is interested in identi-fying and studying these genetic alterations inboth hereditary cancers and their sporadic coun-terparts. Currently we are focusing on threetypes of tumors: kidney tumors, nasopharyngealcarcinoma, and endocrine (hormone-secreting)tumors. We have close and extensive collabora-tions with researchers and clinicians at hospitalsand universities in this country and overseas.

We have initiated a program to study heredi-tary kidney cancer and to date we have identifiedseveral families with the disease. Both cytogenet-ic and molecular studies have been performed toelucidate this cancer’s genetic basis. We haveidentified a family with a chromosome 3 translo-cation and currently we are trying to clone thebreakpoint genes. We also have mapped the genefor Birt-Hogg-Dubé syndrome, a hereditary can-cer, to chromosome 17. This autosomal dominantdisease is characterized by skin and kidney tumorsand cysts in the lung. In addition, we have beenworking on another hereditary disease, hyper-parathyroidism–jaw tumor syndrome, which ischaracterized by parathyroid tumors, jaw tumors,and kidney cysts and tumors. In sporadic kidney

tumors, we have studied the gene expression pro-files of renal cell carcinoma and correlated themwith clinical outcomes. We have also character-ized the biological profiles of kidney tumors hav-ing different histopathologies, including pediatrickidney tumors (Wilms tumor). We confirm ourmicroarray findings by both real-time polymerasechain reaction (RT-PCR) and immunohistochemi-cal studies on kidney cancer tissue arrays.

Nasopharyngeal carcinoma (NPC) is one ofthe most common cancers in southern China andSoutheast Asia. We are undertaking studies toidentify cancer-related genes in NPC cell linesand primary tumors and to correlate their expres-sion with clinical parameters. In endocrine orhormone-secreting tumors, we continue to workon multiple endocrine neoplasia type 1 and havefound new mutations in new MEN1 families. Weare currently focusing on the mapping of thegene for familial acromegaly, a hereditary condi-tion characterized by tumors in the pituitaryglands that secrete growth hormone.

In addition to carrying out these researchprojects, our laboratory has provided coresequencing and cytogenetic services to theInstitute. To date over 15,000 sequences havebeen performed. We have also performed cyto-genetics studies including FISH, conventionalCGH, and SKY in collaboration with internaland external researchers.

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CResearch Projects

External CollaboratorsWe have extensive collaborations with researchers and clinicians from this country and overseas.

PublicationsDwight, T., S. Kytola, B.T. Teh, G. Theodosopoulos, A.L. Richardson, J. Philips, S. Twigg, L. Delbridge,

D.J. Marsh, A.E. Nelson, C. Larsson, and B.G. Robinson. 2002. Genetic analysis of lithium-associ-ated parathyroid tumors. European Journal of Endocrinology 146(5): 619–627.

Dwight, T., A.E. Nelson, D.J. Marsh, B.T. Teh, C. Larsson, and B.G. Robinson. In press. Parathyroidtumorigenesis in association with primary hyperparathyroidism. Current Opinion in Endocrinologyand Diabetes.

Dwight, T., A.E. Nelson, G. Theodosopoulos, A.L. Richardson, D.L. Learoyd, J. Philips, L. Delbridge, J.Zedenius, B.T. Teh, C. Larsson, D. Marsh, and B.G. Robinson. In press. Independent genetic eventsassociated with the development of multiple parathyroid tumors in patients with primary hyper-parathyroidism. American Journal of Pathology.

Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, B.T. Teh, L. Barr, andCraig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adeno-carcinoma and associated intraperitoneal metastases reveals consistent cytogenetic and transcriptionalaberrations associated with progression of multiple metastases. Applied Genomics and Proteomics.

Lui, Weng-Onn, Jindong Chen, Sven Glasker, Bernhad U. Bender, Casey Madura, Sok Kean Khoo, EricKort, Catharina Larsson, Harmut P.H. Neumann, and Bin T. Teh. 2002. Selective loss of chromo-some 11 in pheochromocytomas associated with the VHL syndrome. Oncogene 21(7): 1117–1122.

Perrier, N.D., A. Villablanca, C. Larsson, M. Wong, B.T. Teh, and O.H. Clark. In press. Genetic screen-ing for MEN1 in “familial isolated hyperparathyroidism.” World Journal of Surgery.

Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric Kort, Chong-Chou Lee, Jindong Chen, Ling-Mei Wang,Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H. Resau, andBin T. Teh. 2002. Met protein expression level correlates with survival in patients with late-stagenasopharyngeal carcinoma. Cancer Research 62(2): 589–596.

Takahashi, M., R. Kahnoski, D. Gross, D. Nicol, and B.T. Teh. 2002. Familial adult renal neoplasia.Journal of Medical Genetics 39(1): 1–5.

Villablanca, Andrea, Filip Farnebo, Bin T. Teh, Lars-Ove Farnebo, Anders Höög, and Catharina Larsson.2002. Genetic and clinical characterization of sporadic cystic parathyroid tumours. ClinicalEndocrinology 56(2): 261–269.

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From left to right: Teh, Qian, Chen, Yuhas, Motorna, Khoo, Song, Petillo, Zhang, Sugimura

Laboratory of Molecular Oncology

George F. Vande Woude, Ph.D.Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from RutgersUniversity. From 1964–1972, he served first as a postdoctoral research associate,then as a research virologist for the U.S. Department of Agriculture at Plum IslandAnimal Disease Center. In 1972, he joined the National Cancer Institute as Headof the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980,was appointed Chief of the Laboratory of Molecular Oncology. In 1983, he becameDirector of the Advanced Bioscience Laboratories-Basic Research Program at theNational Cancer Institute’s Frederick Cancer Research and Development Center, aposition he held until 1998. From 1995, Dr. Vande Woude first served as SpecialAdvisor to the Director, and then as Director for the Division of Basic Sciences atthe National Cancer Institute. In 1999, he was recruited to the Directorship of theVan Andel Research Institute in Grand Rapids, Michigan.

Laboratory Members

StudentsDaphna AtiasMarketta HassenYasser JimenezJason Johnson Nathan LanningAdi Laser, B.S.Kofi ObengDan Wohns

Visiting Scientists & StaffNariyoshi Shinomiya, M.D., Ph.D.Galia Tsarfaty, M.D.Ilan Tsarfaty, Ph.D.

StaffRick Hay, M.D., Ph.D.Chong-Feng Gao, Ph.D.Chong-Chou Lee, Ph.D.Ling-Mei Wang, Ph.D.Yu-Wen Zhang, Ph.D.Dafna Kaufman, M.S.Meg Gustafson, B.A.Yanli Su, A.M.A.T.Mary Beth Bruch

esearch conducted in the Laboratory ofMolecular Oncology uses a broad rangeof approaches to elucidate the molecu-

lar basis of cancer and to develop new agents forthe diagnosis and therapy of cancer. We are pri-marily interested in the expression and activitiesof the receptor tyrosine kinase known as Met, itsinteractions with the ligand hepatocyte growthfactor/scatter factor (HGF/SF), and the intracellu-lar events influenced by Met activation. Aberrantexpression of this receptor–ligand pair confers aninvasive/metastatic phenotype in model systemsof cancer. Inappropriate HGF/SF-Met expressionoccurs in most types of human solid tumors andis associated with poor clinical prognosis.

Biochemistry and molecular biology

The transcription factor Stat3, implicated incell transformation induced by many oncogenes,is also a downstream signaling molecule activat-ed by HGF/SF-Met signaling. In collaborationwith Richard Jove, we have utilized Stat3β, adominant negative form of Stat3, to show thatStat3 activity is critical both for HGF/SF-Met–mediated cell growth in soft agar and fortumor growth in athymic nude mice. Our current

efforts are designed to use Stat3β to identify thedownstream targets of Stat3 that influenceanchorage-independent growth.

Therapeutics

In collaboration with David Wenkert, we havebeen testing novel derivatives of the antitumor agentgeldanamycin for suppression of Met activity in cul-tured tumor cells. We have observed that the plas-min-inhibitory activity of geldanamycin and itsderivatives extend over a concentration range ofalmost nine logs. The most potent compoundsinhibited plasmin activity at IC50’s of 5–30 fM, instark contrast to the nanomolar concentrationsrequired for the destabilizing effects of gel-danamycin on HSP90. Three of the derivativesare more potent than geldanamycin: 17-[di-(2-chloroethyl)amino]-17-demethoxygeldanamycin,17-amino-17-demethoxygeldanamycin, and themost potent, 7′-bromogeldanoxazinone.

In vivo imaging

In collaboration with Brian Cao, BeatriceKnudsen, and Milton Gross, we are developingmonoclonal antibodies raised against compo-nents of the Met-HGF/SF receptor–ligand pair aspotential diagnostic and therapeutic agents. We

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RResearch Projects

have undertaken a detailed characterization ofthe single anti-Met monoclonal antibody desig-nated Met3. By immunofluorescence we haveshown that Met3 binds with high avidity to cul-tured tumor cells expressing human Met. Wehave also shown that Met3 binds to cultured nor-mal prostate epithelial cells and to prostateepithelium in human tissue sections. We haveconfirmed by FACS analysis that Met3 binds tocells of the human prostate carcinoma lines PC-3 and DU145, which are known to express Met.

We have examined the ability of Met3 to imagehuman tumors of different tissue origins. Tumorxenografts were raised subcutaneously in hind limbsof athymic nude mice, and animals were injectedintravenously with [125I]Met3. Total-body gammacamera images were acquired and analyzed. Theautocrine Met-expressing tumors S-114 (3T3 murinecells transformed with human Met and HGF/SF) andSK-LMS-1 (human leiomyosarcoma) and theparacrine Met-expressing human prostate carcinomaPC-3 were all readily imaged with [125I]Met3; peaktumor-to-control hind limb asymmetry was observedat about 3 days postinjection (Figure 1).

From these observations we conclude that anti-Met monoclonal antibodies—Met3 in particular—are robust reagents for detecting human Met-expressing cells. They are worthy of further evalu-ation as potential diagnostic and therapeutic agentsfor human cancers, including prostate cancer.

In collaboration with Brian Ross, our visit-ing scientists Ilan and Galia Tsarfaty are leadingan effort to develop a noninvasive tumor molec-ular imaging program. In order to study themetabolic effects of Met-HGF/SF signaling invivo, we recently demonstrated functionalmolecular imaging of Met receptor activity. DA3mammary adenocarcinoma cells were injectedinto the mammary glands of mice, formingtumors expressing high levels of Met. Weshowed that Met activation in vivo by HGF/SFalters the hemodynamics of normal and malig-nant Met-expressing tissues. Organs and tumorsexpressing high levels of Met showed the great-est alteration in blood oxygenation levels asmeasured by BOLD-MRI (blood oxygenationlevel–dependent MRI). Met-expressing tumorsshowed a 30% change in signal by BOLD-MRI,while no significant alteration was observed intumors or organs that do not express Met. Theextent of MRI signal alteration correlated withthe dose of HGF/SF administered.

In autocrine tumors, the hemodynamicchanges in the tumors are greatly enhanced com-pared with those in tumors that do not expressHGF/SF. Moreover, the kidneys and livers ofmice bearing autocrine tumors demonstrateincreased hemodynamic activity that is propor-tional to the tumor size.

These results indicate that functional molec-ular imaging of Met expression can serve as apowerful tool for understanding the metabolicactivities affected by its signal transduction, andthis approach could be used to understand thedifferent molecular mechanisms of receptor acti-vation. Moreover, functional molecular imagingof Met expression may be useful for the detec-tion, analysis, and prognosis of a wide spectrumof human solid tumors.

Human pathology collaborative studies

In collaboration with Beatrice Knudsen, wehave shown an overall 52% Met positivity amongprimary prostate cancers in a cohort of 90

Figure 1. Leiomyosarcoma in mouse thigh imagedwith radioactive anti-Met monoclonal antibody

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patients and confirmed that Met expression waspresent in all of 41 prostate cancer metastases tobone within this cohort (Figure 2).

In collaboration with Nadia Harbeck andErnest Lengyel, we have conducted a retrospec-tive pilot study using breast cancer tissueobtained from about 40 patients to investigatewhether Met-HGF/SF and HER-2/neu are coex-pressed in HER-2-positive tumors. We correlat-ed the results of immunohistochemical evalua-tion of tissue samples in our two laboratorieswith other clinicopathological data. We foundthat neither Met nor HER-2 expression in pri-mary tumors correlated with established prog-nostic factors such as age, lymph node involve-ment, estrogen receptor expression, progesteronereceptor expression, tumor size, or tumor grad-ing. However, Met overexpression alone identi-fied high-risk patients independent of HER-2expression. Median disease-free survival associ-ated with c-Met overexpressing tumors was 8months, compared to 53 months in remaining

patients (p = 0.031; RR 3.1). We conclude thatMet overexpression is associated with signifi-cantly diminished disease-free survival (DFS)and, in the majority of cases, this is independentof HER-2 overexpression.

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Figure 2. Met expression (arrow, brown) byprostate cancer metastasis in human bone

External Collaborators

Michael Clague, University of Liverpool, United Kingdom

Milton Gross, Department of Veterans Affairs Healthcare System and University of Michigan, Ann Arbor

Nadia Harbeck and Ernest Lengyel, Technische Universität, Munich, Germany

Ruth Heimann and Samuel Hellman, University of Chicago, Illinois

Richard Jove, H. Lee Moffitt Cancer and Research Institute, Tampa, Florida

Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington

Len Neckers and Bert Zbar, National Cancer Institute, Bethesda, Maryland

Brian Ross, University of Michigan, Ann Arbor

Peter Schirmacher, University of Cologne, Germany

David Waters, Gerald P. Murphy Cancer Foundation, Seattle, Washington

David Wenkert, Michigan State University, East Lansing

Robert Wondergem, East Tennessee State University, Johnson City

Publications

Collazo, Carmen M., George S. Yap, Gregory D. Sempowski, Kimberly C. Lusby, Lino Tessarollo,George F. Vande Woude, Alan Sher, and Gregory A. Taylor. 2001. Inactivation of LRG-47 andIRG-47 reveals a family of interferon g–inducible genes with essential, pathogen-specific rolesin resistance to infection. Journal of Experimental Medicine 194(2): 181–188.

Furge, Kyle A., David Kiewlich, Phuong Le, My Nga Vo, Michel Faure, Anthony R. Howlett,Kenneth E. Lipson, George F. Vande Woude, and Craig P. Webb. 2001. Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase.Proceedings of the National Academy of Sciences U.S.A. 98(19): 10722–10727.

Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, George F.Vande Woude, and Milton Gross. 2002. Radioimmunoscintigraphy of tumors autocrine forhuman Met and hepatocyte growth factor/scatter factor. Molecular Imaging 1(1): 56–62.

Knudsen, Beatrice S., Glenn A. Gmyrek, J. Inra, D.S. Scherr, E. Darracott Vaughan, D.M. Nanus,M.W. Kattan, W.L. Gerald, and George F. Vande Woude. In press. High expression of the Metreceptor in prostate cancer metastasis to bone. Urology.

Koo, Han-Mo, Matt VanBrocklin, MaryJane McWilliams, Stephan H. Leppla, Nicholas S. Duesbery,and George F. Vande Woude. 2002. Apoptosis and melanogenesis in human melanoma cellsinduced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase.Proceedings of the National Academy of Sciences U.S.A. 99(5): 3052–3057.

Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-MeiWang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H.Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patientswith late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596.

Webb, Craig P., and George F. Vande Woude. 2002. Met gene. In Wiley Encyclopedia of MolecularMedicine, Haig H. Kazazian, ed. New York: Wiley, pp. 2049–2051.

Zhang, Yu-Wen, Ling-Mei Wang, R. Jove, and George F. Vande Woude. 2002. Requirement ofStat3 signaling for HGF/SF-Met-mediated tumorigenesis. Oncogene 21(2): 217–226.

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From left to right: Kaufman, Gao, Zhang, I. Tsarfaty, Shinomiya, Hay, G. Tsarfaty, Su, Vande Woude,Bruch, Gustafson

Tumor Metastasis and Angiogenesis Laboratory

Craig P. Webb, Ph.D.Dr. Webb received his Ph.D. in cell biology from the University of East Anglia,England, in 1995. He then served as a Postdoctoral Fellow in the laboratory ofGeorge Vande Woude in the Molecular Oncology Section of the AdvancedBioScience Laboratories–Basic Research Program at the National CancerInstitute–Frederick Cancer Research and Development Center, Maryland(1995–1999). Dr. Webb joined VARI as a Scientific Investigator in October 1999.

Laboratory Members

StaffJeremy Miller, Ph.D.David Monsma, Ph.D.Emily Eugster, M.S.

Guest workerLonson Barr, D.O.

StudentsKelly Ballast, B.S.Donald ChaffeeMeghan Sheehan

umor metastasis, the process by whichcancer spreads throughout a host to sec-ondary tissues, accounts for the majori-

ty of cancer-related mortalities. The active recruit-ment of tumor vasculature, generally termedangiogenesis, is integral to both tumor growth andmetastasis. Our laboratory focuses on identifyingthe key cellular and molecular determinants ofmetastatic progression in order to improve ourconceptual understanding of the process, with thegoal of developing diagnostics and therapeuticsthat target this most damaging aspect of cancer.

Our lab currently utilizes various systems tostudy metastasis and angiogenesis both in vitro andin vivo. For example, we have previously describedmurine cell lines that display various metastaticpropensities after ectopic expression of effectordomain mutants of the ras oncogene. Thesemutants are particularly useful, because they differ-entially activate signaling pathways downstream ofRas and hence can be used to dissect the pathwaysand subsequent genetic/epigenetic events thatmediate metastasis in this experimental setting. Intandem with our fluorescent imaging capabilities,we are now able to follow individual tumor cells asthey undergo the various stages of metastaticspread. We are using these imaging strategies withother mouse models to follow the progression ofmetastases, such as metastasis to the liver after theformation of primary pancreatic carcinomas.

Of particular importance, we are beginningto determine the factors that contribute tometastatic dormancy, a frequent occurrence inwhich individual metastatic cells within second-ary tissues fail to progress to macroscopic dis-ease, but instead lie dormant until a later time.Very little is known about the factors that con-tribute to this phenomenon, yet this aspect ofmetastasis likely accounts for the minimal resid-ual disease and metastatic relapse observed inmany patients. We are now using a combina-tion of some state-of-the-art technologies,including laser capture microdissection, pro-teomics, and gene chip arrays, to integrate thegenomic and proteomic events that occur duringtumor-host interactions throughout the metasta-tic process in mouse models and human patientmaterial. In this fashion, we have identified anumber of candidate genes and proteins thatappear to play prominent roles in metastasis tospecific secondary sites. Our lab has now begunto systematically validate these targets for theirprecise functional roles. These types of studieswill continue to generate essential informationabout the factors that regulate metastatic pro-gression and will identify diagnostic/therapeutictargets for the future. In collaboration with localclinicians and some industrial partners, we arestriving toward the early diagnosis and treat-ment of malignant disease.

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TResearch Projects

External Collaborators

Lonson Barr, Donald Kim, Martin Luchtefeld, and Thomas Monroe, Spectrum Health, Grand Rapids,Michigan

Yihai Cao, Karolinska Institute, Stockholm, SwedenAnn Chambers, University of Western Ontario, London, CanadaSamir Hanash and Gil Omenn, University of Michigan, Ann ArborRobert Hoffman, Anticancer Inc., San Diego, CaliforniaBeatrice Knudsen, Cornell University, Ithaca, New YorkKen Lipson, SUGEN, Inc., South San Francisco, CaliforniaMartin McMahon, University of San Francisco, CaliforniaAnthony Schaeffer, Northwestern University, Evanston, IllinoisBin S. Teh, Baylor College of Medicine, Waco, TexasAnnette Thelen, Michigan State University, Lansing

Affymetrix, Santa Clara, CaliforniaMicromass, Beverly, MassachusettsPharmacia (SUGEN Inc), California

Publications

Furge, Kyle A., David Kiewlich, Phuong Le, My Nga Vo, Michel Faure, Anthony R. Howlett, KennethE. Lipson, George F. Vande Woude, and Craig P. Webb. 2001. Suppression of Ras-mediatedtumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase. Proceedingsof the National Academy of Sciences U.S.A. 98(19): 10722–10727.

Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, Bin T. Teh, LonsonBarr, and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of humancolon adenocarcinoma and associated intraperitoneal metastases reveals consistent cytogeneticand transcriptional aberrations associated with progression of multiple metastases. AppliedGenomics and Proteomics.

Haddad, Ramsi, and Craig P. Webb. 2001. Hepatocyte growth factor expression in human cancer andtherapy with specific inhibitors. Anticancer Research 21(6B): 4243–4252.

Webb, Craig P., and George F. Vande Woude. 2002. Met gene. In Wiley Encyclopedia of MolecularMedicine, Haig H. Kazazian, ed. New York: Wiley, pp. 2049–2051.

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From left to right: Webb, Monsma, Miller, Sheehan, Eugster, Chaffee, Ballast, Barr

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CapturedAfterBefore

NormalEpithelia

ColonAdenocarcinoma

Laser capture of normal colon crypts and adjacent adenocarcinoma

Laser capture microdissection of normal colon crypts and adjacent adenocarcinoma of the colon, within a sin-gle histopathological section from a patient with metastatic colon cancer. Following capture, the smallamounts of RNA and protein obtained can be applied to a global genomic and proteomic analysis usingAffymetrix gene chips, cDNA microarrays, and mass spectrometry. In this fashion, genes and proteins that areexpressed within the different subcompartments of a heterogeneous tumor (epithelia, endothelia, stroma,inflammatory cells, etc.) can be analyzed. This technology provides an excellent means for identifying thekey genes and proteins that are associated with tumor progression and will likely yield targets for the futurediagnosis and treatment of cancer.(Webb and Monsma)

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Immunofluorescent analysis of blood vasculature in the eye of an Lrp5-deficient mouse

Mutations that inactivate the gene encoding the low-density lipoprotein receptor–related protein 5 (Lrp5) causethe human syndrome osteoporosis pseudoglioma (OPG). Patients suffering from this syndrome develop early-onset osteoporosis and have vision problems due to inappropriate vascularization of the eye. We have foundthat Lrp5-deficient mice model both the osteoporotic and eye problems seen in humans. Shown is animmunofluorescence analysis of a paraffin-embedded eye section from an Lrp5-deficient mouse stained withantibodies to Factor VIII (red) and CD31 (green), two molecules associated with endothelial cells. Theprominent vascularization is absent in normal mice of this age.(Williams)

Laboratory of Chromosome Replication

Michael Weinreich, Ph.D.Dr. Weinreich received his Ph.D. in biochemistry from the University ofWisconsin–Madison in 1993. He then served as a Postdoctoral Fellow in the labora-tory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000.

Laboratory Members

StaffAndrei Blokhin, Ph.D.Don Pappas, Ph.D.Carrie Gabrielse, B.S.Marleah Russo, B.S.

StudentAshley Mynsberge

ne critical step after the commitment tocell division is chromosome replica-tion. Our laboratory is interested inunderstanding how the initiation of

DNA replication occurs at the molecular leveland how initiation events at each chromosomalorigin are restricted to once per cell cycle. Ascells exit mitosis and enter the G1 phase, theyassemble a “pre-replicative complex” (pre-RC)at multiple replication origins. Additional less-well-defined complexes formed at the origin inG1 are then activated to form bidirectional repli-cation forks within a very short time (S-phase).Restricting the assembly of pre-RCs to G1 is akey regulatory event insuring that replication ori-gins become competent only after completion ofthe previous cell cycle. In Saccharomyces cere-visiae, cyclin-dependent kinases inhibit pre-RCformation throughout the cell cycle, but as theirlevels fall during exit from mitosis, pre-RCs areable to form.

Eukaryotic origins of replication have beenprecisely defined only in budding yeast. An ini-tiator protein (ORC) has also been extensivelycharacterized. ORC is a six-subunit complexthat recognizes conserved sequence elements inall origins and is required for the initiation ofreplication. ORC is bound to origins throughoutthe cell cycle; however, in late mitosis, Cdc6pbinds to ORC and promotes loading of the MCMcomplex (a helicase) at the origin. ORC, Cdc6p,and the MCM complex are required to form thepre-RC in vivo. Subsequent events occurring inG1 are much less understood, including the asso-ciation of Cdc45p, the loading of DNA poly-

merases, and the activation of replication bycyclin-dependent kinases and the Cdc7 proteinkinase.

Our long-term goal is to define the proteincomponents of the replication complexes thatform in G1, and particularly to understand howcomplex assembly is regulated. Cdc6p is a criti-cal limiting factor for assembly of the pre-RC.We have previously shown that Cdc6p interactswith ORC and that its essential activity requiresa functional ATP-binding domain. Cdc6p alsocouples replication initiation with progressionthrough the remainder of the cell cycle. If initi-ation does not occur, a nonessential N-terminaldomain of approximately 50 amino acids isrequired for preventing a “reductional mitosis,”in which the unreplicated chromosomes are ran-domly segregated. Cdc6p very likely regulatespassage through mitosis by inhibiting cyclin-dependent kinases.

We have taken a genetic approach to identi-fying additional factors that are important for ini-tiation. Using a temperature-sensitive mutationin CDC6, we have isolated a number of dosage-dependent and extragenic suppressors thatrestore growth at high temperature. These sup-pressors define several novel pathways influenc-ing replication. For example, we isolated oneclass of extragenic suppressors that containedmutations in the silent information regulatorsSIR2, SIR3, and SIR4. The Sir proteins arerequired for the formation of heterochromaticregions at the silent mating-type loci and attelomeres. In addition, Sir2p suppresses recom-bination at the rDNA locus and promotes

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OResearch Projects

increased life span in yeast and Caenorhabditiselegans. SIR2 encodes a histone-dependentdeacetylase and has at least seven orthologues inhuman cells. No evidence has been reported thatthe Sir proteins influence replication globally, asour data suggest. We are testing whether the Sirproteins act directly at origins of replication andnegatively regulate initiation events. If this isoccurring, it could provide a mechanism for theestablishment of transcriptional or developmen-tal states that were coupled to replication of cer-tain chromosomal domains.

We are also studying the Cdc7p-Dbf4pkinase, which is a conserved, two-subunit ser-ine/threonine protein kinase required for a latestep in replication initiation. We are interested inunderstanding the regulation of Cdc7p-Dbf4pkinase activity and determining its critical in vivosubstrates. Cdc7p subunit abundance is constantthroughout the cell cycle, but the Dbf4p subunitis cyclically expressed and is degraded duringmitosis. The Cdc7p-Dbf4p kinase is required forDNA replication, but it has less-well-definedroles in promoting error-prone DNA repair andprogression through meiosis. In response toDNA damage, Dbf4p is phosphorylated in acheckpoint-dependent manner and this decreasesCdc7p-Dbf4p kinase activity. CDC7 mutants are

hypomutable and fail to respond normally to sig-nals generated by stalled replication forks.Therefore, Cdc7p-Dbf4p is emerging as perhapsa more global regulator of chromosome mainte-nance and stability than previously thought.

For this reason we are studying this proteinboth in yeast and in human cells. We have gen-erated wild-type human cDNA clones and haveconstructed baculoviruses for purification ofboth the human and yeast enzymes. We haveraised monoclonal antibodies against humanCdc7 and are now raising antibodies against theDbf4 subunit. We hope to gain valuable reagentsfor examining the regulation and localization ofthe human kinase, both during the normal cellcycle and during periods of genomic stress. TheDbf4 protein has two classical D-box motifs andalso a KEN-box motif. Both of these sequencesare known to promote polyubiquitylation andproteasome-dependent degradation of cyclinsand other unstable proteins. We are examining ifthese sequences function similarly in the humanand yeast kinases.

We are most interested in determining therole of the Cdc7p-Dbf4p kinase during periods ofDNA damage or replication-fork arrest. Both thehuman and yeast Dbf4 proteins contain a singleBRCT domain at the amino terminus. BRCTdomains (first defined as a tandem repeat at theC-terminus of BRCA1) are present in a large fam-ily of proteins involved in DNA repair. Publishedand unpublished work indicates that yeast Cdc7p-Dbf4p is an important target of the S-phasecheckpoint. The S-phase checkpoint in yeastresponds to stalled replication forks that occurthrough a variety of insults. Since abrogatingcheckpoints are thought to facilitate tumorigene-sis, we are examining if the human Cdc7 kinase issimilarly a target of checkpoint kinases followingDNA damage. Also, we are taking a geneticapproach in yeast to more accurately determineits effect on DNA repair and replication.

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Figure 1. S. cerevisiae replication cycle

External Collaborators

Catherine Fox, University of Wisconsin–Madison

Chun Liang, Hong Kong University

Publications

Weinreich, Michael, Chun Liang, Hsu-Hsin Chen, and Bruce Stillman. 2001. Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle. Proceedingsof the National Academy of Sciences U.S.A. 98(20): 11211–11217.

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From left to right: Mynsberge, Weinreich, Russo, Gabrielse, Pappas, Blokhin

Laboratory of Cell Signaling and Carcinogenesis

Bart O. Williams, Ph.D.Dr. Williams received his Ph.D. in biology from Massachusetts Institute ofTechnology in 1996. For three years, he was a Postdoctoral Fellow at the NationalInstitutes of Health in the laboratory of Harold Varmus, former Director of NIH. Dr.Williams joined VARI as a Scientific Investigator in July 1999.

Laboratory Members

StaffTroy Giambernardi, Ph.D.Sheri Holmen, Ph.D.Scott Robertson, B.S.Cassandra Zylstra, B.S.

StudentsHolli CharbonneauJennifer DaughertyJennifer Mieras

ur laboratory is focused on understand-ing how alterations in the Wnt signalingpathway cause human disease.

Alterations in the pathway are among the mostcommon changes associated with human cancerand have also been linked to other disorders,including osteoporosis. A very completeoverview of this pathway can be found on a Website (http://www.stanford.edu/~rnusse/wntwin-dow.html) developed by Dr. Roel Nusse.

We are particularly interested in elucidatingthe mechanisms by which the secreted Wnt lig-and activates the pathway by binding to thereceptor complex at the plasma membrane. Wntbinds to a receptor complex that includes a mem-ber of the frizzled family and LRP5 or LRP6.The activation of this complex then inhibits thetargeting of β-catenin and plakoglobin for ubiq-uitin-dependent proteolysis. This inhibitionresults in the accumulation of these proteins inthe cytosol, where they interact with theLEF/TCF family of DNA binding proteins andsubsequently translocate to the nucleus wherethey alter gene expression. In other contexts,Wnt ligands can activate protein kinase C orRho-dependent pathways.

There are many levels of regulating thereception of Wnt signals. The completion of thehuman genome project has shown that there are19 different genes that encode Wnt proteins, 9encoding Frizzled proteins, and two LDL recep-tor–related proteins that function in Wnt signal-ing (LRP5 and LRP6). In addition, there are sev-eral proteins that can inhibit Wnt signaling bybinding to components of the receptor complexand interfering with normal signaling (Figure 1).

These include Dickkopfs (Dkks) and Frizzled-related proteins (FRPs). One of the long termgoals of our laboratory is to understand howspecificity is generated for the different signalingpathways. The following projects are currentlybeing pursued in the laboratory.

Analysis of Lrp5-deficient mice

Recently, several laboratories have demon-strated that Lrp5 is required for the maintenanceof normal bone density in humans. Consistentwith the work of other laboratories, we haveshown that Lrp5-deficient mice are viable andfertile but have decreased bone density and per-sistent vascularization of the lens. We have cre-ated Lrp5-deficient mice on several genetic

OResearch Projects

Figure 1. Overview of the Wnt signaling pathway(Reprinted by permission from Nature Cell Biology4(7): E172-E173, © (2002) Macmillan Publishers Ltd.)

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backgrounds to assess whether alleles that maymodify bone density and lens vascularization(and perhaps Wnt signaling) can be identified inthe mouse genome. In addition, we are assessingwhether Lrp5 is required for mammary tumori-genesis in MMTV-Wnt1 transgenic mice.

Analysis of mice deficient for both Lrp5 and Lrp6

Mice carrying a mutated allele of Lrp6 (gen-erously provided by Bill Skarnes) are beingcrossed to mice deficient for Lrp5. We are inter-ested in potential phenotypes of Lrp6+/–;Lrp5–/–mice. One possibility is that these mice will havemore severe defects in bone density and eye vas-cularization. In addition, we are determining thephenotype of mice homozygously deficient forboth genes. We expect these mice will die veryearly in gestation. To further characterize thefunctions of these genes, we are creating mouseembryonic stem cell lines deficient for bothgenes and using them to generate chimeric micefor analysis (in collaboration with the VARI lab-oratory of Pamela Swiatek).

Wnt-Fz fusion constructs and specificity inthe Wnt signaling pathway

We have recently published an analysis offusion proteins between selected Wnt andFrizzled molecules in collaboration with AdrianSalic and Marc Kirschner of Harvard MedicalSchool. We found that expression of several suchfusion proteins with Lrp6 could significantly acti-vate a Wnt/β-catenin–responsive reporter geneand stabilize cytoplasmic levels of β-catenin. Weare continuing to utilize these constructs toaddress questions about Wnt signaling specificity.

Expression of Wnt receptor components intumorigenesis and development

We have developed probes for RT-PCRanalysis of Wnt, Frizzled, Lrps, Dkks, Kremens,and FRPs. We are systematically examining theexpression of these components in various tumorcell lines and in tissue samples.

Mouse models for melanoma

Our laboratory is also interested in the broadgoal of improving mouse models of carcinogen-esis. Given our interest in Wnt signaling, we areparticularly focused on developing models in

which alterations in this signaling pathway havebeen introduced into the mouse genome.

An in vivo model is being developed for study-ing melanoma using a retroviral-based gene target-ing system. We have generated a mouse strainexpressing the avian leukosis virus receptor, TV-A,under the control of a promoter that is only active inmelanocyte precursor cells. These cells can then beinfected by avian leukosis virus A (AVL-A) which isharboring genes of interest that have been intro-duced into AVL-A vectors. This system allows mul-tiple genes to be delivered to a single TV-A+ cell.We can then study the effects of multiple oncogenesand tumor suppressor genes on melanoma develop-ment and progression. In addition, this model willallow comparison of melanomas induced by differ-ent genetic changes. These can then be used to eval-uate the efficacy of different therapeutic strategies.

We are also developing a system to regulatethe expression of an activated version of β-cateninin melanocytes. Given that mutations in the β-catenin gene have been identified in melanomas,we feel that this system will allow insight into therole of Wnt signaling in melanocyte differentia-tion and melanoma-genesis.

The role of Wnt signaling in prostate cancer

We are developing a tetracycline-regulatedsystem to control the expression of an activatedversion of β-catenin specifically in the prostate.The rationale for doing this is that up-regulationof β-catenin activity is observed in a significantpercentage of prostate tumors. In addition, the β-catenin protein physically interacts with theandrogen receptor and alters its activity. Thiswork is done with in collaboration with WadeBushman of the University of Wisconsin.

The role of MMP8 in melanoma

Expression of matrix metalloproteinase 8(MMP8), previously thought to be restricted inexpression to neutrophils, was recently detectedin melanomas but not normal melanocytes. Inaddition, expression was also detected in neuralcrest cells during embryonic development. Weare currently creating mice deficient in MMP8 tofurther define its role in development. We arealso examining various stages of melanoma toidentify the point in melanoma progression atwhich MMP8 is turned on.

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External Collaborators

Wade Bushman, University of Wisconsin – Madison

J. Fred Hess, Merck Pharmaceuticals

Marc Kirschner and Adrian Salic, Harvard Medical School, Boston, Massachusetts

Publications

Holmen, Sheri L., Adrian Salic, Cassandra R. Zylstra, Marc W. Kirschner, and Bart O. Williams. Inpress. A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate β-catenin-dependent signaling. Journal of Biological Chemistry

Yang, Hong, Bart O. Williams, Phillip W. Hinds, T. Shane Shih, Tyler Jacks, Roderick T. Bronson, andDavid M. Livingston. 2002. Tumor suppression by a severely truncated species of the retinoblas-toma protein. Molecular and Cellular Biology 22(9): 3103–3110.

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From left to right, back row: Williams, Daugherty, Robertson, Zylstramiddle row: Giambernardi, Holmenfront row: Charbonneau, Mieras

Laboratory of Structural Sciences

H. Eric Xu, Ph.D.Dr. Xu went to Duke University and the University of Texas Southwestern MedicalCenter, where he earned his Ph.D. in molecular biology and biochemistry.Following a postdoctoral fellowship with Carl Pabo at MIT, Dr. Xu moved toGlaxoWellcome in 1996, where he solved the crystal structures of a number ofnuclear hormone receptors. Until recently he was a senior research investigator ofnuclear receptor drug discovery at GlaxoSmithKline in Research Triangle Park,North Carolina. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002.

ur laboratory is using x-ray crystallog-raphy to study structures of key proteincomplexes that are important in various

signaling pathways, as well as for drug discoveryrelevant to human diseases such as cancers anddiabetes. Our major focus is on the superfamilyof nuclear hormone receptors, which includesreceptors for classic steroid hormones such asestrogen, progesterone, androgens, and glucocor-ticoids, as well as receptors for proxisome prolif-erator activators, vitamin D, vitamin A, and thy-roid hormones. These receptors are DNA-bind-ing and ligand-dependent transcriptional factorsthat regulate genes essential for a broad aspect ofhuman physiology, ranging from developmentand differentiation to metabolic homeostasis.

A typical nuclear receptor contains threemajor domains: an N-terminal activation func-tion-1 domain (AF-1), a central DNA-bindingdomain (DBD), and a C-terminal ligand-bindingdomain (LBD). In addition to its role in ligandrecognition, the LBD also contains dimerizationmotifs and an activation function-2 domain thatis highly dependent on the bound ligand. TheLBD is thus the key to the ligand-dependent reg-ulation of nuclear receptor signaling and as suchhas been the focus of intense structural studies.

One set of receptors we will study is that ofthe peroxisome proliferator–activated receptors,alpha, delta, and gamma, (PPARα, δ, and γ). Asnuclear receptors, PPARs are regulated by thebinding of small-molecule ligands. In responseto ligand binding, PPARs affect a wide range ofbiological activities, including the regulation oflipid metabolism and of glucose homeostasis.Thus, PPARs are important therapeutic targetsfor common diseases such as diabetes, cancer,

and cardiovascular disease. Millions of peoplehave benefited from treatment with novel PPARγmedicines for type II diabetes; two of the top 100drugs in 2001 were PPARγ ligands, having com-bined sales of over $2.4 billion. This demon-strates that studying PPARs can have a majoreffect on human health and disease treatment. Tounderstand the molecular basis of ligand-mediat-ed signaling by PPARs, we have determinedcrystal structures of each PPAR’s LBD (Figure 1)

bound to many diverse ligands, including fattyacids, the lipid-lowering drugs called fibrates,and a new generation of antidiabetic drugs, glita-zones. These structures have provided a frame-work for understanding the mechanisms of ago-

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OResearch Projects

Figure 1. Crystal structure of the PPAR LBDsand their ligand-binding pocket. Each PPAR con-tains 13 α-helices and 4 small β-strands. The helicesare arranged into a three-layer sandwich fold to createa ligand-binding pocket (white surface). The ability ofPPARs to recognize so many and such diverse lig-ands can be accounted by the enormous size of thePPAR pocket, which is over 1,300 Å3 and is much larg-er than the pocket seen in any other nuclear receptor.

nists and antagonists, as well as the recruitmentof co-activators and co-repressors in gene activa-tion and repression. Furthermore, these struc-tures also serve as a molecular basis for under-standing potency, selectivity, and binding modeof diverse ligands, information that has providedcritical insights for designing the next generationof PPAR medicines.

The other nuclear receptor we will study isthe human glucocorticoid receptor (GR). GRplays key roles in the metabolism of lipids andcarbohydrates, development of the central nerv-ous system, and homeostasis of the immune sys-tem. GR is also associated with numerous patho-logical pathways and as such is an importantdrug target. In fact, GR has a rich history in drugdiscovery and human medicine. There are morethan 10 GR ligands (including dexamethasone)that are currently used for treatment of suchdiverse medical conditions as asthma, allergies,autoimmune diseases, and cancer. At the molec-ular level, GR can function either as a transcrip-tion activator or a transcription repressor. Bothof these functions of GR are tightly regulated bysmall ligands that bind to the GR ligand-bindingdomain. To explore the molecular mechanism ofligand binding and signaling of GR, we havedetermined a crystal structure of the GR LBDbound to dexamethasone and a co-activator motiffrom TIF2. The structure reveals a novelLBD–LBD dimer interface (Figure 2) that is cru-cial for GR-mediated transactivation but not tran-srepression, suggesting a novel role of LBDdimerization in the GR signaling pathways. Thestructure also contains an unexpected chargeclamp responsible for sequence-specific bindingof co-activators and a unique ligand-bindingpocket to account for specific recognition ofdiverse GR ligands. The detailed molecular inter-actions between the receptor and dexamethasoneshould also facilitate the discovery of new gluco-

corticoid receptor molecules that would reducethe side effects of current GR drugs.

Beside PPARs and GR, the human genomecontains 44 other nuclear receptors. X-ray struc-tures have now been solved for more than a dozenLBDs, bound to agonists and antagonists, co-acti-vators and co-repressors, and in forms ofmonomers, homodimers, heterodimers, andtetramers. These structures have illustrated thedetails of ligand binding, the conformationalchanges induced by agonists and antagonists, thebasis of dimerization, and the mechanism of co-activator and co-repressor binding. All of thenuclear receptors studied to date have broadlysimilar structures and mechanisms of activation,but functionally significant differences have aris-en among the various receptors over the course ofevolution. We can expect more surprises as struc-tural work continues on the remaining nuclearreceptors, and as crystallographers tackle higher-order complexes involving the LBD with the AF-1 domain, the DBD, and other proteins and nucle-ic acids involved in gene transcription.

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Figure 2. Crystal structure of the human GR LBDbound to dexamethasone and a TIF2 co-activator.This structure reveals a novel dimer configuration, asecond charge clamp, and a unique GR pocket. TheGR LBD dimer interface, composed of β-loops andturns, is distinct from the helix-10 interface of theRXR/PPARγ heterodimer or the RXR homodimer.

Nian Zhang, Ph.D.Dr. Zhang received his M.S. in entomology from Southwest Agricultural University,People’s Republic of China, in 1985 and his Ph.D. in molecular biology from theUniversity of Edinburgh, Scotland, in 1992. From 1992 to 1996, he was aPostdoctoral Fellow at the Roche Institute of Molecular Biology. He next served asa Postdoctoral Fellow (1996) and a Research Associate (1997–1999) in the labora-tory of Tom Gridley in mammalian developmental genetics at the JacksonLaboratory, Bar Harbor, Maine. Dr. Zhang joined VARI as a Scientific Investigatorin December 1999.

Laboratory Members

StaffJun Chen, M.D., Ph.D.Jihong Ma, Ph.D.Lan Wang, Ph.D.Liang Kang

e are interested in understanding thecellular and molecular mechanismsunderlying pattern formation during

embryonic development. We previously clonedand targeted the mouse Lunatic fringe (Lfng)gene, which plays an important role in embryosegmentation. Mice homozygous for the Lfngmutation suffer from severe malformation intheir axial skeletons as a result of irregularsomite formation during embryonic develop-ment. Lfng encodes a secreted signaling mole-cule essential for regulating the Notch signalingpathway in mice. We showed that Lfng expres-sion was in response to a biological clock thatoscillated once during the formation of each seg-ment, and the failure of the Lfng mutants inresponding to this clock resulted in the abnormalsegmentation phenotype.

We want to understand how the rhythmicexpression of Lfng is controlled. Using trans-genic animals, we are analyzing regulatory ele-ments that control Lfng expression, and we arealso isolating genes that regulate Lfng expres-sion. In addition, we are identifying proteinsthat interact with Lfng during embryo segmenta-tion. Another approach that we are taking tounderstand somitogenesis is to examine a spon-taneous mutant with a phenotype similar to thatof the Lfng mutant. We plan to clone this muta-tion positionally and test if this mutation inter-acts with Lfng and other mutations that affectsomitogenesis.

The second focus of our laboratory is germcell development, particularly the mechanismsthat govern germ cell migration, survival, sper-matogonial stem cell renewal and differentiation,and their implications for human disease. It isunclear how spermatogonial stem cells areregenerated during the entire reproductive life inmammals. Previous studies on the nematodeCaenorhabditis elegans have shown that theNotch/lin12-mediated signal transduction path-way is important for germ cells to remain in anundifferentiated state. Mutations that compro-mise this pathway force germ cells to enter meio-sis earlier than normal. A constitutively activat-ed signal prevents germ cells from enteringmeiosis, resulting in overproliferation of germcells, a phenotype called “germ cell tumor.”Given the fact that some members in the Notchsignaling pathway are expressed in the testis, wespeculate that Notch signaling may play a simi-lar role in spermatogonial differentiation inmammals. We will further examine the roleNotch signaling may play during spermatogene-sis using transgenic animals and conditionalgene targeting.

We are also studying a spontaneous mutationthat causes sterility. Preliminary data suggestthat this mutation arrests the development ofspermatogenic cells at meiosis II. We are inter-ested in identifying the gene that is disrupted bythis mutation and understanding the role that thisgene plays in meiosis.

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WResearch Projects

Laboratory of Developmental Genetics

External Collaborators

Xiang Gao, Center of Model Animal Genetics, Nanjing University, People’s Republic of China

Douglas L. Pittman, Medical College of Ohio, Toledo

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From left to right: Chen, Kang, Wang, Zhang

Daniel Nathans Memorial Award

Daniel Nathans Memorial Award

The Daniel Nathans Memorial Award was established in memory of Dr.Daniel Nathans, a distinguished member of our scientific community and afounding member of VARI’s Board of Scientific Advisors. We establishedthis award to recognize individuals who emulate Dan and his contributionsto biomedical and cancer research. It is our way of thanking and honoringhim for his help and guidance in bringing Jay and Betty Van Andel’s dreamto reality. The Daniel Nathans Memorial Award was announced at our inau-gural symposium, “Cancer & Molecular Genetics in the Twenty-FirstCentury,” in September 2000.

Award Recipients

2000 Richard D. Klausner, M.D.2001 Francis S. Collins, M.D., Ph.D.

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Francis S. Collins, October 2, 2001

Postdoctoral Fellowship Program

Postdoctoral Fellowship Program

The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientistsbeginning their research careers. The fellowships help promising scientists advance their knowledgeand research experience, while at the same time supporting the research endeavors of VARI. The fel-lowships are funded in three ways: 1) by the laboratories to which the fellow is assigned; 2) by theVARI Office of the Director; or 3) by outside agencies. Each postdoctoral fellow is assigned to a sci-entific investigator who oversees the progress and direction of research. Postdoctoral fellows whoworked in VARI laboratories over the past year are listed below.

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Eduardo AzucenaWayne State University, Detroit, MichiganVARI mentor: Sara Courtneidge

Andrei BlokhinInstitute of Biochemical Physics, Moscow,

RussiaVARI mentor: Michael Weinreich

Jean-François BodartUniversity of Science and Technology, Lille,

FranceVARI mentor: Nicholas Duesbery

Jun ChenWest China University of Medical Sciences,

Chengdu, ChinaVARI mentor: Nian Zhang

Jindong ChenKarolinska Institute, Stockholm, SwedenVARI mentor: Bin Teh

Suganthi ChinnaswamySouthern Illinois University, CarbondaleVARI mentor: Cindy Miranti

Arun ChopraJiwaji University, Gwalior, IndiaVARI mentor: Nicholas Duesbery

Kathryn EisenmannUniversity of Minnesota, MinneapolisVARI mentor: Han-Mo Koo

Chongfeng GaoTokyo Medical and Dental University, JapanVARI mentor: George Vande Woude

Troy GiambernardiUniversity of Texas Health Science Center, San

AntonioVARI mentor: Bart Williams

Steven GrayKarolinska Institute, Stockholm, SwedenVARI mentor: Bin Teh

Xiang GuoSun Yat-Sen University of Medical Sciences,

Guangzhou, ChinaVARI mentor: Bin Teh

Sheri HolmenMayo Graduate School, Rochester, MinnesotaVARI mentor: Bart Williams

Sok Kean KhooTokyo University of Fisheries, JapanVARI mentor: Bin Teh

Hasan KorkayaInternational Center for Genetic Engineering

and Biotechnology, New Delhi, IndiaVARI mentor: Sara Courtneidge

Jihong MaJiamusi Medical College, Jiamusi, ChinaVARI mentor: Nian Zhang

Jeremy MillerUniversity of Michigan, Ann ArborVARI mentor: Craig Webb

Donald Pappas, Jr.Louisiana State University, Baton RougeVARI mentor: Michael Weinreich

Andrew PutnamUniversity of Michigan, Ann ArborVARI mentor: Cindy Miranti

Chao-Nan QianSun Yat-Sen University of Medical Sciences,

Guangzhou, ChinaVARI mentor: Bin Teh

Libing SongSun Yat-Sen University of Medical Sciences,

Guangzhou, ChinaVARI mentor: Bin Teh

Jun SugimuraIwate Medical University, Morioka, JapanVARI mentor: Bin Teh

Rebecca UzarskiMichigan State University, LansingVARI mentor: Sara Courtneidge

Sridhar VenkataramanMichigan State University, LansingVARI mentor: Michael Weinreich

Bradley WallarUniversity of Minnesota, MinneapolisVARI mentor: Arthur Alberts

Chun ZhangTokyo Medical and Dental University, JapanVARI mentor: Bin Teh

Huiying ZhangInstitute of Microbiology and Epidemiology,

Beijing, ChinaVARI mentor: Brian Cao

Heping ZhouFudan University, Shanghai, ChinaVARI mentor: Brian Haab

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Student Programs

Grand Rapids Area Pre-College Engineering Program

The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered byDavenport College and jointly sponsored and funded by Pfizer, Inc., and VARI. The program isdesigned to provide selected high school students who have plans to major in science or genetic engi-neering in college the opportunity to work in a research laboratory. In addition to training in researchmethods, the students also learn workplace success skills such as teamwork and leadership. The three2002 GRAPCEP students are

Marie GravesUnion High School

Grace MiguelCentral High School

Jeanine MylesOttawa Hills High School

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Summer Student Internship Program

The VARI summer student internships were established to provide college students with an oppor-tunity to work with professional researchers in their fields of interest, to use state-of-the-art equipmentand technology, and to learn invaluable people and presentation skills. At the completion of the 10-week program, the students summarize their projects in oral presentations.

From September 2001 through August 2002, VARI hosted 38 students from 13 colleges and uni-versities, both in formal summer internships and in other student positions during the year.

Albion College, Albion, MichiganCassandra Van Dunk

Aquinas College, Grand Rapids, MichiganDonald ChaffeeHolli CharbonneauAshley MynsbergeSarah Scollon

Calvin College, Grand Rapids, MichiganKelly BallastDan DiephouseTodd LaveryAndrea PearsonMeghan Sheehan

Duke University, Durham, North CarolinaJoe Crawley

Grand Rapids Community College, MichiganMarketta HassenYasser JimenezKofi Obeng

Grand Valley State University, Allendale, MichiganHeather BillJenn DaughertyDavina GutierrezKatie KahnoskiSusan KitchenNate LanningAdi LaserBrandon LeeserTherese Roth

Harvard University, Cambridge, MassachusettsChristine Moore

Hope College, Holland, MichiganJason Johnson

Kenyon College, Gambier, OhioLisa Maurer

Michigan State University, LansingErik FreiterSara KienzleTony KokxCasey MaduraTracey Millard

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Michigan Technological University, HoughtonHien DangRadoslav Nickolov

Spring Arbor University, Spring Arbor, MichiganMatthew Main

Stanford UniversityDan Wohns

University of Chicago, IllinoisJon Douglas

University of Michigan, Ann ArborDaphna AtiasJennie Edgar

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VARI Seminar Series

VARI Seminar Series

2001

September Phillippe Soriano, Hutchinson Cancer Research Center, Seattle, Washington

“PDGF signaling in mouse development”

October

Francis Collins, National Institutes of Health, Bethesda, Maryland The Daniel Nathans Lecture: “Decrypting the genome: consequences of the Human GenomeProject for medicine and society”

NovemberTony Wynshaw-Boris, University of California, San Diego

“Modeling human genetic diseases in the mouse”

Mary Ann Handel, University of Tennessee, Knoxville“Genetic models for analysis of chromosome segregation and gametogenesis”

Tayyaba Hasan, Harvard Medical School, Boston, Massachusetts “Therapeutic and diagnostic approaches using light-activatable chemicals”

John Blenis, Harvard University, Cambridge, Massachusetts “Regulation of cell motility, size and proliferation by Ras and PI3-kinase signaling systems”

Sandra Rempel, Henry Ford Hospital, Detroit, Michigan “SPARC modulates glioma growth, attachment, and migration in vitro and in vivo”

December

Gregg Gundersen, Columbia University, New York, New York “Regulation of microtubules by Rho and Cdc42 GTPases in migrating fibroblasts”

Constantine Stratakis, National Institutes of Health, Bethesda, Maryland“Molecular genetics of adrenocortical tumors: Carney complex and PRKAR1A, a novel tumorsuppressor gene”

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2002

January

Don Bottaro, EntreMed, Inc., Rockville, Maryland “Extracellular and intracellular regulation of hepatocyte growth factor signaling”

John Young, University of Wisconsin – Madison “Retrovirus-receptor and anthrax toxin-receptor interactions”

Robert Sigler, Esperion Therapeutics, Inc., Ann Arbor, Michigan “Role of the toxicologic pathologist in drug development”

February

Michael Sheets, University of Wisconsin – Madison “Control of early Xenopus development by regulated cell signaling”

Michael Clague, University of Liverpool, United Kingdom “Linkages between tyrosine kinase receptor trafficking, generation of phosphophosphatidylinosi-tol lipids, and cell signaling”

Michael Brenan and Nick LaRusso, Mayo Clinic, Rochester, Minnesota “The cholangiopathies: from bedside to bench and hopefully back!”

James Herman, John Hopkins University, Baltimore, Maryland “Promoter methylation in cancer: biology and clinical applications”

Robert King, Bristol-Myers Squibb, Wilmington, Delaware “Regulation of hepatitis C virus negative-strand RNA replication by 5′ and 3′ untranslated regions”

March

Steven Frisch, Burnham Institute, La Jolla, California “Cell adhesion, apoptosis, and the epithelial phenotype”

Sue Vande Woude, Colorado State University, Fort Collins “The biology of nondomestic feline lentiviruses”

Peter Sicinski, Harvard University, Cambridge, Massachusetts “Cyclins in development and in breast cancer”

Peter Vogt, Scripps Research Institute, LaJolla, California “The secret life of oncogenes”

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April

David Waters, Purdue University, West Lafayette, Indiana “The role of pet dogs with spontaneous bone and prostate cancers in the development of new can-cer imaging and therapeutic agents”

John Condeelis, Albert Einstein College of Medicine, Bronx, New York “Mechanisms of chemotaxis of carcinoma cells during metastasis from the primary tumor”

Natalie Ahn, University of Colorado, Boulder “Regulation and function of the MAP kinase pathway”

Benjamin Neel, Harvard School of Medicine, Boston, Massachusetts “Tyrosine phosphatases in health and disease”

May

George Klein, Karolinska Institute, Stockholm, Sweden “The role of epigenetic and genetic changes in tumor evolution”

Louis Staudt, National Cancer Institute, Bethesda, Maryland “Molecular diagnosis of cancer by gene expression profiling”

Olga Volpert, Northwestern University, Chicago, Illinois “The cross-talk between inducers and inhibitors of angiogenesis”

Janet Rossant, Mount Sinai Hospital, New York, New York “Stem cells from the mammalian blastocyst”

Bin Sing Teh, Baylor College of Medicine, Houston, Texas “Combined gene therapy and intensity-modulated radiotherapy (IMRT) for prostate cancer”

Jim Woodgett, University of Toronto, Canada “Physiological functions and regulation of protein kinase B and GSK-3”

June

Judith Sebolt-Leopold, Pfizer, Ann Arbor, Michigan “The potential of MEK inhibitors for anticancer therapy”

John Diffley, Cancer Research U.K., Hertfordshire, United Kingdom“DNA replication, genome stability, and cancer: lessons from budding yeast?”

Steve Goff, Columbia University, New York, New York “Host gene products affecting the replication of mammalian retroviruses”

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July

Stan Korsmeyer, Dana-Farber Cancer Institute, Boston, Massachusetts “Mitochondrial gateway to apoptosis”

Valerie Weaver, University of Pennsylvania, Philadelphia“Stromal-epithelial interactions and breast cancer progression: a structural perspective”

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Conference

VIII International Workshop onMultiple Endocrine Neoplasia

The Van Andel Research Institute hosted the VIII International Workshop in Grand Rapids,Michigan, on June 16–18, 2002. Over 180 researchers took part in the sessions. Hosted by Bin T.Teh of VARI, the workshop featured 29 speakers from around the world.

MEN Conference speaker list

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Sunita Argawal National Institutes of Health, U.S.A.

Douglas Ball John Hopkins University, U.S.A.

Albert Beckers,University of Liège, Belgium

John Carpten National Institutes of Health, U.S.A.

Judy Crabtree National Institutes of Health, U.S.A.

Charis Eng The Ohio State University, U.S.A.

Nicholas Hayward Queensland Institute of Medical Research,

Australia

Geoff Hendy McGill University, Canada

Wouter de Herder Erasmus Medical Centre, Netherlands

Robert Jensen National Institutes of Health, U.S.A.

Sissy Jhiang The Ohio State University, U.S.A.

Catharina Larsson Karolinska Institute, Sweden

Irina Lubensky National Cancer Institute, U.S.A.

Eammon Maher University of Birmingham, U.K.

Stephen Marx National Institutes of Health, U.S.A.

Jeffrey F. Moley Washington University School of Medicine,

U.S.A.

Lois Mulligan Queen’s University, Canada

Patricia Niccoli-Sire Hospital of Timone, France

Naganari OhkuraNational Cancer Center Research Institute,

Japan

Bruce Robinson Sydney University, Australia

G. Romeo International Agency for Research on Cancer,

France

James Scheiman University of Michigan, U.S.A.

Joseph Shepherd University of Tasmania, Australia

Constantine Stratakis National Institutes of Health, U.S.A.

Masahide Takahashi Nagoya University School of Medicine, Japan

Bin T. Teh Van Andel Research Institute, U.S.A.

Rajesh V. Thakker University of Oxford, England

Yu Xiong University of North Carolina, U.S.A.

Zhengping Zhuang National Cancer Institute, U.S.A.

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Session chairs

Maria-Luisa Brandi University of Florence, Italy

Settara Chandrasekharappa National Institutes of Health, U.S.A.

Sara Courtneidge Van Andel Research Institute, U.S.A.

Christopher Ellison The Ohio State University, U.S.A.

Charis Eng The Ohio State University, U.S.A.

Robert Gagel M.D. Anderson Cancer Center, U.S.A.

Catharina Larsson Karolinska Institute, Sweden

C.J. Lips University Hospital Utrecht, The Netherlands

Eammon Maher University of Birmingham, U.K.

Harmut HP Neumman Albert-Ludwigs University, Germany

Magnus Nordenskjold Karolinska Institute, Sweden

Britt Skogseid University of Uppsala, Sweden

Constantine Stratakis National Institutes of Health, U.S.A.

Pam Swiatek Van Andel Research Institute, U.S.A.

Norman Thompson University of Michigan, U.S.A.

George Vande Woude Van Andel Research Institute, U.S.A.

Bart Williams Van Andel Research Institute, U.S.A.

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Organization

Van Andel Research Institute

91

George Vande Woude, Ph.D.

Director

Left to right:Shellie KraemerShelly NovakowskiLynn Ritsema

Not shown:Carol HallasKaye Johnson

ResearchAdministration

Group

Sara Courtneidge, Ph.D.

Deputy Director

Roberta Jones

Associate Director forResearch Administration

David E. Nadziejka

Science Editor

Michelle Reed

Administrator to the Director

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Van Andel Research Institute Boards

VARI Board of Trustees

David L. Van Andel, Chairman and CEO

Christian Helmus, M.D.

Fritz M. Rottman, Ph.D.

James B. Wyngaarden, M.D.

David L. Van Andel

Board of Scientific Advisors

The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing recom-mendations and suggestions regarding the overall goals and scientific direction of VARI. The mem-bers are

Michael S. Brown, M.D., Chairman

Richard Axel, M.D.

Joseph J. Goldstein, M.D.

Richard D. Klausner, M.D.

Phillip A. Sharp, Ph.D.

Scientific Advisory Board

The Scientific Advisory Board advises the VARI Director, providing recommendations and sug-gestions specific to the ongoing research, especially in the areas of cancer, genomics, and genetics. Italso coordinates and oversees the scientific review process for the Institute’s research programs. Themembers are

Alan Bernstein, Ph.D.

Malcolm Brenner, M.D., Ph.D.

Patrick O. Brown, M.D., Ph.D.

Webster Cavenee, Ph.D.

Tony Hunter, Ph.D.

Frank McCormick, Ph.D.

Davor Solter, M.D., Ph.D.

Bruce Stillman, Ph.D.

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Van Andel Institute Administrative Organization

The organizational units listed below provide administrative support to both the Van Andel Research Institute and the Van Andel Education Institute.

ExecutiveR. Jack Frick, Chief Financial OfficerAnn Schoen

Communications and DevelopmentCasey Wondergem, Vice PresidentSandra G. KattMargo PrattTina Shelton

Information TechnologyBryon Campbell Chief Information OfficerDavid Drolett, ManagerMichael Roe, ManagerKathleen CerasoliMichael FosterKenneth HoekmanKimberlee JeffriesCandy Wilkerson

Human ResourcesLinda Zarzecki, ManagerMargie HovingPamela MurrayAngela Plutschouw

Grants and ContractsCarolyn Witt, DirectorSara O’Neal

FinanceTimothy Myers, ControllerMatthew Blok, Asst. ControllerRichard HerrickKeri JacksonAngela LawrenceJamie VanPortfleet

PurchasingRichard Disbrow, ManagerDavid ClarkChristian KutchinskiAmy Poplaski

FacilitiesSamuel Pinto, SupervisorJason DawesGerald LaddRichard Ulrich

SecurityKevin Denhof, ChiefKelley Herrick

Glass Washing/ Media PreparationMelissa DonnellyTroy Lawson

Contract SupportValeria Long, Librarian

(Grand Valley State University)Jim Kidder, Safety Manager

(Michigan State University)Stephen Burns, HousekeepingTim Pospisil, HousekeepingRaymond Rupp, Housekeeping

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VARI Photos 2001-2002

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Back cover photo: Genetic ablation of the Drf1 gene gives rise to hypermotile cellsThe Drf1 gene was genetically ablated by homologous recombination. Drf1– cells become hypermotile andproduce large lamellipodia. These observations suggest that Drf1 genes participate in the regulation of motili-ty and that they negatively regulate motility.(Peng, Swiatek, and Alberts)

The Van Andel Institute and/or its affiliated organizations (VARI and VAEI), through its responsible managers,recruits, hires, upgrades, trains, and promotes in all job titles without regard to race, color, religion, sex,national origin, age, disability status, or veteran status, except where an accommodation is unavailable and/oris a bone fide occupational qualification.

Van Andel Research Institute

Scientific Report 2002

333 Bostwick Avenue, N.E., Grand Rapids, MI 49503Phone (616) 234-5000; Fax (616) 234-5001; Web site: www.vai.org

Cover photograph of the Van Andel Institute building, Grand Rapids, Michigan© Jeff Goldberg/Esto