2006 Van Andel Research Institute Scientific Report

VARI | 2006

Phone 616.234.5000 Fax 616.234.5001 www.vai.org

Scientific Report

333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503

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Van Andel Research Institute Scientific Report 2006

Van Andel Research Institute Scientific Report 2006 | Cover Images The images on the cover are products of the research in VARI laboratories.

The cover images are reproduced as full-page illustrations within the book along with captions.

Van Andel Research Institute 333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503 Phone 616.234.5000 Fax 616.234.5001

www.vai.org

VARI | 2006

Van Andel Research Institute Scientific Report 2006

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Table of Contents

VARI | 2006

Director’s Introduction

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Laboratory Reports

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

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Brian Cao, M.D. Antibody Technology

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

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Nicholas S. Duesbery, Ph.D. Cancer and Developmental Cell Biology

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Bryn Eagleson, A.A., RLATG Vivarium and Transgenics

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Kyle A. Furge, Ph.D. Computational Biology

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Brian B. Haab, Ph.D. Cancer Immunodiagnostics

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Rick Hay, Ph.D., M.D., F.A.H.A. Noninvasive Imaging and Radiation Biology

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Sheri L. Holmen, Ph.D. Molecular Medicine and Virology

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Cindy K. Miranti, Ph.D. Integrin Signaling and Tumorigenesis

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James H. Resau, Ph.D. Division of Quantitative Sciences Analytical, Cellular, and Molecular Microscopy Microarray Technology

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Pamela J. Swiatek, Ph.D., M.B.A. Germline Modification

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Bin T. Teh, M.D., Ph.D. Cancer Genetics

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George F. Vande Woude, Ph.D. Molecular Oncology

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

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Michael Weinreich, Ph.D. Chromosome Replication

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Bart O. Williams, Ph.D. Cell Signaling and Carcinogenesis

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H. Eric Xu, Ph.D. Structural Sciences

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Nian Zhang, Ph.D. Mammalian Developmental Genetics

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George F. Vande Woude, Ph.D.

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Daniel Nathans Memorial Award

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Postdoctoral Fellowship Program

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Student Programs

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Tony Hunter, Ph.D., and Tony Pawson, Ph.D.

List of Fellows

Grand Rapids Area Pre-College Engineering Program Summer Student Internship Program

Han-Mo Koo Memorial Seminar Series

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VARI Organization

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2005 | 2006 Seminars

Boards Office of the Director VAI Administrative Organization

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Director’s Introduction

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George F. Vande Woude

Director’s Introduction

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We are now in our sixth year, with the sights and sounds of construction all around us. Easily visible from VARI’s windows—as well as sometimes felt through the floor—are major projects on the other side of Michigan Avenue. Directly north of us is a large excavation destined to become a parking ramp/hotel/retail site. One block east is another that will become the Lemmen-Holton Cancer Pavilion of Spectrum Health. And immediately west, cores have been drilled over the past two months in preparation for construction of the Phase II addition to our own VAI building. That addition, when complete, will provide us with the space to increase our research programs and staff substantially, with major benefits to our existing cancer studies as well as our newer Parkinson disease initiative.

Personnel notes We congratulate Jim Resau, who heads up our newly organized Division of Quantitative Sciences. The purpose of the reorganization was to provide coordinated and integrated support for investigators and special programs within VARI. The PIs of the programs will continue to develop core resources and independent research projects to advance their respective fields. The Division includes the laboratories of Analytical, Cellular, and Molecular Microscopy (J. Resau); Microarray Technology (J. Resau); Computational Biology (K. Furge); Mass Spectrometry and Proteomics (G. Cavey); and a new laboratory under development, Molecular Epidemiology.

Dr. Resau will also continue to serve as Deputy Director for Special Programs.

Congratulations also to Rick Hay, Ph.D., M.D., who was listed as one of “America’s Top Physicians” for 2004–2005 by the Consumers’ Research Council of America. Dr. Hay is engaged in establishing his new Laboratory of Noninvasive Imaging and Radiation Biology.

As Deputy Director for Clinical Programs, he is establishing the Office of Translational Programs, charged with developing relationships between VARI and clinical entities. He is also overseeing the establishment of a Good Manufacturing Practices (GMP) facility.

Bin Teh recently told me of his need to spend more time directing his laboratory and involving himself more deeply in his scientific collaborations and studies. I completely understand his position, yet it was with regret that I acceded to his resignation as a Deputy Director. Both his lab and VARI will benefit from his renewed efforts to uncover how cancer originates and develops.

I have appointed Nick Duesbery as our new Deputy Director for Research Operations. Some of the activities that fall within that area are space allocations, the library, and our seminars and symposia. From VARI’s beginning, Nick has been an active participant in our operations and committees, including the IACUC, the Postdoc Advisory Committee, and the Promotion Review Committee. I am pleased that he has agreed to take on this additional responsibility, and I’m sure his contributions in this role will benefit us all.

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It is also my pleasure to announce that Pamela Swiatek has been promoted to Senior Scientific Investigator. Pam joined VARI in August 2000 to establish the Germline Modification Program, and she has done an outstanding job providing both services and technology to support the development, analysis, maintenance, and preservation of mouse models for the study of human diseases. She has secured or helped secure more than $4 million of extramural funding, and she had a key role in the development of the Core Technology Alliance (CTA). Her efforts to support the planning and development of a continuation proposal for submission under the 21st Century Jobs Fund Program have been invaluable to VARI as well as to our CTA partner institutions. Pam had a keen interest in broadening her administrative skills, so she enrolled in the MBA program in executive management at Purdue University’s Krannert School of Management. Pam completed the program while fully engaged in her responsibilities at VARI and was awarded the MBA degree in 2005. We are fortunate to have Pam as a colleague.

Grants Our investigators have continued to compete successfully for external funding for their research, both from the government and from private organizations. In 2005, Eric Xu was awarded a five-year R01 grant from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, for his project “Structural Genomics of Orphan Nuclear Receptors.” In addition, Brian Haab received an NIH/National Cancer Institute R21 award for the study of “Longitudinal Cancer-Specific Serum Protein Signatures” over two years.

Cindy Miranti was awarded a Research Scholar Grant from the American Cancer Society for the study of “Integrin and RTK Signaling and Crosstalk in Prostate Epithelial Cells” over four years. Cindy also received a two-year grant from the Elsa U. Pardee Foundation for work on the metastasis suppressor KAI1/C. Craig Webb was funded under an FY2005 appropriation from the Health Resources and Services Administration, an agency of the U.S. Department of Health and Human Services, to support the Multiple Myeloma Research Program initiative at the Institute. And, Nick Duesbery has received a three-year NIH/NCI award from the Underrepresented Minorities Program to support a research trainee on his MEK signaling R01 project.

Sok Kean Khoo, of Bin Teh’s Laboratory of Cancer Genetics, received a grant in 2005 from the National Kidney Foundation of Michigan to study the genetics of familial renal cell carcinoma. My own Laboratory of Molecular Oncology has received funding from two firms for sponsored research studies of proprietary compounds they have developed. We will be investigating the potential of these compounds in pre-clinical in vitro and in vivo systems as anticancer therapeutics.

It is important to note that, in addition to success in competing for grant funding, the work done under the grants we have received is being translated into publishable results. VARI has published 226 peer-reviewed articles through April 1, 2006, with another 13 articles in press. I am proud of the productivity of our labs and their dedicated personnel.

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Future directions The VAI Graduate School is coming closer to matriculating its first class of students, and its existence is more tangible now that we have a Dean. Steven Triezenberg, Ph.D., has been named Graduate Dean of the school and will start at VAI in May 2006. He is currently a Professor in the Department of Biochemistry and Molecular Biology at Michigan State University and Associate Director of the graduate program in Cell and Molecular Biology. In addition to leading the Graduate School, he will continue his research on gene expression and transcription using herpes simplex virus as an investigational model. We look forward to watching the Graduate School progress under his leadership.

We are establishing a joint venture with Spectrum Health to set up a Center for Molecular Medicine in West Michigan. This will be a valuable opportunity to develop the clinical benefits of molecular diagnostics growing from our cutting-edge research in areas such as microarrays and gene sequencing, and it will build on Spectrum’s existing facilities and experience in molecular diagnostic services.

We look forward to building this opportunity into a world-class diagnostics service, providing biomedical and health benefits for the population of West Michigan and beyond.

The move of the College of Human Medicine from Michigan State University to the Grand Rapids area has taken form over the past year.

In April the MSU Board of Trustees passed a resolution approving the move. Within the coming months, the detailed structure of the School—involving MSU, VAI, Spectrum Health, Grand Valley State University, St. Mary’s, and others—will be defined and established.

This will be a major addition to the biomedical community here in Grand Rapids, and VAI will have a large and mutually beneficial role to play in the school’s founding and development in the coming years.

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Truly, the Institute finds itself in the midst of a large-scale initiative to develop a regional biomedical center (U.S.A. Today, April 25, 2006).

We are proud to be a major and early stimulus for this initiative.

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Laboratory Reports

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

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In 1993, Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of California, San Diego, where he studied with James Feramisco. From 1994 to 1997, he served as a postdoctoral fellow in Richard Treisman’s laboratory at the Imperial Cancer Research Fund in London, England. From 1997 through 1999, he was an Assistant Research Biochemist in the laboratory of Frank McCormick at the Cancer Research Institute, University of California, San Francisco. Dr. Alberts joined VARI as a Scientific Investigator in January 2000.

Staff Laboratory Staff Jun Peng, M.D. Yunju Chen, Ph.D. Kathryn Eisenmann, Ph.D. Holly Holman, Ph.D. Susan Kitchen, B.S.

Students Students Aaron DeWard, B.S. Dagmar Hildebrand, B.S. Yaojian Liu, B.S.

Visiting Scientists

Visiting Scientists

Stephen Matheson, Ph.D. Brad Wallar, Ph.D.

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Research Interests The actin cytoskeleton is a dynamic, tightly regulated protein network that plays a crucial role in mediating diverse cellular processes including cell division, migration, endocytosis, vesicle trafficking, and cell shape. The research focus of the lab is the genetics and molecular biology of the Rho family of small GTPases and their effectors, which together control multiple aspects of cytoskeletal dynamics. The guiding hypothesis of the laboratory is that cytoskeletal dynamics defines the what, where, and how of signal transduction pathways’ control responses to growth factors and other extracellular cues, and that defects in these tightly controlled dynamics can contribute to cancer pathophysiology. Support for this hypothesis is observed in human cancers that carry mutations in genes encoding regulators of Rho GTPase activity. Ultimately, our goal is to exploit our understanding of the mechanics of GTPase-effector relationships in order to develop anti-cancer therapeutics.

Much of our work focuses on the role of cytoskeletal remodeling mediated by the formin family of actin-nucleating proteins. The formins are highly conserved proteins implicated in a diverse array of cellular functions, including the cytoskeletal remodeling events necessary for cytokinesis, bud formation in yeast, the establishment of cell/organelle polarity, and endocytosis. Formins have the ability to stabilize microtubules, which (like F-actin) are assembled by tightly controlled cycles of polymerization and depolymerization.

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The mDia formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho GTPase binding to mDia amino-terminal GTPase-binding domains (GBDs) sterically hinders the adjacent Dia-inhibitory domain (DID) interaction with the carboxyl-terminal Diaautoregulatory (DAD) domain (Fig. 1). The release of DAD allows the adjacent FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, comprises a core motif (M-D-x-L-L-x-L) and an adjacent region of numerous basic residues (typically R-R-K-R) in the mDia family.

Figure 1.

Figure 1. mDia proteins are autoregulated nucleators of actin. Autoinhibition is mediated by interaction between DID and DAD adjacent to the GBD and FH2 domains, respectively. Activated GTP-bound Rho proteins bind to the GBD where they interfere with DAD binding to DID. The free FH2 domains, which also function as dimerization interfaces, can then nucleate actin monomers and processively elongate actin filaments.

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Recent studies have shown that these specific amino acids within the basic region of DAD (highlighted in blue in Fig. 2) contribute to the binding of DID and therefore to the maintenance of the mDia autoregulatory mechanism. Expression of full-length versions of mDia2, previously shown to have amino acid substitutions in either the DAD core or the basic region, causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge (Fig. 3). These studies further refine the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.

The importance of the observations is severalfold. They further our understanding of how these conserved actin-nucleating proteins are controlled in cells. Since there are numerous drugs that target the cytoskeleton and several (such as paclitaxel [Taxol]) that are effective anti-cancer drugs, the information gleaned from the DAD-DID binding studies will further our efforts to develop drugs that target this family of proteins. Finally, the observation that mDia proteins have a role in filopodia assembly should impact multiple fields beyond that of cancer cell motility. Filopodia and similar structures are important, for example, for the ability of neuronal cells to change shape and form neurites. Neurites are the structures that nerve cells use to contact and communicate with other nerve cells.

Figure 2.

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Figure 2. Formins can be deregulated by disrupting autoinhibition via mutations in DAD that block its ability to interact with DID. The amino acid sequence alignment from Dia-autoregulatory domains from human, mouse, fruit fly, and fungal formins shows the conserved residues in red; similar residues are shown in green. The basic region in DAD is highlighted in blue. Alanine substitutions, such as M1041A in mDia2, disrupt the autoregulatory DID-DAD interaction, thereby leading to a constitutively active protein.

Figure 3.

Figure 3. Constitutively active mDia2-M1041A localizes to the tips of filopodia. YFP-mDia2-M1041A, co-expressed with an interfering form of the GTPase Rac1, causes cells to form extensions (filopodia) resulting from the elongation of F-actin within the filopodia. The result also shows that Rac activity is not involved in mDia2-assembled actin structures.

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External Collaborators Kathy Siminovitch, University of Toronto, Canada Philippe Chavrier, Institut Curie, Paris, France

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Recent Publications

From left: Chen, Holman, DeWard, Eisenmann, Kitchen, Alberts, Hildebrand

Wallar, Bradley J., Brittany N. Stropich, Jessica A. Schoenherr, Holly A. Holman, Susan M. Kitchen, and Arthur S. Alberts. 2006. The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the Diaphanous-related formin inhibitory domain. Journal of Biological Chemistry 281(7): 4300–4307.

Alberts, Arthur S., Huajun Qin, Heather S. Carr, and Jeffery A. Frost. 2005. PAK1 negatively regulates the activity of the Rho exchange factor NET1. Journal of Biological Chemistry 280(13): 12152–12161.

Colucci-Guyon, Emma, Florence Niedergang, Bradley J. Wallar, Jun Peng, Arthur S. Alberts, and Philippe Chavrier. 2005. A role for mammalian Diaphanous-related formins in complement receptor (CR-3)-mediated phagocytosis in macrophages. Current Biology 15: 2007–2012.

Eisenmann, Kathryn M., Jun Peng, Bradley J. Wallar, and Arthur S. Alberts. 2005. Rho GTPase-formin pairs in cytoskeletal remodeling.

In Signalling Networks in Cell Shape and Motility, Novartis Foundation Symp. series, Vol. 269. London, U.K.: Novartis Foundation, pp. 206–230.

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Brian Cao, M.D. Laboratory of Antibody Technology

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Dr. Cao obtained his M.D. from Beijing Medical University, People’s Republic of China, in 1986. On receiving a CDC fellowship award, he was a visiting scientist at the National Center for Infectious Diseases, Centers for Disease Control and Prevention (1991–1994). He next served as a postdoctoral fellow at Harvard (1994–1995) and at Yale (1995–1996). From 1996 to 1999, Dr. Cao was a Scientist Associate in charge of the Monoclonal Antibody Production Laboratory at the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Dr. Cao joined VARI as a Special Program Investigator in June 1999.

Staff Laboratory Staff Ping Zhao, M.S. Tessa Grabinski, B.S.

Students Students Qin Hao Xin Wang Aixia Zhang Jin Zhu

Visiting Scientists

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Research Interests Antibodies are primary tools in several areas of biomedical sciences, including basic research, diagnostics, and molecular therapeutics.

In basic biomedical research, the characterization and analysis of almost any molecule involves the production of specific monoclonal or polyclonal antibodies that react with it. The production and sale of antibodies for research purposes is a multibillion-dollar industry worldwide. Antibodies are also widely used in diagnostic applications for clinical medicine. ELISA and radioimmunoassay systems are antibody-based. Analysis of cells and tissues in pathology laboratories includes the use of antibodies on tissue sections and in flow cytometry analyses. Overall, antibody diagnostics also form a multibillion-dollar industry. Antibodies are making rapid inroads into medical therapeutics as well. The success of anti–tumor necrosis factor in the treatment of rheumatoid arthritis and inflammatory bowel disease; anti–B cell antibodies in the treatment of lymphoma; and antibodies against oncogene products in the treatment of breast cancer are only the first examples of what is emerging as a broad new class of therapeutic agents. The monoclonal antibody market is one of the fastest growing, and it has the potential to reach $30.3 billion by 2010, driven by technological evolution from chimeric and humanized to fully human antibodies.

To speed up antibody-related translational research at VARI, the Antibody Technology laboratory has developed several technologies over the last few years: 1) state-of-the-art monoclonal antibody (mAb) production and characterization, followed by scaled-up production and purification; 2) antibody-binding-site epitope mapping using a phage-display peptide library; 3) construction of a human-antibody-fragment phage-display library and screening of specific fragments from the library; and 4) murine-human chimeric antibody generation and murine antibody humanization.

Functioning as an antibody production core facility, this lab has extensive capabilities. The technologies and services available in the core include antigen preparation and animal immunization; peptide design and coupling to protein carriers; consultation on protein expression and purification; DNA immunization (gene-gun technology); immunization with living or fixed cells; conventional antigen/ adjuvant preparation; immunization of a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Our work also includes the generation of hybridomas from spleen cells of immunized mice, rats, and rabbits; hybridoma expansion and subcloning; cryopreservation of hybridomas secreting mAbs; isotyping of mAbs; ELISA screening of hybridoma supernatants; mAb characterization by immunoprecipitation, Western blot, immunohistochemistry, immunofluorescence staining, FACS, and in vitro bioassays; production of bulk quantities of mAbs using high-density cell culture; purification of mAbs on FPLC affinity columns; generation of bi-specific mAbs by secondary fusion; mAb affinity column preparation to purify antigen; conjugation of mAbs to enzymes, biotin/streptavidin, or fluorescent reporters; and development of detection methods/kits such as sandwich ELISA. Over 100 hybridomas against more than 10 unique proteins have been generated and characterized in the past year. In addition, the Antibody Technology lab has established contract services to local biotechnology companies to generate, characterize, produce, and purify mAbs for their research and for diagnostic kit development.

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The Michigan Core Technology Alliance (CTA), funded by the state government, was created in 2001. The Antibody Technology Core at VARI and the Hybridoma Core at the University of Michigan in Ann Arbor joined together to form the Michigan Antibody Technology Core (MATC) and became the seventh core of CTA in March 2005. Our goals are to provide state-of-the-art antibody technologies and services to research scientists; to generate, characterize, produce, and purify a wide variety of monoclonal antibodies; to make human antibody fragments and humanize murine mAbs for clinical diagnostic/therapeutic applications; and to advance biomedical research and development. The Antibody Technology lab at VARI serves as the core’s hub, and Dr. Brian Cao is director of MATC. Mapping an antibody-binding epitope to a protein-protein or antigen binding site using a phage-display random peptide library is a powerful technology for studying protein-protein interactions and the kinetics of antibody interactions, as well as in screening for small peptides that have potential therapeutic applications. A random peptide library is constructed by genetically fusing oligonucleotides coding for polypeptides to the DNA coding for a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences and the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules such as antibodies.

A library of phage is exposed to a plate coated with mAb. Unbound phage are washed away and then specifically bound phage are eluted by lowering the pH. The eluted pool of phage is amplified, and the process is repeated for two more rounds. Individual clones are isolated, screened by ELISA, and sequenced.

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With this technology, we have been able to screen out and identify a small peptide that specifically binds to Met, the receptor of hepatocyte growth factor/scatter factor (HGF/SF). Activation of Met by HGF/SF affects downstream signaling pathways (including additional protein kinases) responsible for cellular differentiation, motility, proliferation, organogenesis, angiogenesis, and apoptosis. Aberrant expression of the Met-HGF/SF receptor-ligand complex—resulting either from mutations in the complex or in conjunction with mutations in other oncogenes—is associated with an invasive/metastatic phenotype in most solid human tumors. Met-HGF/SF and downstream kinases are therefore attractive targets for new agents aimed at clinical diagnosis, prognosis, and treatment of cancer. We have also successfully epitope-mapped several neutralizing anti-HGF/SF mAbs. The information about the antibody-binding site obtained from these experiments should lead us to new strategies and new reagents for cancer intervention.

In collaboration with Nanjing Medical University, China, we have initiated a project to construct a phage-display antibody fragment library, which will involve the construction and use of human/animal immunized/naïve Fab and scFv antibody gene repertoires. The ability to co-select antibodies and their genes enables the isolation of high-affinity, antigen-specific mAbs derived from either immunized animals or non-immunized humans. Over the past two years, we have closely followed the development of this technology for producing novel recombinant antibody-like molecules. We constructed a human naïve Fab library with a diversity of 2 × 109 in late 2004. In 2005, we screened out several Fab fragments from the library that specifically recognize HGF/SF, Met, and EGFR. Moreover, by modifying and improving biopanning strategies, we have selected Fab fragments that recognize both the Met and EGFR extracellular domains in native confirmation with reasonable affinity and, importantly, with an internalization property that makes these Fabs attractive as conjugate reagents for immuno-chemotherapy or immuno-radiation therapy against cancer.

External Collaborators We have established strong collaborations internationally and locally with universities, institutions, and companies. Some examples are the Key Laboratory of Antibody Technology, Nanjing Medical University, China; the Medical Research Council (MRC), Cambridge, England; the Protein Research Department, Mubarak Scientific City, Egypt; Neogen Corporation, Lansing, Michigan; and Assay Designs, Inc., Ann Arbor, Michigan.

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Recent Publications

From left: Grabinski, Nelson, Cao, Wang, Zhang, Zhao

Tsarfaty, G., G.Y. Stein, S. Moshitch-Moshkovitz, D.W. Kaufman, B. Cao, J. Resau, G.F. Vande Woude, and I. Tsarfaty. In press. HGF/SF increases tumor blood volume: a novel tool for in vivo functional molecular imaging of Met. Neoplasia.

Hay, Rick V., Brian Cao, R. Scot Skinner, Yanli Su, Ping Zhao, Margaret F. Gustafson, Chao-Nan Qian, Bin T. Teh, Beatrice S. Knudsen, James H. Resau, Shuren Shen, David J. Waters, Milton D. Gross, and George F. Vande Woude. 2005. Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek™). Clinical Cancer Research 11(19): 7064s–7069s.

Jiao, Yongjun, Ping Zhao, Jin Zhu, Tessa Grabinski, Zhengqing Feng, Xiaohong Guan, R. Scot Skinner, Milton D. Gross, Rick V. Hay, Hiroshi Tachibana, and Brian Cao. 2005. Construction of human naïve Fab library and characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology 31(1): 41–54.

Ren, Yi, Brian Cao, Simon Law, Yi Xie, Ping Yin Lee, Leo Cheung, Yongxong Chen, Xin Huang, Hiu Man Chan, Ping Zhao, John Luk, George Vande Woude, and John Wong. 2005. Hepatocyte growth factor promotes cancer cell migration and angiogenic factors expression: a prognostic marker of human esophageal squamous cell carcinomas. Clinical Cancer Research 11(17): 6190–6197.

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

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Mr. Cavey received his B.S. degree from Michigan State University in 1990. Prior to joining VARI he was employed at Pharmacia in Kalamazoo, Michigan, for nearly 15 years. As a member of a biotechnology development unit, he was group leader for a protein characterization core laboratory. More recently as a research scientist in discovery research, he was principal in the establishment and application of a state-of-the-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002.

Staff Laboratory Staff Paula Davidson, M.S.

Student Students Wendy Johnson

Visiting Scientists

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Research Interests The Mass Spectrometry and Proteomics program works with many of the research labs at the Institute, using state-of-the-art mass spectrometers in combination with analytical protein separation and purification methods to help answer a wide range of biological questions. Using mass spectrometry data and database search software, proteins can be identified and characterized with unprecedented sensitivity and throughput. Since proteomics is a relatively new scientific discipline, many of the analytical techniques are rapidly changing; therefore our mission involves using established protocols, improving them, and developing new approaches to expand the scope of biological hypotheses being addressed.

Protein-protein interactions Analyzing samples representing different cellular conditions or disease states is a step toward understanding the role of a protein with an unknown function or understanding the regulatory mechanism of several proteins in a given pathway. In this approach, a known protein is affinity-purified from a nondenatured sample using antibodies, affinity tags such as FLAG or TAP, or immobilized small molecules. The purified protein and its binding partners are separated using two-dimensional (2D) electrophoresis gels or SDS-PAGE. After staining, the proteins are cut from the gel, digested into peptides using an enzyme such as trypsin, and then analyzed by nanoscale high-pressure liquid chromatography on line with a mass spectrometer. The mass spectrometer fragments the peptides and the resulting spectra are used to search protein or translated DNA databases. Identifications are made using the amino acid sequences derived from the mass spectrometry data. We have optimized all aspects of this analysis for sample recovery yields and high-sensitivity protein identification.

Recently, we have been evaluating newly developed software that allows us to eliminate the electrophoresis separation step from these analyses, giving the potential to identify more proteins from complex mixtures. With this software, affinity-purified protein complexes are compared to a control sample using a technique known as peptide differential display. The proteins are digested into peptides in solution rather than from gels and are analyzed by high-pressure liquid chromatography–mass spectrometry (LC–MS). Peptides that are unique to the experimental sample relative to the control are used to identify proteins that are part of a protein complex.

Protein characterization Our laboratory also characterizes proteins and their post-translational modifications. Proteins expressed and purified by investigators are analyzed by protein electrospray to confirm the average protein molecular weight before proceeding to labor-intensive studies such as protein crystallization.

Mapping the post-translational modifications of proteins such as phosphorylation is an important yet difficult undertaking in cancer research. Phosphorylation regulates many protein pathways, several of which could serve as potential drug targets for cancer therapy.

In recent years, mass spectrometry has emerged as a primary tool that helps investigators determine exactly which amino acids of a protein are modified. This undertaking is complicated by many factors, but principally by the fact that pathway regulation can occur when only 0.01% of a given protein population is phosphorylated. Thus, we are dealing with an extremely small number of phosphorylated proteins among a huge number of nonphosphorylated proteins. Our lab collaborates with investigators to map protein phosphorylation using a variety of techniques, including metal affinity purification, immunoaffinity purification of phosphoproteins and peptides, and phosphorylation-specific mass spectrometry detection.

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Protein expression As mass spectrometry instruments and protein separation methods develop, proteomics techniques allow researchers to identify and quantitate protein samples of increasing complexity. The ultimate goal is to catalog all proteins expressed in a given cell or tissue, as a means of evaluating all of the physiological processes occurring within. This approach, termed systems biology, aims at understanding how all proteins interact to effect a biological outcome. Traditionally this has been done using 2D gel electrophoresis, image analysis of stained proteins, and identification of proteins from gels using mass spectrometry. Due to the labor-intensive nature of 2D gels and the underrepresentation of some classes of proteins (such as membrane proteins), the field of proteomics has been moving toward solution-based separations and direct mass spectrometry.

To that end, our laboratory recently purchased and installed a Waters Corporation Protein Expression System for non-gel-based protein expression analysis. This system represents a paradigm shift in the field of proteomics because it provides both quantitative and qualitative data on complex mixtures of proteins in a single LC–MS analysis. To perform this analysis, proteins are enzymatically digested using trypsin and, without any chemical or isotopic labeling, the resulting peptides are analyzed by LC–MS. The combination of molecular mass and LC retention time establishes a signature for each peptide and allows comparison across samples. The mass spectrometer signal intensity of each peptide is used for quantitation. Qualitative protein identification data is obtained by fragmenting all peptides eluting into the mass spectrometer, a feature unique to the Waters instrument. This system at VARI puts us in an elite group of institutions that have this powerful new technology; fewer than 20 Protein Expression Systems of this type are in operation worldwide.

This system will be used to map protein changes under a systems biology approach and to discover biomarkers for early detection and diagnosis of cancer and other diseases.

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External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Michael Hollingsworth, Eppley Cancer Center, Omaha, Nebraska

From left: Davidson, Johnson, Cavey

Core Technology Alliance (CTA) This laboratory participates in the CTA as a member of the Michigan Proteomics Consortium (MPC).

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Nicholas S. Duesbery, Ph.D. Laboratory of Cancer and Developmental Cell Biology

Dr. Duesbery received both his M.Sc. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Dr. Duesbery was appointed Deputy Director for Research Operations in March 2006.

Staff

Laboratory Staff Philippe Depeille, Ph.D. Yan Ding, Ph.D. Hilary Wagner, M.S. John Young, M.S. David Slager, B.S. Elissa Boguslawski

Students Students Chia-Shia Lee, M.S. Lisa Orcasitas Zafar Qadir

Visiting Scientists

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Research Interests The overall goal of the Laboratory of Cancer and Developmental Cell Biology is to advance the study of mitogen-activated protein kinase kinase (MEK) signaling in health and disease. Currently, work performed in the lab is organized into three projects to explore 1) MEK signaling and tumor biology, 2) the therapeutic potential of anthrax lethal toxin (LeTx), and 3) molecular mechanisms of anthrax lethal toxin action.

MEK signaling and tumor biology Many malignant sarcomas such as angiosarcomas are refractory to currently available treatments. However, sarcomas possess unique vascular properties which indicate they may be more responsive to therapeutic agents that target endothelial function. MEKs have been demonstrated to play an essential role in the growth and vascularization of carcinomas. We hypothesize that signaling through multiple MEK pathways is also essential for sarcoma growth and vascularization. The objective of this research is to define the role of MEK signaling in the growth and vascularization of human sarcoma and to determine whether inhibition of multiple MEKs by agents such as LeTx, a proteolytic inhibitor of MEKs, may form the basis of a novel and innovative approach in the treatment of human sarcoma.

The therapeutic potential of anthrax lethal toxin 18

Data from the National Cancer Institute’s Anti-Neoplastic Drug Screen indicates that several tumor types, notably melanomas and colorectal adenocarcinomas, are sensitive to LeTx. In addition, we have noted that angio-proliferative tumors are also very sensitive to LeTx treatment. Consequently we have undertaken a systematic evaluation of the effects of LeTx upon human tumor-derived melanoma, colorectal adenocarcinoma, and Kaposi sarcoma cell lines. The goal of this project is to develop novel therapeutic agents that may be efficacious in the treatment of human malignancies. In collaboration with Art Frankel at Scott & White Memorial Hospital Cancer Research Institute in Temple, Texas, we have begun a project to manufacture clinical-grade LeTx. The first results of these studies were published this year in Molecular Cancer Therapeutics.

Molecular mechanisms of anthrax LeTx action The lethal effects of Bacillus anthracis have been attributed to an exotoxin which it produces. This exotoxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). EF is an adenylate cyclase and together with PA forms a toxin referred to as edema toxin. LF is a Zn2+-metalloprotease which together with PA forms a toxin referred to as lethal toxin (LeTx). LeTx is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals. The goal of this project is to develop a detailed molecular understanding of LF function that will improve our understanding of the biology underlying anthrax.

Lab notes This year our lab welcomed two new postdoctoral fellows, Philippe Depeille and Yan Ding, who will be studying the role of MEK signaling in vascularization and the growth of Kaposi sarcoma and fibrosarcoma, respectively. Hilary Wagner and David Slager joined the lab as technicians. Each will perform molecular studies of anthrax toxin function in tissue culture and in mice. Two new students also joined our lab this year. Chih-Shia Lee is a graduate student who, along with Zafar Qadir, a visiting student from the University of Bath, is investigating MEK function in cells following exposure to LeTx. As well, we hosted Courtney Banks and Anna Fairchild, two Grand Rapids area high school students interested in careers in biological research. Courtney and Anna evaluated the sensitivity of macrophage cells to LeTx. Paul Spilotro, a postdoctoral fellow who joined us in 2004, moved on to do his medical residency at Spectrum Health in Grand Rapids.

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Recent Publications

From left: Holman, Lee, Duesbery, Depeille, Young, Qadir, Ding, Boguslawski

Duesbery, Nick, and George Vande Woude. 2006. BRAF and MEK mutations make a late entrance. Science’s STKE 328: pe15.

Abi-Habib, Ralph J., Jeffrey O. Urieto, Shihui Liu, Stephen H. Leppla, Nicholas S. Duesbery, and Arthur E. Frankel. 2005. BRAF status and mitogen-activated protein/extracellular signal–regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin. Molecular Cancer Therapeutics 4(9): 1303–1310.

Bodart, J.-F.L., F.Y. Baert, C. Sellier, N.S. Duesbery, S. Flament, and J.-P. Vilain. 2005. Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes. Developmental Biology 283(2): 373–383.

Bodart, Jean-François L., and Nicholas S. Duesbery. 2005. Xenopus tropicalis oocytes: more than just a beautiful genome. In Xenopus Protocols: Cell Biology and Signal Transduction, X.J. Liu, ed. Methods in Cell Biology series, Totowa, N.J.: Humana Press.

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Photo taken by Veronique Schulz.

VARI | 2006

Metastatic melanoma cells, C8161.9, were fixed and stained to visualize actin (red), focal adhesions (green specks), and nuclei (blue).Â

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Bryn Eagleson, A.A., RLATG Vivarium and Laboratory of Transgenics

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Bryn Eagleson began her career in laboratory animal services in 1981 with Litton Bionetics at the National Cancer Institute’s Frederick Cancer Research and Development Center (NCI-FCRDC) in Maryland. In 1983, she joined the Johnson & Johnson Biotechnology Center in San Diego, California. In 1988, she returned to the NCI-FCRDC, where she continued to develop her skills in transgenic technology and managed the transgenic mouse colony. During this time Ms. Eagleson attended Frederick Community College and Hood College in Frederick, Maryland. In 1999, she joined VARI as the Vivarium Director and Transgenics Special Program Manager.

Technical Staff Laboratory Staff Synthya Roys, M.S., LAT Eric Collier, B.S. Lisa DeCamp, B.S. Dawna Dylewski, B.S. Audra Guikema, B.S., L.V.T. Jamie Bondsfield, A.S. Elissa Boguslawski, RALAT

Animal Caretaker Staff Visiting Scientists

Students

Sylvia Marinelli, Team leader Keri Tice, B.S. Crystal Brady Kathy Geil Jarred Grams Tina Schumaker Erin Tippett

VARI | 2006

Research Interests The goal of the vivarium and the transgenics laboratory is to develop, provide, and support high-quality mouse modeling services for the Van Andel Research Institute investigators, Michigan Technology Tri-Corridor collaborators, and the greater research community.

We use two Topaz Technologies software products, Granite and Scion, for integrated management of the vivarium finances, the mouse breeding colony, and the Institutional Animal Care and Use Committee (IACUC) protocols and records. Imaging equipment, such as the PIXImus mouse densitometer and the ACUSON Sequoia 512 ultrasound machine, is available for noninvasive imaging of mice. VetScan blood chemistry and hematology analyzers are now available for blood analysis. Also provided by the vivarium technical staff are an extensive xenograft model development and analysis service, rederivation, surgery, dissection, necropsy, breeding, and health-status monitoring.

Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the maternal genetic material and one derived from the sperm that contains the paternal genetic material. As development proceeds, these two pronuclei fuse, the genetic material mixes, and the cell proceeds to divide and develop into an embryo. Transgenic mice are produced by injecting small quantities of foreign DNA (the transgene) into a pronucleus of a one-cell fertilized egg. DNA microinjected into a pronucleus randomly integrates into the mouse genome and will theoretically be present in every cell of the resulting organism. Expression of the transgene is controlled by elements called promoters that are genetically engineered into the transgenic DNA. Depending on the selection of the promoter, the transgene can be expressed in every cell of the mouse or in specific cell populations such as neurons, skin cells, or blood cells. Temporal expression of the transgene during development can also be controlled by genetic engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in vivo.

From left to right, standing: Jason, Eagleson, Brady, Roys, Tice, Marinelli, DeCamp, Collier, Grams, Bondsfield; seated, Guikema, Boguslawski, Schumaker, Dylewski, Geil

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Kyle A. Furge, Ph.D. Laboratory of Computational Biology

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Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University School of Medicine in 2000.

Prior to obtaining his degree, he worked as a software engineer at YSI, Inc., where he wrote operating systems for embedded computer devices. Dr. Furge did his postdoctoral work in the laboratory of Dr. George Vande Woude. He became a Bioinformatics Scientist at VARI in June of 2001 and a Scientific Investigator in May of 2005.

Staff Laboratory Staff Karl Dykema, B.A.

VARI | 2006

Research Interests As high-throughput technologies such as DNA sequencing, gene expression microarrays, and genotyping become more available to researchers, analyzing the large amount of data produced by these technologies becomes difficult. Computational disciplines such as bioinformatics and computational biology have emerged to develop methods that assist in the storage, distribution, integration, and analysis of these large data sets. The Computational Biology laboratory at VARI currently focuses on using mathematical and computer science approaches to analyze and integrate complex data sets in order to develop a better understanding of how cancer cells differ from normal cells at the molecular level. In addition, members of the lab provide assistance in data analysis and other computational projects on a collaborative and/or fee-for-service basis.

In the past year the laboratory has worked on many projects to further the research efforts at VARI. For example, we continue to work closely with the Microarray Technology laboratory (MT) on the analysis and management of gene expression microarray chips and data sets. The MT lab manages more than 100,000 unique DNA fragments that are used to monitor gene expression in organisms including canines, rats, mice, and humans. As the knowledge of the sequence information from each species evolves, our group helps provide up-to-date information for each of these fragments. In addition, we constructed a quality assurance program to help the MT lab monitor the gene expression data that is being generated by their group. In another project, we assisted VARI’s Michael Weinreich in performing sequence analysis across multiple species to identify a potentially new protein-binding domain. We also continue our active involvement in larger projects that have resulted in collaborative grants with Jim Resau, Nick Duesbury, and Bin Teh. Providing assistance to other investigators is one focus of the laboratory. We also have a special research focus on understanding how cytogenetic abnormalities influence cancer development and progression. Many cancer types are associated with defined sets of DNA gains and losses. For example, the majority of hepatocellular carcinomas contain extra copies of chromosome 1p and lack copies of chromosome 4q. In contrast, the majority of clear cell renal cell carcinomas contain an extra copy of chromosome 5q and lack a copy of chromosome 3p. Because of the recurrent appearance of certain chromosomal gains and losses, it is reasonable to hypothesize that those disruptions are important to tumor development and progression. Over the past years, we have developed and evaluated computational methods to indirectly identify the chromosomal gains and losses that occur in tumor samples by examining the gene expression data. Then we examine regions of chromosomal gain or loss for misregulation of single genes within the region of abnormality. In addition, we are developing and evaluating methods to determine if a large number of genes that are associated with a particular oncogenic pathway show changes in expression following a chromosomal abnormality. From this type of analysis, we then infer which pathways may be affected by the abnormality.

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Recent Publications

From left: Dykema, Furge

Yang, X.J., J. Sugimura, K.T. Schafernak, M. Tretiakova, N. Vogelzang, K. Furge, and B.T. Teh. In press. Classification of sixteen renal neoplasms based on molecular signatures. Journal of Urology.

Furge, Kyle A., Karl J. Dykema, Coral Ho, and Xin Chen. 2005. Comparison of array-based comparative genomic hybridization with gene expression–based regional expression biases to identify genetic abnormalities in hepatocellular carcinoma. BMC Genomics 6: 67.

Patil, Mohini A., Ines Gütgemann, Ji Zhang, Coral Ho, Siu-Tim Cheung, David Ginzinger, Rui Li, Karl J. Dykema, Samuel So, Sheung-Tat Fan, Sanjay Kakar, Kyle A. Furge, Reinhard Büttner, and Xin Chen. 2005. Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma. Carcinogenesis 26(12): 2050–2057.

Yang, Ximing J., Min-Han Tan, Hyung L. Kim, Jonathon A. Ditlev, Mark W. Betten, Carolina E. Png, Eric J. Kort, Kunihiko Futami, Kyle A. Furge, Masayuki Takahashi, Hiro-omi Kanayama, Puay Hoon Tan, Bin Sing Teh, Chunyan Luan, Kim Wang, et al. 2005. A molecular classification of papillary renal cell carcinoma. Cancer Research 65(13): 5628–5637.

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Brian B. Haab, Ph.D. Laboratory of Cancer Immunodiagnostics

Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000 and became a Scientific Investigator in 2003.

Staff Laboratory Staff

Students

Songming Chen, Ph.D. Michael Shafer, Ph.D. Yi-Mi Wu, Ph.D. Sara Forrester, B.S. Darren Hamelinck, B.S. Thomas LaRoche, B.S. Randal Orchekowski, B.S.

Derek Bergsma Jennifer Lunger Devin Mistry Richard Schildhouse

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Research Interests Cancer induces a variety of changes in the protein composition of blood. Increased cell breakdown in the tumor environment, oxidative stress, inflammation, new blood vessel formation, secretions from tumors, and immune responses against tumors all may contribute to such changes. Measurements of these various alterations may be useful in understanding the roles of secreted and circulating proteins during cancer progression and in developing improved blood tests for cancer diagnosis and management. We are characterizing protein changes that are prevalent in the blood of cancer patients, developing and testing hypotheses relating to the origin and functional consequences of the changes, and exploring the use of these measurements for cancer diagnostics.

We have developed several array-based protein analysis methods to look at various aspects of proteins, including their abundance, glycosylation, immunoreactivity, and protein-protein interactions. Array-based methods allow the analysis of multiple proteins simultaneously in very small sample volumes, which is useful for efficiently gathering information from small clinical samples or for testing hypotheses that may not be practical to test using conventional technologies. We use high-precision robotics to deposit tiny droplets of protein or antibody solutions onto the surfaces of coated microscope slides, which are then incubated with biological samples.

Interactions between species in the samples and the spotted proteins are observed in a variety of ways. Conventional protein analysis methods are used to complement the information obtained from our newly developed platforms.

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Glycosylation (the attachment of carbohydrate structures to proteins) is an important determinant of protein function, and changes in glycosylation are thought to play roles in certain disease processes. Glycosylation changes occur, for example, on cancer-cell adhesion molecules, on secreted proteins that regulate the environment of the cancer cells, and on stress-response proteins. Those changes may have roles in modulating the adhesion or migratory capabilities of cancer cells or in regulating protein-protein interactions and signaling. We have developed a novel array-based strategy to probe the levels of specific glycan structures on multiple different proteins.

Serum samples are incubated on antibody microarrays and, after washing away unbound serum proteins, the glycan levels on the captured proteins are detected with a biotin-labeled lectin (Fig. 1A). (Lectins are plant and animal proteins with natural carbohydrate-binding functionality.) A variant of the method allows the measurement of both the protein level and the glycan level in a single experiment (Fig. 1B).

In that method, serum proteins are labeled with digoxigenin prior to application to an array, and the biotinylated lectin and digoxigenin-labeled proteins are detected with streptavidin-linked phycoerythrin (green fluorescence) and Cy5-labeled anti-digoxigenin antibody (red fluorescence), respectively. The detection of glycans on proteins captured by immobilized antibodies was confirmed using both methods (Fig. 1C). This method for profiling variation in specific glycans on multiple proteins should be useful in diverse areas of glycobiology research.

Figure 1.

Figure 1. Detection of glycans on antibody arrays. a) One-color glycan detection. b) Two-color detection of glycans and proteins, using digoxigeninlabeled proteins. c) Antibody arrays were incubated with no serum, unlabeled serum, or digoxigenin-labeled serum. The arrays were then incubated with biotinylated SNA lectin, followed by detection with Cy5-labeled anti-digoxigenin (red, 633 nm) and Cy3-labeled anti-biotin (green, 543 nm). The arrays were scanned at both 543 nm and 633 nm, and fluorescence from the spots is shown in both color channels from the indicated antibodies.

VARI | 2006

Pancreatic cancer The glycan-detection arrays were used to find glycosylation changes on specific proteins in the sera of pancreatic cancer patients. The proteins CEA and MUC1 are thought to be involved in controlling the tumor cell environment, and glycan alterations on those proteins may be involved in altering cancer cell migratory or adhesive properties. A blood-group-related carbohydrate structure—the sialyl-Lewis structure, targeted by the CA19-9 monoclonal antibody—is elevated on both CEA and MUC1 in cancer patients (Fig. 2). That structure is the ligand for the endothelial cell-surface receptor E-selectin, which controls the homing of white blood cells to sites of inflammation and has been implicated in cancer-cell invasion. We are currently investigating that glycan on secreted MUC1 and CEA molecules in terms of its possible role in modulating cell migration or in signaling to cell surfaces. We also are investigating the roles of glycan structures in mediating interactions between inflammatory cytokines and MUC1, CEA, and other proteins.

Our screens have turned up other cancer-associated glycan changes that we are pursuing, and additional glycan structures continue to be probed.

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Figure 2.

Figure 2. Comparison of protein and glycan levels using parallel sandwich and glycan-detection assays. A) Representative array images. A pool of 30 cancer serum samples was incubated on the left pair of arrays, and a healthy-patient serum sample and cancer-patient serum sample were incubated on the middle and right pairs of arrays (same two samples in each pair). The arrays were detected using the indicated antibodies.

The spots appearing at the lower right of each array were biotinylated control proteins. The other spots that showed signals were anti-haptoglobin in the array detected with anti-MUC1 (second image from left), and anti-CA19-9, anti-pan CEACAM, and the WGA lectin in the array detected with anti-CA19-9 (right-most image). B) Scatter-plot comparison of protein and glycan levels. Forty-six serum samples (23 from cancer patients and 23 from controls) were incubated on replicate sets of antibody arrays. One set of 46 arrays was detected with antibodies against CEA and MUC1 to obtain the variation in levels of these two proteins, and the other set of arrays was detected with the glycan-binding antibody anti-CA19-9.

The graphs depict the levels detected at the CEA- (left graph) or MUC1- (right graph) capture antibodies by either the anti-protein antibodies (y-axis) or the anti-CA19-9 antibody (x-axis) for each control-patient serum sample (dark triangles) and each cancer-patient serum sample (open circles).

The protein alterations that we are discovering in the sera of cancer patients could be used to develop improved diagnostic tests.

Blood tests could be an important improvement in diagnostics because they can be routinely and inexpensively administered for early detection of disease, to aid in diagnosis or staging, or to follow the progress of treatment. Early detection tests for pancreatic cancer could greatly improve outcomes for many patients, since that cancer is usually not detected until it has advanced past a treatable stage. Our previous studies have shown that a number of proteins have altered abundances in the sera of pancreatic cancer patients and that the measurements of multiple proteins simultaneously, as is possible with array-based tools, can be used in combination to form diagnostic tests having improved accuracy over tests based on single protein measurements. In collaboration with Randall Brand (Evanston Northwestern Healthcare), Diane Simeone (University of Michigan), and Michael Hollingsworth (University of Nebraska), as well as with the Early Detection Research Network (EDRN), we are combining our results with other discoveries of protein alterations in pancreatic cancer to further develop, refine, test, and validate these new approaches.

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Prostate cancer A great need also exists for improved tests for prostate cancer, which is the most prevalent cancer among men. A blood test—the prostate-specific antigen (PSA) test—is in wide use for prostate cancer detection, but it suffers from a high rate of false positives and the inability to detect many fast-growing cancers at early stages. We are taking several approaches toward improving prostate cancer diagnostics. In a collaboration with Alan Partin (Johns Hopkins University), we have used array-based screening to identify proteins having altered abundances in prostate cancer. One of the proteins identified in our screens, thrombospondin-1 (TSP-1), may be valuable in differentiating prostate cancer from benign prostatic disease, which are sometimes indistinguishable by conventional methods. We have found that TSP-1 is elevated in 80% of patients with benign disease relative to patients with malignant disease, and it is repressed in prostate cancer patients relative to healthy individuals. One of the functions of TSP-1 is to suppress the growth of new blood vessels (angiogenesis), which is a crucial process in the growth and progression of tumors. We are investigating the glycosylation states and protein-protein interactions of this molecule and the functional consequences of those states.

In another approach to improving prostate cancer diagnostics, we are investigating the use of longitudinal measurements (serial measurements over time) to develop and improve existing diagnostic markers. We hypothesize that the use of individualized thresholds of “abnormal” protein levels, defined by each person’s history of measurements, will yield improved diagnostic accuracy over the use of single thresholds based on levels across the population. In a collaboration with William Catalona (Northwestern University), Robert Vessella (University of Washington), and Ziding Feng (Fred Hutchinson Cancer Research Center), we are looking at changes over time in the concentration of a number of serum proteins leading up to disease recurrence in prostate cancer patients. Our hypothesis has been supported in some individual demonstrations, but we now have a means for systematically exploring it for a large number of proteins and many patients, laying the groundwork for broader application of the approach.

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Our cell culture studies complement the above work, using clinical samples from cancer patients in order to study in a more controlled environment the protein alterations observed in the blood. We are investigating the conditions that reproduce what is observed in the blood, and we are studying the functional consequences of certain cancer-related alterations. This mechanistic understanding will be important in identifying additional protein alterations that have diagnostic value and in devising blood-based strategies to interfere with cancer processes. We hope to translate these developments into improved care for cancer patients.

From left to right: Nelson, Mistry, Wu, Bergsma, Chen, LaRoche, Shafer, Haab

VARI | 2006

Recent Publications Haab, B.B., and P.M. Lizardi. 2006. RCA-enhanced protein detection arrays. In New and Emerging Proteomic Techniques, D. Nedelkov and R.W. Nelson, eds. Methods in Molecular Biology series, Totowa, N.J.: Humana Press, pp. 15–29.

Sanchez-Cabayo, M., N.D. Socci, J.J. Lozano, B.B. Haab, and C. Cordon-Cardo. 2006. Profiling bladder cancer using targeted antibody arrays. American Journal of Pathology 168(1): 93–103.

Gao, Wei-Min, Rork Kuick, Randal P. Orchekowski, David E. Misek, Ji Qiu, Alissa K. Greenberg, William N. Rom, Dean E. Brenner, Gilbert S. Omenn, Brian B. Haab, and Samir M. Hanash. 2005. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis. BMC Cancer 5: 110.

Haab, Brian B. 2005. Antibody arrays in cancer research. Molecular & Cellular Proteomics 4(4): 377–383.

Haab, Brian B. 2005. Multiplexed protein analysis using antibody microarrays and label-based detection. In Microarrays in Clinical Diagnostics, T.O. Joos and P. Fortina, eds. Methods in Molecular Medicine series, Totowa, N.J.: Humana Press, pp. 183–194.

Haab, Brian B., Bernhard H. Geierstanger, George Michailidis, Frank Vitzthum, Sara Forrester, Ryan Okon, Petri Saviranta, Achim Brinker, Martin Sorette, Lorah Perlee, Shubha Suresh, Garry Drwal, Joshua N. Adkins, and Gilbert S. Omenn. 2005. Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: systematic variation between sample types and calibration of mass spectrometry data. Proteomics 5(13): 3278–3291.

Hamelinck, Darren, Heping Zhou, Lin Li, Cornelius Verweij, Deborah Dillon, Ziding Feng, Jose Costa, and Brian B. Haab. 2005.

Optimized normalization for antibody microarrays and application to serum-protein profiling. Molecular & Cellular Proteomics 4(6): 773–784.

Omenn, Gilbert S., David J. States, Marcin Adamski, Thomas W. Blackwell, Rajasree Menon, Henning Hermjakob, Rolf Apweiler, Brian B. Haab, Richard J. Simpson, James S. Eddes, Eugene A. Kapp, Robert L. Moritz, Daniel W. Chan, Alex J. Rai, Arie Admon, et al. 2005.

Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly available database. Proteomics 5(13): 3226–3245.

Orchekowski, Randal, Darren Hamelinck, Lin Li, Ewa Gliwa, Matt VanBrocklin, Jorge A. Marrero, George F. Vande Woude, Ziding Feng, Randall Brand, and Brian B. Haab. 2005. Antibody microarray profiling reveals individual and combined serum proteins associated with pancreatic cancer. Cancer Research 65(23): 11193–11202.

Rai, Alex J., Craig A. Gelfand, Bruce C. Haywood, David J. Warunek, Jizu Yi, Mark D. Schuchard, Richard J. Mehigh, Steven L. Cockrill, Graham B.I. Scott, Harald Tammen, Peter Schulz-Knappe, David W. Speicher, Frank Vitzthum, Brian B. Haab, Gerard Siest, and Daniel W. Chan. 2005. HUPO-PPP Specimen Collection and Handling Committee report: towards the standardization of parameters for proteomic analyses. Proteomics 5(13): 3262–3277.

Steel, Laura F., Brian B. Haab, and Samir M. Hanash. 2005. Methods of comparative proteomic profiling for disease diagnostics.

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 815(1–2): 275–284.

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Photo taken by Mathew Edick.

Scientific Report

VARI | 2006

Colocalization of cytochrome C and mitochondria in primary prostate epithelial cells.

Primary prostate epithelial cells were starved of growth factors for 48 hours and then plated on glass coverslips in growth factor–free medium.

Cells were incubated with MitoTracker (red) for 24 hours to stain the mitochondria, fixed in paraformaldehyde, and then incubated with anti-cytochrome C–specific antibody. Cells were then incubated with Alexa Fluor 488 conjugated secondary antibody (green) to visualize cytochrome C and DAPI (blue) to visualize the nucleus. Colocalization of cytochrome C and mitochondria is shown in yellow. Cells were viewed by fluorescence microscopy.

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Rick Hay, Ph.D., M.D., F.A.H.A. Laboratory of Noninvasive Imaging and Radiation Biology

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Dr. Rick Hay is Senior Scientific Investigator, Director of the Laboratory of Noninvasive Imaging and Radiation Biology, and Deputy Director for Clinical Programs at the Van Andel Research Institute. He earned Ph.D. and M.D. degrees at the University of Chicago and received advanced training in pathology, biochemistry, and nuclear medicine at Chicago, the University of Basel (Switzerland), and the University of Michigan. He has worked as a medical scientist and practicing physician at academic, private, and governmental institutions, and is an expert in the development of radiopharmaceutical agents from benchtop to clinical use. He has previously worked in the fields of cardiovascular pathology, cardiovascular imaging, and imaging of inflammatory disease. His current research emphasizes the evaluation of radiolabeled monoclonal antibodies and derivatives for imaging and therapeutic applications.

Staff Laboratory Staff Troy Giambernardi, Ph.D. Yue Guo, B.S. Catherine Walker, B.S.

Visiting Scientist Visiting Student Students ScientistsConsultants Nigel Crompton, Ph.D., D.Sc.

Jose Toro

Helayne Sherman, M.D., Ph.D., F.A.C.C. Milton Gross, M.D., F.A.C.N.P.

VARI | 2006

Research Interests In July 2005, the Laboratory of Noninvasive Imaging and Radiation Biology originated as an outgrowth and expansion of activities previously supported by the Laboratory of Molecular Oncology. This new laboratory is devoted to both noninvasive imaging (generation and analysis of images depicting structure and selected functions in living organisms without surgically or mechanically penetrating a body cavity) and radiation biology (analysis of the consequences of external and internal radiation exposure in living organisms).

The laboratory’s work is along three major themes:

• Development and use of laboratory models that address medical imaging and radiation exposure problems

• Advancement of technology in imaging and radiation biology, including novel agents, probes, and reporters; new strategies for tackling research problems; and new instrumentation

• Pursuit of two-way translation between the laboratory and the clinical setting, i.e., using examples of human disease to design and improve laboratory model systems for study, as well as moving new discoveries from the laboratory benchtop to the patient’s bedside Both locally and through our ongoing collaborations, we depend upon access to sophisticated instruments and equipment, including nuclear imaging cameras; planar and tomographic X-ray units; clinical and research ultrasonography units; fluorescence detection systems; and cell and organism irradiation capability. We have major instruments either in place or scheduled to be obtained and installed within the coming year. During our first year of operation, we have established research projects in radiation biology and nuclear imaging, and we are in the process of acquiring new equipment and staff and of creating new projects in the areas of ultrasonography, fiberoptic imaging, and fusion imaging (combined nuclear imaging and computed tomography).

One established research project continues work begun by Nigel Crompton at the Paul Scherrer Institute in Switzerland, now performed in collaboration with the radiation oncology service at St. Mary’s Mercy Medical Center. This project seeks to predict the sensitivity of a patient’s normal tissues to irradiation that is being administered for treatment of a serious condition such as cancer. For this project a sample of the patient’s blood is drawn before radiation therapy. The blood sample is then irradiated (outside the patient) under precise conditions of exposure, treated with fluorescent molecules that detect certain types of blood cells (lymphocytes), and then analyzed by fluorescence-activated cell sorting (FACS) for evidence of lymphocyte death. In Switzerland, Dr. Crompton established a close correlation between lymphocyte death and a patient’s normal tissue tolerance to irradiation; we are now determining whether western Michigan patients respond similarly.

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A second established project continues work initiated by Rick Hay and colleagues while he was a member of the Laboratory of Molecular Oncology. Since 2001 we have been evaluating a group of antibodies (designated MetSeek™) that recognize the Met receptor tyrosine kinase. Met plays a key role in causing cancers to become more aggressive, so that they spread to nearby tissues (invasion) and/or travel through the bloodstream or lymph channels to distant organs (metastasis). We previously showed that two MetSeek agents used as originally produced in the laboratory (by hybridoma cells) are useful for nuclear imaging of Met-expressing human tumors (xenografts) grown under the skin of immunodeficient mice. In the past year, in collaboration with our colleagues at VARI and with our outside collaborators in Ann Arbor, Detroit, and East Lansing, we have shown further that smaller versions of MetSeek such as Fab fragments also generate satisfactory nuclear images of Met-expressing xenografts. We are currently characterizing other smaller versions of MetSeek as potential nuclear imaging agents and as carriers of anticancer molecules; exploring new ways of complexing radioactive atoms with MetSeek agents for improved ease of use and future clinical application; and systematically evaluating the use of MetSeek in tumors having the highest prevalence of abnormal Met expression.

External Collaborators

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Our laboratory depends critically on intramural and extramural collaborations to address our research themes. Our extramural collaborators encompass scientists and physicians at the Department of Veterans Affairs Healthcare System in Ann Arbor; the University of Michigan in Ann Arbor; Michigan State University in East Lansing; ApoLife, Inc., in Detroit; Henry Ford Hospital in Detroit; West Michigan Heart, P.C., in Grand Rapids; St. Mary’s Mercy Medical Center in Grand Rapids; Fred Hutchinson Cancer Research Center in Seattle; the Gerald P. Murphy Foundation in West Lafayette, Indiana; the National Cancer Institute in Bethesda, Maryland; and the University of Illinois in Champaign-Urbana.

Recent Publications Hay, Rick V., Brian Cao, R. Scot Skinner, Yanli Su, Ping Zhao, Margaret F. Gustafson, Chao-Nan Qian, Bin T. Teh, Beatrice S. Knudsen, James H. Resau, Shuren Shen, David J. Waters, Milton D. Gross, and George F. Vande Woude. 2005. Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek™). Clinical Cancer Research 11(19): 7064s–7069s.

Jiao, Yongjun, Ping Zhao, Jin Zhu, Tessa Grabinski, Zhengqing Feng, Xiaohong Guan, R. Scot Skinner, Milton D. Gross, Rick V. Hay, Hiroshi Tachibana, and Brian Cao. 2005. Construction of human naïve Fab library and characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology 31(1): 41–54.

VARI | 2006

Office of Translational Programs In July 2005 the Office of Translational Programs (OTP) became the administrative home base for activities overseen by the Deputy Director for Clinical Programs (DDCP). The role of OTP is to promote and facilitate collaborative programs involving the Van Andel Research Institute and other institutions in the realm of translational medicine.

OTP has accomplished the following during our inaugural year of formal operation.

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Serving as the interim administrative home for the Grand Rapids-GMP project: With funding from the state of Michigan and the federal Health Resources and Services Administration, this project—a partnership venture between VARI and Grand Valley State University (GVSU)—will build and operate a Good Manufacturing Practices facility for preparing biological agents and small molecules to be used in early phase clinical trials, primarily serving academic and commercial investigators in Michigan and the Midwest. In December 2004, administrative oversight of the project was assigned to DDCP. First a planning group and then a GR-GMP Board of Directors (five representatives from VARI and GVSU) have directed project progress during the past year. A facility director has been recruited and brought on board, and a dedicated business entity is being incorporated to manage details of facility construction and operation.

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Hosting regional meetings of the Michigan Cancer Consortium (MCC): As an active member of the MCC, VARI is committed to participating in statewide programs to reduce the burden of cancer in Michigan. In 2005, we and other MCC members gained approval to launch a series of meetings with the purpose of identifying fresh initiatives that better suited the needs of western Michigan and, by doing so, to reinvigorate member participation. Since December 2005 we have held monthly meetings of a focus group comprising MCC members within Kent County. We have identified specific regional deficiencies in delivery of cancer-related patient care, and we are preparing a formal funding proposal to support a pilot project on this front.

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Promoting new inter-institutional collaborations and providing resources for funding proposals: OTP has provided personnel, logistical support, grant preparation expertise, meeting venues, and seed funding for new inter-institutional collaborations seeking extramural funding through the first round of proposals submitted to the 21st Century Jobs Fund (Michigan Economic Development Corporation), as well as administrative assistance for generating funding requests to other agencies. The two largest programs we are promoting in this fashion are the Innovative Clinical Research Alliance (ICRA), spearheaded by Dr. Craig Webb and collaborators from regional healthcare and clinical trial entities; and a biotrust project initiated by Drs. Clark Radcliffe and Nigel Paneth of Michigan State University. At this writing, we are awaiting the results of peer review on these and our smaller applications.

Staff Rick Hay, Ph.D., M.D., F.A.H.A. Troy Carrigan

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Sheri L. Holmen, Ph.D. Laboratory of Molecular Medicine and Virology

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Dr. Holmen received her M.S. in biomedical science from Western Michigan University in 1995 and her Ph.D. in tumor biology from the Mayo Clinic College of Medicine in 2000. She did her postdoctoral work at VARI from 2000 to 2003 and became a Junior Investigator at VARI in December 2003.

Staff Laboratory Staff Todd Whitwam, Ph.D. Marleah Russo Sarah Warlick

Students

Visiting Scientists

VARI | 2006

Research Interests My laboratory is focused on defining critical targets in the signaling pathways of cancer cells that can become the focus for therapeutic intervention. Because of the high cost of developing new therapies, it is essential that we first identify which genetic alterations can be productively targeted. We are concentrating our initial efforts on melanoma and glioblastoma, tumors which demonstrate constitutive activation of Ras and Akt signaling. We plan to further validate the roles of these pathways using pharmacological inhibitors of clinical importance.

The RCAS system We use a series of replication-competent retroviral vectors based on Rous sarcoma virus (RSV), a member of the avian leukosis virus family, to study the roles of various genes in tumor initiation and progression. RSV is the only known naturally occurring, replication-competent retrovirus that carries an oncogene, src. In RCAS vectors, the region encoding src (which is dispensable for viral replication) has been replaced by a synthetic DNA linker. Foreign genes inserted into this linker are expressed from the viral LTR promoter via a subgenomic splice site (just as src is in RSV). RCAN vectors differ from RCAS vectors in that they lack the src splice acceptor, so the gene of interest is inserted along with an internal promoter. Higher-titer viruses subsequently have been generated by replacing the RSV pol gene with the pol gene of the Bryan strain of RSV; these vectors are termed RCASBP or RCANBP. The ability of these vectors to infect non-avian cells relies on expression of the corresponding receptor on the cell surface. The viral receptor is typically introduced into mammalian cells (or mice) via an inducible and/or tissue-specific transgene. Therefore, this system allows for tissue- and cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. Alternatively, when targeted infection of mammalian cells is not required (e.g., in cell culture), infection can be achieved through the use of non-avian envelopes, such as the amphotropic envelope from murine leukemia virus. The receptor for this envelope is endogenously expressed on almost all mammalian cells.

We have used the RCASBP/RCANBP family of retroviral vectors extensively in both cultured cells and live animals for studies of viral replication and for cancer modeling in mice. Most of these studies have analyzed gain-of-function phenotypes by delivering and overexpressing a particular gene of interest. Recently we engineered the RCANBP vector to reduce the expression of specific genes through the delivery of short hairpin RNA sequences. We also engineered this vector to control the expression of the inserted gene using the tetracycline (tet)-regulated system. Sequences inserted into this region are transcribed from a tet-responsive element and not the viral LTR. This virus allows inserted genes to be turned on and off, in order to determine if expression of the gene is required for tumor initiation, maintenance, or progression. The ability to turn off gene expression will help determine if that gene is a good target for therapy.

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Melanoma Activated NRAS oncogenes, which turn on mitogen-activated protein kinase (MAPK) signaling, are detected in approximately 20% of human melanomas. Recently, activating mutations in the BRAF gene, which also activate MAPK signaling, have been found in more than 65% of malignant melanomas. With mutually exclusive mutations in RAS and BRAF, the MAPK signaling pathway is constitutively activated in over 85% of cases of malignant melanoma, indicating its importance. Interestingly, it was recently observed that tumors harboring BRAF mutations are much more sensitive to pharmacological inhibition of downstream MAPK signaling than those with NRAS mutations. This suggests that single-agent therapeutic strategies may be ineffective in tumors containing RAS mutations and that rational combination therapeutic strategies will be required. The RAS subfamily consists of H (Harvey) RAS, N (neuroblastoma) RAS, and two splice variants of K (Kirsten) RAS (KRAS4A and KRAS4B). In many tumors, oncogenic mutations have been identified at positions 12, 13, or 61, which cause RAS to remain constitutively active. With the exception of thyroid cancers, most tumors are associated with mutation of only one isoform of RAS and this association cannot be explained solely by differential regulation of RAS gene expression in different tissues. HRAS mutations are more commonly associated with bladder and kidney cancers; KRAS mutations are found in lung, colorectal, ovarian, and pancreatic cancers; and NRAS mutations are most commonly associated with melanoma and hematologic malignancies.

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We have been characterizing the transforming capabilities of the different Ras isoforms in a nontransformed, immortal Ink4a/Arfdeficient mouse melanocyte cell line. We have observed that activated NRas is able to transform these cells much more efficiently than either activated HRas or KRas; and whereas expression of NRas increases the proliferation of the melanocytes, expression of KRas does not. Furthermore, coexpression of c-myc with KRas in these cells mimics the proliferation and transformation capabilities of NRas alone, whereas coexpression of Akt with KRas in these cells mimics the proliferation and transformation capabilities of HRas alone.

These data suggest that the different Ras isoforms have distinct, nonredundant functions in melanocytes and may explain why most melanomas are associated with mutation of only one isoform of Ras.

Glioblastoma Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. It is also the most fatal: mean survival is less than one year from the time of diagnosis, with less than 10% survival after two years. Despite major improvements in imaging, radiation, and surgery, the prognosis for patients with this disease has not changed in the last 20 years. Recently, genes that are differentially expressed in tumor tissue relative to normal brain tissue have been found. However, those that can be productively targeted for therapeutic intervention in human patients remain to be identified.

A mouse model of human GBM based on the avian RCAS/TVA system has been developed by Eric Holland. In this model, the retroviral receptor TVA is expressed under the control of the Nestin promoter, which is active in neural and glial progenitors. Malignant gliomas can be induced in these mice through the combined expression of activated forms of both KRas and Akt in glial progenitor cells. To determine the reliance of these tumors on continued KRas signaling in vivo, we generated a viral vector that allows the expression of KRas to be controlled post-delivery. Tumor-free survival rates were compared between those animals with continued KRas expression and animals in which KRas expression was suppressed. KRas signaling was found to be required for the maintenance of these tumors in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and increased survival. Subsequent reexpression of KRas reinitiated tumor growth, indicating that a percentage of the progenitor cells survived and retained tumorigenic properties. This model shows the crucial importance of the Ras pathway in glioblastoma maintenance and indicates that continuous suppression of Ras signaling is necessary and sufficient to suppress the tumorigenic potential of the glial progenitor cells. In addition, this regulated expression system will allow the evaluation of the role of other genes and pathways in this context. This has important clinical implications for pharmacologic agents targeting these pathways in GBM patients.

VARI | 2006

Antiviral strategies A second, related focus of our group is the identification of effective antiviral strategies using the RCASBP/RCANBP family of retroviral vectors as a model system. Viral diseases pose a major risk to the food supply and to animal welfare, especially in today’s highintensity animal agriculture. Many viruses are highly communicable and are capable of rapid mutation to escape immune surveillance; few effective antiviral drugs are available. Over the past several years, we have developed strategies aimed at conferring dominant resistance to viral pathogens in chickens. These strategies have focused on manipulating viral (“pathogen-derived resistance”) and/or cellular genes (“host-derived resistance”) to express new proteins capable of disrupting the viral life cycle. The results have provided valuable information on the biology of avian viruses, as well as having the potential for practical application. We are now working to adapt the new RNA interference (RNAi) technology to the development of new antiviral strategies for two important chicken pathogens, avian leukosis virus and avian influenza. These two targets have been chosen because both are economically important and because they have distinctly different infectious cycles that provide different challenges for RNAi.

External Collaborators John Brigande, Oregon Health & Science University, Portland Jerry Dodgson, Michigan State University, East Lansing Henry Hunt and Huanmin Zhang, Avian Disease and Oncology Laboratory, East Lansing, Michigan Nita Maihle, Yale University School of Medicine, New Haven, Connecticut Phillipe Monnier, University of Toronto, Canada Bill Pavan, National Institutes of Health, Bethesda, Maryland Maria Soengas, University of Michigan, Ann Arbor Richard Vile, Mayo Clinic, Rochester, Minnesota

Recent Publications

From left to right, rear: Whitwam, Holmen; front: Russo, Warlick

Park, Ki-Sook, Soung Hoo Jeon, Sung-Eun Kim, Young-Yil Bahk, Sheri L. Holmen, Bart O. Williams, Kwang-Chul Chung, Young-Joon Surh, and Kang-Yell Choi. 2006. APC inhibits ERK pathway activation and cellular proliferation induced by Ras. Journal of Cell Science 119(5): 819–827.

Wang, PengFei, Dong Kong, Matthew W. VanBrocklin, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams, and Sheri L. Holmen. 2006. Simplified method for the construction of gene targeting vectors for conditional gene inactivation in mice. Transgenics 4: 215–228.

Holmen, Sheri L., and Bart O. Williams. 2005. Essential role for Ras signaling in glioblastoma maintenance. Cancer Research 65(18): 8250–8255.

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

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Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982 and her Ph.D. in biochemistry from Harvard Medical School in 1995. She was a postdoctoral fellow in the laboratory of Dr. Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Massachusetts, from 1995 to 1997 and in the Department of Cell Biology at Harvard Medical School from 1997 to 2000. Dr. Miranti joined VARI as a Scientific Investigator in January 2000. She is also an Adjunct Assistant Professor in the Department of Physiology at Michigan State University.

Staff Laboratory Staff

Students Students

Mathew Edick, Ph.D. Suganthi Sridhar, Ph.D. Kristin Saari, M.S. Lia Tesfay, M.S. Laura Lamb, B.S. Veronique Schulz, B.S.

Erik Freiter Stefan Kutscheidt Katie Sian

Visiting Scientists

VARI | 2006

Research Interests Our laboratory is interested in understanding the mechanisms by which integrin receptors, interacting with the extracellular matrix, regulate cell processes involved in the development and progression of cancer. Using tissue culture models, biochemistry, molecular genetics, and mouse models, we are defining the cellular and molecular events involved in integrin-dependent adhesion and downstream signaling that are important for prostate tumorigenesis and metastasis.

Integrins are transmembrane proteins that serve as receptors for extracellular matrix (ECM) proteins. By interacting with ECM, integrins stimulate intracellular signaling transduction pathways to regulate cell shape, proliferation, migration, survival, gene expression, and differentiation. Integrins do not act autonomously, but “crosstalk” with receptor tyrosine kinases (RTKs) to regulate many of these cellular processes. Studies in our lab indicate that integrin-mediated adhesion to ECM proteins activates epidermal growth factor receptors EGFR/ErbB2 and the HGF/SF receptor c-Met. Integrin-mediated activation of these RTKs is ligand-independent and is required for the activation of a subset of intracellular signaling molecules in response to cell adhesion.

The prostate gland and cancer Tumors that develop in cells of epithelial origin, i.e., carcinomas, represent the largest tumor burden in the United States. Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer death in men. Patients who present at the time of diagnosis with androgen-dependent and organ-confined prostate cancer are relatively easy to cure through radical prostatectomy or localized radiotherapy. However, patients with aggressive and metastatic disease have fewer options. Androgen ablation can significantly reduce the tumor burden in these patients, but the potential for relapse and the development of androgen-independent cancer is high. Currently there are no effective treatments for patients who reach this stage of disease.

In the human prostate gland, α3β1 and α6β4 integrins bind to the ECM protein laminin 5 in the basement membrane. In tumor cells, however, the α3 and α4 integrin subunits disappear—as does laminin 5—and the tumor cells express primarily α6β1 and adhere to a basement membrane containing laminin 10. There is also an increase in expression of the RTKs EGFR and c-Met in the tumor cells. Two fundamental questions in our lab are whether the changes in integrin and matrix interactions that occur in tumor cells are required for or help to drive the survival of tumor cells, and whether crosstalk with RTKs is important.

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Role of integrins and RTKs in prostate epithelial cell survival Increased cell survival and resistance to cell death is a prerequisite for tumorigenesis. Several reports have suggested that the signaling pathways that regulate cell survival in normal prostate epithelial cells are different from the pathways regulating cell survival in prostate tumor cells. How integrin engagement of different ECMs regulates survival pathways in normal and tumor cells is not known. We have recently demonstrated that integrin-induced activation of EGFR in primary prostate epithelial cells is required for cell survival on laminin 5. The ability of EGFR to support integrin-mediated cell survival on laminin is mediated through ﾎｱ3ﾎｲ1 integrin and requires signaling downstream to Erk. Inhibition of integrin-induced EGFR/Erk signaling results in decreased expression of the anti-apoptosis regulator Bcl-XL and increased expression of the pro-apoptosis regulator BimEL (Fig. 1). Inhibition of Bcl-XL expression with siRNA induces cell death. However, despite this apparent regulation of the apoptotic machinery, cell death mediated by inhibition of EGFR or Bcl-XL occurs through a non窶田ytochrome C, non-caspase-dependent mechanism. Future studies in our lab are aimed at deciphering this cell death pathway.

In contrast to the normal cells, EGFR and Erk are not activated by integrins in the metastatic PC3 prostate tumor cell line. Accordingly, integrin-mediated survival of PC3 cells does not depend on EGFR or Erk, but is instead dependent on PI-3K. Integrin-mediated survival through PI-3K in PC3 cells controls phosphorylation of the pro-apoptosis regulator Bad, which when blocked induces classic apoptotic cell death (Fig. 1). We will be testing additional prostate tumor cells lines to determine if this switch in matrix-mediated survival pathways is universally observed.

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Figure 1. Laminin-mediated survival signaling pathways in normal and prostate tumor cells.

Figure 1.

Role of androgen receptor in integrin-mediated survival All primary and metastatic prostate cancers express the intracellular steroid receptor for androgen, AR. In the normal gland, the AR-expressing cells do not interact with the basement membrane; however, all AR-expressing tumor cells do adhere to the ECM in the basement membrane. In normal cells, AR expression suppresses growth and promotes differentiation, but in tumor cells AR expression promotes cell growth and is required for cell survival. The mechanisms that lead to the switch from growth inhibition and differentiation to growth promotion and survival are unknown. Our hypothesis is that adhesion to the ECM by the tumor cells is responsible for driving the switch in AR function.

When prostate tumor cells are placed in culture, they lose expression of AR. The reason for this is not clear, but it may have something to do with loss of the appropriate ECM-containing basement membrane. When we introduce AR into prostate tumor cells, it actually suppresses their growth and induces cell death. However, if we place the AR-expressing tumor cells on laminin (the ECM found in tumors) and block the PI-3K signaling pathway, these cells do not die. The mechanisms responsible for this change in survival are unknown. Future work in the lab will be aimed at determining which cell survival pathways are activated by integrins.

VARI | 2006

Role of CD82 and integrin signaling in prostate cancer metastasis Prostate cancer death is due to the development of metastatic disease, which is difficult to control. The mechanisms involved in progression to metastatic disease are not understood. One approach that we are taking is to characterize genes that are specifically associated with metastatic prostate cancer. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer, but not in primary tumors. Interestingly, CD82/KAI1 is known to associate with both integrins and RTKs. Our goal has been to determine how loss of CD82/KAI1 expression promotes metastasis by studying the role of CD82/KAI1 in integrin and RTK crosstalk.

We have found that reexpression of CD82/KAI1 in metastatic tumor cells suppresses laminin-specific migration and invasion; integrininduced c-Met receptor and Src activation; and activation of the Src substrates Cas and FAK. Inhibition of either c-Met or Src also suppresses laminin-dependent invasion. In addition, CD82 suppresses HGF-induced activation of c-Met; however, if c-Met is overexpressed at sufficiently high levels, CD82 can no longer inhibit that activation. Thus, these data indicate that CD82/KAI1 normally acts to regulate both integrin- and HGF-mediated signaling to c-Met such that upon CD82 loss in tumor cells, signaling through c-Met is increased, leading to increased invasion. We are currently determining the mechanism by which CD82/KAI1 down-regulates c-Met signaling. So far our investigations indicate that c-Met and CD82 do not directly interact, and CD82 may act to suppress c-Met signaling by dispersing c-Met aggregates present on metastatic tumor cells into monomers and thus blocking signaling (Fig. 2).

T=0

T = 1h

T = 4h

Vector

Figure 2. CD82 reexpression in metastatic prostate cancer cells alters the distribution of c-Met (orange) on the surface of cells.

CD82

Figure 2.

We have also initiated several mouse studies to demonstrate the importance of CD82 in regulating metastasis in vivo. Using orthotopic injection of wild-type or CD82-expressing metastatic prostate tumor cells directly into the prostate, we found that CD82 also suppresses metastasis in vivo. We are continuing these studies using HGF-overexpressing mice and we hope to demonstrate that CD82 expression in metastatic prostate cells will suppress HGF-induced tumor formation in vivo. This will provide support for the role of CD82 in regulating c-Met function in vivo.

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External Collaborators Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York Nita Maihle, Yale University, New Haven, Connecticut Ilan Tsarfaty, Tel Aviv University, Israel

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Recent Publications

Left to right, back row: Freiter, Edick, Schulz, Saari, Kutscheidt, Sridhar; front: Lamb, Tesfay, Sian, Miranti

Knudsen, Beatrice S., and Cindy K. Miranti. In press. Impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression. Journal of Cellular Biochemistry.

Sridhar, Suganthi C., and Cindy K. Miranti. In press. Tumor metastasis suppressor KAI1/CD82 is a tetraspanin. In Contemporary Cancer Research: Metastasis, C. Rinker-Schaeffer, M. Sokoloff, and D. Yamada, eds.

Sridhar, Suganthi C., and Cindy K. Miranti. 2006. Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene 25(16): 2367–2378.

VARI | 2006

James H. Resau, Ph.D. Division of Quantitative Sciences Laboratory of Analytical, Cellular, and Molecular Microscopy Laboratory of Microarray Technology

Dr. Resau received his Ph.D. from the University of Maryland School of Medicine in 1985. He has been involved in clinical and basic science imaging and pathology-related research since 1972. Between 1968 and 1994, he was in the U.S. Army (active duty and reserve assignments) and served in Vietnam. From 1985 until 1992, Dr. Resau was a tenured faculty member at the University of Maryland School of Medicine, Department of Pathology. Dr. Resau was the Director of the Analytical, Cellular and Molecular Microscopy Laboratory in the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland, from 1992 to 1999. He joined VARI as a Special Program Senior Scientific Investigator in June 1999 and in 2003 was promoted to Deputy Director. In 2004, Dr. Resau assumed as well the direction of the Laboratory of Microarray Technology to consolidate the imaging and quantification of clinical samples in a CLIA-type research laboratory program. In 2005, Dr. Resau was made the Division Director of the quantitative laboratories (pathology-histology, microarray, proteomics, epidemiology, and computational biology).

Staff Laboratory Staff

Students

Eric Kort, M.D. Yair Andegeko, Ph.D. Bree Berghuis, B.S., HTL(ASCP), QIHC Pete Haak, B.S. Eric Hudson, B.S. Mitch Machiela, B.S. Paul Norton, B.S. J.C. Goolsby

Courtney Banks Devrim Bilgili Alex Camenzuli Hien Dang Anna Fairchild Nick Miltgen Ken Olinger Amy Percival

Rebecca Roe Eddy Solomon Jourdan Stuart Huong Tran Grant Van Eerden

Consulting Veterinarian

Consultant

Robert Sigler, D.V.M., Ph.D.

Brandon Leeser, B.S.

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Research Interests The laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM) and of Microarray Technology (MT) are members of the Division of Quantitative Sciences. Other members of the Division are the Mass Spectrometry and Proteomics, Computational Biology, and Molecular Epidemiology laboratories.

The ACMM is responsible for the Van Andel Tissue Repository (VATR), which is an expanding collection of human pathology tissues containing approximately 200,000 paraffin blocks (SPIN) and 550 frozen tissues (TAS) obtained from local area hospitals. This program provides a tissue bank of human samples for the study of disease. The paraffin blocks are also used to prepare a wide variety of tissue microarrays for research and potentially for diagnostic uses. We are continuously entering tissue data and now have 55,900 clinical narrative pathology reports that explain and describe the tissue samples archived. These reports are not directly linked to any personal identifiers or names and meet all HIPAA/CLIA regulations. This year we have used the Aperio ScanScope to image representative regions of the TAS tissues and have added digital images and area measurements to the VATR file; currently we have information on 1300 samples. These tissues may be used in cellular and molecular protocols that have been approved by our Institutional Review Board.

This is a collaboration with VARI’s Rick Hay (TAS) and Eric Kort (epidemiology). We organize and provide consultative histopathology through our consulting veterinarian, Robert Sigler. We have added additional services to the ACCM during this year, a major one being the plastic embedding and sectioning of bone tissues. We use methyl methacrylate to increase our resolution (reduced thickness). Our first bone sample was sectioned on site this year. This is a collaboration with VARI’s Bart Williams to analyze mouse bones stained with safranin and Weigert’s Iron Hematoxylin B.

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Hauenstein Parkinson’s Center Clinical sample collection has begun in collaboration with Drs. Berryhill and Neuman at the Hauenstein Parkinson’s Center (HPC) at St. Mary’s Hospital. Consenting Parkinson disease patients and their relatives enter the study by donating a blood and urine sample. The samples are divided into aliquots and frozen for future genetic and proteomic study of the RNA, DNA, plasma, serum, and urine. We hope that these samples, for example, will allow future researchers at the Institute and HPC to pursue a blood-based diagnostic marker for Parkinson disease or to develop individualized pharmacogenetic applications. All aliquots are barcode-labeled and tracked in the VATR. To date, 33 patients have been enrolled.

Australia Parkinson cases VARI’s Laboratory of Microarray Technology and Laboratory of Cancer Genetics (Bin Teh) have begun collaborations with Alan Mackay-Sim and Peter Silburn of Griffith University, Australia. Dr. Mackay-Sim has been gaining recognition for his work on adult stem cells, including receiving the Queenslander of the Year award for 2003. His lab harvests neurons from the olfactory bulb and cultures the cells under conditions that select for adult neural stem cells. VARI is involved in characterizing the gene expression profiles of these “neurosphere” cell cultures. It is hoped that this collaboration will lead to gene-based methods for diagnosis of neural disorders such as Parkinson and Alzheimer disease, as well as potential therapeutic uses for the stem cell cultures. Adult stem cells hold the possibility of regenerating damaged neurons in the patient without fear of immune-system rejection.

VARI | 2006

ThinPrep The ThinPrep method for gynecologic cancer screening has significantly changed the practice of cytopathology. In many hospitals, ThinPrep has replaced the traditional but highly variable Pap smear as a method for consistently and accurately evaluating cervical pathology. In collaboration with Holland Hospital, we have obtained samples (cells) from the ThinPrep procedure that have been fixed in buffered methanol. Optimal RNA is normally obtained from frozen, unfixed cells, but recent experiments in our lab indicate that RNA isolation is possible from ThinPrep fixed cervical cells. A reconstruction experiment investigating the RNA preservation of the ThinPrep preservative is in progress; the findings will be used to optimize the yield and quality of RNA extraction. If RNA obtained from the ThinPreps is amenable to use in characterizing gene expression by cDNA microarrays, we stand to add molecular definitions to traditional cervical pathology. Analysis of gene expression profiles of normal, ASCUS, LSIL, HSIL, and HPV samples will provide useful insights into the prevention, diagnosis, and treatment of cervical cancer.

Linkagene Ltd. The ACMM and MT labs have completed phase 1 of a collaboration with Sylvia Kalchesky of the Israeli company Linkagene Ltd. Together we have developed an asthma diagnostic kit based on the gene expression profiles of leukocytes. White blood cell samples were collected from asthmatic and healthy members of the open Israeli population and from within the Cochin Israelis. The Cochin cohort members experience normal asthma rates in their former home in India and abnormally high asthma rates upon returning to Israel. VARI gene expression analysis of leukocytes showed a group of 199 genes that differentiate asthmatic patients from healthy individuals within both the Cochin and the open Israeli populations. Linkagene filed an asthma diagnosis patent in 2005 based on these genes and is currently working with VARI to validate the experiments and set up a clinical trial in the West Michigan area.

NASA collaboration We are collaborating with the Kennedy Space Center/NASA, Brookhaven National Laboratory, and VARI’s Bart Williams, as well as with a number of other institutions, to study the early effects of low-dose radiation on gene expression in the neuronal cells of live mice. To this point we have completed pilot studies that have identified a number of genes that discriminate between cosmic radiation–exposed and control mice despite a lack of overt light-microscopic evidence of injury, suggesting that microarray-based gene expression analysis may serve as an early detection method for the effects of cosmic radiation in human beings.

Sperm toxicology Fully 25% of cases of human infertility have unknown causes. We are investigating the genomic aspect of this problem. In collaboration with Stephen Krawetz of Wayne State University, we have been funded to develop a diagnostic for male infertility caused by changes in the sperm mRNA content. This analysis and development of a diagnostic will be a significant improvement of existing methods that analyze sperm cell number and motility only, ignoring the importance of RNA molecules that code for proteins required in the very early stages of fertilization and development.

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SKY analysis The Laboratory of Microarray Technology is currently examining the correlation between chromosomal abnormalities and gene expression profiles. We are able to quantify differential gene expression between similar cells having differing karyotypes and correlate this with the chromosomal differences of the cells as shown by spectral karyotyping (SKY). For example, when we observe an overexpression of genes found on a specific chromosome (e.g., 7q), SKY analysis confirms the same degree of amplification of that chromosome or region of that chromosome. We are expanding on this finding in collaboration with Julie Koeman of the Swiatek laboratory to examine gene expression patterns in cells with highly unstable karyotypes, and we hypothesize that the patterns will very closely mirror the chromosomal changes shown by SKY. This data will then serve as powerful evidence of the accuracy of the microarrays produced and the techniques employed by the laboratory.

LMT renovations The Laboratory of Microarray Technology has relocated to a new area within the Institute that is specifically designed to meet the needs of the microarray facility as a CLIA-certifiable clinical laboratory. This area consists of two rooms, one of which is designated as administrative and data analysis space. The second is reserved for experimental work involving microarray production and gene expression analysis and contains all instrumentation required for these tasks. This room is maintained as a semi-clean room and allows for the isolation needed for analyzing clinical samples. We have continued to improve the accuracy and reliability of our cDNA arrays and are documenting our improvements for future publication.

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Educational highlights In 2005 we had two students from the Grand Rapids Area Pre-College Engineering Program (GRAPCEP), one from the Bridges to the Baccalaureate program, and one from the College of Veterinary Medicine at Michigan State University, as well as guest students from the University of Bath in the United Kingdom. The Bridges program is a collaboration to support the recruitment of women and minorities into science careers; Dr. Resau is a co-investigator and the VAI site coordinator for the program. In addition, this year we partnered with GRAPCEP to build a school within a school for science education and instruction within the Grand Rapids Public School system.

Our GRAPCEP mentorship program continues to be funded by Pfizer for a sixth year. Dr. Resau is a member of the graduate school committee that in the past year formalized the establishment of the VAEI Graduate School and recruited a Dean, which will increase our research and educational opportunities.

From left: Norton, Haak, Kort, Machiela, Hudson, Berghuis, Goolsby, Andegeko, Jason, Camenzuli, Resau

VARI | 2006

External Collaborators Eric Arnoys and John Ubels, Calvin College, Grand Rapids, Michigan Stephan Baldus, University of Duesseldorf, Germany Lonson Barr, Marcos Dantus, Matti Koeppel, and J. Webster, Michigan State University, East Lansing D. Chamberlain and M. Vazquez, National Aeronautics and Space Administration E. Fody, Holland Hospital, Holland, Michigan Steve Gronsman, Rose Technologies, Grand Rapids, Michigan Nadia Harbeck, Ludwig-Maximilians-Universität, Munich, Germany Christine Hughes and O. Orit Rosen, Harvard University, Cambridge, Massachusetts Sylvia Kachalsky, Linkagene, Lod, Israel Stephen Krawetz, James P. McAllister II, and Golam Newaz, Wayne State University, Detroit, Michigan K. Lindemann, Munich, Germany M. Magnusson, Lund University, Lund, Sweden R. McClintock, and M. Staves, Grand Valley State University, Allendale, Michigan Peter Silburn and Alan Mackay-Sim, Griffith University, Australia M. Suckow, Notre Dame University, South Bend, Indiana Ilan Tsarfaty, Tel Aviv University, Israel

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Recent Publications Kort, E.J., M.R. Moore, E.A. Hudson, B. Leeser, G.M. Yeruhalmi, R. Leibowitz-Amit, G. Tsarfaty, I. Tsarfaty, S. Moshkovitz, and J.H. Resau.

In press. Use of organ explant and cell culture. In Mechanisms of Carcinogenesis, Hans Kaiser, ed. Dordrecht, The Netherlands: Kluwer Academic.

Moshitch-Moshkovitz, S., G. Tsarfaty, D.W. Kaufman, G.Y. Stein, K. Shichrur, R. Sigler, J. Resau, G.F. Vande Woude, and I. Tsarfaty.

In press. In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met. Neoplasia.

Tsarfaty, G., G.Y. Stein, S. Moshitch-Moshkovitz, D.W. Kaufman, B. Cao, J. Resau, G.F. Vande Woude, and I. Tsarfaty. In press. HGF/SF increases tumor blood volume: a novel tool for in vivo functional molecular imaging of Met. Neoplasia.

Ubels, John L., Holly M. Hoffman, Sujata Srikanth, James H. Resau, and Craig P. Webb. 2006. Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells. Investigative Ophthalmology and Visual Science 47(5): 1876–1885.

Webster, Joshua D., Vilma Yuzbasiyan-Gurkan, John B. Kaneene, RoseAnn Miller, James H. Resau, and Matti Kuipel. 2006. The role of c-KIT in tumorigenesis: evaluation in canine cutaneous mast cell tumors. Neoplasia 8(2): 104–111.

Hay, Rick V., Brian Cao, R. Scot Skinner, Yanli Su, Ping Zhao, Margaret F. Gustafson, Chao-Nan Qian, Bin T. Teh, Beatrice S. Knudsen, James H. Resau, Shuren Shen, David J. Waters, Milton D. Gross, and George F. Vande Woude. 2005. Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek™). Clinical Cancer Research 11(19): 7064s–7069s.

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Pamela J. Swiatek, Ph.D., M.B.A. Laboratory of Germline Modification

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Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology from Indiana University. From 1988 to 1990, she was a postdoctoral fellow at the Tampa Bay Research Institute. From 1990 to 1994, she was a postdoctoral fellow at the Roche Institute of Molecular Biology in the laboratory of Tom Gridley. From 1994 to 2000, Dr. Swiatek was a Research Scientist and Director of the Transgenic Core Facility at the Wadsworth Center in Albany, N.Y., and an Assistant Professor in the Department of Biomedical Sciences at the State University of New York at Albany. She joined VARI as a Special Program Investigator in August 2000. She has been the chair of the Institutional Animal Care and Use Committee since 2002 and is an Adjunct Assistant Professor in the College of Veterinary Medicine at Michigan State University. Dr. Swiatek received her M.B.A. in 2005 from Krannert School of Management at Purdue University. She was promoted to Senior Scientific Investigator in 2006.

Staff Laboratory Staff Julie Koeman, B.S. Kellie Sisson, B.S. Juraj Zahatnansky, B.S. Kaye Johnson, B.A. Diana Lewis

Students

Visiting Scientists

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Research Interests The Germline Modification lab is a full-service lab that functions at the levels of service, research, and teaching to develop, analyze, and maintain mouse models of human disease. Our lab applies a business philosophy to core service offerings and we focus on scientific innovation, customer satisfaction, and service excellence. Mouse models are produced using gene-targeting technology, a well-established, powerful method for inserting specific genetic changes into the mouse genome. The resulting mice can be used to study the effects of these changes in the complex biological environment of a living organism. The genetic changes can include the introduction of a gene into a specific site in the genome (gene “knock-in”) or the inactivation of a gene already in the genome (gene “knock-out”). Since these mutations are introduced into the reproductive cells known as the germline, they can be used to study the developmental aspects of gene function associated with inherited genetic diseases.

In addition to traditional gene-targeting technologies, the Germline Modification lab can produce mouse models in which the gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These types of mouse models, known as conditional knock-outs, are particularly useful in studying genes that, if missing, cause the mouse to die as an embryo. The lab also has the ability, using tetraploid embryo technology, to produce mutant embryos that have a wild-type placenta. This technique is useful when the gene-targeted mutation prevents implantation of the mouse embryo in the uterus. We also assist in the development of embryonic stem (ES) or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation.

Our gene-targeting service encompasses three major procedures: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting procedures are initiated by mutating the genomic DNA of interest and inserting it into ES cells using the electroporation technique. The mutated gene integrates into the genome of the ES cells and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into tissue culture, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced into the mouse by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos (blastocysts). These embryos, containing a mixture of wild-type and mutant ES cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are heterozygotes, i.e., they possess one copy of the mutated gene. The heterozygous mice are bred together to produce mice that completely lack the normal gene. These homozygous mice have two copies of the mutant gene and are called knock-out mice.

Once gene targeting mice are produced, our lab assists in developing breeding schemes and provides for complete analysis of the mutants. The efficiency of mutant mouse production and analysis is enhanced by the AutoGenprep 960, a robotic, high-throughput DNA isolation machine. Tail biopsies from genetically engineered mice are processed in a 96-well format and the DNA samples are delivered to the client for analysis.

Our lab also directs the VARI cytogenetics core, which offers a variety of custom services. Mouse, rat, and human cell lines derived from tumors, fibroblasts, blood, or ES cells can be grown in tissue culture, growth-arrested, fixed, and spread onto glass slides. Karyotyping of chromosomes using Leishman- or Giemsa-stained (G-banded) chromosomes is our basic service. However, spectral karyotyping (SKY) analysis of metaphase chromosome spreads, using high-quality, 24-color, whole-chromosome fluorescent paints, can aid in the detection of subtle and complex chromosomal rearrangements. Fluorescence in situ hybridization (FISH) analysis, using indirectly or directly labeled bacterial artificial chromosome (BAC) or plasmid probes, can also be performed on metaphase spreads or on interphase nuclei derived from tissue touch preps of nondividing cells. Sequential staining of identical metaphase spreads using FISH and SKY can assist in identifying the chromosome integration site of a randomly integrated transgene.

Finally, we provide cryopreservation services for archiving and reconstituting valuable mouse strains. These cost-effective procedures decrease the need to continuously breed valuable mouse models, and they provide added insurance against the loss of custom mouse lines due to disease outbreak or a catastrophic event. Mouse sperm or eight-cell mouse embryos can be cryopreserved and stored in liquid nitrogen; the strains can be reconstituted by in vitro fertilization of oocytes or by implantation into the oviducts of recipient mice, respectively.

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The VARI Germline Modification lab directs the Michigan Animal Model Consortium (MAMC) of the Core Technology Alliance (CTA) Corp.

MAMC is one of eight collaborative core facilities located at the University of Michigan, Michigan State University, Wayne State University, Western Michigan University, Kalamazoo Valley Community College, and VARI; the others offer research services in proteomics, bioinformatics, structural biology, genomics, biological imaging, high-throughput compound screening, and antibody technology. The MAMC labs receive funding from the Michigan Economic Development Corporation to provide efficient mouse modeling services to researchers studying human diseases.

Michigan Technology Tri-Corridor/Michigan Life Science Corridor (MTTC/MLSC) funding has enabled MAMC to grow from 4 initial service offerings to a broader platform of 15 customized scientific services that maximize research productivity and catalyze life science product development in an efficient and effective manner. The long-term goal of MAMC is to offer a comprehensive set of cutting-edge services that, through continuous enhancements and development, will define our organization as a single point-of-service site for animal models research. These services are described more completely on the MAMC website at <www.vai.org/core/mouse>.

Centralized provision of services decreases time to discovery and is in high demand by academia and pharmaceutical and biotechnology companies, which are increasingly looking to outsource to service centers. Through its well-organized service structure, staff of experts, and proven service record, MAMC can be valuable to such entities and support the growth of the life science industry in Michigan, which is congruent with the CTA goals.

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Animal model development

Mouse transgenics – Transgenic technology is used to produce genetically engineered mice and rats expressing foreign genes and serving as models for human disease research. Transgenic technology uses microinjection to deliver the foreign DNA into the pronucleus of a one-cell fertilized egg. Service is provided using various strains of laboratory mice, with production of three transgenic founder mice guaranteed from each procedure.

Gene targeting – By transfecting mouse embryonic stem cells with inactivating, homologous DNAs, target gene expression can be shut down. Genetically engineered mice are produced by microinjecting mutant stem cells into mouse embryos and breeding the progeny to mutant homozygosity. This service is provided using 129 or C57BL/6 embryonic stem cells.

Xenotransplantation – Human cancer cells are injected into immunodeficient mice to produce human-derived tumors. Protocols are designed to test anti-tumor treatment regimens that can lead to prognostic, diagnostic, or therapeutic procedures in humans.

Animal model analysis

Cytogenetics – Mouse and rat chromosomal abnormalities and genetic loci are visually observed using Giemsa stain, SKY, and FISH techniques.

Blood analysis – VetScan instrumentation is used to perform differential cell counts and biochemical analysis of mouse blood.

Necropsy – Mice are dissected post-mortem and tissues are fixed for histological analysis. Necropsy reports are generated using voice-recognition software.

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Histology – Histological sections are prepared from mouse tissues using microtomes and cryostats, processed and stained using automated instruments, and then microscopically analyzed.

Veterinary pathology – A board-certified veterinary pathologist holding the D.V.M. and Ph.D. degrees provides expert microscopic analysis and project consultation.

DNA isolation – DNA is isolated from mouse tail biopsies using the AutoGenprep 960 instrument.

Animal model maintenance and preservation

Mouse rederivation – All mouse strains entering the specific pathogen–free breeding facility are rederived to specific pathogen–free mouse status using embryo transfer techniques.

Animal housing services – Mice are housed in a specific pathogen–free vivarium.

Animal technical services – Veterinary services such as injections, measurements, mating set-up, and tail biopsies are performed by the animal technician staff.

Contract breeding services – Wild-type mouse strains and genetically engineered animal models are maintained for research purposes by breeding the strains in a specific pathogen–free environment.

Embryo/sperm cryopreservation – Genetically engineered mice are preserved for archival purposes, disease control, genetic stability, and economic efficiency using germplasm cryopreservation techniques.

Cancer model repository – Mouse cancer models of research interest are maintained through breeding strategies.

Recent Publications

From left: Johnson, Lewis, Sisson, Swiatek, Koeman, Zahatnansky

Zhang, Qing-Yu, Jun Gu, Ting Su, Huadong Cui, Xiuling Zhang, Jaime D’Agostino, Xiaoliang Zhuo, Weizhu Yang, Pamela J. Swiatek, and Xinxin Ding. 2005. Generation and characterization of a transgenic mouse model with hepatic expression of human CYP2A6.

Biochemical and Biophysical Research Communications 338(1): 318–324.

Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Pamela J. Swiatek, Roderick T. Bronson, Robert Sigler, Richard W. Martin, and George F. Vande Woude. 2005. Targeted disruption of Mig-6 in the mouse genome leads to early onset degenerative joint disease. Proceedings of the National Academy of Sciences U.S.A. 102(33): 11740–11745.

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Photo taken by Susan Kitchen.

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VARI | 2006

Cellular localization of cytoskeletal remodeling proteins in an adherent fibroblastic cell.

The protein vinculin (green) is thought to be involved in the attachment of the actin (blue) microfilaments to the cell membrane. mDia2 (red) is a canonical member of the formin family of actin filament assembly proteins.

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

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Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, and his Ph.D. from the Karolinska Institute, Sweden, in 1997. Before joining the Van Andel Research Institute (VARI), he was an Associate Professor of medical genetics at the Karolinska Institute. Dr. Teh joined VARI as a Senior Scientific Investigator in January 2000. Dr. Tehâ&#x20AC;&#x2122;s research mainly focuses on kidney cancer, and he is currently on the Medical Advisory Board of the Kidney Cancer Association. He was promoted to Distinguished Scientific Investigator in 2005.

Staff Laboratory Staff

Students

Chao-Nan (Miles) Qian, M.D., Ph.D. Peng-Fei Wang, M.D., Ph.D. Xin Yao, M.D., Ph.D. Eric Kort, M.D. Daisuke Matsuda, M.D. Jindong Chen, Ph.D. Leslie Farber, Ph.D. Kunihiko Futami, Ph.D. Dan Huang, Ph.D.

Visiting Scientists

Sok Kean Khoo, Ph.D. Douglas Luccio-Camelo, Ph.D. David Petillo, Ph.D. Zhongfa (Jacob) Zhang, Ph.D. Stephanie Bender, M.S. Wangmei Luo, M.S. Mark Betten, B.S. Aaron Massie, B.S. Sabrina Antio

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Research Interests Kidney cancer, or renal cell carcinoma (RCC), is the tenth most common cancer in the United States (39,000 new cases and more than 13,000 deaths a year). Its incidence has been increasing, a phenomenon that cannot be accounted for by the wider use of imaging procedures. We have established a comprehensive and integrated kidney research program, and our major research goals are 1) to identify the molecular signatures of different subtypes of kidney tumors, both hereditary and sporadic, and to understand how genes function and interact in giving rise to the tumors and their progression; 2) to identify novel biomarkers and key drug targets; and 3) to develop animal models for drug testing and preclinical bioimaging.

Our program to date has established a worldwide network of collaborators, a tissue bank containing fresh-frozen tumor pairs (over 1,100 cases) and serum, and a gene expression profiling database of 500 tumors with long-term clinical follow-up information for half of them. Our program includes positional cloning of hereditary RCC syndromes and functional studies of their related genes, microarray and bioinformatic analysis, generation of RCC mouse models, and more recently, molecular therapeutic studies.

Hereditary RCC syndromes â&#x20AC;&#x201C; positional cloning and functional studies We are currently focusing on the cloning of the gene responsible for familial clear cell renal cell carcinoma, which is a separate entity from von Hippel-Lindau (VHL) disease and from familial RCC with a chromosome-3 translocation. These efforts involve the use of high-density, single nucleotide polymorphism (SNP) microarrays and correlation with our existing gene expression profiles. We also have established that the HRPT2 homozygote is embryonically lethal, and we have generated kidney- and parathyroid-specific HRPT2 conditional knock-outs using the Cre-lox system.

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Microarray gene expression profiling and bioinformatics We continue to mine our RCC expression database, which today includes over 500 tumors, cell lines, and xenografts. High-density SNP genotyping was also performed on some of these materials. We are currently focusing on analysis and data mining. Clinically, we continue to subclassify the tumors by correlation with clinicopathological information. One example is the study of the unclassified group of tumors, for which the histological diagnosis is â&#x20AC;&#x153;unknown.â&#x20AC;? We have also identified a specific set of genes that can distinguish chromophobe (malignant) from oncocytoma (benign), two types that share a high degree of similarity in their expression profiles. We also plan to carry out a multi-center, prospective clinical trial for our RenoChip. Our database has proven to be very useful in RCC research, since we can obtain differential expression of any gene in seconds; this has led to numerous collaborations. We are currently combining SNP and expression data to identify novel RCC-related genes.

Mouse models of kidney cancer and molecular therapeutic studies We have generated several kidney-specific conditional knock-outs including APC, PTEN, and VHL. The first two give rise to renal cysts and tumors, whereas VHL remains neoplasia free. Double knock-outs are being studied. We have successfully generated nine xenograft RCC models via subcapsular injection, all of which have various characteristic clinical features and outcomes. Tumors and serum have been harvested for a baseline data set. We are currently performing in vitro and in vivo studies on several new drugs for kidney cancer.

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From left to right, standing: Khoo, Petillo, Qian, Luccio-Camelo, Farber, Kort, Wang, Matsuda, Betten, Massie, Zhang, Yao, Chen; seated: Bender, Huang, Teh, Antio, Luo

External Collaborators We have extensive collaborations with researchers and clinicians in the United States and overseas.

VARI | 2006

Recent Publications Adley, B.P., A. Gupta, F. Lin, C. Luan, B.T. Teh, and X.J. Yang. In press. Expression of kidney-specific cadherin in chromophobe renal cell carcinoma and renal oncocytoma. American Journal of Clinical Pathology.

Pimenta, F.J., G.C. Silveira, G.C. Taveres, A.C. Silva, P.F. Perdigao, W.H. Castro, W.V. Gomez, B.T. Teh, L. De Marco, and R.S. Gomez.

In press. HRPT2 gene alterations in ossifying fibroma of the jaws. Oral Oncology.

Tretiakova, M., M. Turkyilmaz, T. Grushko, M. Kocherginsky, C. Rubin, O. Olopade, B.T. Teh, and X.J. Yang. In press. Topoisomerase IIα expression in Wilms tumors. Journal of Clinical Pathology.

Yang, X.J., J. Sugimura, K.T. Schafernak, M. Tretiakova, N. Vogelzang, K. Furge, and B.T. Teh. In press. Classification of sixteen renal neoplasms based on molecular signatures. Journal of Urology.

Yang, X.J., M. Takahashi, K.T. Schafernak, M.S. Tretiakova, J. Sugimura, N. Vogelzang, H.-O. Kanayama, and B.T. Teh. In press.

Does “granular cell type” renal cell carcinoma exist? — Molecular and histopathological reclassification of renal tumors with previous diagnoses of granular renal cell carcinoma. Applied Immunohistochemistry and Molecular Morphology.

Gimm, O., K. Lorenz, P. Nguyen Thanh, U. Schneyer, M. Bloching, V.M. Howell, D.J. March, B.T. Teh, U. Krause, and H. Dralle. 2006.

Prophylactic parathyroidectomy for familial parathyroid carcinoma [in German]. Chirurg 77(1): 15–24.

Lim, L.C., M.-H. Tan, C. Eng, B.T. Teh, and R.C. Rajasoorya. 2006. Thymic carcinoid in multiple endocrine neoplasia 1 — genotypephenotype correlation and prevention. Journal of Internal Medicine 259(4): 428–432.

Lin, Fan, Wannian Yang, Mark Betten, Bin Tean Teh, Ximing J. Yang, and the French Kidney Cancer Study Group. 2006. Expression of S-100 protein in renal cell neoplasms. Human Pathology 37(4): 462–470.

Takahashi, M., Bin T. Teh, and H.-O. Kanayama. 2006. Elucidation of the molecular signatures of renal cell carcinoma by gene expression profiling. Journal of Medical Investigation 53(1–2): 9–19.

Wang, PengFei, Dong Kong, Matthew W. VanBrocklin, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams, and Sheri L. Holmen. 2006. Simplified method for the construction of gene targeting vectors for conditional gene inactivation in mice. Transgenics 4: 215–228.

Warner, J.V., D.R. Nyholt, F. Busfield, M. Epstein, J. Burgess, S. Stranks, P. Hill, D. Perry-Keene, D. Learoyd, B. Robinson, B.T. Teh, J.B. Prins, and J.W. Cardinal. 2006. Familial isolated hyperparathyroidism is linked to a 1.7-Mb region on chromosome 2p13.3-14. Journal of Medical Genetics 43(3): e12.

Zhuang, Z., S. Huang, J.A. Kowalak, Y. Shi, J. Lei, I.A. Lubensky, G.P. Rodgers, A.S. Cornelius, R.J. Weil, B.T. Teh, and A.O. Vortmeyer.

2006. From tissue phenotype to proteotype: sensitive protein identification in microdissected tumor tissue. International Journal of Oncology 28(1): 103–110.

Davies, Helen, Chris Hunter, Raffaella Smith, Philip Stephens, Chris Greenman, Graham Bignell, Jon Teague, Adam Butler, Sarah Edkins, Claire Stevens, Adrian Parker, Sarah O’Meara, Tim Avis, Syd Barthorpe, Lisa Brackenbury, et al. 2005. Somatic mutations of the protein kinase gene family in human lung cancer. Cancer Research 65(17): 7591–7595.

Gray, Steven G., Antonio H. Iglesias, Fernando Lizcano, Raul Villanueva, Sandra Camelo, Hisaka Jingu, Bin T. Teh, Noriyuki Koibuchi, William W. Chin, Efi Kokkotou, and Fernando Dangond. 2005. Functional characterization of JMJD2A, a histone deacetylase– and retinoblastoma-binding protein. Journal of Biological Chemistry 280(31): 28507–28518.

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Hay, Rick V., Brian Cao, R. Scot Skinner, Yanli Su, Ping Zhao, Margaret F. Gustafson, Chao-Nan Qian, Bin T. Teh, Beatrice S. Knudsen, James H. Resau, Shuren Shen, David J. Waters, Milton D. Gross, and George F. Vande Woude. 2005. Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek™). Clinical Cancer Research 11(19): 7064s–7069s.

Huo, Lei, Jun Sugimura, Maria S. Tretiakova, Kurt T. Patton, Rohit Gupta, Boris Popov, William B. Laskin, Anjana Yeldandi, Bin Tean Teh, and Ximing J. Yang. 2005. C-kit expression in renal oncocytomas and chromophobe renal cell carcinomas. Human Pathology 36(3): 262–268.

Morris, Mark R., Dean Gentle, Mahera Abdulrahman, Esther N. Maina, Kunal Gupta, Rosamonde E. Banks, Michael S. Wiesener, Takeshi Kishida, Masahiro Yao, Bin Teh, Farida Latif, and Eamonn R. Maher. 2005. Tumor suppressor activity and epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2/SPINT2 in papillary and clear cell renal cell carcinoma. Cancer Research 65(11): 4598–4606.

Stephens, Philip, Sarah Edkins, Helen Davies, Chris Greenman, Charles Cox, Chris Hunter, Graham Bignell, Jon Teague, Raffaella Smith, Claire Stevens, Sarah O’Meara, Adrian Parker, Patrick Tarpey, Tim Avis, Andy Barthorpe, et al. 2005. A screen of the complete protein kinase gene family identifies diverse patterns of somatic mutations in human breast cancer. Nature Genetics 37(6): 590–592.

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Sweet, Kevin, Joseph Willis, Xiao-Ping Zhou, Carol Gallione, Takeshi Sawada, Pia Alhopuro, Sok Kean Khoo, Attila Patocs, Cossette Martin, Scott Bridgeman, John Heinz, Robert Pilarski, Rainer Lehtonen, Thomas W. Prior, Thierry Frebourg, et al. 2005. Molecular classification of patients with unexplained hamartomatous and hyperplastic polyposis. Journal of the American Medical Association 294(19): 2465–2473.

Takahashi, Masayuki, Veronica Papavero, Jason Yuhas, Eric Kort, Hiro-omi Kanayama, Susumu Kagawa, Robert C. Baxter, Ximing J. Yang, Steven G. Gray, and Bin T. Teh. 2005. Altered expression of members of the IGF-axis in clear cell renal cell carcinoma.

International Journal of Oncology 26(4): 923–931.

Takahashi, M., X.J. Yang, S. McWhinney, N. Sano, C. Eng, S. Kagawa, B.T. Teh, and H.-O. Kanayama. 2005. cDNA microarray analysis assists in diagnosis of malignant intrarenal pheochromocytoma originally masquerading as a renal cell carcinoma. Journal of Medical Genetics 42(8): e48.

Wang, P.F., M.-H. Tan, C. Zhang, H. Morreau, and B.T. Teh. 2005. HRPT2, a tumor suppressor gene for hyperparathyroidism-jaw tumor syndrome. Hormone and Metabolic Research 37(6): 380–383.

Yang, Ximing J., Min-Han Tan, Hyung L. Kim, Jonathon A. Ditlev, Mark W. Betten, Carolina E. Png, Eric J. Kort, Kunihiko Futami, Kyle A. Furge, Masayuki Takahashi, Hiro-omi Kanayama, Puay Hoon Tan, Bin Sing Teh, Chunyan Luan, Kim Wang, et al. 2005. A molecular classification of papillary renal cell carcinoma. Cancer Research 65(13): 5628–5637.

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

Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from Rutgers University. 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 Island Animal Disease Center. In 1972, he joined the National Cancer Institute as Head of the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980, was appointed Chief of the Laboratory of Molecular Oncology. In 1983, he became Director of the Advanced Bioscience Laboratories–Basic Research Program at the National Cancer Institute’s Frederick Cancer Research and Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as Special Advisor to the Director, and then as Director for the Division of Basic Sciences at the National Cancer Institute.

In 1999, he was recruited to the Directorship of the Van Andel Research Institute in Grand Rapids, Michigan.

Staff

Student

Laboratory Staff Yu-Wen Zhang, M.D., Ph.D. Chongfeng Gao, Ph.D. Carrie Graveel, Ph.D. Sharon Moshkovitz, Ph.D. Qian Xie, Ph.D. Dafna Kaufman, M.Sc. Matt VanBrocklin, M.S.

Jack DeGroot, B.S. Rachel Kuznar, B.S. Benjamin Staal, B.S. Yanli Su, A.M.A.T.

Angelique Berens

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Research Interests Activating Met mutations produce unique tumor profiles Signaling through Met and its ligand, HGF/SF, has been implicated in most types of human cancer. Compelling genetic evidence for the role of Met stems from the discovery that activating gain-of-function mutations are found in human kidney cancers and in other cancer types (http://www.vai.org/vari/metandcancer/). To study how Met activating mutations are involved in tumor development, we generated mice bearing mutations in the endogenous Met locus representative of both the inherited and sporadic mutations found in human cancers. On a C57/B6 background, the different mutant Met lines developed unique tumor profiles, including carcinomas, sarcomas, and lymphomas. We have found that the differences in tumor types and latency, depending on the mutation, may be due to signaling differences triggered by the specific mutation in a tissue- or stem cellâ&#x20AC;&#x201C;specific pattern. Cytogenetic analysis of all tumor types shows trisomy in the Met locus on chromosome 6 in all cases, and it is the mutant met allele that is amplified and likely to be required for tumor progression. Understanding the signaling specificity of these mutations is essential for developing successful therapeutics. However, the genetic background also has a profound influence, since transferring the mutant Met to another mouse background results in a dramatic shift in the disease pattern, to mammary cancer. Our mutant mice provide a valuable model for testing Met inhibitors and for understanding the molecular events crucial for Met-mediated tumorigenesis.

Direct, intravital imaging of Met oncogene activation

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We have generated mice carrying a murine GFP-Met transgene that emits intense fluorescence to reveal where in the animal Met is expressed. Founder mice were selected that had GFP-Met expression levels from high to moderate. About 30% of the transgenic GFP-Met male mice, but no females, developed tumors in the preputial sebaceous glands. GFP-Met expression is always higher in the sebaceous gland tumors than in normal skin, and mice expressing the highest levels of the GFP-Met transgene develop tumors earliest, but tumors originating from the different founder lines have similar pathological phenotypes. The mice present with sebaceous tumors ranging from adenomas, to adenocarcinomas, to angiosarcomas. Metastases developed in 71% of GFP-Met transgenic mice with adenocarcinoma and in 18% of mice with angiosarcoma. The metastases were found locally in the skin and in distant organs such as the liver, lung, and kidney. Image analyses of unfixed frozen tumor metastases showed large numbers of cells overexpressing GFP-Met, and tissue arrays of these tumors revealed higher GFP-Met levels relative to the primary tumors.

Recently, we have been able to image GFP-Met in vivo in response to HGF/SF treatment. We observed that Met is both activated and internalized. Moreover, we can quantitatively monitor Met in vivo by imaging the subcellular localization of GFP-Met, and we can detect single cells expressing GFP-Met in and around the tumor. These cells correlate in immunohistochemical analysis with single cells and micrometastases expressing high levels of the receptor. This provides us with an in vivo system for high-resolution, real-time evaluation of Met-targeted drugs.

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Immunocompromised transgenic mice with Met-expressing xenografts We have generated a severe combined immune deficiency (SCID) mouse strain carrying a human HGF/SF transgene. This mouse provides a species-compatible ligand for propagating human tumor cells expressing human Met receptors. The growth of Met-expressing human tumor xenografts can be significantly enhanced in this transgenic mouse relative to those in nontransgenic hosts. This immunocompromised strain is useful for investigating the role of the Met tyrosine kinase receptor in tumor malignancy. Currently, we are developing metastasis models and generating orthotopic xenografts of human tumor cells. This model will also be used for preclinical testing of drugs or compounds targeting the HGF/SF-Met complex and downstream signaling pathways.

HGF-induced proliferation and invasion We are interested in how HGF/SF-induced proliferation and invasion contribute to tumor progression. We have established in vitro methods for selecting highly proliferative or invasive tumor cell populations that may mimic the in vivo process of clonal selection during tumor progression. Our studies have shown that most tumor cells display both invasive and proliferative phenotypes and that they can reversibly change from one phenotype to the other. We have determined that chromosomal instability correlates with the different tumor phenotypes. Using spectral karyotyping (SKY) and Fish, we have found significant changes in chromosome content with each phenotype. These changes show remarkable concordance with changes in gene expression, and the changes in regional gene expression appear to favor the expression of genes appropriate for the specific phenotype of invasion or proliferation. Moreover, the ratio of chromosomal changes closely parallels the ratio of gene expression in the chromosome. These results show that chromosome instability and the resulting tumor cell heterogeneity in chromosome composition provide the diversity in gene expression that allows tumor cell clonal evolution.

Geldanamycin inhibits tumor cell invasion at femtomolar concentrations Our lab has been studying the mechanism by which geldanamycin (GA) inhibits urokinase activation of plasmin from plasminogen (uPA). Previously, we have shown that a subset of GA derivatives at femtomolar concentration inhibits HGF/SF-induced activation of plasmin in canine MDCK cells. We have found that such inhibition also occurs in several human glioblastoma tumor cell lines. Curiously, these GA drugs inhibit HGF/SF-induced uPA activation and block MDCK cell scattering and glioblastoma tumor cell invasion in vitro only at femtomolar concentrations, well below the concentration required to exhibit a measurable effect on Met expression. This inhibition is observed only with HGF/SF-mediated activation and only when the magnitude of HGF/SF-uPA induction is 1.5 times basal uPA-plasmin activity. Our studies provide circumstantial evidence for a novel non-HSP90 molecular target that is involved in HGF/SF-mediated tumor cell invasion.

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MAPK in melanoma Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades, potentiating biological activities such as cell proliferation, differentiation, and survival. Constitutive activation of MAPK signaling pathways is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRas or BRAF are found in about 85% of all melanomas, resulting in constitutive activation of the MAPK pathway (Ras-BRaf-MEK-Erk-Rsk). We have previously demonstrated that inhibition of this pathway with small-molecule MEK inhibitors selectively induces apoptosis in human melanoma cells both in vitro and in vivo, but not in normal melanocytes. These results support the concept that the MAPK pathway represents a tumor-specific survival signaling pathway in melanoma cells and that targeting members of this pathway may be an effective therapeutic strategy. Understanding the mechanisms by which constitutive MAPK promotes survival and defining the minimal vital MAPK pathway components required for the development and progression of melanoma may have direct translational implications. The overall objective is to define the molecular mechanism(s) by which the MAPK pathway mediates melanoma-specific survival signals. Preliminary data suggest that MAPK activation actively suppresses several pro-apoptotic Bcl-2 family members. We are currently using the specific small-molecule MEK inhibitor PD184352, together with molecular biological approaches, to selectively modulate the expression and function of these molecules in order to validate and develop them as novel therapeutic targets for treating melanoma and other MAPK-associated cancers.

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From left to right: Reed, Vande Woude, Moshkovitz, Kaufman, Graveel, Gao, Su, Staal, Nelson, VanBrocklin, Zhang, DeGroot, Xie

External Collaborators Ermanno Gherardi, MRC Center, Cambridge, England Nadia Harbeck, Technische Universit채t, Munich, Germany Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Ernest Lengyel, University of Chicago, Illinois Alnawaz Rehemtulla, Brian Ross, and Richard Simon, University of Michigan, Ann Arbor Yuehai Shen and David Wenkert, Michigan State University, East Lansing Ilan Tsarfaty, Tel Aviv University, Israel Robert Wondergem, East Tennessee State University, Johnson City

VARI | 2006

Recent Publications Moshitch-Moshkovitz, S., G. Tsarfaty, D.W. Kaufman, G.Y. Stein, K. Shichrur, R. Sigler, J. Resau, G.F. Vande Woude, and I. Tsarfaty.

In press. In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met. Neoplasia.

Tsarfaty, G., G.Y. Stein, S. Moshitch-Moshkovitz, D.W. Kaufman, B. Cao, J. Resau, G.F. Vande Woude, and I. Tsarfaty. In press. HGF/SF increases tumor blood volume: a novel tool for in vivo functional molecular imaging of Met. Neoplasia.

Duesbery, Nick, and George Vande Woude. 2006. BRAF and MEK mutations make a late entrance. Science’s STKE 328: pe15.

Gao, Chong-Feng, Qian Xie, Yan-li Su, Julie Koeman, Sok Kean Khoo, Margaret Gustafson, Beatrice S. Knudsen, Rick Hay, Nariyoshi Shinomiya, and George F. Vande Woude. 2005. Proliferation and invasion: plasticity in tumor cells. Proceedings of the National Academy of Sciences U.S.A. 102(30): 10528–10533.

Graveel, Carrie R., Cheryl A. London, and George F. Vande Woude. 2005. A mouse model of activating Met mutations. Cell Cycle 4(4): 518–520.

Hay, Rick V., Brian Cao, R. Scot Skinner, Yanli Su, Ping Zhao, Margaret F. Gustafson, Chao-Nan Qian, Bin T. Teh, Beatrice S. Knudsen, James H. Resau, Shuren Shen, David J. Waters, Milton D. Gross, and George F. Vande Woude. 2005. Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek™). Clinical Cancer Research 11(19): 7064s–7069s.

Orchekowski, Randal, Darren Hamelinck, Lin Li, Ewa Gliwa, Matt VanBrocklin, Jorge A. Marrero, George F. Vande Woude, Ziding Feng, Randall Brand, and Brian B. Haab. 2005. Antibody microarray profiling reveals individual and combined serum proteins associated with pancreatic cancer. Cancer Research 65(23): 11193–11202.

Ren, Yi, Brian Cao, Simon Law, Yi Xie, Ping Yin Lee, Leo Cheung, Yongxong Chen, Xin Huang, Hiu Man Chan, Ping Zhao, John Luk, George Vande Woude, and John Wong. 2005. Hepatocyte growth factor promotes cancer cell migration and angiogenic factors expression: a prognostic marker of human esophageal squamous cell carcinomas. Clinical Cancer Research 11(17): 6190–6197.

Shen, Yuehai, Qian Xie, Monica Norberg, Edward Sausville, George F. Vande Woude, and David Wenkert. 2005. Geldanamycin derivative inhibition of HGF/SF-mediated Met tyrosine kinase receptor–dependent urokinase-plasminogen activation. Bioorganic & Medicinal Chemistry 13(16): 4960–4971.

Xie, Qian, Chong-Feng Gao, Nariyoshi Shinomiya, Edward Sausville, Rick Hay, Margaret Gustafson, Yuehai Shen, David Wenkert, and George F. Vande Woude. 2005. Geldanamycins exquisitely inhibit HGF/SF-mediated tumor cell invasion. Oncogene 24(23): 3697–3707.

Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Pamela J. Swiatek, Roderick T. Bronson, Robert Sigler, Richard W. Martin, and George F. Vande Woude. 2005. Targeted disruption of Mig-6 in the mouse genome leads to early onset degenerative joint disease. Proceedings of the National Academy of Sciences U.S.A. 102(33): 11740–11745.

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

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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 of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland (1995–1999). Dr. Webb joined VARI as a Scientific Investigator in October 1999.

Staff Laboratory Staff

Visiting Scentists Student Visiting Scientists

Students

Jennifer Bromberg-White, Ph.D. Jeremy Miller, Ph.D. David Monsma, Ph.D. Emily Eugster, M.S. Sujata Srikanth, M.Phil.

Marcy Ross, B.S. Danielle Welch, B.A.

Gustavo Cumbo-Nacheli, M.D. Mecker Moller, M.D. John Ubels, Ph.D.

Molly Dobb

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Research Interests We use cell-based assays, preclinical animal models, and clinical research subjects to identify and validate biomarkers and therapeutic targets for metastatic and drug-refractory cancers. The ability to navigate seamlessly across these diverse model systems requires the efficient capture, management, analysis, and reporting of integrated data between species and across disparate technologies. This has been greatly enhanced through the development of our XenoBase-BioIntegration Suite (XB-BIS™), a central informatics solution that has been successfully distributed to pharmaceutical and biotech companies, as well as to academic and clinical collaborators.

Our goal is to efficiently translate our investigational findings into clinical practice, initially in the areas of improved molecular-based diagnostics and biomarker-driven therapeutic strategies.

In the research lab, we use molecular technologies in conjunction with XB-BIS and systems biology to identify and validate diagnostic signatures for prospective evaluation within the Center for Molecular Medicine (CMM). Potential therapeutic intervention strategies are also identified and validated in the laboratory using RNA interference and/or existing therapeutic agents in the appropriate preclinical models. Finally, our molecular-based technologies are now being applied in the area of personalized medicine, with an initial focus on adult and pediatric patients with aggressive or refractory cancers. While our lab’s research is focused in oncology, the translational infrastructure can be and has been applied to other disease areas. The optimal therapeutic target is no longer the specific disease, but rather the molecular networks at play within the disease and the individual.

Tumor metastasis Metastasis accounts for the majority of cancer-related mortalities. The active recruitment of tumor vasculature, termed angiogenesis, is integral to both tumor growth and metastasis. Our laboratory currently uses both in vitro and in vivo systems to study metastasis and angiogenesis. We continue to identify molecular correlates of metastatic disease that may represent future biomarkers and/or molecular targets for diagnosis and treatment. At this time, we are focused on highly aggressive solid tumor malignancies, including cancers of the pancreas, colon and rectum, breast, lung, and mesothelium.

One study with Spectrum Health Hospital and the Digestive Disease Institute is underway to identify gene expression signatures in primary colorectal tumors. A multiplexed gene expression signature has been identified that predicts with 85% accuracy the likelihood of subsequent development of widespread metastatic disease. Dr. Mecker Moller, a resident who rotated through the lab, presented a paper detailing these findings to the 22nd Annual Michigan State University Research Forum and received a first-prize award. The paper was also accepted for presentation at last year’s Annual Society of Surgical Oncology meeting. The ability of this genomic signature to predict clinical progression in an independent patient cohort is currently being assessed within the CMM. In addition, potential blood markers of colorectal cancer have been identified, and we are currently screening blood plasma and urine samples from patients having colorectal cancer—as well as other inflammatory diseases including Crohn disease and ulcerative colitis—using protein microarrays in collaboration with Brian Haab’s laboratory at VARI. In a more recent study in association with local physicians and research staff from Spectrum Health’s Research Department, we have accrued over 100 patients with breast cancer to a clinical protocol that is investigating the use of genome-wide Affymetrix arrays to predict clinically important phenotypes. Early data looks highly promising with respect to identifying new diagnostic and therapeutic opportunities in breast cancer.

Our efforts in pancreatic cancer include both clinical and preclinical studies involving local physicians and collaborators at the University of California, San Francisco. While our collection of pancreatic tumors and associated specimens is ongoing in collaboration with Spectrum Health and Saint Mary’s hospitals, we have developed an excellent orthotopic xenograft murine model of pancreatic cancer. Immune-compromised mice implanted with a variety of human pancreatic cancer cell lines develop different extents of liver metastases that can be quantified using a variety of means, including recently established enhanced fluorescent imaging techniques.

The molecular correlates of metastatic propensity in these cell lines are currently being identified, and predicted therapeutic strategies are evaluated in both in vitro cell-based assays and our preclinical models.

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Our ongoing collaboration with the Proteomics Alliance for Cancer Research, which includes clinical and basic researchers from the University of Michigan, continues to generate exciting data relating to identifying biomarkers of lung cancer. In our lab, we are using a variety of RNAi technologies to determine the therapeutic potential of selected molecular targets (including SMAD2/3, Met, EGFR, and VEGF-A) in the progression of lung cancer, as well as identifying genomic and proteomic signatures associated with target disruption.

These targeting strategies are also being tested in an orthotopic murine model of non-small-cell lung carcinoma in which A549 cells are implanted in the pleural cavity of immune-compromised mice, resulting in bilateral lung disease and widespread metastasis. Proteomic markers of disease burden in these mice have been found in the serum, and using mass spectrometry techniques, the species origin (tumor or host) of these biomarkers can be identified. The majority of such markers appear to be derived from proteolytic cleavage of host tissue, rather than from secretion by the tumor cells. These findings are currently being validated using more traditional proteomic techniques.

Our continued collaboration with Harvey Pass at New York University remains focused on determining the molecular mechanisms of mesothelioma etiology and progression, as well as developing molecular-based early screening and diagnostic tests. For example, osteopontin was identified as a potential early detection biomarker in at-risk (asbestos-exposed) patient cohorts and was the topic of a recent New England Journal of Medicine article. We have developed a parallel preclinical xenograft model of mesothelioma for the evaluation of potential therapeutic agents, biomarkers of disease, and surrogate markers of therapeutic index.

Multiple myeloma 70

Some 40,000 Americans are living with multiple myeloma (MM), and deaths—which usually occur within three years of diagnosis—are over 11,000 per year. Treatment of this highly aggressive cancer is extremely limited. Without in-depth study into the molecular causes of multiple myeloma, near-term advances in diagnosis and treatment are unlikely. At the end of 2002, with generous support from VAI, the McCarty Foundation, and Ralph Hauenstein, as well as a postdoctoral fellowship from the Multiple Myeloma Research Foundation awarded to Jennifer Bromberg-White, we initiated the development of a multiple myeloma research initiative. We have also recently received an HRSA award to equip the myeloma laboratory. Our specific goal is to use our integrated approach to identify optimal treatments for myeloma patients.

Through collaboration with local physicians, we initiated a clinical protocol to collect bone marrow aspirates and/or core biopsies, blood, and urine from patients with multiple myeloma or the precursor condition, monoclonal gammopathy of undetermined significance. These clinical specimens are subjected to complete immunophenotyping in collaboration with Spectrum Health’s flow cytometry laboratory, and we perform molecular analysis on the purified myeloma cell populations. A panel of myeloma cell lines provided by Dr. Keith Stewart has provided us with a means to screen for sensitivity to the drugs currently used to treat MM, allowing us to rapidly identify potential molecular-based diagnostics that may predict likely response. These cell lines have also been used to develop an excellent murine model of MM in which mice exhibit the classic signs of the disease. Having successfully established the initiative, early research has focused on the role of CXCR4 in myeloma progression and drug resistance, and the preclinical models are now being used to test experimental treatment strategies identified through genomic analysis of myelomas from patients. Our XB-BIS informatics solution is being used to track each (mouse or human) subject’s response to administered therapies, and as the MM database expands over time, our predictive models will increase in validity. Ongoing discussions with the Karmanos Cancer Institute present us with a unique opportunity to expand our clinical specimen collection and to collaborate on potential clinical trials.

VARI | 2006

Integrated informatics XB-BIS (patent pending) is a fully integrated genomic/proteomic/medical informatics system that permits efficient data capture (e.g., from medical electronic records), management (e.g., sample tracking), analysis (visualization tools, statistical analysis, systems biology, literature mining) and reporting (e.g., predictive diagnostic reports). Data from various molecular experiments can be associated with specimens derived from subjects of interest, and comparative analysis can be performed on data across platforms and between species. Results can be automatically submitted to literature- and patent-mining tools, and fully integrated network analysis can be performed through our systems biology partnerships with Ingenuity and GeneGo. The accumulation of knowledge can be applied through the predictive modeling interface for developing diagnostics that incorporate trends within the data. XB-BIS also incorporates a drug-target database, linking results to potential clinical trials and agent databases. Collectively, the XB-BIS tool represents a fully integrated solution that we use for accelerating the translation of bimolecular research data into clinical diagnostic and intervention strategies. Information on XB-BIS licensing can be obtained from the VARI Business Development Office.

Community Initiatives As our research discoveries move closer to the point of clinical application, we continue working to increase the readiness of the community to offer advances in molecular medicine. To translate our discoveries into human benefit, we must work in highly coordinated, mutually beneficial partnerships with community institutions. The synergistic goals are to benefit human health and promote Grand Rapids as a leader in translational medicine. The community will be viewed as a leading destination for clinicians, researchers, industrial partners, and patients.

The three initiatives outlined below are in the final planning stages and are scheduled to launch in late 2006.

The Center for Molecular Medicine (CMM) â&#x20AC;&#x201C; A joint venture with Spectrum Health that will bring cutting-edge, molecularbased diagnostics tests to physicians and their patients. The program has also negotiated a service contract with Affymetrix that provides cost-effective genomic services to VARI investigators and academic/industry partners.

Innovative Clinical Research Alliance (ICRA) â&#x20AC;&#x201C; A multi-institutional alliance that will offer new biomarker-driven clinical trials to patients and physicians, and will be an obvious destination for pharmaceutical and biotech companies wishing to carry out clinical development.

Personalized Medicine Initiative â&#x20AC;&#x201C; With an initial focus in oncology, we will continue to develop the ability to predict optimal therapies in patients with cancer. This effort includes international collaborators taking part in the World Initiative for Personalized Medicine, as well as local adult and pediatric oncologists in conjunction with the Spectrum Health IRB and Research Department.

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External Collaborators James R. Baker and Gilbert Omenn, University of Michigan, Ann Arbor Lonson L. Barr, Annette Thelen, and Alan Davis, Michigan State University, East Lansing Marvin Baynes, Schering-Plough Julie Bryant and Andrej Bugrim, GeneGo, Inc., St. Joseph, Michigan Alan Campbell, Mark Campbell, and Timothy J. O’Rourke, Cancer & Hematology Centers of Western Michigan, P.C.,

Grand Rapids, Michigan Nadine Cohen, Johnson & Johnson Sandra L. Cottingham, Michael Dobbs, Anthony J. Foster, Pamela G. Kidd, John O’Donnell, Leo Oostendorp, Timothy J. Pelkey, Linda

C. Pool, Deborah Ritz-Holland, Marcy Ross, Michael Warzynski, and Marian Lange, Spectrum Health, Grand Rapids, Michigan Timothy Fitzgerald, Chad Stouffer, Susan Hoppough, and Michael Hoag, St. Mary’s Mercy Medical Center, Grand Rapids, Michigan Keith Johnson, Pfizer Donald G. Kim, Martin A. Luchtefeld, and Pamela Grady, Digestive Disease Institute, Grand Rapids, Michigan David E. Langholz, Richard F. McNamara, and David Wohns, West Michigan Heart, Grand Rapids, Michigan Eric P. Lester, Oncology Care Associates, P.L.L.C., St. Joseph, Michigan Francesco Marincola, Journal of Translational Medicine, Bethesda, Maryland Martin McMahon and Karen Kimura, University of California, San Francisco Deanna Mitchell, DeVos Children’s Hospital, Grand Rapids, Michigan Harvey I. Pass, New York University, New York Guenter Tusch, Grand Valley State University, Allendale, Michigan 72

Recent Publications

From left to right, standing: Webb, Miller, Ross, Monsma, Bromberg-White, Dobb, Eugster; seated: Welch, Srikanth

Ubels, John L., Holly M. Hoffman, Sujata Srikanth, James H. Resau, and Craig P. Webb. 2006. Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells. Investigative Opthalmology and Visual Science 47(5): 1876–1885.

Creighton, Chad J., Jennifer L. Bromberg-White, David E. Misek, David J. Monsma, Frank Brichory, Rork Kuick, Thomas J. Giordano, Weimin Gao, Gilbert S. Omenn, Craig P. Webb, and Samir M. Hanash. 2005. Analysis of tumor-host interactions by gene expression profiling of lung adenocarcinoma xenografts identifies genes involved in tumor formation. Molecular Cancer Research 3(3): 119–129.

Pass, Harvey I., Dan Lott, Fulvio Lonardo, Michael Harbut, Zhandong Liu, Naimei Tang, Michele Carbone, Craig Webb, and Anil Wali. 2005. Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. New England Journal of Medicine 353(15): 1564–1573.

VARI | 2006

Michael Weinreich, Ph.D. Laboratory of Chromosome Replication

Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsinâ&#x20AC;&#x201C;Madison in 1993. He then was a postdoctoral fellow in the laboratory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000.

Staff Laboratory Staff Dorine Savreux, Ph.D. FuJung Chang, M.S. Carrie Gabrielse, B.S. Vickie Harkins, B.S. Jeffrey Kasperski, B.S. Jessica Lanning, B.S.

Students Students Charles Miller, B.S. Amber Crampton Kate Leese Graham Roberts

Visiting Scientists

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Research Interests Our laboratory is interested in determining how chromosome replication occurs only once per cell cycle. Chromosome replication begins at specific DNA sequences termed replication origins. There are hundreds of replication origins in the budding yeast and perhaps 10,000 origins in human cells; those origins must be coordinately regulated to initiate DNA synthesis during the S-phase of each cell cycle. How is this cell cycle synchrony achieved and what are the molecular requirements for initiating DNA replication? The answer to both questions involves the regulated assembly of perhaps 30 polypeptides at replication origins during G1 phase as a prerequisite for DNA synthesis. We know that the multi-subunit origin recognition complex (ORC), which binds to DNA, determines origin position in all eukaryotes. However, the ORC is not sufficient for initiating DNA synthesis. After ORC binding, the Cdc6 protein (Cdc6p) binds to ORC and helps load the MCM DNA helicase at the origin. Both Cdc6p and the MCM complex are essential for initiating DNA replication. The MCM helicase is a multi-subunit complex that is thought to encircle the double-stranded DNA and is required to unwind the origin DNA to a single-stranded form. We are studying the role of Cdc6p during the initiation of DNA synthesis and have found that its activity is influenced by chromatin structure.

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Because Cdc6p is required for DNA replication, it is essential for cell survival. We used its essential character to select for genetic suppressors of a cdc6-4 temperature-sensitive mutant that could not grow at 37 °C. We recovered mutations disrupting a non-essential (but conserved) protein called Sir2p; in other words, a loss of Sir2p activity allowed the cdc6-4 cells to grow at 37 °C. Sir2p belongs to a class of enzymes called histone deacetylases and as such is a chromatin-modifying enzyme. We further found that the loss of Sir2p allowed Cdc6p to load the MCM helicase at some origins. We have exploited this origin specificity to systematically screen the budding yeast chromosome III to determine those origins that are sensitive to Sir2p. We found that Sir2p inhibited the activity at 4 of 13 chromosome III origins when Cdc6p activity was compromised. An examination of several of these origins in detail showed that they share a common organization. All origins in Saccharomyces cerevisiae described to date contain a binding site for ORC composed of the so-called A and B1 elements (Fig. 1). Origins also generally contain a preferred binding site for the MCM helicase termed the B2 element. We have found a novel inhibitory sequence, which we have termed the Is element, located downstream of B2 that is responsible for Sir2p inhibition at these origins. We are now studying how this element functions on the chromosome and are determining its molecular role during the process of initiation. Based on our genetic findings, we propose that this element acts to inhibit MCM helicase loading during G1.

Figure 1.

Figure 1. Structure of several yeast replication origins. The structure of ARS1 is as reported by Mararhens and Stillman. Structures of the ARS305 and ARS315 origins were determined by a linker scan analysis. “ACS” denotes the ARS core consensus sequence; the approximate positions of the ORC and MCM binding sites are also indicated. IS is the inhibitory element defined in our study.

VARI | 2006

A second project in the lab is to investigate the biology of Cdc7p-Dbf4p kinase. Cdc7p-Dbf4p is a two-subunit serine/threonine kinase required for the onset of DNA synthesis. Cdc7p is the kinase subunit and Dbf4p is a regulatory subunit that is absolutely required for Cdc7p kinase activity. We undertook a structure-function analysis to determine the residues in the Dbf4p N-terminus required for its essential role in DNA replication. We found that the first 265 residues of Dbf4p (out of 704 residues) are dispensable for its essential replication function other than to provide nuclear localization signals. Within these 265 amino acids, we identified a 100-amino-acid region of similarity among all Dbf4 orthologs that includes a previously described 40-amino-acid “motif N”, which resembles the N-terminus of the BRCT motif. BRCT motifs encode domains of about 100 amino acids and are found primarily in proteins that function in the DNA damage response. Although the expanded region of homology that we have uncovered does not encode a bona fide BRCT motif, it appears to encode a variant of that motif unique to Dbf4 proteins (Fig. 2). We have therefore called this the BRDF motif, for BRCT and Dbf4p similarity. Importantly, we found that yeast mutants altering this motif are sensitive to replication fork arrest but not to methylmethane sulfonate (MMS)–induced DNA damage or double-stranded DNA breaks caused by the cancer therapeutic bleomycin (Fig. 3).

It appears, therefore, that Dbf4p has a role in maintaining replication fork stability that is separable from that of promoting the initiation of DNA replication. Exactly how this is occurring is the subject of active investigation in the lab. We know that Rad53p phosphorylates Dbf4p—probably directly—in response to replication fork arrest. Rad53p (human Chk2) is an essential kinase required to coordinate repair of DNA damage or of replication fork stalling during the S-phase. We are beginning to understand the molecular role of the BRDF motif and how it might influence Cdc7p-Dbf4p phosphorylation of proteins at stalled replication forks.

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Figure 2A.

Figure 2B.

Figure 2. A single BRCT-like motif in Dbf4p. A) An alignment of Dbf4 orthologs compared with the BRCT consensus sequence for regions I–III. In the row labels at the left, the species abbreviations used are Hs (Homo sapiens), Mm (Mus musculus), Xl (Xenopus laevis), Dm (Drosophila melanogaster), An (Aspergillus nidulans), Sp (Schizosaccharomyces pombe), and Sc (Saccharomyces cerevisiae). The alignment across region IVa revealed a non-BRCT consensus sequence. Identical Dbf4 residues are in red (with K = R and E = D) and similar residues are blue. In the consensus line, “h” indicates a preferred hydrophobic residue, “p” a polar residue, and a capital letter, a preferred amino acid. Open arrows indicate the endpoints of viable N-terminal deletions; solid arrows, nonviable deletions (from Fig. 3A). The gray bars indicate the boundaries of motif N for S. cerevisiae and above that, the other orthologs. Above regions I–III, the secondary structural elements from the first Brca1 BRCT repeat (shown in B) are indicated. Above region IVa, a secondary structure prediction is indicated for the Hs, Mm, and Xl Dbf4 proteins.

B) A ribbon diagram representing the Brca1 BRCT repeat crystal structure using PDB coordinate ID 1JNX. Secondary structural elements are labeled for the first repeat.

Van Andel Research Institute |

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Lastly, we have found that human Cdc7 protein is up-regulated in many cancer cell lines and that knockdown of CDC7 using RNAi results in an apoptotic response in some cancer cell lines but not in normal cells. We have further determined that Cdc7 protein is highly expressed in some primary tumors. Since Cdc7 is an essential kinase required for DNA replication and apparently plays a role in other aspects of chromosome metabolism, we think that these findings have significance for understanding the biology of certain types of tumor cells.

Figure 3.

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Figure 3. The BRDF motif is required for response to DNA-damaging agents. A) pRS415-DBF4 plasmids containing the indicated deletions were transformed into M895 (dbf4∆::kanMX6) and tested for dbf4 complementation by streaking onto FOA plates at 25 °C. Motifs N, M, and C are numbered according to Masai and Arai. B) For representative deletions in A, tenfold serial dilutions of saturated cultures were spotted onto YPD plates at the indicated temperatures or YPD plates containing 0.01% MMS, 2 μg/ml bleomycin, or 0.1 M hydroxyurea at 25 °C and were scored after three days.

Recent Publications

From left: Weinreich, Harkins, Leese, Chang, Miller, Roberts, Savreux, Gabrielse

Gabrielse, C., C.T. Miller, K.H. McConnell, A. DeWard, C.A. Fox, and M. Weinreich. In press. A Dbf4p BRCT-like domain required for the response to replication fork arrest in budding yeast. Genetics.

VARI | 2006

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

Dr. Williams received his Ph.D. in biology from Massachusetts Institute of Technology in 1996. For three years, he was a postdoctoral fellow at the National Institutes of Health in the laboratory of Harold Varmus, former Director of NIH. Dr. Williams joined VARI as a Scientific Investigator in July 1999.

Staff Laboratory Staff

Students

Charlotta Lindvall-Weinreich, M.D., Ph.D. Dan Robinson, Ph.D. Katia Bruxvoort, B.S. Nicole Evans, B.S. Cassandra Zylstra, B.S.

Student Visiting Scientists Sarah Mange

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Research Interests Our laboratory is interested in understanding how alterations in the Wnt signaling pathway cause human disease. Wnt signaling is an evolutionarily conserved process that has been adapted to function in the differentiation of most tissues within the body. Given its central role in growth and differentiation, it is not surprising that alterations in the pathway are among the most common events associated with human cancer. In addition, several other human diseases, including osteoporosis, have been linked to alterations in the regulation of this pathway. One of the long-term goals of our laboratory is to understand how specificity is generated for the different Wnt signaling pathway components, with a specific focus on understanding the molecular functions of Lrp5 and Lrp6.

Recently, my laboratory has become focused on understanding the role of Wnt signaling in bone formation. We are interested not only from the perspective of normal bone development, but also in trying to understand whether aberrant Wnt signaling plays any role in the predisposition of some common tumor types (for example prostate, breast, lung, and renal tumors) to metastasize to and grow in bone.

The long-term goal of this work is to provide insights that could be used to develop strategies to lessen the morbidity and mortality associated with skeletal metastasis.

Wnt signaling in normal bone development

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Mutations in the Wnt receptor, Lrp5, have been causally linked to alterations in human bone development. We have characterized a mouse strain deficient for Lrp5 and shown that it recapitulates the low-bone-density phenotype seen in human patients deficient for Lrp5. We have furthered this study by showing that mice carrying mutations in both Lrp5 and the related Lrp6 protein have even more severe defects in bone density.

We also tested whether Lrp5 deficiency causes changes in bone density due to aberrant signaling through ß-catenin. To do this, we created mice carrying an osteoblast-specific deletion of ß-catenin (OC-cre;ß-catenin-flox/flox mice). These mice die within five weeks of birth due to profound deficiencies in bone development. In collaboration with Tom Clemens of the University of Alabama at Birmingham, we found that alterations in Wnt/ß-catenin signaling in osteoblasts lead to changes in the expression of RANKL and osteoprotegerin (OPG). Consistent with this, histomorphometric evaluation of bone in the mice with osteoblast-specific deletions of either Apc or ßcatenin revealed significant alterations in osteoclastogenesis.

VARI | 2006

We are currently pursuing work aimed at addressing how other genetic alterations linked to Wnt/ß-catenin signaling affect bone development and osteoblast function. We are particularly interested in understanding why Lrp5-deficient mice, while showing low bone mass, live a normal lifespan, while mice carrying an osteoblast-specific deletion of the ß-catenin gene fail to live beyond four weeks of age.

We are pursuing several lines of experiments aimed at addressing this question. First, we are generating mice with a conditional allele of Lrp6 that can be inactivated via cre-mediated recombination. We will use these mice to assess the role of Lrp6 in terminal osteoblast differentiation as well as to generate mice lacking both Lrp5 and Lrp6 in osteoblasts. A second possible explanation for the difference in severity between the phenotypes of Lrp5-deficient and OC-cre;ß-catenin-flox/flox mice is that the latter may have disruptions in cadherin-mediated cell adhesion. To address this possibility, we have generated mice carrying a conditional deletion of ß-catenin in osteoblasts, and we are currently examining their phenotype. Finally, we have initiated experiments aimed at determining what other signaling pathways in osteoblasts may impinge on ß-catenin signaling to control osteoblast differentiation and function. For example, we have created mice carrying an osteoblast-specific deletion of Pten in order to activate Akt signaling; these mice display a progressive increase in bone mass throughout life (Fig. 1). We are currently determining the molecular mechanisms underlying these observations and are examining whether the activation of Akt can increase bone mass in mice with alterations in the Wnt signaling pathway.

Figure 1.

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Control

∆Pten

Figure 1. Conditional deletion of Pten in osteoblasts leads to increased bone mass. Representative microCT analysis of femurs isolated from a 12-month-old OC-cre;Pten-flox/flox mouse (∆pten) and a control littermate.

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Wnt signaling in prostate development and cancer Activation of the Wnt signaling pathway occurs in a significant percentage of prostate carcinomas. In some cases, this is associated with activating mutations in the Ă&#x;-catenin genes, while in other cases a loss of APC has been demonstrated. Two hallmarks of advanced prostate cancer are the development of skeletal osteoblastic metastasis and the ability of the tumor cells to become independent of androgen for survival. The association of Wnt signaling with bone growth, plus the fact that Ă&#x;-catenin can bind to the androgen receptor and make it more susceptible to activation with steroid hormones other than DHT, make Wnt signaling an attractive candidate for explaining some phenotypes associated with advanced prostate cancer.

We have created mice with a prostate-specific deletion of the Apc gene. These mice develop fully penetrant prostate hyperplasia by four months of age, and these tumors progress to frank carcinomas by seven months. We have found that these tumors initially regress under androgen ablation but show signs of androgen-independent growth some months later.

Wnt signaling in mammary development and cancer

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We are also addressing the relative roles of Lrp6 and Lrp5 in Wnt1-induced mammary carcinogenesis. We have found that deficiency in Lrp5 dramatically inhibits the development of mammary tumors in this context (Fig. 2). Further work has focused on examining the effects of Lrp5 and/or Lrp6 deficiency on normal mammary development. We have found that germline deficiency for either gene results in delayed mammary development. As Lrp5-deficient mice are viable and fertile, we have focused our initial efforts on these mice. In collaboration with Caroline Alexanderâ&#x20AC;&#x2122;s laboratory, we have found dramatic reductions in the number of mammary progenitor cells in these mice. We are currently examining the mechanisms underlying this reduction.

Figure 2.

Figure 2. Lrp5 deficiency delays tumor onset in MMTV-Wnt1 females. Twenty to twenty-five female mice of each Lrp5 genotype were aged and sacrificed when tumors became palpable. Tumor-free survival is plotted as a function of age.

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VARI mutant mouse repository With partial support from the Michigan Animal Models Consortium, my laboratory maintains a repository of mutant mouse strains to support the general development of animal models of human disease. We distribute these strains at a nominal cost to interested laboratories.

External Collaborators Bone development Thomas Clemens, University of Alabama-Birmingham Matthew Warman, Case Western Reserve University, Cleveland, Ohio Mary Bouxsein, Beth Israel Deaconness Medical Center, Boston Marie Claude Faugere, University of Kentucky, Lexington Prostate cancer Wade Bushman and Ruth Sullivan, University of Wisconsin–Madison Mammary development Caroline Alexander, University of Wisconsin–Madison Yi Li, Baylor Breast Center, Houston Jeffrey Rubin, National Cancer Institute, Bethesda, Maryland Mechanisms of Wnt signaling Silvio Gutkind, National Institute of Dental and Craniofacial Research, Bethesda, Maryland Kun-Liang Guan, University of Michigan, Ann Arbor Kang-Yell Choi, Yansei University, Seoul, South Korea

Recent Publications

From left: Evans, Lindvall-Weinreich, Williams, Zylstra, Bruxvoort

Park, Ki-Sook, Soung Hoo Jeon, Sung-Eun Kim, Young-Yil Bahk, Sheri L. Holmen, Bart O. Williams, Kwang-Chul Chung, Young-Joon Surh, and Kang-Yell Choi. 2006. APC inhibits ERK pathway activation and cellular proliferation induced by Ras. Journal of Cell Science 119(5): 819–827.

Wang, PengFei, Dong Kong, Matthew W. VanBrocklin, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams, and Sheri L. Holmen. 2006. Simplified method for the construction of gene targeting vectors for conditional gene inactivation in mice. Transgenics 4: 215–228.

Castellone, Maria Domenica, Hidemi Teramoto, Bart O. Williams, Kirk M. Druey, and J. Silvio Gutkind. 2005. Prostaglandin E2 promotes colon cancer cell growth through a GS-axin-ß-catenin signaling axis. Science 310(5753): 1504–1510.

Holmen, Sheri L., and Bart O. Williams. 2005. Essential role for Ras signaling in glioblastoma maintenance. Cancer Research 65(18): 8250–8255.

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Photo taken by Nian Zhang and James Resau.

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Scrib1 neural tube defect.

This photograph shows a cross section of a day-8.5 embryo (E8.5) homozygous for the Scribble1 (Scrib1) mutation, which is characterized by an open neural tube that forms the two curving sides of the “V” in the photo. Normally the columns of cells forming the V would be parallel and connected at their upper ends. This phenotype represents the most severe form of neural tube defect. The section was stained for β-catenin (green), α-catenin (red), and cell nuclei (blue).

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

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Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl Pabo at MIT, he moved to GlaxoWellcome in 1996 as a research investigator of nuclear receptor drug discovery. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002.

Staff Laboratory Staff Schoen Kruse, Ph.D. Yong Li, Ph.D. Augie Piozak, Ph.D. David Tolbert, Ph.D. Yong Xu, Ph.D.

Students Chenghai Zhang, Ph.D. X. Edward Zhou, Ph.D. Jennifer Daughtery, B.S. Amanda Kovach, B.S. Kelly Suino, B.S.

Visiting Scientist Student Visiting Scientists Ross Reynolds, Ph.D.

Jeffery Lai

VARI | 2006

Research Interests Our laboratory is using modern molecular and biophysical techniques to study the structures and functions of key protein complexes that are important in terms of basic biology, as well as for drug discovery relevant to human diseases such as cancer and diabetes.

Currently we are focusing on nuclear hormone receptors and the Met tyrosine kinase receptor.

Nuclear hormone receptors Nuclear hormone receptors are a large family of ligand-regulated and DNA-binding transcriptional factors, which include receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well as receptors for proxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. One distinguishing fact about these classic receptors is that they are among the most successful targets in the history of drug discovery. Every receptor has one or more cognate synthetic ligands currently being used as medicines. Nuclear receptors also include a class of “orphan” receptors for which no ligand has been identified. In the last two years, we have developed the following projects centering on the structural biology of nuclear receptors.

Peroxisome proliferator–activated receptors

The peroxisome proliferator–activated receptors (PPARα, δ, and γ) are the key regulators of glucose and fatty acid homeostasis and as such are important therapeutic targets for treating cardiovascular disease, diabetes, and cancer. Millions of patients have benefited from treatment with the novel PPARγ ligands rosiglitazone and pioglitazone for type II diabetes. To understand the molecular basis of ligand-mediated signaling by PPARs, we have determined crystal structures of each PPAR’s ligand-binding domain (LBD) bound to many diverse ligands, including fatty acids, the lipid-lowering fibrate drugs, and a new generation of anti-diabetic drugs, the glitazones.

We have also determined the crystal structures of these receptors bound to coactivators or co-repressors. These structures have provided a framework for understanding the mechanisms of agonists and antagonists, as well as the recruitment of coactivators and co-repressors in gene activation and repression. Furthermore, these structures also serve as a molecular basis for understanding the potency, selectivity, and binding modes of diverse ligands, which have provided critical insights for designing the next generation of PPAR medicines. Currently we are developing this project beyond the structures of the ligand-binding domains into the structures of larger PPAR fragments and DNA complexes.

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The human glucocorticoid and mineralocorticoid receptors

The human glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are classic steroid hormone receptors that are crucial for a wide spectrum of human physiology including immune/inflammatory responses, metabolic homeostasis, and control of blood pressure. Importantly, both GR and MR are well-established drug targets. GR ligands such as dexamethasone (Dex) and fluticasone propionate (FP) are used to treat asthma, leukemia, and autoimmune diseases; MR ligands such as spironolactone and eplerenone are used to treat hypertension and heart failure. However, the clinical use of these ligands is limited by undesirable side effects partly associated with their receptor cross-reactivity or low potency. Thus, the discovery of highly potent and more-selective ligands for these receptors remains an intensive goal of pharmaceutical research.

We have determined a crystal structure of the GR LBD bound to dexamethasone and of the MR LBD bound to corticosterone, both of which are in complex with a coactivator peptide motif (Fig. 1). These structures provide a detailed basis for the specificity of hormone recognition and coactivator assembly by GR and MR. Currently we are studying receptor-ligand interactions by crystallizing GR and MR with various steroid or nonsteroid ligands. The detailed information from these structures should provide a rational basis for designing new ligands that would reduce the side effects of current GR and MR drugs. In collaboration with Brad Thompson and Raj Kumar at the University of Texas Medical Branch at Galveston, we are also extending our studies to the structure of a large GR fragment bound to DNA.

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Figure 1.

Figure 1. Crystal Structures of the GR and MR LBDs. The GR and MR are shown in blue and red, respectively, with the coactivator motif shown in yellow. The bound hormones are shown in a space-filling representation, with carbon, oxygen, nitrogen, and phosphate depicted as green, red, blue, and purple, respectively. .

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The human androgen receptor

The androgen receptor (AR) is the central molecule in the development and progression of prostate cancer, and as such it serves as the molecular target of anti-androgen therapy. However, the majority of prostate cancer patients develop resistance to anti-androgen therapy, mostly due to mutations in this hormone receptor. These mutations alter the three-dimensional structure of the receptor and allow AR to escape the repression of anti-androgen treatment. The growth of prostate cancer cells that harbor a mutated AR is then no longer dependent on androgen, making anti-hormone therapy ineffective. This form of hormone-independent prostate cancer is highly aggressive and is responsible for most deaths from prostate cancer. Currently there is no cure for advanced, hormone-independent prostate cancer. It is estimated that more than one million men now over age 50 will die from prostate cancer unless new therapies are developed. The development of effective therapies requires a detailed understanding of the structure and functions of the central molecule, i.e., the androgen receptor, and its interactions with hormones and co-regulators. In this project, we are aiming to determine the structures of mutated AR proteins that alter the response of anti-hormone therapy. In collaboration with Donald MacDonnell at Duke University, we are working on the crystal structure of the full-length AR/DNA complex.

Structural genomics of nuclear receptor ligand-binding domains

The LBD of nuclear receptors contains key structural elements that mediate ligand-dependent regulation of nuclear receptors and as such has been the focus of intense structural study. Crystal structures for more than half of the 48 human nuclear receptors have been determined. These structures have illustrated the details of ligand binding, the conformational changes induced by agonists and antagonists, the basis of dimerization, and the mechanism of coactivator and co-repressor binding. The structures also provide many surprises regarding the identity of ligands, the size and shape of the ligand-binding pockets, and the structural implications of the receptor signaling pathways. There are only a few orphan nuclear receptors for which the LBD structure remains unsolved. In the past two years, we have focused on structural characterization of two orphan receptors: constitutive androstane receptor (CAR) and steroidogenic factor-1 (SF-1). The CAR structure reveals a compact LBD fold which contains a small pocket that is only half the size of the pocket in PXR, a receptor closely related to CAR. The constitutive activity of CAR appears to be mediated by a novel linker helix between the C-terminal AF-2 helix and helix 10. On the other hand, SF-1 is regarded as a ligand-independent receptor, but its LBD structure reveals the presence of a phospholipid ligand in a surprisingly large pocket; its size is more than twice that of the pocket in the mouse LRH-1, a receptor closely related to SF-1. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and reduce SF-1 transcriptional activity. These findings establish that SF-1 is a ligand-dependent receptor and suggest an unexpected link between nuclear receptor and phospholipid signaling pathways. We can expect more surprises as structural work continues on the remaining orphan receptors.

The Met tyrosine kinase receptor The MET receptor is a tyrosine kinase that is activated by hepatocyte growth factor/scatter factor (HGF/SF). Aberrant activation of the Met receptor has been linked with development and metastasis of many types of solid tumors and has been correlated with poor clinical prognosis. HGF/SF has a modular structure with an N-terminal domain, four kringle domains, and an inactive serine protease domain.

The structure of the N-terminal domain with a single kringle domain (NK1) has been determined. Less is known about the structure of the Met extracellular domain. The molecular basis of the MET receptor-HGF/SF interaction and the activation of MET signaling by this interaction remain poorly understood. In collaboration with George Vande Woude and Ermanno Gherardi, we are developing this project to solve the crystal structure of the Met receptor/HGF complex.

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External Collaborators Doug Engel, University of Michigan, Ann Arbor Ermanno Gherardi, University of Cambridge, England Steve Kliewer and David Mangelsdorf, University of Texas Southwestern Medical Center, Dallas Donald MacDonnell, Duke University, Durham, North Carolina Stoney Simmons, National Institutes of Health, Bethesda, Maryland Scott Thacher, Orphagen Pharmaceuticals, San Diego, California Brad Thompson and Raj Kumar, University of Texas Medical Branch at Galveston Ming-Jer Tsai, Baylor College of Medicine, Houston, Texas

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Recent Publications

From left to right, back row: Daugherty, Suino, Lai; middle: Zhou, Li, Tolbert, Kovach, Reynolds, Xu; front: Zhang, Piozak, Kruse

Motola, Daniel L., Carolyn L. Cummins, Veerle Rottiers, Kamalesh K. Sharma, Tingting Ti, Yong Li, Kelly Suino-Powell, H. Eric Xu, Richard J. Auchus, Adam Antebi, and David J. Mangelsdorf. 2006. Identification of hormonal ligands for the orphan nuclear receptor DAF-12 that govern dauer formation and reproduction in C. elegans. Cell 124(6): 1209–1223.

Li, Yong, Mihwa Choi, Kelly Suino, Amanda Kovach, Jennifer Daugherty, Steven A. Kliewer, and H. Eric Xu. 2005. Structural and biochemical basis for selective repression of the orphan nuclear receptor liver receptor homolog 1 by small heterodimer partner.

Proceedings of the National Academy of Sciences U.S.A. 102(27): 9505–9510.

Li, Yong, Kelly Suino, Jennifer Daugherty, and H. Eric Xu. 2005. Structural and biochemical mechanisms for the specificity of hormone binding and coactivator assembly by mineralocorticoid receptor. Molecular Cell 19(3): 367–380.

VARI | 2006

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

Dr. Zhang received his M.S. in entomology from Southwest Agricultural University, Peopleâ&#x20AC;&#x2122;s Republic of China, in 1985 and his Ph.D. in molecular biology from the University of Edinburgh, Scotland, in 1992. From 1992 to 1996, he was a postdoctoral fellow at the Roche Institute of Molecular Biology. He next served as a postdoctoral fellow (1996) and a Research Associate (1997â&#x20AC;&#x201C;1999) in the laboratory of Tom Gridley in mammalian developmental genetics at the Jackson Laboratory, Bar Harbor, Maine. Dr. Zhang joined VARI as a Scientific Investigator in December 1999.

Staff

Student

Wei Ma, Ph.D. Lanlan Yin, Ph.D. Lisheng Zhang, Ph.D. Kate Groh, B.S. Liang Kang

William Bond

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Research Interests Germ cell development One focus of our laboratory is on germ cell development, particularly the mechanisms that govern germ cell migration, survival, and meiosis, as well as their implications for human disease. We are studying spontaneous mutations that cause sterility. In mice, primordial germ cells (PGCs) are differentiated from the epiblasts during an early embryonic stage. After their formation, they migrate through the dorsal mesentery and enter the genital ridge, where they collaborate with the somatic gonad cells to form the gonads. The PGCs proliferate during their migration. We are studying the spontaneous mutation atrichosis (at), which causes male and female sterility.

Preliminary data suggest that this mutation affects fetal germ cell proliferation. We have found that by 10.5 dpc, there already are significantly fewer germ cells in the gonads of atrichosis embryos relative to the wild type. We have mapped the atrichosis mutation to a 270-kb region on chromosome 10. We are now screening the candidate genes by transgenic rescue and direct sequencing.

Another mutation we are studying is in the â&#x20AC;&#x153;skeletal fusion and sterileâ&#x20AC;? (sks) gene; this mutation affects meiosis in both sexes. Our results indicate that sks is required for the metaphase/anaphase transition at meiosis I. In the sks mutant, homologous chromosomes fail to separate; therefore, meiosis is stopped at metaphase I. We have demonstrated that this failure is due to the inability of sks mutants to degrade Securin in the primary spermatocytes and possibly in the oocytes.

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The second focus of the lab is to investigate the role of the Scribble1 (Scrib1) gene, a mouse homologue of the Drosophila scribble (scrib) gene in tumor metastasis and neuronal development. In Drosophila, scrib acts as tumor suppresser gene, the loss of which disrupts epithelial structures in a way reminiscent of neoplasia in mammals. In combination with the oncogene Ras, mutation of Scrib1 leads to the metastasis of solid tumors in flies. The mouse Scrib1 gene encodes a LAP4 protein that contains a leucine-rich domain required for its membrane association in epithelial cells and neurons, as well as four PDZ domains that are likely to act as a scaffold to integrate the actions of other signal molecules on the membrane. By conditional deletion of the Scrib1 gene, we have determined the critical time of action of this gene during neural tube development. Mechanistically, we have found that Scrib1 is required for membrane remodeling during the convergent extension of the neural plate. Our goal is to recapitulate tumor metastasis in mice by creating a conditional knock-out mouse for Scrib1, and at the same time to conditionally activate Ras.

Recent Publications

From left: Kang, N. Zhang, Ma, L. Zhang, Bond, Groh

Chen, J., Liang Kang, and Nian Zhang. In press. A negative feedback loop formed by lunatic fringe and Hes7 controls their oscillatory expression during somitogenesis. Genesis.

Wang, PengFei, Dong Kong, Matthew W. VanBrocklin, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams, and Sheri L. Holmen. 2006. Simplified method for the construction of gene targeting vectors for conditional gene inactivation in mice. Transgenics 4: 215â&#x20AC;&#x201C;228.

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Daniel Nathans Memorial Award

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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 a founding member of VARI’s Board of Scientific Advisors. We established this award to recognize individuals who emulate Dan and his contributions to biomedical and cancer research. It is our way of thanking and honoring him for his help and guidance in bringing Jay and Betty Van Andel’s dream to reality. The Daniel Nathans Memorial Award was announced at our inaugural symposium, “Cancer & Molecular Genetics in the Twenty-First Century,” in September 2000.

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2005 Award to Tony Hunter, Ph.D., and Tony Pawson, Ph.D. The 2005 Daniel Nathans Memorial Award is jointly awarded to Tony Hunter, Ph.D., of the Salk Institute for Biological Studies, San Diego, and to Tony Pawson, Ph.D., of Mount Sinai Hospital Research Institute, Toronto. Dr. Hunter, whose lab identified the tyrosine phosphorylation of proteins, continues to work on how cells regulate their growth and division and how gene mutations lead to cancer.

Dr. Pawson, whose work uncovered the Src homology 2 (SH2) domain that binds to phosphotyrosines, has his lab focused on the protein-protein interactions underlying signal transduction and on the molecular signals involved in axon guidance.

Previous Award Recipients 2000 2001 2002 2003 2004

Richard D. Klausner, M.D. Francis S. Collins, M.D., Ph.D. Lawrence H. Einhorn, M.D. Robert A. Weinberg, Ph.D. Brian Druker, M.D.

Dr. Brian Druker giving his Award lecture in September 2005.

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Postdoctoral Fellowship Program

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Postdoctoral Fellowship Program The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientists beginning their research careers. The fellowships help promising scientists advance their knowledge and research experience while at the same time supporting the research endeavors of VARI. The fellowships are funded in three ways: 1) by the laboratories to which the fellow is assigned; 2) by the VARI Office of the Director; or 3) by outside agencies. Each fellow is assigned to a scientific investigator who oversees the progress and direction of research. Fellows who worked in VARI laboratories in 2005 and early 2006 are listed below.

Yair Andegeko

Holly Holman

Jun Sugimura

Tel Aviv University, Israel VARI mentor: James Resau

University of Glasgow, U.K. VARI mentor: Arthur Alberts

University of Morioka Medical School, Japan VARI mentor: Bin Teh

Jennifer Bromberg-White

Dan Huang

Min-Han Tan

Pennsylvania State University College of Medicine, Hershey VARI mentor: Craig Webb

Peking Union Medical College, China VARI mentor: Bin Teh

Royal College of Physicians, U.K. VARI mentor: Bin Teh

Schoen Kruse

Peng Fei Wang

University of Colorado, Boulder VARI mentor: Eric Xu

Fourth Military Medical University, China VARI mentor: Bin Teh

Douglas Luccio-Camelo

Todd Whitwam

University of Brazil, Rio de Janeiro VARI mentor: Bin Teh

Mayo Clinic College of Medicine, Rochester, Minnesota VARI mentor: Sheri Holmen

Yun-Ju Chen University of Glasgow, U.K. VARI mentor: Arthur Alberts

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Philippe Depeille University of Montpellier, France VARI mentor: Nicholas Duesbery

Yan Ding Peking Union Medical College, China VARI mentor: Nicholas Duesbery

Mathew Edick University of Tennessee, Memphis VARI mentor: Cindy Miranti

Kathryn Eisenmann University of Minnesota, Minneapolis

Wei Ma Chinese Academy of Science, Beijing VARI mentor: Nian Zhang

Daisuke Matsuda Kitasato University, Japan VARI mentor: Bin Teh

Augen Pioszak University of Michigan, Ann Arbor VARI mentor: Eric Xu

VARI mentor: Arthur Alberts

Leslie Farber George Washington University, Washington, D.C. VARI mentor: Bin Teh

Kunihiko Futami Tokyo University of Fisheries, Japan VARI mentor: Bin Teh

Chongfeng Gao Tokyo Medical and Dental University, Japan VARI mentor: George Vande Woude

Carrie Graveel University of Wisconsin â&#x20AC;&#x201C; Madison VARI mentor: George Vande Woude

Dorine Savreux Virology University, France VARI mentor: Michael Weinreich

Michael Shafer Michigan State University, East Lansing VARI mentor: Brian Haab

Paul Spilotro St. George University, Grenada VARI mentor: Nicholas Duesbery

Suganthi Sridhar Southern Illinois University, Carbondale VARI mentor: Cindy Miranti

Qian Xie Fudan University, Shanghai, China VARI mentor: George Vande Woude

Xin Yao Tianjin Medical University, China VARI mentor: Bin Teh

Lanlan Yin Nanjing University, China VARI mentor: Nian Zhang

Chenghai Zhang Virus Institute of the CDC, China VARI mentor: Eric Xu

Chun Zhang Tokyo Medical and Dental University, Japan VARI mentor: Bin Teh

Lisheng Zhang Chinese Academy of Science, Beijing VARI mentor: Nian Zhang

Xiaoyin Zhou University of Alabama â&#x20AC;&#x201C; Birmingham VARI mentor: Eric Xu

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Student Programs

VARI | 2006

Grand Rapids Area Pre-College Engineering Program The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered by Davenport University and jointly sponsored and funded by Pfizer, Inc., and VARI. The program is designed to provide selected high school students, who have plans to major in science or genetic engineering in college, the opportunity to work in a research laboratory. In addition to research methods, the students also learn workplace success skills such as teamwork and leadership. VARI hosted three GRAPCEP students in 2005.

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From left: Fairchild, Banks, Warlick

Courtney Banks (Resau/Duesbery) New Century Montessori School

Anna Fairchild (Resau/Duesbery) New Century Montessori School

Sarah Warlick (Holmen) New Century Montessori School

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Summer Student Internship Program The VARI student internships were established to provide college students with an opportunity to work with professional researchers in their fields of interest, to use state-of-the-art equipment and technologies, and to learn valuable people and presentation skills. At the completion of the 10-week program, the students summarize their projects in an oral presentation.

From January 2005 to March 2006, VARI hosted 45 students from 20 colleges and universities in formal summer internships under the Frederik and Lena Meijer Student Internship Program and in other student positions during the year. An asterisk (*) indicates a Meijer student intern.

Calvin College, Grand Rapids, Michigan

Northern Illinois University, Dekalb

Nicholas Be* (Weinreich) Rachel Cichowski* (Williams) Bill Wondergem* (Teh)

Franciscan University, Steubenville, Ohio

Joan Krilich* (Cavey)

Grand Valley State University, Allendale, Michigan 98

Erik Freiter (Miranti) Nick Miltgen* (Resau) Lisa Orcasitas (Duesbery) Rebecca Roe (Resau) Katie Sian (Miranti) Adam Werts* (Vande Woude)

Hope College, Holland, Michigan

Marie Graves (Cavey) Wendy Johnson (Cavey)

Tel Aviv University, Israel

Patrick Condit (Teh)

Grant Van Eerden* (Resau)

Michigan State University, East Lansing

Aaron DeWard, B.S. (Alberts) Michelle Gilmer* (Xu) Chia-Shia Lee, M.S. (Duesbery) Yaojian Liu, B.S. (Alberts) Charles Miller, B.S. (Weinreich) Lan Tong* (Furge)

Michigan Technological University, Houghton

Hien Dang (Resau)

Nanjing Medical University, China

Xin Wang (Cao) Jin Zhu (Cao)

National Institute for Communicable Disease Control and Prevention, Beijing

Qin Hao (Cao)

Stefan Kutscheidt (Miranti)

University of Bath, United Kingdom

Alex Camenzuli (Resau) Katharine Collins (Alberts) Amber Crampton (Weinreich) Amy Percival (Reasu) Zafar Qadir (Duesbery)

University of Chicago, Illinois Jonathan Douglas (Duesbery)

University of Illinois, Champaign-Urbana

Miami University, Oxford, Ohio

Yair Andegeko (Resau) Eddy Solomon (Resau)

University of Applied Sciences, Mannheim, Germany

Marquette University, Milwaukee

Devrim Bilgili (Resau)

Huong Tran (Resau)

University of Michigan, Ann Arbor

Stephanie Berry* (Williams) Shirley Cohen* (Vande Woude) Myra Epp (Webb) Jennifer Lunger* (Haab) John Whang (Vande Woude)

University of North Carolina, Chapel Hill

Jourdan Stuart (Resau)

University of Notre Dame, South Bend, Indiana

Kristin Buzzitta* (Teh) Joseph Church* (Duesbery)

Western Michigan University, Kalamazoo

Ken Olinger (Resau) Nicole Repair* (Miranti)

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Han-Mo Koo Memorial Seminar Series

VARI | 2006

Han-Mo Koo Memorial Seminar Series This seminar series is dedicated to the memory of Dr. Han-Mo Koo, who was a VARI Scientific Investigator from 1999 until his passing in May of 2004.

January 2005

Ali Shilatifard, St. Louis University

“A COMPASS and a GPS in defining molecular machinery in histone modifications, transcriptional regulation, and human cancer: the coordinates of the genome”

February

Hsueh-Chia Chang, University of Notre Dame

“Micro-fluidic technologies for cancer detection and drug delivery”

Paul A. Krieg, University of Arizona

“Growth factor regulation of vascular development”

March

Max S. Wicha, University of Michigan

“Stem cells in normal human breast development and cancer: implications for prevention and therapy”

Judah Folkman, Harvard Medical School

“Platelet angiogenic profile as an early biomarker for cancer”

Robert J. Amato, The Methodist Hospital, Houston

“Therapeutic updates for the management of patients with renal cell carcinoma”

Aaron M. Zorn, Cincinnati Children’s Hospital Research Foundation

“Sox17 and ß-catenin signaling in development”

April

Martin E. Hemler, Harvard Medical School and Dana-Farber Cancer Institute, Boston

“Cell surface molecular networking: the role of tetraspanin-enriched microdomains”

Alan Mackay-Sim, Griffith University, Australia

“Adult stem cells from olfactory mucosa”

Ravi Salgia, University of Chicago

“The role of c-Met in lung cancer”

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S. Stuart Hwang, Celera

“Targeted vascular genomics in drug development”

John Schimenti, Cornell University

“Identification of new infertility and genomic instability mutations in mice by forward genetic mutagenesis screens”

May

Zhu Chen, Shanghai Institute of Hematology

“Systems biology of leukemia”

Brad St. Croix, National Cancer Institute

“Targeting the tumor vasculature”

David E. Schteingart, University of Michigan Health System

“Studies in adrenal cancer”

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Alain Verreault, London Research Institute

“Tales of histone acetylation and degradation”

June

Susan L. Forsburg, University of Southern California

“Replication, recombination, and genome integrity in S. pombe”

David Sidransky, Johns Hopkins University

“Molecular advances in cancer detection and therapy”

Stephen P. Ethier, Barbara Ann Karmanos Cancer Institute

“Oncogene activation and expression of transformed phenotypes in human breast cancer cells”

July

Michael Way, London Research Institute

“Manipulation of Src and Rho signaling during vaccinia virus infection”

Matthew Meyerson, Harvard Medical School

“Genomic and functional studies of human lung and endocrine cancers”

J. Silvio Gutkind, National Institute of Dental & Craniofacial Research

“AIDS malignancies and signaling networks: a case of molecular hijacking”

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August Amar

Klar, National Cancer Institute

“Fission yeast paradigm used to explain genetics of human hand-use preference, brain laterality,

schizophrenia, and bipolar traits”

Karen Hasty, University of Tennessee

“Cartilage matrix degradation in osteoarthritis”

Michael D. Gershon, Columbia University

“Function and formation of the enteric nervous system”

September

Steven A. Kliewer, University of Texas Southwestern Medical Center

“Bile-ology of nuclear receptors in the gut-liver axis”

Bing-Cheng Wang, Case Western Reserve University

“Novel role of EphA2 receptor tyrosine kinase in epithelial cell biology and tumorigenesis”

Brian Druker, Oregon Health and Science University

The Daniel Nathans Memorial Scientific Lecture: “Imatinib as a paradigm of targeted cancer therapies” The Daniel Nathans Memorial Lay Lecture: “Cancer therapy in the 21st century”

October

Richard Vallee, Columbia University

“Shaping the brain one gene at a time: live-cell imaging of LIS1-deficient neural stem cells”

November

Li-Na Wei, University of Minnesota Medical School

“Proteomic studies of nuclear receptor and co-regulator protein modification in gene regulation: ligand-independent effects”

Rajesh V. Thakker, University of Oxford

“Genetic pathways in parathyroid gland development”

Michael Hollingsworth, University of Nebraska Medical Center

“MUC1 in pancreatic cancer: sensing and signaling as the cells slip away”

Ting Xie, Stowers Institute

“Molecular mechanisms controlling stem cell regulation in the Drosophila ovary”

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Steve Heidemann, Michigan State University

“Mechanical tension: the immediate and limiting stimulus for axonal growth”

January 2006

W. Michael Kuehl, National Cancer Institute

“Molecular pathogenesis of multiple myeloma”

Kenneth Bradley, University of California – Los Angeles

“Anthrax lethal toxin”

Valina L. Dawson, Johns Hopkins University

“Life and death signaling by PAR in the brain”

Ted M. Dawson, Johns Hopkins University

“Genetic clues to the mysteries of Parkinson’s disease”

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Andy Futreal, Wellcome Trust Sanger Institute

“Surveying somatic mutations in human cancer by targeted re-sequencing”

February

Morag Park, McGill University, Montreal

“The Met receptor tyrosine kinase: from tubes to tumorigenesis”

Nicholas J. Vogelzang, Nevada Cancer Institute

“Treatment options in metastatic renal cell carcinoma: an embarrassment of riches”

March

Teresa L. Burgess, Amgen, Inc.

“Fully human monoclonal antibodies to hepatocyte growth factor”

Kenneth L. van Golen, University of Michigan “Understanding the roles of Rho and Rac GTPases in prostate cancer bone metastasis”

Thomas W. Glover, University of Michigan

“Mechanisms and significance of chromosome fragile site instability in cancer”

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Van Andel Research Institute Organization

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David L. Van Andel, Chairman and CEO, Van Andel Institute VARI Board of Trustees

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David L. Van Andel, Chairman and CEO Christian Helmus, M.D. Fritz M. Rottman, Ph.D. James B. Wyngaarden, M.D.

Board of Scientific Advisors The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing recommendations and suggestions regarding the overall goals and scientific direction of VARI. The members are

Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

Scientific Advisory Board The Scientific Advisory Board advises the VARI Director, providing recommendations and suggestions specific to the ongoing research, especially in the areas of cancer, genomics, and genetics. It also coordinates and oversees the scientific review process for the Instituteâ&#x20AC;&#x2122;s research programs. The members are

Alan Bernstein, Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D.

VARI | 2006

Office of the Director

George F. Vande Woude, Ph.D. Director

Deputy Director for Clinical Programs

Deputy Director for Special Programs

Deputy Director for Research Operations

Rick Hay, Ph.D., M.D.

James H. Resau, Ph.D.

Nicholas S. Duesbery, Ph.D.

Director for Research Administration

Administrator to the Director

Science Editor

Roberta Jones

Michelle Reed

David E. Nadziejka

Administration Group From left, standing: McGrail, Antio, Koo, Nelson, Novakowski, Jason, Carrigan; Seated: Johnson, Holman, Lewis, Stougaard

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

Executive

Human Resources

Steven R. Heacock, Chief Administrative Officer and General Counsel R. Jack Frick, Chief Financial Officer Ann Schoen, Executive Assistant

Linda Zarzecki, Director Margie Hoving Pamela Murray Angela Plutschouw Mary Van Gessel

Communications and Development John Van Fossen Andrea Nielsen Kirstin VanderMolen

Facilities

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Samuel Pinto, Manager Jason Dawes Teresa DeMull Ken De Young Richard Sal Richard Ulrich

Finance Timothy Myers, Controller Heather Ly Sandi Essenberg Richard Herrick Keri Jackson Angela Lawrence Susan Raymond Kevin Tafelsky Jamie VanPortfleet

Information Technology Bryon Campbell, Ph.D., Chief Information Officer David Drolett, Manager Michael Roe, Manager Bill Baillod Tom Barney Phil Bott Nathan Bumstead Kenneth Hoekman Kimberlee Jeffries Theo Pretorius Russell Vander Mey Candy Wilkerson

Purchasing Richard Disbrow, Manager Heather Frazee Chris Kutchinski Amy Poplaski Andrew Schmidt John Waldon

Security Glassware and Media Services Bob Sadowski Marlene Sal

Kevin Denhof, CPP, Chief Christen Dingman Sandra Folino Emily Young

Grants and Contracts Carolyn W. Witt, Director Rob Junge Sara Oâ&#x20AC;&#x2122;Neal David Ross

Contract Support Valeria Long, Librarian (Grand Valley State University) Jim Kidder, Safety Manager (Michigan State University)

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Van Andel Institute

Van Andel Institute Board of Trustees David Van Andel, Chairman Peter C. Cook Ralph W. Hauenstein John C. Kennedy

Board of Scientific Advisors Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

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Van Andel Research Institute Board of Trustees

Chief Executive Officer David Van Andel

Van Andel Education Institute Board of Trustees

David Van Andel, Chairman Christian Helmus, M.D. Fritz M. Rottman, Ph.D. James B. Wyngaarden, M.D.

David Van Andel, Chairman Gordon Van Harn, Ph.D. Gordon Van Wylen, Sc.D.

Van Andel Research Institute Director

Van Andel Education Institute Director

George Vande Woude, Ph.D.

Gordon Van Harn, Ph.D.

Chief Administrative Officer and General Counsel

VP Communications and Development

Steven R. Heacock

(Under recruitment)

Chief Financial Officer R. Jack Frick

VARI | 2006

Van Andel Research Institute

DIRECTOR â&#x20AC;&#x201C; George Vande Woude, Ph.D.

Deputy Directors

SCIENTIFIC ADVISORY BOARD

Clinical Programs Rick Hay, Ph.D., M.D. Special Programs James Resau, Ph.D. Research Operations Nick Duesbery, Ph.D.

Director for Research Administration

Alan Bernstein, Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D.

Roberta Jones

BASIC SCIENCE

SPECIAL PROGRAMS 111

DIVISION OF QUANTITATIVE SCIENCES

Cancer Cell Biology

Animal Models

Brian Haab, Ph.D. Cancer Immunodiagnostics

Nicholas Duesbery, Ph.D. Cancer & Developmental Cell Biology

Sheri Holmen, Ph.D. Molecular Medicine & Virology

Bart Williams, Ph.D. Cell Signaling & Carcinogenesis

George Vande Woude, Ph.D. Molecular Oncology

Nian Zhang, Ph.D. Mammalian Developmental Genetics

Craig Webb, Ph.D. Tumor Metastasis & Angiogenesis

James Resau, Ph.D. Analytical, Cellular, & Molecular MIcroscopy

Cancer Genetics

Signal Transduction

Bin Teh, M.D., Ph.D. Cancer Genetics

James Resau, Ph.D. Microarray Technology

Art Alberts, Ph.D. Cell Structure & Signal Integration

Structural Biology

Cindy Miranti, Ph.D. Integrin Signaling & Tumorigenesis

Eric Xu, Ph.D. Structural Sciences

Greg Cavey, B.S. Mass Spectrometry and Proteomics

DNA Replication & Repair

Systems Biology

James Resau, Ph.D. Molecular Epidemiology

Michael Weinreich, Ph.D. Chromosome Replication

Jeffrey MacKeigan, Ph.D. Systems Biology

Animal Imaging Rick Hay, Ph.D., M.D. Noninvasive Imaging & Radiation Biology

(effective June 2006)

Gene Regulation Steven Triezenberg, Ph.D. Transcriptional Regulation Dean of VAI Graduate School (effective May 2006)

James Resau, Ph.D.

Kyle Furge, Ph.D. Computational Biology

Brian Cao, M.D. Antibody Technology Pamela Swiatek, Ph.D., M.B.A. Germline Modification Bryn Eagleson, A.A. Transgenics and Vivarium Pamela Swiatek, Ph.D., M.B.A. Cytogenetics Bin Teh, M.D., Ph.D. Sequencing

Published June 2006 | copyright 2006 by the Van Andel Institute | All rights reserved

Van Andel Institute, 333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503, U.S.A.

Van Andel Research Institute Scientific Report 2006 | Cover Images The images on the cover are products of the research in VARI laboratories.

The cover images are reproduced as full-page illustrations within the book along with captions.

Van Andel Research Institute 333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503 Phone 616.234.5000 Fax 616.234.5001

www.vai.org

VARI | 2006

Phone 616.234.5000 Fax 616.234.5001 www.vai.org

Scientific Report

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Van Andel Research Institute Scientific Report 2006