2005 Scientific Report

Van Andel Research Institute
Scientific Report 2005

®
333 Bostwick Avenue, N.E., Grand Rapids, MI 49503
Phone (616) 234-5000; Fax (616) 234-5001; Web site: www.vai.org
Cover photograph of the Van Andel Institute building, Grand Rapids, Michigan

Van Andel Research Institute
Scientific Report
2005

Title page photo: Podosomes in transformed cells
This image shows podosomes in NIH3T3 mouse fibroblasts transformed with activated Src tyrosine
kinase. Podosomes are the rounded structures at the tips of many of the cell extensions. They are rich in
filamentous actin, which has been stained green with a conjugated phalloidin dye. The cells are co-stained
with an antibody that recognizes the adhesion protein CrkL (red; or where co-localized with actin and its
phalloidin stain, yellow). The protein CrkL is involved in integrin-induced cell adhesion and migration.
The study of these proteins (Src, F-actin, CrkL, integrins) in these and other tumorigenic cell types may
shed light on the mechanisms of podosome-mediated cancer cell metastasis and invasion.
(Eduardo F. Azucena, Jr., Darren Seals, James Resau, and Sara Courtneidge)
Published June 2005
©2005 by the Van Andel Institute
All rights reserved
Van Andel Institute
333 Bostwick Avenue, N.E.
Grand Rapids, Michigan 49503, U.S.A.

Contents
Director’s Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Laboratory Reports
Cell Structure and Signal Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Arthur S. Alberts, Ph.D.
Antibody Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Brian Cao, M.D.
Mass Spectrometry and Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Gregory S. Cavey, B.S.
Signal Regulation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Sara A. Courtneidge, Ph.D.
Cancer and Developmental Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Nicholas S. Duesbery, Ph.D.
Vivarium and Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Bryn Eagleson, A.A., RLATG
Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Kyle Furge, Ph.D.
Cancer Immunodiagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Brian B. Haab, Ph.D.
Molecular Medicine and Virology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Sheri L. Holmen, Ph.D.
Integrin Signaling and Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Cindy K. Miranti, Ph.D.
Analytical, Cellular, and Molecular Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
and
Microarray Technology and Molecular Diagnostics
James H. Resau, Ph.D.
Germline Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Pamela J. Swiatek, Ph.D., M.B.A.
Cancer Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Bin T. Teh, M.D., Ph.D.
iii

Molecular Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
George F. Vande Woude, Ph.D.
Tumor Metastasis and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Craig P. Webb, Ph.D.
Chromosome Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Michael Weinreich, Ph.D.
Cell Signaling and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Bart O. Williams, Ph.D.
Structural Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
H. Eric Xu, Ph.D.
Mammalian Developmental Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Nian Zhang, Ph.D.
Daniel Nathans Memorial Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
Postdoctoral Fellowship Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
Student Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
Han-Mo Koo Memorial Seminar Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
Recent VARI Photos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99
iv

Director’s Introduction

Director’s Introduction
We are now in our
fifth year since the
opening of the Van
Andel Institute. The
Institute was created
because of the
generosity of Jay Van
Andel and his wife
Betty, both of whom
George F. Vande Woude
passed away in 2004.
While deeply saddened by their loss,
we continue our endeavor to fulfill their vision of
a center for research and education excellence in
the heart of Grand Rapids.
I know Jay was able to see the beginning of
what a great contribution he made to society and
the betterment of human health. Sharing his and
Betty’s vision, we have begun to expand our
research beyond cancer biology into the field of
neurological disorders. David and Carol Van
Andel, in Jay’s honor, have created an endowed
chair dedicated to Parkinson disease (PD)
research. The search for a leading scientist to
take this chair is now underway.
Already, Jim Resau and Bin Teh have
initiated a collaboration with Australian
scientists Alan Mackay-Sim and Peter Silburn at
Queensland University in Australia to better
understand Parkinson disease and how it
develops. Alan is a developmental biologist who
has initiated some exciting discoveries using
adult olfactory stem cells. Peter is a neurologist
who studies and treats PD patients. Together
with these new collaborators, we will increase
our understanding of the function, growth, and
death of human nerve cells as models for PD. In
addition, these approaches will be used in the
study of Alzheimer disease pathophysiology.
Our scientists have also partnered with the St.
Mary’s Hauenstein Parkinson Center—named in
honor of lead donor (and VAI trustee) Ralph
Hauenstein—to determine the interactions of the
environment and genes associated with PD. We
will learn how these genes correlate with disease
progression and drug response.
We are pleased to announce that, in
collaboration with the Van Andel Education
Institute, the Research Institute is developing a
graduate school with a program leading to the
Ph.D. degree in cellular and molecular genetics,
with an emphasis on translation. We expect the
graduate program to contribute to the vitality and
creativity of our successful research programs
and to also address the nation’s need for
expertise in the life sciences and biotechnology.
We have received a charter from the state of
Michigan and have appointed a Board of
Directors for the school. Our goal is to have our
first students on board in September 2006.
Personnel
A special occasion occurred this past year
with our first promotion review. We are very
proud to announce the promotion of Bin Tean Teh
to Distinguished Scientific Investigator, our
highest appointment level. Bin has made major
contributions to the understanding of kidney,
nasopharyngeal, and endocrine cancer. In recognition
of his efforts, Bin was recently appointed
to the Medical Advisory Board of the Kidney
Cancer Association. We are also proud to
announce the appointment of Rick Hay as a
Senior Scientific Investigator. Rick will establish
the Laboratory of Animal Imaging. Our congratulations
to both scientists!
Sara Courtneidge has taken a position at the
Burnham Institute and is relocating her
laboratory in early summer 2005. Sara and I
have been friends and colleagues for over 20
years. I was delighted when she joined VARI
and I am grateful for all her contributions to the
Institute, especially her efforts in helping to
establish the graduate program with MSU and
the VARI postdoctoral advisory committee. The
Burnham Institute is very fortunate to have
recruited her, where she will be reunited with her
many West Coast friends. I am sorry to see her
leave, but I wish her great success and look
forward to our continued scientific interactions.
Publications and Competitive Funding
As of April 1, 2005, there have been 190
peer-reviewed articles published by VARI
investigators. In December 2004, a VARI article
was featured on the cover of the Journal of Bone
3

and Mineral Research; in January 2005, we had a
featured article in Molecular Cell; and in
February, a VARI article in Cancer Cell was the
source of the issue’s cover photo. Our
investigators have also demonstrated their
competitive research abilities in terms of receiving
grants for funding of their work. In fiscal year
2004, extramural funding for VARI dramatically
increased over that in 2003. Seventeen of our
scientists and six of our postdoctoral fellows
received funding from 42 grants.
Our new major awards have come from a
variety of sources. The National Institutes of
Health (NIH) National Cancer Institute (NCI)
made two awards to Nick Duesbery. The first
was an R01 grant for studying MEK signaling in
sarcoma growth and vascularization. The second
grant, an R21, was for investigating the antitumor
effects of an anthrax toxin moiety (which
we have termed tumor lethal factor, or “TLF”) on
Kaposi sarcoma. Nick’s lab has been developing
TLF as a potential cancer therapeutic.
Brian Haab was awarded an R21 NCI grant for a
two-year project entitled “Longitudinal Cancer-
Specific Serum Protein Signatures.” This project
seeks to develop protein microarray methods for
detecting and diagnosing prostate cancer by
examining changes over time in several cancerrelated
proteins in serum. And, Art Alberts
received an R21 award from the NIH to exploit a
discovery in his lab with a long-term goal of
developing novel anti-cancer therapies.
The American Cancer Society awarded two
Research Scholar Grants to our researchers.
One grant, to Art Alberts, is for a four-year
project to study Diaphanous-related formins in
myelodysplasia. Art has been studying the role
of formins in cancer. A second American Cancer
Society grant was awarded to Michael Weinreich.
Michael is identifying small-molecule inhibitors
of Cdc7 kinase for study of its regulation in DNA
replication, with a long-term goal of identifying
novel targets for cancer diagnosis and therapy.
Two major grants have also been received
from the Michigan Technology Tri-Corridor
(MTTC). One award went to Rick Hay for the
development of novel agents for nuclear imaging
and therapy of Met-expressing human tumors.
The project is a collaboration among scientists at
VARI, Michigan State University, the Department
of Veterans Affairs Healthcare System in Ann
Arbor, ApoLife, Inc., and the National Cancer
Institute. A second grant was awarded to Bin Teh
for the development of the “RenoChip,”
a diagnostic and prognostic tool for use against
kidney cancer. In addition, the Department of
Defense awarded Eric Xu a grant for a three-year
study of the structure and function of the
androgen receptor in prostate cancer. Eric’s lab
aims to make progress in understanding the
androgen dependence (or independence) of
prostate cancer.
Funding from other sources in the past year
has included Brian Haab’s grant from DHHS/
NCI via the University of Michigan for a project
entitled “Accelerated Cancer Biomarker
Discovery.” This project is being undertaken by
a consortium of laboratories and focuses on the
development and application of new proteomics
technologies for cancer biomarker discovery.
Bin Teh has received a grant from the
Schregardus Family Foundation for a project on
renal cell carcinoma (RCC) in which his lab will
be studying the prognostic value of genes for
improving the clinical management of RCC
patients. Bin is also the recipient of a grant from
the Gerber Foundation for gene expression
profiling in newborns with congenital chromosomal
abnormalities.
We are also proud that three of our
postdoctoral fellows have received awards.
Carrie Graveel (Vande Woude lab) and Kate
Eisenmann (Alberts lab) have received National
Research Service Awards from the NIH, while
Jennifer Bromberg-White (Webb lab) received a
fellowship award from the Multiple Myeloma
Research Foundation.
Looking to the Future
We now look to expanding not only our
research goals but the Institute itself. On May
17th we celebrated our fifth anniversary, and our
CEO, David Van Andel, announced that in 2006
we will begin the second construction phase of
our Institute. The new building, a model of
which is displayed in the Cook-Hauenstein Hall,
will join and mirror our current exceptional
facility, but it will provide two-and-a-half times
the existing laboratory space, or an additional
150,000 square feet.
4

As we plan and begin our expansion, we are
part of the unprecedented growth in the health
industry that Grand Rapids is experiencing.
The area of Grand Rapids in which the Institute is
situated has been appropriately renamed “Medical
Hill.” Our neighbor across Bostwick Avenue,
Spectrum Health, has celebrated the opening of
the new Fred and Lena Meijer Heart Center.
Furthermore, the hospital will soon break ground
for the construction of a new cancer center as well
as a new pediatric hospital. In another project, our
own Rick Hay serves as the chairman of a
combined VARI and Grand Valley State
University group that is planning a good
manufacturing practices (GMP) facility. This
facility is being established with state and federal
funding, and it will produce small quantities of
clinical-grade biological products that can be
tested in patients. Finally, there is the strong
possibility of Michigan State University’s College
of Human Medicine relocating to Grand Rapids,
which would certainly be a major event in the
development of the biomedical community here.
Overall, with the new hospital facilities at
Spectrum Health and St. Mary’s, Grand Valley
State University’s strength in health sciences, and
our own research program and future expansion,
we will witness in the next decade exciting and
dramatic developments in biomedical research,
scientific discovery, and health care delivery
taking place in Grand Rapids. We look forward
to these many exciting changes and to the
formation of a center of excellence in biomedical
disciplines in western Michigan.
5

Van Andel Research Institute
Laboratory Reports

2005 VARI
Scientific Retreat
8

Laboratory of Cell Structure and Signal Integration
Arthur S. Alberts, Ph.D.
Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of
California, San Diego, in 1993, 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
Art Alberts, Ph.D.
Jun Peng, M.D.
Yunju Chen, Ph.D.
Kathryn Eisenmann, Ph.D.
Holly Holman, Ph.D.
Susan Kitchen, B.S.
Laboratory Members
Students
Aaron DeWard, B.S.
Yaojian Liu, B.S.
Katharine Collins
Visiting Scientists
Stephen Matheson, Ph.D.
Brad Wallar, Ph.D.
Research Interests
T
he 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.
PAK1 negatively regulates the activity of the
Rho exchange factor NET1
Rho GTPases act as molecular switches in
cells, alternating between on and off states while
bound to GTP and GDP nucleotides, respectively.
The activated, GTP-bound proteins preferentially
interact with numerous autoregulated downstream
effector proteins. Rho GTPases are activated by
Rho guanine nucleotide exchange factors (Rho
GEFs), one of which is the neuroepithelioma
transforming gene 1 (NET1). Recently it was
demonstrated that wild-type NET1 is localized in
the nucleus and that truncation of the amino
terminus results in relocalization of a fraction of
the NET1 to the cytoplasm. This is at least
partially due to the elimination of two putative
nuclear localization signals within the amino
terminus. Thus, NET1 activity is regulated at least
in part through subcellular localization.
Rho family members can modulate the
activity of other Rho proteins. The protein kinase
PAK1 down-regulates the activity of the RhoAspecific
GEF NET1. Specifically, PAK1
phosphorylates NET1 on three sites in vitro:
serines 152, 153, and 538. Replacement of
serines 152 and 153 with glutamate residues
reduces the activity of NET1 as an exchange
factor in vitro, as well as its ability to stimulate
actin stress fiber formation in cells. Using a
phospho-specific antibody, PAK1 can be shown
to phosphorylate NET1 on serine 152 in cells, and
Rac1, which activates PAK1, stimulates serine
152 phosphorylation in a PAK1-dependent
manner. Furthermore, coexpression of
constitutively active PAK1 inhibits NET1
stimulation of actin polymerization only when
serines 152 and 153 are present. These
observations provide a novel mechanism for the
control of RhoA activity.
9

Signal transduction and spatially controlled
assembly of F-actin networks
One well-characterized actin nucleator, the
Arp2/3 complex, induces the formation of
branched actin filaments. Arp2/3 works by
complexing with G-actin or by binding to the side
of preexisting filaments. Its nucleation and
filament-binding activity is tightly regulated by
interactions with nucleation-promoting factors
(NPFs), the most prominent being the WASp/Scar
family. WASp is an autoregulated molecular
switch controlled by yet other switch-like proteins
such as Cdc42, a Rho family small GTPase. Thus,
NPFs integrate signals controlling growth
factor–stimulated actin nucleation and branching.
Cdc42-activated WASp induction of Arp2/3
activity is shown schematically in Fig. 1A.
The mammalian Diaphanous-related formins
Formins are a highly conserved family of
proteins implicated in a diverse array of cellular
functions including the cytoskeletal remodeling
events necessary for cytokinesis, bud formation
in yeast, 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.
The mammalian Diaphanous-related formin
(mDia) proteins are a subfamily of formins that
share a loosely conserved Rho GTPase-binding
domain (GBD) in the amino terminus and a
highly conserved Diaphanous-autoregulatory
domain (DAD) in the carboxy terminus.
The GBDs can interact with their internal DAD
partners in vitro, leading to the autoregulation
model depicted in Fig. 1B. The model shows that
while the formin proteins dimerize through their
FH2 domains, it is the GBD-DAD interaction that
is the linchpin of autoregulation. GTP-bound
Rho can interact with the GBD and interrupt the
autoinhibited conformation, leading to nucleation
and elongation of nonbranched actin filaments.
It has been unclear whether GTPase binding
simply activates the mDia proteins’ ability to
nucleate actin, or provides other signals that
direct subcellular targeting and recruitment of
mDia-associated proteins. Another question is,
do mDia proteins activated in specific cellular
contexts (i.e., on vesicles or at sites of adhesion)
associate with or work in parallel with other
modifiers of actin polymerization to generate
site-specific F-actin networks? We are studying
such questions using FRET technology.
Site-specific interactions between Rho
GTPases and mDia proteins
Fluorescence resonance energy transfer
(FRET) is a powerful technique that allows us to
assay protein-protein interactions in cells by
using two fluorophores, in this case, cyan
fluorescent protein (CFP) and yellow fluorescent
protein (YFP). When fused to the GTPase and
excited at the appropriate wavelength, CFP acts
as a fluorescent donor which then excites YFP,
which is fused to a Drf protein (Fig. 1B). FRET
Figure 1. Drfs are actin nucleators whose activity is regulated through interactions with small
GTPases. A) A model for formin (mDia1-3) and WASp collaborating in cells with activated Cdc42, in which
Cdc42 interacts with mDia2 in cells at specific sites associated with membrane protrusions. In this model,
activated WASp nucleates branched filaments from the side of mDia2 nucleated “mother” filaments;
alternatively, mDia2 binds to and processively elongates filaments after nucleation by Arp2/3. B) GTP-bound
Rho GTPase binding disrupts intramolecular interactions between the GBD and DAD of a DRF. If the GTPase
and GBD are linked to fluorophores (ECFP and EYFP), the proximity of the two can be determined by FRET.
10

occurs only when the donor/acceptor pair is in
close proximity (less than 30 Å) .
Fusion proteins are expressed following
microinjection of their expression plasmids into
cells, which are then fixed 4 h later. We have
shown that YFP-mDia2 is expressed with CFP-
Cdc42, primarily at the leading cell edge (or
cortex) and at the microtubule-organizing center.
In other experiments, we have shown that this
interaction depends upon the integrity of the
CRIB motif within the mDia2 GBD. CRIB
motifs are necessary for binding to the GTPase.
Our observations indicate that one particular
GTPase-formin pair, Cdc42 and mDia2, may
have a role in remodeling actin at the cell edge.
What this pair contributes to actin or microtubule
dynamics at the MTOC, however, remains an
open question. We speculate that the pair may
participate in microtubule regulation at the minus
(–) end of the tubules, which (like actin) are
assembled and disassembled in a polarized
fashion. Other GTPase-formin pairs may be
working at the plus end to direct them to focal
adhesions or other sites. From collaborative
efforts with the Gundersen lab, it has been shown
that mDia1 and mDia2 can complex with the
MT(+)-end binding proteins APC and EB1.
In contrast to the speculative role of Cdc42
and mDia2, RhoB is known to have a role in
endocytic or vesicular trafficking, and it interacts
with mDia2 on endosomes (Fig. 2). This result
is consistent with our discovery of both mDia1
and mDia2 on endosomes. The expression of
either activated RhoB or deregulated versions of
mDia1, mDia2, and mDia3 blocks the movement
of vesicles and increases their number. One
interpretation is that expression of RhoB or
deregulated mDia1–3 triggers an inappropriate
transition from fast microtubule-dependent
transport to actin-dependent transport.
Formins as anti-cancer drug targets
A dynamic cytoskeleton is required for tumor
cell growth. Drugs that stabilize the cytoskeleton
are emerging as effective anti-cancer
therapeutics. For example, Taxol binds directly
to the components that comprise the microtubule
cytoskeleton and blocks their dynamics. A
cyclopeptide derived from a sea sponge,
jasplakinolide, has similar effects on actin.
Figure 2. RhoB
interacts with mDia2
on vesicles. CFP-fused
activated RhoB-G14V
and YFP-fused mDia2
were co-injected into
cells and FRET was
assessed 4 h later. A
FRET signal is observed
between activated RhoB
and mDia2 upon
endosomes. These data
indicate that individual
GTPase-formin pairs
would appear to be
functionally distinct,
promoting the formation
of different actin
structures at different
sites. Since both RhoB
and Cdc42 have been
implicated in endocytosis
and vesicle trafficking, it
is possible that both
GTPases use mDia2
sequentially or in parallel
as effectors to transport
cargo within cells.
Previously, we found that a peptide derived
from the DAD region of mDia proteins, when
expressed in cells, stabilized both the actin and
microtubule cytoskeletons. Because the
mechanism of DAD action is unique—it binds to
cellular formins and disrupts their normal
autoregulatory mechanism—DAD represents a
novel class of anti-tumor drugs.
Because DAD is unable to enter cells as a
drug, we have begun searching for functional
analogs of DAD that could have similar properties
under a drug-development program funded by the
National Cancer Institute. We hypothesize that by
deregulating specific mDia molecules in tumor
cells, we can arrest dynamic remodeling of the
cytoskeleton, a process required for cell motility
and cytokinesis. We will characterize the
structural and functional requirements for DADinduced
cell death and develop a high-throughput
screen for the identification of novel molecules
that can deregulate mDia proteins and kill tumor
cells. Our objectives are to initiate a drug
discovery program by validating mDia proteins as
molecular targets in cancer and to determine the
physical requirements for DAD interactions with
the mDia GBD.
11

External Collaborators
Philippe Chavrier, Institut Curie, Paris, France
Jeff Frost, University of Houston, Texas
Gregg Gundersen, Columbia University, New York
George Prendergast, Lankenau Institute, Wynnewood, Pennsylvania
Kathy Siminovitch, University of Toronto, Canada
Recent Publications
Alberts, A.S., H. Qin, H.S. Carr, and J.A. Frost. In press. PAK1 negatively regulates the activity of
the Rho exchange factor NET1. Journal of Biological Chemistry.
Eisenmann, K.M., J. Peng, B.J. Wallar, and A.S. Alberts. In press. Rho GTPase-formin pairs in
cytoskeletal remodeling. In Signaling Networks in Cell Shape and Motility, London, U.K.:
Novartis Foundation.
Wen, Ying, Christina H. Eng, Jan Schmoranzer, Noemi Cabrera-Poch, Edward J.S. Morris, Michael
Chen, Bradley J. Wallar, Arthur S. Alberts, and Gregg G. Gundersen. 2004. EB1 and APC bind
to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nature Cell
Biology 6(9): 820–830.
Left to right: Liu, Holman, DeWard, Chen, Peng, Collins, Alberts, Kitchen, Eisenmann
12

Laboratory of Antibody Technology
Brian Cao, M.D.
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
Ping Zhao, M.S.
Tessa Grabinski, B.S.
Laboratory Members
Visiting Scientist
Mei Guo, M.S.
Students
Yong-jun Jiao
Xin Wang
Jin Zhu
Research Interests
H
epatocyte
growth factor/scatter factor
(HGF/SF) is a multifunctional
heterodimeric protein produced by
mesenchymal cells and is an effector of cells
expressing the tyrosine kinase receptor Met. Met,
the protein product of the c-met protooncogene, is
from the same family as epidermal growth factor
(EGF) receptors. The activation of Met by
HGF/SF affects downstream signaling pathways
(including other 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
anti-cancer agents for clinical diagnosis,
prognosis, and treatment.
The aberrant expression of the Met receptor
kinase by two-thirds of localized prostate cancers,
and apparently by all osseous metastases,
suggests that Met provides a strong mechanism of
selection for metastatic development. We have
generated and characterized several anti-Met
murine monoclonal antibodies (mAbs) that have
high affinity for and specifically recognize Met
extracellular domains in their native
conformation. In collaborative studies, we are
using two of these radiolabeled anti-Met mAbs,
designated Met3 and Met5, to study mouse
xenograft and orthotopic models of localized and
metastatic prostate cancer via clinical nuclear
imaging. Moreover, we will soon be testing these
two radiolabeled mAbs on dog spontaneous
prostate cancer and bone metastasis models.
In collaboration with the Nanjing Medical
University of China, we have initiated a project to
construct a phage-display antibody fragment
library. This technique involves the construction
and use of animal/human, immunized/naïve Fab
and scFv antibody gene repertoires by phage
display. The ability to co-select antibodies and
their genes allows the isolation of high-affinity,
antigen-specific mAbs derived from either
immunized animals or non-immunized humans. A
number of procedures for selecting such antibodies
from recombinant libraries have been described,
and some useful antibodies have been produced
with this approach. Over the past two years, we
have closely followed the development of this
technology for producing novel recombinant
antibody-like molecules. We have constructed a
human naïve Fab library with the diversity of 2 ×
10 9 and have screened out some mAb fragments
against tumor marker proteins. In particular, we
have selected from this library and characterized
one specific anti-Met Fab fragment, designated as
Fab-Met-1, using a subtractive whole-cell panning
approach (Figs. 1 and 2).
We have also established the technology of a
phage-display peptide library for mAb epitope
13

A
B
mapping. A random peptide library
is constructed by genetically fusing
oligonucleotides to the coding
sequence of a coat protein of
bacteriophage, resulting in display
of the fused polypeptide 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
phages are washed away, and
specifically bound phages 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. We have successfully
epitope-mapped a variety of important mAbs
including anti-HGF/SF, anti-Met, and anti-anthrax
lethal factor. We are now exploring the use of this
technology on protein-protein interactions; one
example is the mapping of the HGF/SF-Met
binding site in an in vitro system, and several
interesting peptides have been selected from the
library as being potential Met antagonists.
Functioning as an antibody production core
facility at the Van Andel Research Institute, this
lab has extensive capabilities in the generation,
characterization, scaled-up production, and
purification of mAbs using comprehensive
cutting-edge technologies. The technologies and
services available in the core include animal
immunization and antigen preparation; peptide
design; DNA immunization (Gene-gun
technology); 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.
Other services we provide include the generation
of hybridomas from spleen cells of immunized
mice, rats, and rabbits; hybridoma expansion and
subcloning; cryopreservation of hybridomas
secreting mAbs; monoclonal antibody isotyping;
ELSIA screening of hybridoma supernatants;
monoclonal antibody characterization by
immunoprecipitation, Western blot, immunohistochemistry,
immunofluorescence staining,
FACS, or in vitro bioassays; production of bulk
quantities of mAbs using high-density cell culture;
purification of mAbs using FPLC affinity columns;
generation of bi-specific mAbs by secondary
fusion; conjugation of mAbs to detection enzymes
(biotin/streptavidin, fluorescence reporters, etc.);
and the development of detection methods/kits
such as sandwich ELISA. Over the past few years,
this facility has generated more than 200 different
mAbs, 10 of which have been licensed to
commercial companies. We have also contracted
services to local biotechnology companies to
generate, characterize, produce, and purify mAbs
for their research/diagnostic kit development.
S114
Figure 1. A) SDS–PAGE of Fab-
Met-1 fragment purified by affinity
chromatography. Lane 1, standard
molecular weight markers; lane 2,
purified Fab fragment under reducing
conditions. The concentration of the
running gel was 12%.
B) Immunoprecipitation analysis of
Fab-Met-1. Met from cell extracts
was immunoprecipitated with purified
Fab-Met-1 and detected by western
blot analysis. Lane 1 is S114 cell
lysate immunoprecipitated with C-28
(rabbit anti-human Met polyclonal
antibody). Lanes 2–5 are cell lysates
immunoprecipitated with Fab-Met-1:
lane 2, S-114 (Met+); lane 3, MKN45
(Met+); lane 4, M14 cell (Met–); and
lane 5, NIH3T3 (Met–).
M14
Figure 2. The binding affinity of a selected Fab
fragment (Fab-Met-1) was tested by FACS analysis.
Two cell lines, S114 (Met+) and M14 (Met–), were
incubated with purified Fab-Met-1. Bound Fab was
detected by staining the cells with secondary goat
anti-human Fab–FITC conjugate and analyzing by
FACS (black lines). Green lines indicate staining
with the secondary antibody only.
14

External Collaborators
Zhen-qing Feng, Nanjing Medical University, China
Milton Gross, Department of Veterans Affairs Medical Center/University of Michigan, Ann Arbor
Xiao-hong Guan, Nanjing Medical University, China
Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington
Yi Ren, Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio
Kang-lin Wan, Chinese Centers for Disease Control and Prevention, Beijing, China
David Waters, Gerald P. Murphy Cancer Foundation, Seattle, Washington
David Wenkert, Michigan State University, East Lansing
Wei-cheng You, Beijing Institute for Cancer Research, China
Recent Publications
Jiao, Y., P. Zhao, J. Zhu, T. Grabinski, Z. Feng, X. Guan, R.S. Skinner, M.D. Gross, Y. Su,
G.F. Vande Woude, R.V. Hay, and B. Cao. In press. Construction of human naïve Fab
library and characterization of anti-Met Fab fragment generated from the library.
Molecular Biotechnology.
Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Margaret Gustafson, Nariyoshi Shinomiya, Ping Zhao,
Brian Cao, Galia Tsarfaty, Ling-Mei Wang, Rick Hay, and George F. Vande Woude. 2005.
Enhanced growth of human Met-expressing xenografts in a new strain of immunocompromised
mice transgenic for human hepatocyte growth factor/scatter factor. Oncogene 24(1): 101–106.
Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping
Zhao, Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat
Cheah, Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin
immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer
Research 10(19): 6629–6637.
Zhao, Ping, Xudong Liang, Jessica Kalbfleisch, Han-Mo Koo, and Brian Cao. 2003. Neutralizing
monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model.
Human Antibodies 12(4): 129–135.
From left to right: Zhu, Wang, Cao, Ferrell, Grabinski, Zhao
15

Laboratory of Mass Spectrometry and Proteomics
Gregory S. Cavey, B.S.
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 stateof-the-art
proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a
Special Program Investigator in July 2002.
Staff
Paula Davidson, M.S.
Laboratory Members
Student
Wendy Johnson
Research Interests
The Mass Spectrometry and Proteomics
program works with many of the
research labs at the Institute, using stateof-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
challenges 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 affinitypurified
from a nondenatured sample. 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, enzymatically digested into peptides,
and then analyzed by nanoscale high-pressure
liquid chromatography on line with a mass
spectrometer (LC-MS). 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, affinitypurified
protein complexes are compared to a
control sample via peptide differential display. The
proteins are digested into peptides in solution rather
than from gels and are analyzed by LC-MS.
Peptides 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. Purified
proteins 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
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
the molecules of a given protein are
16

phosphorylated. Thus, we are dealing with an
extremely small number of molecules, in addition
to the fact that the purification of phosphopeptides
is always difficult. Our lab collaborates with
investigators to map protein phosphorylation using
techniques including immobilized metal affinity
purification following esterification; immunoaffinity
purification of phosphoproteins and
peptides; and phosphorylation-specific mass
spectrometry detection.
Protein expression
As mass spectrometry instruments and protein
separation methods develop, we hope to identify
and quantitate all the 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 affect a
biological outcome. Traditionally this approach
has used 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), proteomics has been
moving toward solution-based separations and
direct mass spectrometry. Our first approach is to
digest all proteins into peptides and label their
C-terminus with 18 O water to effect a mass shift.
Experimental and control samples are then mixed
and separated by multidimensional high-pressure
liquid chromatography using strong-cation ion
exchange and reverse-phase separation modes.
Peptides that are differentially expressed in
experimental and control samples according to
their 16 O/ 18 O ratio are identified using mass
spectrometry and database searching.
We intend to apply this or other mass
spectrometry–based approaches in the discovery
of biomarkers for early cancer detection, for morespecific
diagnosis, and for more-accurate
prognosis following drug treatment.
External Collaborators
Greg Fraley, Hope College, Holland, Michigan
Gary Gibson, Henry Ford Hospital, Detroit, Michigan
Brett Phinney, Michigan State University, East Lansing
Recent Publications
Li, Yong, Mihwa Choi, Greg Cavey, Jennifer Daugherty, Kelly Suino, Amanda Kovach, Nathan C.
Bingham, Steven A. Kliewer, and H. Eric Xu. 2005. Crystallographic identification and functional
characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1.
Molecular Cell 17(4): 491–502.
From left to right: Davidson, Johnson, Cavey
17

Laboratory of Signal Regulation and Cancer
Sara A. Courtneidge, Ph.D.
Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Research
in London. She began her career in the basic sciences in 1978 as a postdoctoral
fellow in the laboratory of J. Michael Bishop at the University of California School of
Medicine. She later joined her alma mater as a member of the scientific staff. In
1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as group
leader and in 1991 was appointed a senior scientist with tenure. She joined Sugen
in 1994 as Vice President of Research, later becoming Senior Vice President of
Research and then Chief Scientist. Dr. Courtneidge joined VARI in January 2001
as a Distinguished Scientific Investigator.
Staff
Eduardo Azucena, Ph.D.
Paul Bromann, Ph.D.
Hasan Korkaya, Ph.D.
Laboratory Members
Ian Pass, Ph.D.
Darren Seals, Ph.D.
Laila Al-Duwaisan
Research Interests
Our laboratory wants to understand at the
molecular level how proliferation is
controlled in normal cells and by what
mechanisms these controls are subverted in tumor
cells. We largely focus on a family of oncogenic
tyrosine kinases, the Src family. The prototype of
the family, vSrc, originally discovered as the
transforming protein of Rous sarcoma virus, is a
mutated and activated version of a normal cellular
gene product, cSrc. The activity of all members of
the Src family is normally under strict control;
however the enzymes are frequently activated or
overexpressed, or both, in human tumors.
In normal cells, Src family kinases (SFKs) have
been implicated in signaling from many types of
receptors, including receptor tyrosine kinases,
integrin receptors, and G protein–coupled
receptors. Signals generated by SFKs are thought
to play roles in cell cycle entry, cytoskeletal
rearrangements, cell migration, and cell division.
In tumor cells, Src may play a role in growth
factor–independent proliferation or in invasiveness.
Furthermore, some evidence points to a role for
SFKs in angiogenesis. Some of the current projects
in the laboratory are outlined below.
The role of the Src substrate
Tks5/Fish in tumorigenesis
Tks5/Fish is an adaptor protein which has
five SH3 domains and a phox homology (PX)
domain. Tks5/Fish is tyrosine-phosphorylated in
Src-transformed fibroblasts (suggesting that it is a
target of Src in vivo) and in normal cells after
treatment with any of several growth factors.
We recently found that in Src-transformed cells,
Tks5/Fish is localized to specialized regions of
the plasma membrane called podosomes
(sometimes referred to as invadopodia).
These actin-rich protrusions from the plasma
membrane are sites of matrix invasion and
locomotion. The PX domain of Tks5/Fish
associates with phosphatidylinositol 3-phosphate
and phosphatidylinositol 3,4-bisphosphate, and it
is required for targeting Tks5/Fish to podosomes.
The fifth SH3 domain of Tks5/Fish mediates its
association with members of the ADAMs family of
membrane metalloproteases, which in Srctransformed
cells are also localized to podosomes.
We have begun to dissect the role of Tks5/Fish in
transformation. Src-transformed cells with
reduced Tks5/Fish levels no longer form
podosomes and are poorly invasive. We detected
Tks5/Fish expression in podosomes in invasive
human cancer cell lines, as well as in tissue
samples from human breast cancer and melanoma.
Tks5/Fish expression was also required for
invasion of human cancer cells. Furthermore, we
have developed an assay to generate podosomes
upon expression of Tks5/Fish, which will allow us
to dissect the requirements for podosome
formation in more detail. We are also investigating
the potential of both Tks5/Fish and its binding
proteins as markers of invasive disease and as
potential therapeutic targets.
18

The role of Src family kinases in
mitogenic signaling pathways
We have previously shown that Src family
kinases are required for both Myc induction and
DNA synthesis in response to platelet-derived
growth factor (PDGF) stimulation of NIH3T3
fibroblasts. We have also previously identified and
characterized a small-molecule inhibitor of Src
family kinases called SU6656. We wanted to
address whether there is a discrete SFK-specific
pathway leading to enhanced gene expression, or
whether SFKs act to generally enhance PDGFstimulated
gene expression. To do this, we treated
quiescent NIH3T3 cells with PDGF in the
presence or absence of SU6656 and analyzed
global patterns of gene expression. We determined
that a discrete set of immediate early genes was
induced by PDGF and inhibited by SU6656.
We further determined that SFKs did not stimulate
the rate of transcription of these genes, but rather
promoted mRNA stabilization. We are currently
exploring how SFKs signal gene expression by
enhancing mRNA stability.
Breast cancer
Increased Src activity can be demonstrated in
the majority of breast cancers, both estrogendependent
and estrogen-independent, yet the role
of Src in breast tumorigenesis has not been fully
established. We have been characterizing the role
of Src in estrogen-stimulated signal transduction
pathways in breast cancer cell lines. We have
shown that Src family kinase activity is required
for estrogen to stimulate mitogenesis in MCF7
cells. Furthermore, inhibition of Src prevents
estrogen stimulation both of Myc and of MAP
kinase activity. We are currently dissecting which
Src signaling pathways are necessary for estrogenstimulated
growth, as well as how Src activity
results in the activation of MAP kinase and in the
production of Myc.
Recent Publications
Bromann, Paul A., Hasan Korkaya, Craig P. Webb, Jeremy Miller, Tammy L. Calvin, and Sara A.
Courtneidge. 2005. Platelet-derived growth factor stimulates Src-dependent mRNA stabilization of
specific early genes in fibroblasts. Journal of Biological Chemistry 280(11): 10253–10263.
Seals, Darren F., Eduardo F. Azucena, Jr., Ian Pass, Lia Tesfay, Rebecca Gordon, Melissa Woodrow, James
H. Resau, and Sara A. Courtneidge. 2005. The adaptor protein Tks5/Fish is required for podosome
formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell 7(2):
155–165.
Bromann, Paul A., Hasan Korkaya, and Sara A. Courtneidge. 2004. The interplay between Src family
kinases and receptor tyrosine kinases. Oncogene 23(48): 7957–7968.
From left to right: Azucena, Seals, Pass, Al-Duwaisan, Bromann
19

Laboratory of Cancer and Developmental Cell Biology
Nicholas S. Duesbery, Ph.D.
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.
Staff
Paul Spilotro, M.D.
Philippe Depeille, Ph.D.
Hilary Wagner, M.S.
John Young, M.S.
Elissa Boguslawski
Laboratory Members
Students
Chia-Shia Lee
Lisa Orcasitas
Visiting Scientist
Gustavo Nacheli, M.D.
Research Interests
The overall goal of the Laboratory of
Cancer and Developmental Cell Biology
is to further the study of mitogenactivated
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
LeTx 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 growth and
vascularization of sarcomas. 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 anthrax lethal toxin, a
proteolytic inhibitor of MEKs, may form the basis
of a novel therapeutic approach to the treatment of
human sarcoma. In 2004 we successfully
obtained funding from the National Institutes of
Health for this project.
The therapeutic potential of
anthrax lethal toxin
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 melanomas, colorectal adenocarcinomas,
and Kaposi’s sarcoma. The goal of
this project is to develop novel therapeutic agents
that may be efficacious in the treatment of human
malignancies. In 2004 we successfully obtained
funding from the National Institutes of Health to
evaluate the therapeutic potential of LeTx in the
treatment of Kaposi’s sarcoma.
Molecular mechanisms of LeTx action
The lethal effects of Bacillus anthracis have
been attributed to an exotoxin that 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 Zn 2+ -metalloprotease which
together with PA forms a toxin referred to as lethal
toxin. 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
20

the LF/MEK interaction that will facilitate the
development of therapeutic agents for anthrax. In
2004, we identified a cluster of surface-exposed
residues of LF that are distal to the catalytic site
and are essential for its catalytic activity (Fig. 1).
Details of this study were published in the Journal
of Biological Chemistry.
Staff notes
Sherrie Boone, who has served as a technician
in the lab since 2001, has left us. She was replaced
in February 2004 by John Young, an M.Sc.
graduate from the University of Oregon. John has
initiated studies of LeTx and Kaposi’s sarcoma
and has played a significant role in our
identification of novel regions of LF that are
required for its activity. Xudong Liang, a
postdoctoral fellow who joined us in 2002, has
moved on to a new position at the University of
Minnesota. In his place we welcome Paul
Spilotro, who joined us in August. Paul is
currently evaluating MEK signaling in human
fibrosarcoma. Elissa Boguslawski and Lisa
Orcasitas joined our team in September. Elissa
will serve as our vivarium technician in charge of
murine studies, including xenografts. Lisa is a
Bridges to the Baccalaureate student and is
currently making sarcoma tissue microarrays so
that she can evaluate MEK signaling in human
tumor samples. In the summer of 2004, the lab
was joined by Mia Hemmes, an undergraduate
student from Michigan State University, who
undertook a cytogenetic analysis of a primary
human sarcoma-derived cell line. As well, we
hosted Ricky Gonzalez and Lynda Gladding, two
Grand Rapids area high school students interested
in careers in biological research. Ricky and Lynda
evaluated the sensitivity of murine endothelial
cells to LeTx.
Figure 1. A surface plot of anthrax LF highlighting mutagenized residues. A space-filled surface plot of LF
was generated using Protein Explorer freeware. Residues identified as being critical for LF activity are colored
yellow (K294), green (L293), red (L514), purple (N516), and orange (R491). Residues found to play a neutral or
marginal role in LF activity are white. The NH 2 -terminus of MEK is indicated in black. A magnified image of this
region shows that the critical residues are organized side-by-side in a focused band (KLLNR), which lies at one end
of the catalytic groove.
21

External Collaborators
Jean-François Bodart, Université des Sciences et Technologies de Lille, France.
Art Frankel, Wake Forest University, Winston-Salem, North Carolina
Silvio Gutkind, National Institute of Dental and Craniofacial Research, Bethesda, Maryland
Stephen Leppla, National Institute of Allergies and Infectious Diseases, Bethesda, Maryland
Recent Publications
Bodart, J.-F., and N.S. Duesbery. In press. Xenopus tropicalis oocytes: more than just a beautiful genome.
In Cell Biology and Signal Transduction, J. Liu, ed. Humana Press.
Singh, Yogendra, Xudong Liang, and Nicholas S. Duesbery. 2005. Pathogenesis of Bacillus anthracis:
the role of anthrax toxins. In Microbial Toxins: Molecular and Cellular Biology, T. Proft, ed.
Norwich, U.K.: Horizon Scientific, pp. 285–312.
Liang, Xudong, John J. Young, Sherrie A. Boone, David S. Waugh, and Nicholas S. Duesbery. 2004.
Involvement of domain II in toxicity of anthrax lethal factor. Journal of Biological Chemistry
279(50): 52473–52478.
From left to right, standing: Lee, Duesbery, Depeille, Young, Cumbo-Nacheli, Spilotro
seated: Holman, Boguslawski, Wagner
22

Microinjection of embryonic stem cells
In these photos, technicians are microinjecting embryonic stem cells into mouse blastocysts for gene targeting studies.
(Photos by Julie Koeman)
23

Vivarium and Laboratory of Transgenics
Bryn Eagleson, A.A., RLATG
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.
Managerial staff
Jason Martin, RLATG
Laboratory Members
Technical staff
Dawna Dylewski, B.S.
Audra Guikema, B.S., L.V.T.
Elissa Boguslawksi, RALAT
Jamie Bondsfield
Sylvia Marinelli
Vivarium staff
Lisa DeCamp, B.S.
Laura Sixburry, B.S.
Crystal Brady
Kathy Geil
Jarred Grams
Tina Schumaker
Research Interests
T
he
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.
Transgenics
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.
24

Standing from left to right: Bondsfield, Sixbury, Grams, DeCamp, Brady, Guikema, Martin
seated from left to right: Schumaker, Dylewski, Marinelli, Eagleson, Boguslawski
25

Bioinformatics Special Program
Kyle A. Furge, Ph.D.
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 and became a Bioinformatics Scientist at VARI in June of 2001.
Laboratory Members
Staff
Karl Dykema, B.A.
Research Interests
Ashigh-throughput biotechnologies such
as DNA sequencing, gene expression
microarrays, and genotyping become
more available to researchers, analysis of the data
produced by these technologies becomes
increasingly difficult. Disciplines such as
bioinformatics and computational biology have
recently emerged to help develop methods that
assist in the storage, distribution, integration, and
analysis of these data sets. The bioinformatics
program at VARI is currently focused on using
computational approaches to understand how
cancer cells differ from normal cells at the
molecular level. In addition, VARI is also part of
the overall bioinformatics effort in the state of
Michigan through the Michigan Center for
Biological Information.
Laboratory members from the bioinformatics
program have worked on a wide variety of projects
to further the research efforts at VARI in 2004.
Recently, we constructed a program to identify
short sequences within genes that are likely to be
responsive to siRNA interference. siRNAs are
short, double-stranded DNA sequences that when
introduced into living cells bind to the RNA
produced from a gene of interest and inhibit
expression of the gene. As such, the introduction
of siRNAs is becoming a more widely used
technique to examine the role of individual genes
in both cancerous and noncancerous cells.
The program we developed contained a new
algorithm, developed by one of the VARI
investigators, to identify potential sites within
genes that would be sensitive to siRNAs. In
another project, we assisted the Microarray
Technology Laboratory in the placement of quality
control markers on gene expression microarrays.
These microarrays contain more than 20,000
unique DNA fragments that are robotically placed
on a small glass slide. In order to ensure the DNA
fragments are placed correctly, the quality control
markers are robotically placed on the arrays in a
very specific pattern as the arrays are constructed.
As each array is produced, a quick visual
inspection of the pattern of quality control markers
can be used to verify that all the DNA fragments
were placed on the arrays correctly.
In addition to assisting other VARI research
labs, our group has a special focus on
understanding how cytogenetic abnormalities
influence cancer development and progression.
Many cancer types, including liver and kidney
cancers, 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.
Interestingly, we and other groups have noticed
that transcription is dramatically disrupted within
regions of DNA copy number change. We are
currently developing and testing a number of
different algorithms to identify these disrupted
regions using gene expression data. In addition,
we are developing methods to identify key
regulatory genes that lie within the abnormal
region and may be involved in tumor development
and/or progression.
26

External Collaborators
Xin Chen, Stanford University, Stanford, California
Recent Publications
Yang, X.J., M.-H. Tan, H.L. Kim, J.A. Ditlev, M.W. Betten, C.E. Png, E.J. Kort, K. Futami, K.J. Dykema,
K.A. Furge, M. Takahashi, H. Kanayama, P.H. Tan, B.S. Teh, C. Luan, et al. In press. A molecular
classification of papillary renal cell carcinoma. Cancer Research.
Dykema, K.J., and K.A. Furge. 2004. Diminished transcription of chromosome 4q is inversely related to
local spread of hepatocellular carcinoma. Genes, Chromosomes and Cancer 41(4): 390–394.
Furge, Kyle A., Kerry A. Lucas, Masayuki Takahashi, Jun Sugimura, Eric J. Kort, Hiro-omi Kanayama,
Susumu Kagawa, Philip Hoekstra, John Curry, Ximing J. Yang, and Bin T. Teh. 2004. Robust
classification of renal cell carcinoma based on gene expression data and predicted cytogenetic
profiles. Cancer Research 64(12): 4117–4121.
Haven, C.J., V.M. Howell, P.H.C. Eilers, R. Dunne, M. Takahashi, M. van Puijenbroek, K. Furge, J. Kievit,
M.-H. Tan, G.J. Fleuren, B.G. Robinson, L.W. Delbridge, J. Philips, A.E. Nelson, U. Krause, et al.
2004. Gene expression of parathyroid tumors: molecular subclassification and identification of the
potential malignant phenotype. Cancer Research 64(20): 7405–7411.
Lindvall, Charlotta, Kyle Furge, Magnus Björkholm, Xiang Guo, Brian Haab, Elisabeth Blennow, Magnus
Nordenskjöld, and Bin Tean Teh. 2004. Combined genetic and transcriptional profiling of acute
myeloid leukemia with normal and complex karyotypes. Haematologica 89(9): 1072–1081.
Sugimura, Jun, Richard S. Foster, Oscar W. Cummings, Eric J. Kort, Masayuki Takahashi, Todd T. Lavery,
Kyle A. Furge, Lawrence H. Einhorn, and Bin Tean Teh. 2004. Gene expression profiling of earlyand
late-relapse nonseminomatous germ cell tumor and primitive neuroectodermal tumor of the testis.
Clinical Cancer Research 10(7): 2368–2378.
Tan, Min-Han, Craig G. Rogers, Jeffrey T. Cooper, Jonathon A. Ditlev, Thomas J. Maatman, Ximing Yang,
Kyle A. Furge, and Bin Tean Teh. 2004. Gene expression profiling of renal cell carcinoma. Clinical
Cancer Research 10(18): 6315S–6321S.
Yang, Ximing J., Jun Sugimura, Maria S. Tretiakova, Kyle Furge, Gregory Zagaja, Mitchell Sokoloff,
Michael Pins, Raymond Bergan, David J. Grignon, Walter M. Stadler, Nicholas J. Vogelzang, and Bin
Tean Teh. 2004. Gene expression profiling of renal medullary carcinoma: potential clinical relevance.
Cancer 100(5): 976–985.
From left to right: Furge, Dykema
27

Laboratory of Cancer Immunodiagnostics
Brian B. Haab, Ph.D.
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.
Staff
Songming Chen, Ph.D.
Michael Shafer, Ph.D.
Sara Forrester, B.S.
Darren Hamelinck, B.S.
Randall Orchekowski, B.S.
Laboratory Members
Students
Thomas LaRoche
Richard Schildhouse
Research Interests
Many cancers are difficult to detect at
early stages and are often diagnosed too
late to allow curative treatment. Earlier
and more accurate detection of cancer could lead
to better outcomes for many patients. A greater
knowledge of the molecular changes associated
with the development of cancer could lead to a
better understanding of disease mechanisms and
improved diagnostic tests. The Haab laboratory
is developing novel experimental approaches to
gather such molecular information and to use it
for the diagnosis of cancer. Our goal is that these
studies will produce measurable benefits to
cancer patients.
We are taking a variety of approaches to
identify changes in blood protein composition that
define cancer and that could be diagnostically
useful. Microarray methods have features that are
particularly useful for this research. We have been
developing several related antibody and protein
microarray methods—such as two-color
competition assays, sandwich assays, glycan
detection, and antigen detection of antibodies—to
analyze serum samples from cancer patients and
controls. With these methods (Fig. 1), we can
efficiently probe the binding to many different
antibodies and proteins and explore the use of
multiple measurements for classifying samples.
Previous technological developments that are now
in routine use include a high-sensitivity detection
method (two-color rolling-circle amplification)
and a new method for isolating multiple
microarrays on a single slide, allowing highthroughput
sample processing. Our research also
makes use of gel and chromatographic separations
and mass spectrometry to provide complementary
experimental information.
An ongoing study of protein profiles from the
sera of pancreatic cancer patients and controls (in
collaboration with Randall Brand and George
Vande Woude) shows the value of antibody
microarrays for diagnostics research. Protein
profiles from antibody microarrays targeting a
wide variety of proteins—such as those previously
associated with cancer, involved in biological
systems altered by cancer, or having elevated levels
in the tumor environment—are revealing many
proteins at either higher or lower abundances in the
cancer patients. Some of the proteins were not
known to be associated with pancreatic cancer and
may contribute to improved early detection of the
disease. The serum samples can be classified as
from cancer or control patients using the antibody
measurements. The accuracy of the classifications
was greatly improved with multiple measurements
in combination (relative to using individual
measurements); we need to further develop this
approach for cancer diagnostics.
A recent modification to this technology
measures alterations in the glycosylation state of
the proteins. Glycosylation—the attachment of
specific carbohydrate structures to proteins—
plays a major role in determining protein function,
and glycosylation alterations have been associated
with the development and progression of cancer.
The ability to conveniently measure changes in
specific carbohydrates on different proteins could
be valuable in identifying the changes most
28

associated with cancer and thus of use for
diagnostics. We have methods for detecting
changes in glycosylation on proteins captured by
antibody microarrays (see Fig. 1C) and are using
those methods to profile the glycosylation
alterations on serum proteins from cancer patients
and control subjects.
We use protein microarrays (see Fig. 1D) to
gather additional information about changes
occurring in the blood of cancer patients. Some
tumor proteins elicit the production of antibodies
targeting those proteins. The identification and
measurement of tumor-recognizing antibodies
could provide information about molecular
alterations in the tumor and be valuable in cancer
detection. The protein microarray is an ideal
screening tool for that purpose. In collaboration
with Gilbert Omenn and Samir Hanash,
microarrays of tumor-derived proteins from
cancer cell lines are probed with sera from cancer
patients to identify proteins recognized by the
patients’ antibodies. An ongoing study of serum
from prostate cancer patients (in collaboration
with Alan Partin) has identified several proteins
that could be involved in an immune response.
We are characterizing the nature of the responses
and the proteins involved.
The above methods may be applied in a novel
way to further their effectiveness. We have begun
to develop and improve existing diagnostic markers
through the use of longitudinal measurements
(serial measurements over time). In a collaboration
with William Catalona, Robert Vessella, and Ziding
Feng, we are looking at changes over time in the
concentrations of several serum proteins leading up
to disease recurrence in prostate cancer patients.
We hypothesize that the use of individualized
thresholds defining abnormal protein levels,
defined by each person’s history of measurements,
will yield improved diagnostic accuracy over the
use of single, population-wide thresholds. Our
hypothesis has been supported in some individual
demonstrations, and we now have an experimental
system for systematically exploring it for a large
number of proteins and many patients.
Figure 1. Antibody and protein microarray
formats. A) Two-color competition assay. Two pools
of proteins, respectively labeled with biotin and
digoxigenin tags, are mixed and co-incubated on
antibody microarrays. The relative amount of binding
to each antibody from the two pools is determined
through detection of the biotin and digoxigenin tags.
B) Sandwich assay. After incubation of a protein
sample on an antibody microarray, the amount of
protein binding to each antibody is measured using a
second antibody that targets the captured proteins.
C) Glycan detection. A pool of digoxigenin-labeled
proteins is incubated on antibody microarrays, and the
level of protein binding at each antibody is determined
by detection of the digoxigenin tag. The amount of a
particular glycan on the captured proteins is detected
using a biotin-labeled protein that specifically binds to
that glycan. D) Protein array detection of antibodies.
Serum samples are incubated on arrays containing a
variety of tumor-derived proteins. Antibodies in the
serum samples that recognize and bind to the
proteins are detected using a secondary antibody that
binds to human antibodies.
We are seeking to apply the methods
described above in ways that will have a positive
result in terms of cancer care. The incorporation
of mass spectrometry methods, through
collaborations with Greg Cavey at VARI and
members of the Michigan Proteome Consortium,
will provide more opportunities for discovery.
The further testing of the value of these tools is
being pursued through local clinical oncology
programs. The access to clinical practice is
especially valuable for translating the
development and discovery in our laboratory into
benefits for cancer patients.
External Collaborators
Phil Andrews, Gilbert Omenn, and Diane Simeone, University of Michigan, Ann Arbor
Randall Brand, Evanston Northwestern Healthcare, Illinois
E. Brian Butler and Bin S. Teh, Baylor College of Medicine, Houston, Texas
William Catalona, John Grayhack, and Anthony Schaeffer, Northwestern University, Evanston, Illinois
29

Jose Costa and Paul Lizardi, Yale University School of Medicine, New Haven, Connecticut
Deborah Dillon, Brigham and Women’s Hospital, Boston, Massachusetts
Ziding Feng and Samir Hanash, Fred Hutchinson Cancer Research Center, Seattle, Washington
Jorge Marrero, University of Michigan Hospital, Ann Arbor
Alan Partin, Johns Hopkins University, Baltimore, Maryland
Peter Schirmacher, University of Cologne, Germany
Robert Vessella, University of Washington, Seattle
Cornelius Verweij, University of Amsterdam, The Netherlands
Recent Publications
Hamelinck, D., H. Zhou, L. Li, Z. Feng, C. Verweij, D. Dillon, J. Costa, and B.B. Haab. In press.
“Optimized normalization for antibody microarrays and the identification of serum protein
alterations associated with pancreatic cancer.” Molecular & Cellular Proteomics.
Haab, B.B., and P.M. Lizardi. In press. “RCA-enhanced protein detection arrays.” Methods in
Molecular Biology.
Breuhahn, Kai, Sebastian Vreden, Ramsi Haddad, Susanne Beckebaum, Dirk Stippel, Peer Flemming,
Tanja Nussbaum, Wolfgang H. Caselmann, Brian B. Haab, and Peter Schirmacher. 2004.
Molecular profiling of human hepatocellular carcinoma defines mutually exclusive interferon
regulation and insulin-like growth factor II overexpression. Cancer Research 64(17): 6058–6064.
Konwinski, R., R. Haddad, J.A. Chun, S. Klenow, S. Larson, B.B. Haab, and L.L. Furge. 2004.
Oltipraz, 3H-1,2-dithiole-3-thione and sulforaphane induce overlapping and protective antioxidant
responses in murine microglial cells. Toxicology Letters 153(3): 343–355.
Lindvall, Charlotta, Kyle Furge, Magnus Björkholm, Xiang Guo, Brian Haab, Elisabeth Blennow,
Magnus Nordenskjöld, and Bin Tean Teh. 2004. Combined genetic and transcriptional profiling
of acute myeloid leukemia with normal and complex karyotypes. Haematologica 89(9):
1072–1081.
Qiu, Ji, Juan Madoz-Gurpide, David E. Misek, Rork Kuick, Dean E. Brenner, George Michailidis,
Brian B. Haab, Gilbert S. Omenn, and Sam Hanash. 2004. Development of natural protein
microarrays for diagnosing cancer based on an antibody response to tumor antigens. Journal of
Proteome Research 3(2): 261–267.
Zhou, Heping, Kerri Bouwman, Mark Schotanus, Cornelius Verweij, Jorge A. Marrero, Deborah
Dillon, Jose Costa, Paul Lizaardi, and Brian B. Haab. 2004. Two-color, rolling-circle
amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements.
Genome Biology 5(4): R28.
From left to right: Haab, Hamelinck, Orchekowski, Chen, Shafer, Forrester
30

Molecular Medicine and Virology Group
Sheri L. Holmen, Ph.D.
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 in the laboratory of Bart Williams from
2000 to 2003 and became a Junior Investigator at VARI in December 2003.
Laboratory Members
Staff
Marleah Russo
Research Interests
T
he
primary focus of the Molecular
Medicine and Virology Group is to
identify molecules that can be effective
targets for cancer therapy, with the goal of
developing better therapies with fewer side effects.
The sequencing of the human genome has yielded
a wealth of biological data, but we still know
relatively little about which genes are causally
associated with tumor development and which are
only markers of cancer. Because of the high cost
of developing new therapies, it is important that we
identify which genetic changes can be
productively targeted. We are concentrating our
initial efforts on melanoma and glioblastoma,
tumors which demonstrate constitutive activation
of Ras signaling.
The RCAS system
We use a series of replication-competent
retroviral vectors based on the SR-A strain of Rous
sarcoma virus (RSV), a member of the avian
leukosis virus (ALV) family, to study the roles of
different genes in tumor initiation and progression.
RSV is the only known naturally occurring,
replication-competent retrovirus that carries an
oncogene, src. In the 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; the gene of
interest is inserted along with an internal promoter.
Higher-titer viruses subsequently have been
generated by replacing the RSV SR-A 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 tissueand
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 of 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, and progression.
The ability to turn off gene expression will help
determine if that gene is a good target for therapy.
31

Melanoma
Melanoma is the most rapidly increasing
malignancy among young people in the United
States. If detected early, the disease is easily
treated, but once the disease has metastasized it
has a high mortality rate. Current systemic
therapy for advanced, metastatic melanoma
includes dacarbazine (DTIC) chemotherapy,
either alone or in combination with other agents,
and biological therapy using recombinant
interferon-α (IFN-α) and/or interleukin-2 (IL-2).
However, except on rare occasions, none of these
treatments has produced long-term control of the
disease, and cytokine therapy is associated with
significant toxicities.
We have developed a mouse model of
melanoma based on the avian RCAS/TVA system.
In this model, the retroviral receptor TVA is
expressed under the control of the tyrosinaserelated
protein 2 (TRP2) promoter, which is
expressed in melanocytes. In replicating
mammalian cells that express TVA, the viral vector
is capable of stably integrating into the DNA and
expressing the experimental gene at high levels,
but the virus is replication-defective because viral
RNA and proteins are inefficiently produced.
Therefore, the viral vectors cannot spread in the
target animals; in addition, since little viral
envelope protein is produced, there is no
interference to superinfection. Theoretically, there
is no limit to the number of experimental genes
that can be introduced into a TVA-expressing
mammalian cell. The ability of these cells to be
infected by multiple viruses allows efficient
modeling of melanoma, because multiple
oncogenic alterations can be introduced into the
same cell or animal without the costs associated
with mating multiple strains of transgenic mice.
Activated ras oncogenes, which turn on
mitogen-activated protein kinase (MAPK)
signaling, are detected in approximately 20% of
human melanomas. Recently, activating mutations
in the B-raf gene, which also activate MAPK
signaling, have been found in more than 65% of
malignant melanomas. With mutually exclusive
mutations in ras and B-raf, the MAPK signaling
pathway is constitutively activated in over 85% of
cases of malignant melanoma, indicating its
importance. Overexpression of activated H-ras
(G12V) specifically in melanocytes of Ink4adeficient
mice results in the development of
multiple spontaneous cutaneous melanomas.
However, unlike in the human disease, these
tumors fail to metastasize. A conditional Ink4a/Arf
knockout allele, Ink4a-lox, has been introduced
into the germline of FVB/N mice. The lox sites
flank exons 2 and 3 of this locus such that Cremediated
excision eliminates both p16 INK4A and
p19 ARF . We have recently obtained two
homozygous Ink4a-lox/lox breeding pairs, which
will be crossed to our TRP2-TVA transgenic mice.
We plan to use the mice to study the role of
different genes in melanoma initiation,
maintenance, and progression.
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. Intracranial infection of Nestin-TVA
mice with RCASBP(A)-Akt and RCASBP(A)-
KRas induces glioblastomas that are histologically
similar to human GBM. We have generated an
RCANBP(A)TRE-KRas virus in which expression
of K-Ras can be controlled post-delivery. We are
using the Nestin-TVA model to test the function of
this virus in vivo. We plan to use these vectors to
determine if K-Ras expression is required for
tumor maintenance in this system and to identify
which genes are appropriate targets for therapy.
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
32

major risk to the food supply and to animal
welfare, especially in today’s high-intensity 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 anti-viral strategies for two
important chicken pathogens, ALV and Marek’s
Disease virus (MDV). 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
Jerry Dodgson, Michigan State University, East Lansing
Henry Hunt and Huanmin Zhang, Avian Disease and Oncology Laboratory, East Lansing, Michigan
Recent Publications
Ai, Minrong, Sheri L. Holmen, Wim van Hul, Bart O. Williams, and Matthew W. Warman. 2005.
Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone
mass–associated missense mutations in LRP5 affect canonical Wnt signaling. Molecular and
Cellular Biology 25(12): 4946–4955.
Holmen, Sheri L., Scott A. Robertson, Cassandra R. Zylstra, and Bart O. Williams. 2005.
Wnt-independent activation of ß-catenin mediated by a Dkk-1-Frizzled 5 fusion protein.
Biochemical and Biophysical Research Communications 328(2): 533–539.
Holmen, Sheri L., Cassandra R. Zylstra, Aditi Mukherjee, Robert Sigler, Marie-Claude Faugere,
Mary Bouxsein, Lianfu Deng, Thomas Clemens, and Bart O. Williams. 2005. Essential role of
ß-catenin in postnatal bone acquisition. Journal of Biological Chemistry 280(22): 21162–21168.
Sanchez-Perez, Luis, Timothy Kottke, Rosa Maria Diaz, Atique Ahmed, Jill Thompson, Heung
Chong, Alan Melcher, Sheri Holmen, Gregory Daniels, and Richard G. Vile. 2005. Potent
selection of antigen loss variants of B16 melanoma following inflammatory killing of
melanocytes in vivo. Cancer Research 65(5): 2009–2017.
Bromberg-White, Jennifer L., Craig P. Webb, Veronique
S. Patacsil, Cindy K. Miranti, Bart O. Williams, and
Sheri L. Holmen. 2004. Delivery of short hairpin
RNA sequences by using a replication-competent
avian retroviral vector. Journal of Virology 78(9):
4914–4916.
Holmen, Sheri L., Troy A. Giambernardi, Cassandra R.
Zylstra, Bree D. Buckner-Berghuis, James H. Resau,
J. Fred Hess, Vaida Glatt, Mary L. Bouxsein,
Minrong Ai, Matthew L. Warman, and Bart O.
Williams. 2004. Decreased BMD and limb
deformities in mice carrying mutations in both Lrp5
and Lrp6. Journal of Bone and Mineral Research
19(12): 2033–2040.
From left to right: Russo, Holmen
33

Laboratory of Integrin Signaling and Tumorigenesis
Cindy K. Miranti, Ph.D.
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, Mass., 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.
Laboratory Members
Staff
Suganthi Chinnaswamy, Ph.D.
Mathew Edick, Ph.D.
Robert Long, B.A.
Veronique V. Schulz, B.S.
Student
Erik Freiter
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 of cancer. Using
tissue culture models, biochemistry, molecular
genetics, and mouse models, we are defining the
cellular and molecular events of integrindependent
adhesion and downstream signaling that
are important in melanoma and prostate
tumorigenesis and metastasis.
Integrins are transmembrane proteins that
serve as receptors for extracellular matrix (ECM)
proteins. By interacting with the ECM, integrins
stimulate intracellular signaling transduction
pathways that regulate cell shape, proliferation,
migration, survival, gene expression, and
differentiation. Integrins do not act autonomously;
they are involved in “crosstalk” with receptor
tyrosine kinases (RTKs) to regulate many cellular
processes. Studies in our lab, for example, indicate
that integrin-mediated adhesion to ECM proteins
activates the epidermal growth factor receptors
EGFR and ErbB2 and the HGF/SF receptor Met.
Integrin-mediated activation of these RTKs is
ligand-independent and is required for 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 U.S. men and the
second leading cause of cancer death in men.
Eighty percent of human prostate tumors arise in the
peripheral zone of the gland and are primarily
confined to the intermediate basal and secretory
epithelial cells. Patients who at the time of diagnosis
have 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 secretory glands, basal
epithelial cells form a contiguous layer adjacent to
the basement membrane. Upon them rests a layer of
secretory luminal cells, forming a stratified
epithelium. The basal cells express a broad
repertoire of integrins, including α2, α3, α6, β1, and
β4. The secretory cells express primarily α6 and β1,
with some α2. In vivo, basal cells secrete and
organize a laminin 5–containing basement
membrane that also contains collagens IV and VII
and laminin 10. The basal cells bind to laminin 5
and collegen VII through α6β4 to form
hemidesmosomal complexes at the basal surface.
Basal cells adhering to this matrix respond to growth
factors secreted by the surrounding stroma,
including EGF and HGF. They proliferate and give
rise to the nonproliferating secretory cells through
34

the generation of an intermediate, transiently
amplifying cell population that has traits of both cell
types. In primary prostate tumors, α6β4 integrin
and its ligands, laminin 5 and collagen VII, are lost.
The tumor cells, unlike normal secretory cells,
develop the ability to adhere, via α2β1 and α6β1, to
an altered basement membrane consisting of
collagen IV and laminin 10. Thus, tumor cells have
characteristics of both basal and secretory cells, but
not all the properties of either.
Whether the tumor cells are derived from the
transient differentiating population of basal cells or
from differentiated secretory cells has not been
unequivocally determined, but it is clear that the
way in which these cells interact with the ECM has
been changed. If tumor cells are derived from
basal cells, they are now interacting with an altered
matrix and using different integrins to engage it. If
they are derived from the secretory cells, the tumor
cells are now engaging a matrix, which they did
not do previously. A fundamental question in our
lab is whether the changes in integrin/matrix
interactions that occur in tumor cells are required
for or help to drive the survival of tumor cells.
The role of integrins and RTKs in
prostate epithelial cell survival
Increased cell survival due to 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 those in prostate
tumor cells. How integrin engagement of different
ECMs regulates survival pathways is not known.
We have recently found that integrin-induced
activation of both EGFR and c-Met in primary
prostate epithelial cells is required for cell survival
in the absence of growth factors. Our goal is to
determine how integrin activation of EGFR and
c-Met regulate cell survival.
We have previously shown that integrin
activation of EGFR is required for integrinmediated
induction of the Ras/Erk and PI3K/Akt
signaling pathways. Recent studies indicate that
integrin activation of Src depends on c-Met.
Interestingly, inhibition of either Src or Ras/Erk
signaling—but not PI3K/Akt signaling—induces
cell death in primary prostate cells even when they
are still adherent to matrix. Together these data
indicate that Src signals generated by c-Met, as well
as Ras/Erk signals generated by EGFR, are driving
cell survival in primary cells (Fig. 1). We are
currently exploring the downstream events that Src
and Erk regulate to maintain cell survival on ECM.
Figure 1. Met and EGFR both act independently
to regulate integrin-mediated survival of primary
prostate epithelial cells. Activation of Src and Erk
through Met and EGFR, respectively, are proposed to
be involved in integrin-mediated survival.
We are also exploring the mechanisms by
which integrins activate EGFR and ErbB2. This
activation is ligand independent, requires only the
cytoplasmic domain of EGFR, and stimulates the
phosphorylation of only a subset of sites on EGFR.
ErbB2 activation depends on EGFR, suggesting
that integrins induce the formation of an
EGFR/ErbB2 heterodimer. Integrin activation of
Src or FAK is not required, but integrin activation
of a phosphatase may be involved; we are currently
investigating the role of several phosphatases.
Integrin and RTK crosstalk in
prostate cancer metastasis
Death from prostate cancer is due to the
development of metastatic disease, which is
difficult to control. The mechanisms involved in
progression to metastatic disease are not
understood. Our approach 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.
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.
35

During prostate cancer progression there is a
shift in the expression of laminin-specific integrins:
β4 integrins are lost, and there is a concomitant
increase in α6β1 and α3β1, which both interact
with CD82/KAI1. We predict that the loss of
CD82/KAI1 alters the function of α6β1 and α3β1.
Using primary prostate epithelial cells, which
express high levels of CD82/KAI1, as well as
several prostate tumor cell lines that do not, we are
exploring the role of CD82/KAI1 in regulating
α6β1- and α3β1-mediated cell adhesion, migration,
and integrin signaling. We have found that
overexpression of CD82/KAI1 in tumor cells
suppresses laminin-specific migration and invasion;
integrin-induced EGFR and c-Met receptor
activation; Src and Lyn activation; and activation of
the Src/Lyn substrates Cas and FAK. Furthermore,
integrin activation of Src is dependent on c-Met, and
laminin-mediated invasion depends on both Src and
c-Met. Together these data indicate that
CD82/KAI1 normally acts to regulate integrin
signaling to c-Met such that upon its loss of
expression in tumor cells, signaling through c-Met
to Src is increased, leading to increased motility and
invasion (Fig. 2). We are currently determining the
mechanism by which CD82/KAI1 down-regulates
c-Met signaling. In reciprocal experiments, we are
inhibiting the expression of CD82/KAI1 in primary
cells using siRNA and mouse models.
Integrin regulation of melanoma
progression by PKC
The incidence of melanoma has been steadily
increasing over the last 10 years. If diagnosed at
an early stage, melanoma is curable, but once it has
become invasive, it progresses very rapidly and is
virtually untreatable. Metastasis and invasion by
tumor cells require the activity of integrins, and in
melanoma, expression of the αvβ3 integrin is
induced during the development of invasive
disease. Therefore, an understanding of how the
αvβ3 integrin functions to regulate invasion will
help our understanding of melanoma metastasis.
We have been focusing our attention on two
signaling molecules, PKCα and Src, both of which
are regulated by the αvβ3 integrin and whose
activities are enhanced in metastatic melanoma.
Adhesion of normal melanocytes to extracellular
matrix induces the formation of focal adhesion
complexes and actin stress fibers. However, in a
highly invasive metastatic melanoma cell line,
C8161.9, these structures are absent. We have
shown that the levels of Src activity and PKCα
protein are elevated in these cells, and
overexpression of PKCα in immortalized normal
melanocytes is sufficient to confer an invasive
phenotype in vitro. We have found that the activity
of Rac, a small GTPase that is required for the
formation of lamellipodia, is elevated in these cells
and that inhibition of PKCα blocks Rac activity.
The activity of the small GTPase Rho, which is
involved in stress fiber and focal adhesion
formation, is negatively regulated by active Src.
Thus PKCα, acting to enhance Rac, and active Src,
acting to inhibit Rho, together drive the formation
of lamellipodia and the dissolution of stress fibers
and focal adhesions, leading to increased motility
(Fig. 3). We are currently exploring how Src is
activated in melanoma cells, as well as the effects
of blocking Src and PKCα expression with siRNA
on migration and invasion.
Figure 2. CD82 reexpression in prostate tumor
cells inhibits invasion by blocking integrinmediated
signaling to Met and Src.
Figure 3. PKCa and Src cooperate to enhance
migration and invasion in melanoma cells by
differentially targeting Rac and Rho.
36

External Collaborators
Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington
Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York
Recent Publications
Sridhar, S.C., and C.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. Totowa, N.J.: Humana Press.
Bill, Heather M., Beatrice Knudsen, Sheri L. Moores, Senthil K. Muthuswamy, Vikram R. Rao, Joan S.
Brugge, and Cindy K. Miranti. 2004. Epidermal growth factor receptor–dependent regulation of
integrin-mediated signaling and cell cycle entry in epithelial cells. Molecular and Cellular Biology
24(19): 8586–8599.
Lee, Chong-Chou, Andrew J. Putnam, Cindy K. Miranti, Margaret Gustafson, Ling-Mei Wang, George F.
Vande Woude, and Chong-Feng Gao. 2004. Overexpression of sprouty-2 inhibits HGF/SF-mediated
cell growth, invasion, migration, and cytokinesis. Oncogene 23(30): 5193–5202.
From left to right: Schulz, Edick, Miranti, Long, Freiter, Sridhar
37

Laboratory of Analytical, Cellular, and Molecular Microscopy
and
Laboratory of Microarray Technology and Molecular Diagnostics
James H. Resau, Ph.D.
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 pathologyrelated
research since 1972. Between 1968 and 1994, he was in the U.S. Army
(active duty and reserve) 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.
Staff
Eric Kort, M.S.
Bree Berghuis, B.S.,
HTL (ASCP), QIHC
Pete Haak, B.S.
Eric Hudson, B.S.
Paul Norton, B.S.
J.C. Goolsby
Laboratory Members
Consulting Veterinarian
Robert Sigler, D.V.M., Ph.D.
Students
Hien Dang
Brandon Leeser
Amy Percival
Huang Tran
Research Interests
T
he
Laboratory of Analytical, Cellular,
and Molecular Microscopy (ACMM)
and the Laboratory of Microarray
Technology and Molecular Diagnostics (MTMD)
are organized and equipped to produce highresolution
images, genomic arrays, and
bioinformatics data that support the cellular and
molecular biology programs of the Institute. Our
laboratories collaborate with the investigators to
improve the understanding, diagnosis, and
characterization of disease, injury, and
differentiation. Although we primarily study
cancer, we work with investigators in and out of
the Institute on a variety of diseases and
processes. As examples, during the past year, we
have used our microscopes to produce intravital
images of the Met protein in living cells and
animals; localized and quantified a unique
transcriptional protein in human cells; worked
closely with VARI’s Bart Williams to characterize
the phenotypic changes in transgenic mice
expressing mutations related to Wnt and the
Lrp5/Lrp6 genes; began to image Art Alberts’
small GTPase proteins in living cells; and
collaborated in studies of the expression of c-Met
in a series of primary human breast cancers.
During 2004, we added two significant
instruments to the ACMM laboratory. The first is
the Aperio Scanscope, which enables us to
digitize a 1 × 3-inch microscope slide in full
color such that it can be analyzed, quantified, and
shared throughout the digital network. The
second is the Ventana automated immunostainer,
which can carry out FISH, ISH, IHC, and
array spotting in a programmed, specific, and
accurate fashion. These two instruments have
allowed us to increase our productivity, shorten
our turnaround time, and improve the quality of
our preparations without any increase in
personnel. In the last calendar year, we
processed 403 requests for histopathologic
services that required 4,100 blocks and more
than 31,600 glass slides. Using the Scanscope
during December 2004, we scanned over 300
cases in high resolution for inclusion in the
VATR database. We anticipate generating digital
Scanscope files for 3,000–4,000 cases in 2005
that will be available on network servers. The
MTMD laboratory is preparing gene expression
data from cells and tissues and is correlating that
with histology, tissue volume, and nuclear
density to determine an effective and accurate
38

screen for applications in molecular diagnostic
assays. We have developed a QC and QA
protocol for evaluating specimens for array
analysis. During the past year we have prepared
1,500 cDNA arrays for 20 collaborators from
both in and outside the Institute.
Our laboratories are primarily responsible for
the archived clinical histopathology program
called the Van Andel Tissue Repository (VATR).
This program allows investigators to use existing
human clinical samples to assess the expression
of proteins. We also use these blocks to prepare a
wide variety of tissue microarrays for research.
We have increased the number of specimens in
VATR to nearly 200,000 tissue samples. We are
continuously entering data from the 200,000
blocks and now have 54,473 reports that further
explain and describe the archives. The reports are
not directly linked to any personal identifiers or
names and meet all HIPAA/CLIA regulations.
During this year we have begun the process of
imaging representative blocks from the cases and
adding demographic data. The material from
future years will have digital information on age,
sex, and diagnosis and will be linked to image
files. These samples will be used in cellular and
molecular protocols approved by our Institutional
Review Board. Since its inception, the VATR
program has been used by 24 registered users
who have submitted 534 requests for searches and
101 subsequent tissue requests.
In collaboration with Rick Hay, we have
augmented the VATR program with freshly frozen
tissues from the tissue acquisition program. This
HIPAA-compliant program involves the active
cooperation of patients, surgeons, and pathologists
from area hospitals, and the surgical tissues
collected are used primarily in cDNA and
Affymetrix gene expression studies. This
collection also provides the participating
physicians with access to research collaborations,
with the aim of facilitating the translation of
research results into clinical practice. The goal of
this project is to develop genetics-based
diagnostic classification of human disease. There
is a Scientific Advisory Board for this project
comprising members of VARI and of the
Spectrum Health pathology, surgical and medical
oncology, and surgery departments.
Original research within our labs focuses on
quantification of images or arrays and the
development of objective measurable data from
images. A recent paper by Rozenblatt-Rosen et
al. used a program written by Eric Kort for
determining the co-localization of pixels that
express unique fluorescent properties as well as
the number of lumens or vessels in IHC-stained
preparations. This is a direct extension of our
Cytometry paper of 2003.
We have recently obtained funding to
advance our understanding of breast cancer in
collaboration with Ilan Tsarfaty, George Vande
Woude, and Craig Webb. We have begun
molecular imaging of breast cancer and
correlation of the findings with Affymetrix gene
expression. Other collaborations within VARI
involve Met and HGF/SF in cells and tissues; the
location of gene-targeted proteins in rodents; and
the evaluation of monoclonal antibodies as
diagnostic reagents. We have been funded by the
Michigan Technology Tri-Corridor to develop
the commercialization of diagnostic gene
expression (collaboration with Bin Teh), imaging
of primary tumors (Rick Hay), and expression of
mRNA in human sperm as an indicator of
fertility or sterility (Stephen Krawetz). We are in
the early stages of developing a program in
neuropathology with a concentration on
Parkinson’s disease, starting with the
morphological and genomic characterization of
human adult stem-cell populations of specific
neurons.
This year we have a student from Bath
University in the U.K. The Bridges to the
Baccalaureate program is a collaboration to
support the recruitment of women and minorities
into science careers; Dr. Resau is a coinvestigator
and the VAI site coordinator for the
program. In addition this year, we partnered with
the Grand Rapids Area Pre-College Engineering
Program (GRAPCEP) to build a school within a
school for science education and instruction in
Creston High School of the Grand Rapids Public
School system. Our GRAPCEP mentorship
program continues to be funded by Pfizer for a
fifth year. Seven high school students have
trained in the laboratory and are now in
baccalaureate programs.
39

External Collaborators
Eric Arnoys and John Ubels, Calvin College, Grand Rapids, Michigan
Stephan Baldus, University of Cologne, Germany
Lonson Barr, Marcos Dantus, and Matti Koeppel, Michigan State University, East Lansing
Nadia Harbeck, Ludwig-Maximilians-Universität, Munich, Germany
Christine Hughes and O. Orit Rosen, Harvard University, Cambridge, Massachusetts
Sylvia Kachalsky, Linkagene, Lod, Israel
Iafa Keydar, Tel Aviv University, Israel
Stephen Krawetz, Wayne State University, Detroit, Michigan
Ernst Lengyel, University of Chicago, Illinois
Maria Roberts, National Cancer Institute, Frederick, Maryland
Rulong Shen, Ohio State University, Columbus
Ilan Tsarfaty, Tel Aviv University, Israel
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.
Lengyel, Ernst, Dieter Prechtel, James H. Resau, Katja Gauger, Anita Welk, Kristina Lindemann,
Georgia Salanti, Thomas Richter, Beatrice Knudsen, George F. Vande Woude, and Nadia Harbeck.
2005. c-Met overexpression in node-positive breast cancer identifies patients with poor clinical
outcome independent of Her2/neu. International Journal of Cancer 113(4): 678–682.
Rozenblatt-Rosen, Orit, Christina M. Hughes, Suraj J. Nannepaga, Kalai Selvi Shanmugam, Terry D.
Copeland, Tad Guszczynski, James H. Resau, and Matthew Meyerson. 2005. The parafibromin
tumor suppressor protein is part of a human Paf1 complex. Molecular and Cellular Biology 25(2):
612–620.
Seals, Darren F., Eduardo F. Azucena, Jr., Ian Pass, Lia Tesfay, Rebecca Gordon, Melissa Woodrow,
James H. Resau, and Sara A. Courtneidge. 2005. The adaptor protein Tks5/Fish is required for
podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer
Cell 7(2): 155–165.
Akervall, Jan, Xiang Guo, Chao-Nan Qian, Jacqueline Schoumans, Brandon Leeser, Eric Kort,
Andrew Cole, James Resau, Carol Bradford, Thomas Carey, Johan Wennerberg, Harald Anderson,
Jan Tennvall, and Bin T. Teh. 2004. Genetic and expression profiles of squamous cell carcinoma
of the head and neck correlate with cisplatin sensitivity and resistance in cell lines and patients.
Clinical Cancer Research 10(24): 8204–8213.
Birchenall-Roberts, Maria C., Tao Fu, Ok-sun Bang, Michael Dambach, James H. Resau, Cari L.
Sadowski, Daniel C. Bertolette, Ho-Jae Lee, Seong-Jin Kim, and Francis W. Ruscetti. 2004.
Tuberous sclerosis complex 2 gene product interacts with human SMAD proteins. Journal of
Biological Chemistry 279(24): 25605–25613.
Holmen, Sheri L., Troy A. Giambernardi, Cassandra R. Zylstra, Bree D. Buckner-Berghuis, James H.
Resau, J. Fred Hess, Vaida Glatt, Mary L. Bouxsein, Minrong Ai, Matthew L. Warman, and Bart
O. Williams. 2004. Decreased BMD and limb deformities in mice carrying mutations in both
Lrp5 and Lrp6. Journal of Bone and Mineral Research 19(12): 2033–2040.
Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping Zhao,
Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat Cheah,
Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin
immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research
10(19): 6629–663
40

From left to right: Resau, Goolsby, Haak, Berghuis, Norton, Percival, Hudson, Ferrell
41

Laboratory of Germline Modification
Pamela J. Swiatek, Ph.D., M.B.A.
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.
Staff
Julie Koeman, B.S.
Kellie Sisson, B.S.
Juraj Zahatnansky, B.S.
Laboratory Members
IACUC Coordinator
Kaye Johnson, B.S.
Research Interests
The germline modification lab is a fullservice
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 wellestablished,
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 “knockin”)
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 capability to produce mutant
embryos that have a wild-type placenta using
tetraploid embryo technology. 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
42

and mutant ES cells, develop into mice called
chimeras. The offspring of chimeras that inherit
the mutated gene are heterozygotes, because 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. It
is our future plan that the DNA analysis be fully
automated, with samples moving directly from the
AutoGenprep 960 to a high-throughput
genotyping platform, eliminating the need for
clients to perform this labor-intensive analysis.
The germline modification 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, growtharrested,
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, wholechromosome
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 or 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 the germline modification lab
provides 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. Eight-cell mouse embryos or
mouse sperm can be cryopreserved and stored in
liquid nitrogen; they can be reconstituted by
implantation into the oviducts of recipient mice or
by in vitro fertilization of oocytes, respectively.
The VARI germline modification lab directs
the Michigan Animal Model Consortium
(MAMC) of the Core Technology Alliance (CTA)
Corp. MAMC is one of six collaborative core
facilities located at the University of Michigan,
Michigan State University, Wayne State
University, Kalamazoo Community College, and
VARI, offering research services in proteomics,
structural biology, genomics, bioinformatics,
high-throughput compound screening, and animal
modeling. These labs receive funding from the
Michigan Economic Development Corporation to
efficiently provide mouse modeling services to
researchers studying human diseases and to
promote the commercialization of the core
services in order to stimulate the development of
biomedical research in Michigan.
The MAMC services are classified into three
major categories: mouse model development;
analysis; and maintenance and preservation.
Model development services consist of gene
targeting, transgenic, TVA transgenic, and
xenotransplantation procedures. Analytical
procedures are performed on the animal models to
determine the nature and extent of any phenotypic
consequences in the models and their correlation
with human disease. Mouse analysis consists of
histology, necropsy, veterinary pathology,
cytogenetics, blood chemistry, blood hematology,
and imaging. The DNA isolation service supports
the genotyping analysis of the models, and the
monoclonal antibody core supports the molecular
analysis of the mutant mice and xenotransplantation
models. Finally, the maintenance
and preservation services include a mouse
repository, mouse breeding services, mouse
rederivation, and embryo/sperm cryopreservation.
These services are described more completely on
the MAMC website at .
43

Recent Publications
Robertson, Scott A., Jacqueline Schoumans, Brendan D. Looyenga, Jason A. Yuhas, Cassandra R.
Zylstra, Julie M. Koeman, Pamela J. Swiatek, Bin T. Teh, and Bart O. Williams. 2005. Spectral
karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal
instability in vitro. International Journal of Oncology 26(3): 629–634.
Wu, Lin, Jun Gu, Huadong Cui, Qing-Yu Zhang, Melissa Behr, Cheng Fang, Yan Weng, Kerri
Kluetzman, Pamela J. Swiatek, Weizhu Yang, Laurence Kaminsky, and Xinxin Ding. 2005.
Transgenic mice with a hypomorphic NADPH-cytochrome P450 reductase gene: effects on
development, reproduction, and microsomal cytochrome P450. Journal of Pharmacology and
Experimental Therapeutics 312(1): 35–43.
Graveel, Carrie, Yanli Su, Julie Koeman, Ling-Mei Wang, Lino Tessarollo, Michelle Fiscella, Carmen
Birchmeier, Pamela Swiatek, Roderick Bronson, and George Vande Woude. 2004. Activating Met
mutations produce unique tumor profiles in mice with selective duplication of the mutant allele.
Proceedings of the National Academy of Sciences U.S.A. 101(49): 17198–17203.
From left to right: Swiatck, Koeman, Zahatnansky, Sisson
44

Laboratory of Cancer Genetics
Bin T. Teh, M.D., Ph.D.
Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, 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. Teh’s research mainly focuses on kidney cancer,
and he is currently on the Medical Advisory Board of the Kidney Cancer Association.
He became VARI’s Deputy Director for Research Operations in the fall of 2003 and
was promoted to Distinguished Scientific Investigator in 2005.
Staff
Miles Chao-Nan Qian, M.D., Ph.D.
Pengfei Wang, M.D., Ph.D.
Xin Yao, M.D., Ph.D.
Jindong Chen, Ph.D.
Kunihiko Futami, Ph.D.
Laboratory Members
Sok Kean Khoo, Ph.D.
Douglas Luccio-Camelo, Ph.D.
David Petillo, Ph.D.
Eric Kort, M.S.
Stephanie Potter, M.S.
Jeff Bates, B.S.
Timothy Yaw Bediako, B.S.
Mark Betten, B.S.
Aaron Massie, B.S.
Research Interests
Kidney cancer, or renal cell carcinoma
(RCC), is the tenth most common
cancer in the United States (34,000 new
cases and more than 12,000 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 these genes
function and interact in giving rise to the tumors
and their progression; 2) to identify and develop
novel biomarkers and key drug targets; and 3) to
generate 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 700
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, and generation of RCC mouse models.
Hereditary RCC syndromes – positional
cloning and functional studies
Following the identification of the HRPT2,
BHD, and two familial RCC breakpoint genes
(NORE1 and LSAMP) by us and others, we are
now focusing on functional studies of these genes,
including generating mouse models that harbor
mutations of these genes (see below). We
established the role of HRPT2 in parathyroid
carcinoma by mutation analysis and
immunohistochemical staining. We have shown
that its protein, parafibromin, accumulates
predominantly in the nucleus, and this nuclear
localization is essential to maintaining its antiproliferative
function.
Microarray gene expression profiling
and bioinformatics
Based on microarray profiling of 400 kidney
tumors using both our own spotted arrays and
Affymetrix microarrays, we have identified the
molecular signatures for 1) different subtypes of
kidney tumors; 2) the prognosis for clear cell RCC
and papillary RCC; and 3) the prediction of drug
response (immunotherapy). Our studies have been
validated by RT-PCR and immunohistochemical
staining. We have also been working closely with
VARI’s Kyle Furge in analyzing our microarray
data. Using a program developed by his team,
comparative genomic microarray analysis
(CGMA), we correlated gene expression profiles
with the predicted chromosomal imbalances for
different RCC histological subtypes, which may
facilitate our efforts in identifying RCC-related
genes from these chromosomal regions. More
information and details can be found in several of
our review articles and book chapters.
45

Mouse models of kidney cancer
In collaboration with VARI’s Bart Williams
and Pam Swiatek, we have set up several mouse
models for kidney tumors. These include
conventional and conditional knock-outs for
BHD, HRPT2, VHL, PTEN, and APC. For the
latter three cases, we collaborated with Peter
Igarashi of the University of Texas Southwestern
Medical Center and obtained from him
Ksp1.3/Cre transgenic mice expressing Cre
recombinase exclusively in the kidney and
developing GU tract, which can mediate
epithelial-specific Cre/lox recombination in these
tissues. We then crossed these mice with the
APC-, VHL-, and PTEN-floxed mice. To date, we
have completed the studies on APC conditional
knock-outs, which give the phenotype of
polycystic kidney disease and renal adenoma.
We are currently characterizing the phenotypes of
the other mice. In addition, we have established
xenograft subcapsular models using five different
RCC cell lines. The natural course of these
models has been documented and studied by
microarrary gene expression profiling.
External Collaborators
We have extensive collaborations with researchers and clinicians in the United States and overseas.
Recent Publications
Morris, M.R., D. Gentle, M. Abdulrahman, E.N. Maina, K. Gupta, R.E. Banks, M.S. Wiesener, T.
Kishida, M. Yao, B. Teh, F. Latif, and E.R. Maher. In press. Tumor suppressor activity and
epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2 (HAI-2/SPINT2) in
papillary and clear cell renal cell carcinoma. Cancer Research.
Takahashi, M., X.J. Yang, S. McWhinney, N. Sano, C.H. Eng, S. Kagawa, B.T. Teh, and H. Kanayama.
In press. cDNA microarray analysis assists in diagnosis of malignant intrarenal pheochromocytoma
originally masquerading as a renal cell carcinoma. Journal of Medical Genetics.
Tretiakova, M., M. Turkyilmaz, T. Grushko, M. Kocherginsky, C. Rubin, O. Olopade, B.T. Teh, and
X.J. Yang. In press. Topoisomerase II[α] expression in Wilms tumors. Clinical Cancer Research.
Wang, P., M.-H. Tan, C. Zhang, H. Morreau, and B.T. Teh. In press. HRPT2, a tumor suppressor gene
for hyperparathyroidism-jaw tumor syndrome. Hormone and Metabolic Research.
Yang, X.J., M.-H. Tan, H.L. Kim, J.A. Ditlev, M.W. Betten, C.E. Png, E.J. Kort, K. Futami, K.J.
Dykema, K.A. Furge, M. Takahashi, H. Kanayama, P.H. Tan, B.S. Teh, C. Luan, et al. In press.
A molecular classification of papillary renal cell carcinoma. Cancer Research.
Cardinal, J., L. Bergman, N. Hayward, A. Sweet, J. Warner, L. Marks, D. Learoyd, T. Dwight, B.
Robinson, M. Epstein, M. Smith, B.T. Teh, D. Cameron, and J. Prins. 2005. A report of a national
mutation testing service for the MEN1 gene: clinical presentations and implications for mutation
testing. Journal of Medical Genetics 42(1): 69–74.
Chuang, S.T., P. Chu, J. Sugimura, M.S. Tretiakova, V. Papavero, K. Wang, M.-H. Tan, F. Lin, B.T.
Teh, and X.J. Yang. 2005. Overexpression of glutathione S-transferase α in clear cell renal cell
carcinoma. American Journal of Clinical Pathology 123(3): 421–429.
Lee, Youn-Soo, Alexander O. Vortmeyer, Irina A. Lubensky, Timothy W.A. Vogel, Barbara Ikejiri,
Sophie Ferlicot, Gérard Benoît, Sophie Giraud, Edward H. Oldfield, W. Marston Linehan, Bin T.
Teh, Stéphane Richard, and Zhengping Zhuang. 2005. Coexpression of erythropoietin and
erythropoietin receptor in Von Hippel-Lindau disease–associated renal cysts and renal cell
carcinoma. Clinical Cancer Research 11(3): 1059–1064.
Qian, Chao-Nan, Jared Knol, Peter Igarashi, Fangmin Lin, Uko Zylstra, Bin Tean Teh, and Bart O.
Williams. 2005. Cystic renal neoplasia following conditional inactivation of Apc in mouse renal
tubular epithelium. Journal of Biological Chemistry 280(5): 3938–3945.
Robertson, Scott A., Jacqueline Schoumans, Brendan D. Looyenga, Jason A. Yuhas, Cassandra R.
Zylstra, Julie M. Koeman, Pamela J. Swiatek, Bin T. Teh, and Bart O. Williams. 2005. Spectral
46

karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals
chromosomal instability in vitro. International Journal of Oncology 26(3): 629–634.
Rogers, C., M.-H. Tan, and B.T. Teh. 2005. Gene expression profiling of renal cell carcinoma and
clinical implications. Urology 65(2): 231–237.
Schoumans, Jacqueline, Ann Nordgren, Claudia Ruivenkamp, Karen Brøndum-Nielsen, Bin Tean Teh,
Göran Annéren, Eva Holmberg, Magnus Nordenskjöld, and Britt-Marie Anderlid. 2005.
Genome-wide screening using array-CGH does not reveal microdeletions/microduplications in
children with Kabuki syndrome. European Journal of Human Genetics 13(2): 260–263.
Takahashi, Masayuki, Veronica Papavero, Jason Yuhas, Eric Kort, Hiro-omi Kanayama, Susumu Kagawa,
R.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.
Akervall, Jan, Xiang Guo, Chao-Nan Qian, Jacqueline Schoumans, Brandon Leeser, Eric Kort,
Andrew Cole, James Resau, Carol Bradford, Thomas Carey, Johan Wennerberg, Harald Anderson,
Jan Tennvall, and Bin T. Teh. 2004. Genetic and expression profiles of squamous cell carcinoma
of the head and neck correlate with cisplatin sensitivity and resistance in cell lines and patients.
Clinical Cancer Research 10(24): 8204–8213.
Atkins, Michael B., David E. Avigan, Ronald M. Bukowski, Richard W. Childs, Janice P. Dutcher, Tim
G. Eisen, Robert A. Figlin, James H. Finke, Robert C. Flanigan, Daniel J. George, S. Nahum
Goldberg, Michael S. Gordon, Othon Iliopoulos, William G. Kaelin, Jr., W. Marston Linehan, et
al. 2004. Innovations and challenges in renal cancer: consensus statement. Clinical Cancer
Research 10(18, Pt.2): 6277S–6281S.
Calender, A., C. Morrison, P. Komminoth, J.Y. Scaoazec, K. Sweet, and B.T. Teh. 2004. Multiple
endocrine neoplasia type 1. In Pathology and Genetics of Tumours of the Endocrine Organs,
DeLellis, Heitz, Lloyd, and Eng, eds. WHO Classification of Tumours series, Vol. 8. Lyon,
France: IARC Press, pp. 218–227.
Furge, Kyle A., Kerry A. Lucas, Masayuki Takahashi, Jun Sugimura, Eric J. Kort, Hiro-omi
Kanayama, Susumu Kagawa, Philip Hoekstra, John Curry, Ximing J. Yang, and Bin T. Teh. 2004.
Robust classification of renal cell carcinoma based on gene expression data and predicted
cytogenetic profiles. Cancer Research 64(12): 4117–4121.
Haven, Carola J., Viive M. Howell, Paul H.C. Eilers, Robert Dunne, Masayuki Takahashi, Marjo van
Puijenbroek, Kyle Furge, Job Kievit, Min-Han Tan, Gert Jan Fleuren, Bruce G. Robinson, Leigh
W. Delbridge, Jeanette Philips, Anne E. Nelson, Ulf Krause, et al. 2004. Gene expression of
parathyroid tumors: molecular subclassification and identification of the potential malignant
phenotype. Cancer Research 64(20): 7405–7411.
Lindvall, Charlotta, Kyle Furge, Magnus Björkholm, Xiang Guo, Brian Haab, Elisabeth Blennow,
Magnus Nordenskjöld, and Bin Tean Teh. 2004. Combined genetic and transcriptional profiling of
acute myeloid leukemia with normal and complex karyotypes. Haematologica 89(9): 1072–1081.
Luccio-Camelo, Douglas C., Karina N. Une, Rafael E.S. Ferreira, Sok Kean Khoo, Radoslav Nickolov,
Marcello D. Bronstein, Mario Vaisman, Bin Tean Teh, Lawrence A. Frohman, Berenice B.
Mendonca, and Monica R. Gadelha. 2004. A meiotic recombination in a new isolated familial
somatotropinoma kindred. European Journal of Endocrinology 150(5): 643–648.
Marsh, Deborah J., Hans Morreau, and Bin T. Teh. 2004. HRPT2 and parathyroid cancer. Lancet
Oncology 5(2): 78.
Morrison, C., K. Sweet, and B.T. Teh. 2004. Hyperthyroidism-jaw tumor syndrome. In Pathology
and Genetics of Tumours of the Endocrine Organs, DeLellis, Heitz, Lloyd, and Eng, eds. WHO
Classification of Tumours series, Vol. 8. Lyon, France: IARC Press, pp. 228–237.
Patton, Kurt T., Maria S. Tretiakova, Jorge L. Yao, Veronica Papavero, Lei Huo, Brian P. Adley, Guan
Wu, Jiaoti Huang, Michael R. Pins, Bin T. Teh, and Ximing J. Yang. 2004. Expression of RON
proto-oncogene in renal oncocytoma and chromophobe renal cell carcinoma. American Journal
of Surgical Pathology 28(8): 1045–1050.
47

Stephens, P., C. Hunter, G. Bignell, S. Edkins, H. Davies, J. Teague, C. Stevens, S. O’Meara, R. Smith,
A. Parker, A. Barthorpe, M. Blow, L. Brackenbury, A. Butler, O. Clarke, et al. 2004. Lung cancer:
intragenic ERBB2 kinase mutations in tumours. Nature 431(7008): 525–526.
Sugimura, Jun, Richard S. Foster, Oscar W. Cummings, Eric J. Kort, Masayuki Takahashi, Todd T.
Lavery, Kyle A. Furge, Lawrence H. Einhorn, and Bin Tean Teh. 2004. Gene expression profiling
of early- and late-relapse nonseminomatous germ cell tumor and primitive neuroectodermal tumor
of the testis. Clinical Cancer Research 10(7): 2368–2378.
Sugimura, Jun, Ximing J. Yang, Maria S. Tretiakova, Masayuki Takahashi, Eric J. Kort, Barbara
Fulton, Tomoaki Fujioka, Nicholas J. Vogelzang, and Bin Tean Teh. 2004. Gene expression
profiling of mesoblastic nephroma and Wilms tumors—comparison and clinical implications.
Urology 64(2): 362–368.
Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping Zhao,
Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat Cheah,
Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin
immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research
10(19): 6629–6637.
Tan, Min-Han, Craig G. Rogers, Jeffrey T. Cooper, Jonathon A. Ditlev, Thomas J. Maatman, Ximing
Yang, Kyle A. Furge, and Bin Tean Teh. 2004. Gene expression profiling of renal cell carcinoma.
Clinical Cancer Research 10(18): 6315S–6321S.
Tan, M.-H., and B.T. Teh. 2004. Renal neoplasia in the hyperparathyroidism-jaw tumor syndrome.
Current Molecular Medicine 4(8): 895–897.
Teh, B.T. 2004. Gene expression profiling begins to fulfill promise in differential diagnosis and
prognosis of renal cell carcinomas. Kidney Cancer Journal 2(3): 15–18.
Standing, left to right: Qian, Wang, Massie, Petillo, Bates, Bediako, Chen
seated, left to right: Betten, Khoo, Antio, Potter, Teh, Futami
48

Laboratory of Molecular Oncology
George F. Vande Woude, Ph.D.
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
George Vande Woude, Ph.D.
Rick Hay, Ph.D., M.D.
Yu-Wen Zhang, M.D., Ph.D.
Chongfeng Gao, Ph.D.
Carrie Graveel, Ph.D.
Laboratory Members
Sharon Moshkovitz, Ph.D.
Qian Xie, Ph.D.
Dafna Kaufman, M.Sc.
Lia Tesfay, M.S.
Matt VanBrocklin, M.S.
Benjamin Staal, B.S.
Yanli Su, A.M.A.T.
Visiting Scientists
Galia Tsarfaty, M.D.
Ilan Tsarfaty, Ph.D.
Student
Jack DeGroot
Research Interests
Activating mutations in Met are found in
human kidney and gastric cancers,
providing compelling genetic evidence
that Met is an important oncogene
(http://www.vai.org/vari/metandcancer/). To study
how activating mutations are involved in tumor
development, we generated mice bearing Met
with mutations representing both inherited and
sporadic mutations found in human cancers. The
different mutant Met lines developed unique
tumor profiles including carcinomas, sarcomas,
and lymphomas. Cytogenetic analysis of the
tumors in Met mutant mice shows that in all cases
amplification of the mutant met allele is observed.
Selective chromosomal amplification is also
found in patients with renal cancer, indicating
that amplification of the mutant met allele may be
required for tumor progression.
The differences in tumor types and latency,
depending on the mutation, may be due to
signaling differences triggered by the specific
mutation. Studies are underway to identify the
signaling pathways selectively activated by these
mutations. Understanding the signaling specificity
of these mutations is essential to identifying and
developing successful therapeutics. Our mutant
mice provide a valuable model for testing Met
inhibitors and for understanding the molecular
events critical for Met-mediated tumorigenesis.
Proliferation and invasion
We are interested in how HGF/SF-induced
proliferation and invasion contribute to tumor
progression. We have established in vitro methods
to select highly proliferative or invasive cell
populations that may mimic the in vivo process of
clonal selection during tumor progression. Our
studies show that most tumor cells display both
invasive and proliferative phenotypes and that they
can reversibly change from invasive to proliferative
phenotypes. We are currently studying the genetic
and epigenetic factors that determine the different
cellular responses.
HGF/SF-Met-mediated tumor angiogenesis
HGF/SF acts as an angiogenic switch by
simultaneously up-regulating vascular endothelial
growth factor (VEGF) and down-regulating
thrombospondin 1 (TSP-1) expression in the same
tumor cells, mediated through the MAPK
pathway. In addition, HGF/SF also downregulates
TSP-1 expression in normal human
umbilical vascular endothelial cells, in which
VEGF expression is undetectable. TSP-1 and
inhibitors of VEGF (such as Avastin) have been
shown to inhibit tumor angiogenesis and growth.
Our data raise the question of whether TSP-1 in
combination with VEGF inhibitors would
enhance inhibition of HGF/SF-Met-mediated
49

tumor angiogenesis and growth relative to each
tested separately, and whether these inhibitors are
better than those specifically directed against
HGF/SF or Met receptor. We are testing these
approaches.
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. Our data strongly
suggest that this immunocompromised strain will
be a useful tool for investigating the role of Met
in tumor malignancy. Currently, we are testing
experimental metastases and 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.
Geldanamycin inhibits tumor cell
invasion at femtomolar concentrations
Our lab has been studying the mechanism of
geldanamycin (GA) inhibition of urokinase
activation of plasmin from plasminogen (uPA).
Previously, we have shown that a subset of GA
derivatives at femtomolar concentrations (fM-
GAi) inhibit HGF/SF-induced activation of
plasmin in canine MDCK cells. We have
recently found that such inhibition also occurs in
several human glioblastoma cell lines (DBTRG,
U373, and SNB19) and in SK-LMS-1 human
leiomyosarcoma cells. Curiously, fM-GAi drugs
only inhibit HGF/SF-induced uPA activity, and
only when the magnitude of HGF/SF-uPA
induction is above 1.5 times basal uPA activity.
These fM-GAi derivatives also block MDCK cell
scattering and glioblastoma tumor cell invasion
in vitro at concentrations well below those
required to exhibit a measurable effect on Met
expression. Other experiments using radicicol
and macbecin II provide circumstantial evidence
for a novel non-HSP90 molecular target that is
involved in HGF/SF-mediated tumor cell
invasion.
Imaging of Met oncogene activation
We have generated mice carrying a murine
GFP-Met transgene that emits intense
fluorescence to reveal where in the animal Met is
expressed. Five founder mice were selected that
had GFP-Met expression levels from high to
moderate. All of the transgenic GFP-Met male
mice—but no females—develop tumors in the
preputial sebaceous glands. Moreover, mice
expressing the highest levels of the GFP-Met
transgene develop tumors earlier. However,
tumors originating from the different founder
lines had similar pathological phenotypes; the
mice presented with cystic sebaceous tumors
having a rapid growth rate. GFP-Met expression
is always higher in the sebaceous gland tumors
relative to normal skin, with tumors covering the
spectrum of adenomas, adenocarcinomas, and
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 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.
MAPK in melanoma
Constitutive activation of MAPK signaling
contributes to many human cancers, including
melanoma, with activating mutations in Nras or
BRAF found in 80% of the tumors. While most
cancer cells exhibit a cytostatic response to
disruption of MAPK signaling, melanomas show
a cytotoxic response. The Koo laboratory
showed that inhibition of MAPK signaling
efficiently triggers cell death by apoptosis in
human melanoma cells. Normal epidermal
melanocytes, however, do not undergo apoptosis
in response to MAPK inhibition, but arrest in the
G1 phase of the cell cycle. Moreover, in vivo
interference of MAPK signaling produces either
significant or complete regression of human
melanoma xenograft tumors in athymic nude
mice. These results indicate that the MAPK
pathway represents tumor-specific survival
signaling in melanoma, and that inhibition of this
pathway may be a therapeutic strategy.
50

Nuclear oncology
During the past year our laboratory has made
progress in the area of nuclear oncology on two
fronts: the continued development of full-length
anti-Met monoclonal antibodies (mAbs) for
radioimmunodiagnostic and therapeutic applications
and the new development of genetically
engineered Met-directed antibody fragments.
We call our biological agents that recognize
human Met “MetSeek” and we continue to
evaluate two full-length mAbs, Met3 and Met5,
in animal models of cancer. Met3 recognizes an
epitope on the extracellular domain of human
Met, but not on canine Met, whereas Met5
recognizes an epitope common to human and
canine Met. Experiments on the usefulness of
radiolabeled Met3 for imaging human
glioblastoma multiforme tumor xenografts
(because of their exquisite sensitivity to
geldanamycins) are in progress. We have a new
collaboration with David Schteingart to study
Met expression by human adrenocortical
carcinomas and to evaluate MetSeek mAbs as
diagnostic tools in this setting. Preliminary
experiments (performed in collaboration with
David Wenkert and Milton Gross) have shown
that radiometal chelation (e.g., with Tc-99m
pertechnetate) may be used instead of direct
radioiodination for labeling MetSeek mAbs. In
collaboration with David Waters, we have
conducted a short-term analysis of nonradioactive
Met5 in normal dogs. No clinical,
hematological, or chemical evidence of toxicity
has been found, and histopathological analysis of
tissues harvested at necropsy is pending.
We have launched a collaborative project to
develop and evaluate genetically engineered
Met-directed antibody fragments as alternatives
to full-length MetSeek mAbs for diagnostic and
therapeutic applications. Single-chain versions
of Met3 and Met5 (scFv) are under construction
at ApoLife, Inc., a biotechnology firm in Detroit,
and we have shown that a new Met-directed
human-Fab-expressing phage display product
can be used to image autocrine human
leiomyosarcoma (SK-LMS-1/HGF) tumor
xenografts in nude mice.
External Collaborators
Milton Gross, Department of Veterans Affairs Healthcare System, Ann Arbor, Michigan
Nadia Harbeck, Technische Universität, Munich, Germany
Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington
Ernest Lengyel, University of Chicago, Illinois
Alnawaz Rehemtulla, Brian Ross, and David Schteingart, University of Michigan, Ann Arbor
Yuehai Shen and David Wenkert, Michigan State University, East Lansing
Olga Volpert, Northwestern University, Evanston, Illinois
David Waters, Gerald P. Murphy Cancer Foundation, West Lafayette, Indiana
Robert Wondergem, East Tennessee State University, Johnson City
Recent Publications
Jiao, Y., P. Zhao, J. Zhu, T. Grabinski, Z. Feng, X. Guan, R.S. Skinner, M.D. Gross, Y. Su, G.F. Vande
Woude, R.V. Hay, and B. Cao. In press. Construction of human naïve Fab library and
characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology.
Gao, Chong Feng, and George F. Vande Woude. 2005. HGF/SF signaling in tumor progression. Cell
Research 15(1): 49–51.
Islam, Azharul, Yoko Sakamoto, Kazuhisa Kosaka, Satoshi Yoshitome, Isamu Sugimoto, Kazuo
Yamada, Ellen Shibuya, George F. Vande Woude, and Eikichi Hashimoto. 2005. The distinct
stage-specific effects of 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid on the activation of
MAP kinase and Cdc2 kinase in Xenopus oocyte maturation. Cellular Signalling 17(4): 507–523.
Lengyel, Ernst, Dieter Prechtel, James H. Resau, Katja Gauger, Anita Welk, Kristina Lindemann,
Georgia Salanti, Thomas Richter, Beatrice Knudsen, George F. Vande Woude, and Nadia Harbeck.
51

2005. c-Met overexpression in node-positive breast cancer identifies patients with poor clinical
outcome independent of Her2/neu. International Journal of Cancer 113(4): 678–682.
Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Margaret Gustafson, Nariyoshi Shinomiya, Ping Zhao,
Brian Cao, Galia Tsarfaty, Ling-Mei Wang, Rick Hay, and George F. Vande Woude. 2005.
Enhanced growth of human Met-expressing xenografts in a new strain of immunocompromised
mice transgenic for human hepatocyte growth factor/scatter factor. Oncogene 24(1): 101–106.
Graveel, Carrie, Yanli Su, Julie Koeman, Ling-Mei Wang, Lino Tessarollo, Michelle Fiscella, Carmen
Birchmeier, Pamela Swiatek, Roderick Bronson, and George Vande Woude. 2004. Activating Met
mutations produce unique tumor profiles in mice with selective duplication of the mutant allele.
Proceedings of the National Academy of Sciences U.S.A. 101(49): 17198–17203.
Kelloff, Gary J., Robert C. Bast, Jr., Donald S. Coffey, Anthony V. D’Amico, Robert S. Kerbel, John
W. Park, Raymond W. Ruddon, Gordon J.S. Rustin, Richard L. Schilsky, Caroline C. Sigman, and
George F. Vande Woude. 2004. Biomarkers, surrogate end points, and the acceleration of drug
development for cancer prevention and treatment: an update. Clinical Cancer Research 10(11):
3881–3884.
Lee, Chong-Chou, Andrew J. Putnam, Cindy K. Miranti, Margaret Gustafson, Ling-Mei Wang,
George F. Vande Woude, and Chong-Feng Gao. 2004. Overexpression of sprouty-2 inhibits
HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene 23(30):
5193–5202.
Shinomiya, Nariyoshi, Chong Feng Gao, Qian Xie, Margaret Gustafson, David J. Waters, Yu-Wen
Zhang, and George F. Vande Woude. 2004. RNA interference reveals that ligand-independent
Met activity is required for tumor cell signaling and survival. Cancer Research 64(21):
7962–7970.
Vande Woude, George F., Gary J. Kelloff, Raymond W. Ruddon, Han-Mo Koo, Caroline C. Sigman,
J. Carl Barrett, Robert W. Day, Adam P. Dicker, Robert S. Kerbel, David R. Parkinson, and
William J. Slichenmyer. 2004. Reanalysis of cancer drugs: old drugs, new tricks. Clinical
Cancer Research 10(11): 3897–3907.
Zhang, Yu-Wen, Carrie Graveel, Nariyoshi Shinomiya, and George F. Vande Woude. 2004.
Met decoys: will cancer take the bait? Cancer Cell 6(1): 5–6.
From left to right, back row: Zhang, Su, Gao, DeGroot, Staal
middle row: Hay, Xie, Tesfay, Kaufman, Moshkovitz
front row: Reed, Vande Woude, Graveel
52

Laboratory of Tumor Metastasis and Angiogenesis
Craig P. Webb, Ph.D.
Dr. Webb received his Ph.D. in cell biology from the University of East Anglia,
England, in 1995. He then served as a postdoctoral fellow in the laboratory 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
Jennifer Bromberg-White, Ph.D.
Jeremy Miller, Ph.D.
David Monsma, Ph.D.
Emily Eugster, M.S.
Sujata Srikanth, M.Phil.
Meghan Sheehan, B.S.
Laboratory Members
Visiting Scientist
Gustavo Cumbo-Nacheli, M.D.
Research Interests
W
e
use model systems ranging from cellbased
assays to preclinical animal
models and clinical subjects to identify
and validate biomarkers and therapeutic targets of
aggressive cancer. The ability to navigate
seamlessly across these diverse model systems
(for example, comparing data from mouse
models to that from human clinical trials) and
between the various molecular platforms is
essential in optimizing the translational research
pipeline (Fig. 1).
translate our investigational findings
into clinical practice, initially in the
areas of improved diagnostics and
pharmacogenomics. Our discoveries
of new, potentially “drugable”
targets is being coupled with siRNA
technologies to verify the functional
validity of targets in preclinical
models. Companion diagnostics are
also being developed that could be
used to assess drug efficacy in
patients. We are beginning to partner
with pharmaceutical companies to
validate new therapeutic areas for
existing drugs, as well as to test
predictions of optimal drug
combinations. At some point in the
near future, accurate diagnosis of
disease will naturally transition to
appropriate treatment.
Our goal is to efficiently
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.
Using a variety of molecular technologies, we
have identified genomic and proteomic correlates
of metastatic disease that may be future
biomarkers and/or molecular targets for diagnosis
Figure 1. Overview of translational research. Commencing with
human research subjects, we can readily transition through specimen
and data collection, data analysis, preclinical models, and ultimately
clinical trials.
53

and treatment. Using our proprietary informatics
system (described below), we have identified
several genes that distinguish normal from
abnormal colon tissue and predict the metastatic
outcome of patients with colorectal cancer.
The potential diagnostic applications of these data
are currently being pursued in a larger cohort of
patients. Our findings to date suggest we can
accurately diagnose colon cancer in pathological
samples and, moreover, predict the likelihood
of metastatic relapse well in advance of
clinical presentation.
In addition, we are using laser capture
microdissection in conjunction with genomic and
proteomic technologies to identify key tumor-host
interactions during metastatic progression, with
emphasis on identifying the molecular factors that
contribute to metastatic dormancy in the liver. A
number of candidate genes are now being pursued
as potential targets of future treatments. For this
purpose, we have developed retroviral and
lentiviral systems for delivering small hairpin RNA
(shRNA) molecules that target candidate genes.
We are knocking-out the expression of several
potential mediators of the metastatic phenotype in
human tumor cell lines and assessing the effects in
orthotopic murine xenografts.
Multiple myeloma
Some 40,000 Americans are living with
multiple myeloma, and deaths are over 11,000 per
year, usually within three years of diagnosis.
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 the generous support
of the McCarty Foundation and Ralph Hauenstein,
we initiated the development of a dedicated
multiple myeloma research laboratory (MMRL).
Our specific goal is to use our unique integrated
approach to identify optimal treatments for
patients. We are collaborating with Keith Stewart
at the Princess Margaret Hospital, Toronto, as well
as with local hematologists, oncologists,
pathologists, and other specialists. Through the
collection of detailed clinical information such as
treatment response (efficacy and toxicity), coupled
with single nucleotide polymorphism, gene
expression, and proteomic analysis of collected
samples, we aim to identify molecular correlates of
therapeutic response and disease progression.
Systems biology
XenoBase (patent pending) is a fully integrated
genomic/proteomic/medical informatics system
that includes analysis and annotation tools. Raw
data from molecular analyses can be associated
with specimens and subjects of interest, and
comparative analysis can be performed on data
across platforms and species. XenoBase allows for
direct correlation between the subject, sample, and
experimental parameters and the molecular data.
Literature-based and gene ontology annotation
software have been incorporated, along with
specific metrics for biomarker and target discovery.
Current therapeutics that specifically target
molecular aberrations can also readily be identified
(Fig. 2). Thus, XenoBase represents an integrated
system for basic bimolecular research, clinical
diagnostics, and new pharmacogenomic strategies
for the future.
XenoBase is written primarily in C#.NET and
uses a rich Windows GUI for robust client
interaction. All statistical algorithms are written
in C++ and optimized for a Windows 32-bit
operating system. This approach combines the
speed of C++ with the easy maintenance of the
.NET framework. The application itself is
structured into four primary layers. The GUI layer
allows for client interaction with the system. The
Controller layer provides the bulk of the code and
is responsible for controlling interactions between
the GUI, the algorithms, and the database. The
Algorithm components are part of the controller
layer but are segmented to work independently
and are optimized for performance using C++.
Finally, the Database layer houses the database
I/O code and “plumbing” necessary to keep the
application database independent and allow the
transactional objects to interact without the
overhead of database-specific calls. The code
itself uses a standard .NET exception handling
framework and is documented internally using
code-blocks and inline comments. The use of
C#.NET allows for the automatic generation of
both an object model and API documentation.
The system was built with expandability in
mind. The layered approach allows new user
interfaces (Internet application, PDA, web
54

Figure 2. Molecular
networks associated
with aggressive
mesothelioma.
This figure shows the
interconnectivity
between genes
associated with rapidly
progressing
mesothelioma.
Mapping genomic
correlates of disease to
highly curated
molecular pathways can
identify the underlying
molecular mechanisms
of the disease. This
information could be
used for diagnostic
applications, as well as
identification of key
steps that may
represent intervention
points for treatment.
Here, EGFR was
identified as a key
convergence point that
appears hyperactivated
in aggressive
mesothelioma. Pathway
mapping was generated
using MetaCore
(GeneGo, Inc., St.
Joseph, MI). For more
information on this tool,
see http://genego.com).
services, etc.) to be developed without
redeveloping core functionality. The .NET
framework provides ready access to development
tools and plug-ins, speeding development of new
features and allowing easy integration with
virtually any software platform. The use of
ADO.NET allows for seamless integration with a
variety of databases, including Microsoft SQL
Server, IBM DB/2, Oracle, and MySQL. Finally,
the system makes use of a database-driven storage
system and is database-independent. Its design
includes scripts for database creation and baseline
data setup and uses ODBC (OLEDB) for rapid
execution and flexible database binding.
External Collaborators
James R. Baker, University of Michigan Health System, Ann Arbor, Michigan
Lonson L. Barr, Michigan State University College of Osteopathic Medicine, Grand Rapids, Michigan
Andrej Bugrim, GeneGo, Inc., St. Joseph, Michigan
Alan D. Campbell, Spectrum Health Cancer Center, Grand Rapids, Michigan
Sandra L. Cottingham, Pamela G. Kidd, Susan M. Mammina, and Timothy J. Pelkey, Spectrum Health,
Pathology & Laboratory Medicine, Grand Rapids, Michigan
Alan T. Davis, Michigan State University and Spectrum Health, Grand Rapids, Michigan
Michael Dobbs, Anthony J. Foster, Pamela Grady, Thomas J. Monroe, Linda C. Pool, Deborah Ritz-
Holland, Marcy Ross, Angela R. Tiberio, and Michael J. Warzynski, Spectrum Health, Grand
Rapids, Michigan
55

Timothy Fitzgerald, St. Mary’s Mercy Medical Center, Grand Rapids, Michigan
Neal Goodwin, ProNAi, Kalamazoo, Michigan
Jason Joseph, Order Streams Management, Inc., Grand Rapids, Michigan
Donald G. Kim and Martin A. Luchtefeld, The Ferguson Clinic, Grand Rapids, Michigan
David E. Langholz, Richard F. McNamara, and Timothy C. Vander Meulen, West Michigan Heart,
Grand Rapids, Michigan
Eric P. Lester, Oncology Care Associates, P.L.L.C., St. Joseph, Michigan
Martin McMahon, University of California, San Francisco
John B. O’Donnell, Grand Rapids Medical Education & Research Center, Grand Rapids, Michigan
Gilbert S. Omenn, University of Michigan Medical School, Ann Arbor, Michigan
Leon Oostendorp, Towers Surgeons, P.C., Grand Rapids, Michigan
Timothy J. O’Rourke, Cancer & Hematology Centers of Western Michigan, P.C., Grand Rapids,
Michigan
Harvey I. Pass, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan
Keith Stewart, McLaughlin Centre for Molecular Medicine, Toronto, Canada
Annette Thelen, Michigan State University, East Lansing
Guenter Tusch, Grand Valley State University, Allendale, Michigan
Recent Publications
Creighton, C.J., J.L. Bromberg-White, D. Misek, D. Monsma, F. Brichory, R. Kuick, T.J. Giordano,
W. Gao, G.S. Omenn, C.P. Webb, and S.M. Hanash. In press. Reciprocal tumor-host
interactions revealed by gene expression profiling of lung adenocarcinoma xenografts.
Molecular Cancer Therapeutics.
Bromann, Paul A., Hasan Korkaya, Craig P. Webb, Jeremy Miller, Tammy L. Calvin, and Sara A.
Courtneidge. 2005. Platelet-derived growth factor stimulates Src-dependent mRNA
stabilization of specific early genes in fibroblasts. Journal of Biological Chemistry 280(11):
10253–10263.
Webb, Craig P., and Harvey I. Pass. 2004. Translational research: from accurate diagnosis to
appropriate treatment. Journal of Translational Medicine 2: 22 pp.
From left to right: Bromberg-White, Monsma, Sheehan, Cumbo-Nacheli, Miller,
Eugster, Srikanth, Webb
56

High-throughput processing of antibody microarrays.
Microscope slides containing 12 (A) or 48 (B) identical microarrays per slide were produced, and each
microarray was incubated with a different sample. The insets show microarrays containing 288 spots (from A)
and 96 spots (from B). The arrays were separated by a hydrophobic wax border that was imprinted onto the
slide using a custom-built device (patent pending). The device uses an interchangable printing cartridge that
can be designed to imprint any user-defined pattern onto a slide. The ability to process multiple samples on one
slide increases the throughput and decreases the cost of the experiments and enables large-scale projects such as
clinical biomarker studies.
(Brian Haab)
57

Laboratory of Chromosome Replication
Michael Weinreich, Ph.D.
Dr. Weinreich received his Ph.D. in biochemistry from the University of
Wisconsin–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
Carrie Gabrielse, B.S.
Jeffrey Kasperski, B.S.
Laboratory Members
Jessica Lanning, B.S.
Students
Charles Miller, B.S.
Amber Crampton
Research Interests
Figure 1. Model for the initiation of DNA replication.
We are studying the initiation of
chromosomal DNA replication in
budding yeast and in human cells. The
initiation of DNA synthesis occurs at multiple
replication origins throughout the genome within
S-phase, but each replication origin is activated
only once per cell cycle. Initiation is precisely
controlled because errors (such as initiating
replication multiple times from a single origin)
would cause genome amplification and instability.
Since DNA replication is essential for cell division
and genome instability is a property of many
cancer cells, we are also investigating the aberrant
regulation of initiation factors in cancer.
Initiation occurs at very well defined
sequences in the budding yeast, so we are using
this organism as a genetic model. Initiation
requires at least three steps (see Fig. 1). The first
is origin marking by ORC, a six-subunit complex
that recognizes conserved DNA sequence elements
in all origins. The second step is the assembly of a
large macromolecular complex called the prereplicative
complex, or pre-RC. ORC, Cdc6p,
Cdt1p, and the MCM complex are together
required to form the pre-RC. Additional proteins
associate with the origin during G1, and then this
large complex of proteins is activated to form bidirectional
replication forks by the Cdc7p-Dbf4p
protein kinase. The process of origin activation is
not understood.
Does chromatin structure affect initiation?
Cdc6p is a critical, limiting factor for assembly
of the pre-RC. We have previously shown that
Cdc6p interacts with ORC and that its essential
activity requires a functional ATP-binding motif. A
cdc6-4 mutant that alters the ATP binding domain is
temperature sensitive (ts), and we have used this
property to isolate suppressors of the cdc6-4 ts.
Loss-of-function alleles within the silent
information regulators (SIR2–4) suppress the cdc6-
4 ts in varying degrees. Deletion of SIR2 gives the
best suppression (Fig. 2). We have also shown that
the loss of SIR2 suppresses several replication
initiation mutants but that its function is originspecific.
Sir2p is a histone
deacetylase required for the
formation of an altered
(“silenced”) chromatin domain at
several transcriptionally silent loci
in the budding yeast. Therefore,
one model to explain our finding
proposes that histone acetylation
stimulates pre-RC assembly. We
are currently investigating the
potential mechanism by which
Sir2p inhibits Cdc6p function and
also how the Sir2p origin
specificity is achieved.
58

CDC7 kinase is required for DNA
replication and repair
Another effort in the lab is to
understand how the two-subunit
Cdc7p-Dbf4p kinase triggers
replication initiation. We have
isolated mutants of the kinase that are
proficient in DNA replication but
defective in DNA repair. Dbf4p is
phosphorylated following inhibition
of DNA replication in a MEC1- and
RAD53-dependent manner and this
phosphorylation appears to inhibit
Cdc7p-Dbf4p kinase activity. MEC1
is a homologue of the human
ATM/ATR (PI 3
-related) kinases that
are key regulators of the response to
DNA damage. RAD53 is the homologue
of the human Cds1/Chk2
checkpoint kinase. Genetic data
suggest that RAD53 is required for
survival of our Cdc7p-Dbf4p kinase
mutants and may promote an
alternative function of this kinase,
i.e., to aid recovery from DNA
damage-induced replication arrest.
Determining how the DNA damage
checkpoint pathway alters the activity
of Cdc7p-Dbf4p kinase or modulates
DNA replication and repair is an
ongoing and exciting area of research.
A.
B.
WT
cdc6-4
cdc6-4 orc1∆N
cdc6-4 sir1∆
cdc6-4 sir2∆
cdc6-4 sir3∆
cdc6-4 sir4∆
25˚C
37˚C
Figure 2. A) Representation of Cdc6p showing the conservation of eight
motifs (II through SensII) within the AAA+ family of ATP binding proteins.
A change of K114A within the “Walker A” ATP binding motif results in the
cdc6-4 ts allele. B) Deletion of SIR2, SIR3, or SIR4 suppresses the
cdc6-4 ts. Serial dilutions of wild-type, cdc6-4, and various cdc6-4
double-mutant strains were spotted onto plates at low and high
temperature to determine growth.
From left to right, standing: Kasperski, Weinreich, Miller; seated: Crampton, Gabrielse
59

Laboratory of Cell Signaling and Carcinogenesis
Bart O. Williams, Ph.D.
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.
Laboratory Members
Staff
Charlotta Lindvall-Weinreich, M.D., Ph.D.
Troy A. Giambernardi, Ph.D.
Katia Bruxvoort, B.S.
Holli Charbonneau, B.S.
Cassandra Zylstra, B.S.
Students
Nicole Evans
Jose Toro
Research Interests
My 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. One of the long-term goals of
our laboratory is to understand how specificity is
generated for the different signaling pathways.
Recently, we have focused on understanding
the role of Wnt signaling in bone formation. We
are interested not only in normal bone
development, but also in whether aberrant Wnt
signaling plays a role in the predisposition of
some common tumor types (for example prostate,
breast, lung, and renal tumors) to metastasize to
and grow in the bone. The long-term goal is to
provide insights that could be used in developing
strategies to lessen the morbidity and mortality
associated with skeletal metastasis.
Wnt signaling in normal bone development
Mutations in the Wnt receptor, Lrp5, have
been causally linked to alterations in human bone
development. We have characterized a mouse
strain deficient in Lrp5 that recapitulates the lowbone-density
phenotype seen in human patients
having this deficiency. We have further shown
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 β-catenin (Fig. 1). To do this,
we created mice carrying an osteoblast-specific
deletion of β-catenin. These mice die within five
weeks of birth due to profound deficiencies in
bone development. A reciprocal experiment, in
which mice missing the Apc gene specifically in
osteoblasts (and therefore expressing elevated
levels of β-catenin), was also performed. These
mutant mice again died very early after birth, and
Figure 1. Bone defects in mice carrying
osteoblast-specific mutations in β-catenin and/or
Apc. Top set of three: micro-CT of femurs from
a) a 30-day-old wild-type mouse, b) a mouse with an
osteoblast-specific deletion of β-catenin or c) a mouse
lacking both Apc and β-catenin in osteoblasts. Lower
two: micro-CT of femurs from d) 12-day-old mice
either wild type or e) carrying an osteoblast-specific
deletion of Apc. Note the presence of severe
osteoporosis in mice lacking β-catenin (b) and
osteopetrosis in those lacking Apc in osteoblasts (e).
The image in (c) demonstrates that loss of β-catenin
is epistatic to the loss of Apc in osteoblasts.
60

they showed a dramatic overgrowth of bone to the
point where very little marrow cavity was present.
Our current work in this project is aimed at
addressing the molecular mechanisms that
underlie these phenotypes. We have isolated
osteoblasts from these mice and are analyzing
them in tissue culture to determine their abilities
to produce and mineralize osteoid. Also, we are
identifying potential downstream mediators of
Wnt signaling in osteoblasts via microarraybased
expression analysis. As a result of this
work (done in collaboration with Tom Clemens),
we found that alterations in Wnt/β-catenin
signaling in osteoblasts led to changes in the
expression of RANKL and osteoprotegerin
(OPG). Consistent with this, histomorphometric
evaluation of bone in the mice with osteoblastspecific
deletions of either Apc or β-catenin
revealed significant alterations in osteoclastogenesis.
We are currently working to address
how other genetic alterations linked to Wnt/βcatenin
signaling affect bone development and
osteoblast function.
Wnt signaling in 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 β-catenin genes, while
in others there is a loss of APC. Two hallmarks
of advanced prostate cancer are skeletal
osteoblastic metastasis and the androgenindependent
survival of tumor cells. The
association of Wnt signaling with bone growth,
plus the fact that β-catenin can bind to the
androgen receptor and make it more susceptible
to activation with steroid hormones other than
dihydrotestosterone, make Wnt signaling an
attractive candidate to explain some phenotypes
associated with advanced prostate cancer.
We have created mice with a prostatespecific
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 are
currently assessing whether these tumors are
androgen-independent, and we are evaluating
whether loss of Apc can synergize with other
genetic alterations to produce higher-grade
prostate carcinoma. It is our ultimate goal to test
whether loss of activation of β-catenin signaling
can lead to a mouse model of prostate cancer
with skeletal metastases.
We are also examining the roles of Lrp5 and
Lrp6 in normal mammary development. We have
found that mice lacking these genes have delays in
normal mammary development. We are currently
assessing the temporal and spatial organization of
Lrp6 and Lrp5 expression during this process.
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. The tumors
that do develop have altered morphology. We are
testing the hypothesis that this inhibition of
tumorigenesis is associated with changes in
mammary stem cells.
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. The repository
includes strains that can be used with the
RCAS/TVA retroviral gene targeting system,
which my laboratory has used to develop more
efficient ways of accurately and efficiently
modeling human diseases in mice.
External Collaborators
Caroline Alexander, University of Wisconsin–Madison
Mary Bouxsein, Beth Israel Deaconess Medical Center, Boston, Massachusetts
Thomas Clemens, University of Alabama–Birmingham
Marie-Claude Faugere, University of Kentucky, Lexington
Peter Igarashi, University of Texas–Southwestern Medical School, Dallas
Michael T. Lewis and Yi Li, Baylor Breast Center, Houston, Texas
Matthew Warman, Case Western Reserve University, Cleveland, Ohio
61

Recent Publications
Ai, Minrong, Sheri L. Holmen, Wim van Hul, Bart O. Williams, and Matthew W. Warman. 2005.
Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone
mass–associated missense mutations in LRP5 affect canonical Wnt signaling. Molecular and
Cellular Biology 25(12): 4946–4955.
Holmen, Sheri L., Scott A. Robertson, Cassandra R. Zylstra, and Bart O. Williams. 2005.
Wnt-independent activation of ß-catenin mediated by a Dkk-1-Frizzled 5 fusion protein.
Biochemical and Biophysical Research Communications 328(2): 533–539.
Holmen, Sheri L., Cassandra R. Zylstra, Aditi Mukherjee, Robert Sigler, Marie-Claude Faugere,
Mary Bouxsein, Lianfu Deng, Thomas Clemens, and Bart O. Williams. 2005. Essential role of
ß-catenin in postnatal bone acquisition. Journal of Biological Chemistry 280(22): 21162–21168.
Qian, Chao-Nan, Jared Knol, Peter Igarashi, Fangmin Lin, Uko Zylstra, Bin Tean Teh, and Bart O.
Williams. 2005. Cystic renal neoplasia following conditional inactivation of Apc in mouse
renal tubular epithelium. Journal of Biological Chemistry 280(5): 3938–3945.
Robertson, Scott A., Jacqueline Schoumans, Brendan D. Looyenga, Jason A. Yuhas, Cassandra R.
Zylstra, Julie M. Koeman, Pamela J. Swiatek, Bin T. Teh, and Bart O. Williams. 2005. Spectral
karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals
chromosomal instability in vitro. International Journal of Oncology 26(3): 629–634.
Holmen, Sheri L., Troy A. Giambernardi, Cassandra R. Zylstra, Bree D. Buckner-Berghuis, James
H. Resau, J. Fred Hess, Vaida Glatt, Mary L. Bouxsein, Minrong Ai, Matthew L. Warman, and
Bart O. Williams. 2004. Decreased BMD and limb deformities in mice carrying mutations in
both Lrp5 and Lrp6. Journal of Bone and Mineral Research 19(12): 2033–2040.
Spike, Benjamin T., Alexandra Dirlam, Benjamin C. Dibling, James Marvin, Bart O. Williams, Tyler
Jacks, and Kay F. Macleod. 2004. The Rb tumor suppressor is required for stress
erythropoiesis. EMBO Journal 23(21): 4319–4329.
From left to right: Williams, Charbonneau, Toro, Zylstra, Giambernardi, Bruxvoort
62

Laboratory of Structural Sciences
H. Eric Xu, Ph.D.
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
Schoen Kruse, Ph.D.
Yong Li, Ph.D.
David Tolbert, Ph.D.
X. Edward Zhou, Ph.D.
Laboratory Members
Jennifer Daughtery, B.S.
Amanda Kovach, B.S.
Kelly Suino, B.S.
Tricia Velting, B.S.
Visiting Scientist
Ross Reynolds, Ph.D.
Research Interests
Our laboratory is using x-ray
crystallography and molecular biology
to study structures and functions of key
protein complexes that are important in basic
biology and in drug discovery relevant to human
diseases such as cancers and diabetes. Currently
we are focusing on nuclear hormone receptors
and the Met tyrosine kinase receptor.
The nuclear hormone receptors
Nuclear hormone receptors form 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 peroxisome proliferator
activators, vitamin D, vitamin A, and thyroid
hormones. These classic receptors are among the
most successful targets in the history of drug
discovery. Almost every one has one or more
synthetic ligands currently being used as
medicines. 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 cardiovascular
disease, diabetes, and cancer.
To understand the molecular basis of ligandmediated
signaling, we have determined crystal
structures of each PPAR’s ligand-binding domain
(LBD) bound to diverse ligands including fatty
acids, the lipid-lowering fibrate drugs, and the
new anti-diabetic drugs called glitazones. We
have also determined crystal structures of these
receptors bound to co-activators or co-repressors.
These structures have provided a framework for
understanding the mechanisms of agonists and
antagonists, as well as the mechanisms for
recruitment of co-activators and co-repressors by
nuclear receptors. We are now developing this
project beyond the structures of the ligandbinding
domains and into defining the structures
of large PPAR fragment/DNA complexes.
The human glucocorticoid receptor
The human glucocorticoid receptor (GR) is a
key regulator of energy metabolism and of
homeostasis of the immune system. The GR is
also a classic target of drug discovery due to its
association with numerous pathological
conditions. There are more than 10 GR ligands
(including dexamethasone) that are currently
used for treating such diverse medical conditions
as asthma, allergy, autoimmune diseases, and
cancer. At the molecular level, GR can function
either as a transcriptional activator or repressor.
Both functions are tightly regulated by small
ligands that bind to the GR LBD. To explore the
molecular mechanisms of GR ligand binding and
signaling, we determined a crystal structure of
the GR LBD bound to dexamethasone and a coactivator
motif from TIF2. The structure
revealed a novel LBD-LBD dimer interface, an
unexpected charge clamp responsible for
sequence-specific binding of co-activators, and a
unique ligand-binding pocket that accounts for
63

the specific recognition of diverse GR ligands.
Currently we are studying receptor-ligand
interactions by crystallizing GR with various
steroid or nonsteroid molecules.
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 that alter
the three-dimensional structure of the receptor.
These hormone-independent cells are highly
aggressive and are responsible for most deaths from
prostate cancer. In this project, we are aiming to
determine the structures of the mutated AR proteins
that alter the response to anti-hormone therapy. In
collaboration with Donald MacDonnell, 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 the receptors’
ligand-dependent regulation, and as such, the
LBD has been the focus of intense structural
studies. There are only a few nuclear receptors for
which the LBD structure remains unsolved. In the
past two years, we have focused on structural
characterization of two of these “orphan”
receptors: constitutive androstane receptor (CAR)
and steroidogenic factor-1 (SF-1). The CAR
structure reveals a compact LBD fold that
contains a small pocket only half the size of the
pocket in PXR, a closely related receptor (Fig. 1).
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, more than
twice the size of the pocket in the mouse LRH-1,
a closely related receptor (Fig. 2). The bound
phospholipid is readily exchanged and modulates
SF-1 interactions with co-activators. Mutations
designed to reduce the size of the SF-1 pocket or
to disrupt hydrogen bonds with the phospholipid
Figure 1. Structural comparison of CAR vs. PXR.
The ligand binding pockets are shown as the pink
surface. The AF-2 helix of the receptors is in red and
the co-activator helix is in purple.
abolish SF-1/co-activator 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
receptors and phospholipid signaling pathways.
We can expect more surprises as structural work
continues on the remaining orphan receptors.
The Met tyrosine kinase receptor
Met is a tyrosine kinase receptor that is
activated by hepatocyte growth factor/scatter
factor (HGF/SF). Aberrant activation of the Met
receptor has been linked with the 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
Figure 2. Ribbon representation of the SF-1/
phospholipid/SHP complex in two views separated
by 90˚. SF-1 is in red and the SHP ID1 motif is in
yellow. The bound phospholipid ligand is shown in a
space-filling representation, with carbon, oxygen,
nitrogen, and phosphate depicted as green, red, blue,
and purple, respectively.
64

extracellular domain. The molecular basis of the
Met-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.
External Collaborators
Doug Engel, University of Michigan, Ann Arbor
Ermanno Gherardi, University of Cambridge, U.K.
Steve Kliewer, University of Texas Southwestern Medical Center, Dallas
Millard Lambert, GlaxoSmithKline Inc., Research Triangle Park, North Carolina
Donald MacDonnell, Duke University, Durham, North Carolina
Stoney Simmons, National Institutes of Health, Bethesda, Maryland
Scott Thacher, Orphagen Pharmaceuticals, San Diego, California
Brad Thompson, University of Texas Medical Branch at Galveston
Ming-Jer Tsai, Baylor College of Medicine, Houston, Texas
Recent Publications
Li, Y., M. Choi, K. Suino, A. Kovach, J. Daugherty, S.A. Kliewer, and H.E. Xu. In press. Structural
and biochemical basis for selective repression of the orphan nuclear receptor LRH-1 by SHP.
Proceedings of the National Academy of Sciences U.S.A.
Li, Yong, Mihwa Choi, Greg Cavey, Jennifer Daugherty, Kelly Suino, Amanda Kovach, Nathan C.
Bingham, Steven A. Kliewer, and H. Eric Xu. 2005. Crystallographic identification and
functional characterization of phospholipids as ligands for the orphan nuclear receptor
steroidogenic factor-1. Molecular Cell 17(4): 491–502.
Haffner, Curt D., James M. Lenhard, Aaron B. Miller, Darryl L. McDougals, Kate Dwornik, Olivia R.
Ittoop, Robert T. Gampe, Jr., H. Eric Xu, Steve Blanchard, Valerie G. Montana, Tom G. Consler,
Randy K. Bledsoe, Andrea Ayscue, and Dallas Croom. 2004. Structure-based design of potent
retinoid X receptor α agonists. Journal of Medicinal Chemistry 47(8): 2010–2029.
Suino, Kelly, Li Peng, Ross Reynolds, Yong Li, Ji-Young Cha, Joyce J. Repa, Steven A. Kliewer, and
H. Eric Xu. 2004. The nuclear xenobiotic receptor CAR: structural determinants of constitutive
activation and heterodimerization. Molecular Cell 16(6): 893–906.
From left to right: Li, Zhou, Tolbert, Daugherty, Xu, Velting, Kruse, Suino, Kovach
65

Laboratory of Mammalian Developmental Genetics
Nian Zhang, Ph.D.
Dr. Zhang received his M.S. in entomology from Southwest Agricultural University,
People’s Republic of China, in 1985 and his Ph.D. in molecular biology from 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 (1997–1999) 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
Wei Ma, Ph.D.
Lisheng Zhang, Ph.D.
Kate Groh, B.S.
Liang Kang
Laboratory Members
Student
William Bond
Research Interests
We are interested in understanding the
cellular and molecular mechanisms
underlying pattern formation during
embryonic development. We previously cloned
and targeted the mouse Lunatic fringe (Lfng) gene,
which plays an important role in embryo
segmentation. Mice homozygous for the Lfng
mutation suffer from severe malformation of their
axial skeleton as a result of irregular somite
formation during embryonic development. Lfng
encodes a secreted signaling molecule essential for
regulating the Notch signaling pathway in mice.
We showed that Lfng expression was in response to
a biological clock that oscillated once during the
formation of each segment, and the failure of the
Lfng mutants in responding to this clock resulted in
the abnormal segmentation phenotype. We want to
understand how the rhythmic expression of Lfng is
controlled. Our recent studies indicate that the
cyclic expression of Lfng is controlled by a
negative feedback loop. The signals transmitted
through the loop are mediated by the components
in the Notch signaling pathway. We found that
Hes7 can directly bind to the N-boxes in the 5′-
regulatory region of the Lfng gene and its own
promoter and repress their transcription in vitro.
Hes7 can also override the Notch-mediated
activation of the Lfng promoter. However, our
results suggest that Hes7 needs a co-factor to
achieve this suppression in vivo. Our data suggests
that when NOTCH1 is modified by LFNG, it
becomes receptive to the signal from DLL3, thus
activating its downstream target. This target
interacts with Hes7 to turn down Hes7 and Lfng.
When the level of HES7 is down, it relieves its
repression on Lfng and its own promoter, and thus
the next cycle begins. We have also demonstrated
that the 3′-untranslated region (UTR) is important
for the rapid degradation of Lfng mRNA, which
ensures accurate oscillation.
Germ cell development
The second focus of our laboratory is on germ
cell development, particularly the mechanisms that
govern germ cell migration, survival,
spermatogonial stem cell renewal, and
differentiation, as well as their implications for
human disease. It is unclear how spermatogonial
stem cells are regenerated during the entire
reproductive life in mammals. Previous studies on
the nematode Caenorhabditis elegans have shown
that the Notch/lin12-mediated signal transduction
pathway is important if germ cells are to remain in
an undifferentiated state. Mutations that
compromise this pathway force germ cells to enter
meiosis earlier than normal. A constitutively
activated signal prevents germ cells from entering
meiosis, resulting in overproliferation of germ
cells, a phenotype called “germ cell tumor.” Given
the fact that some members in the Notch signaling
pathway are expressed in the testis, we speculate
that Notch signaling may play a similar role in
spermatogonial differentiation in mammals. We
will further examine the role Notch signaling may
play during spermatogenesis using transgenic
animals and conditional gene targeting.
66

We are also 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 found that by 12.5
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 sks
gene; this mutation affects normal meiosis in both
sexes. Our results indicate that sks is required for
the metaphase/anaphase transition in meiosis I. In
the sks mutant, homologous chromosomes fail to
separate; therefore, meiosis is stopped at MI. We
have demonstrated that this failure is due to the
inability of the sks mutant to degrade securin in the
primary spermatocytes and possibly in the oocytes.
From left to right, back row: Kang, Ma, L. Zhang, Groh;
front row: N. Zhang, Bond
67

Double staining of protein and RNA in mouse testis
This is a section of mouse testis stained with anti-MPM-2 antibody (green) and an RNA probe (red) to label the
spermatocytes. This image is from studies of germ cell development and the implications for human disease.
(Nian Zhang)
68

Daniel Nathans Memorial Award

Daniel Nathans Memorial Award
The Daniel Nathans Memorial Award was established in memory
of Dr. Daniel Nathans, a distinguished member of our scientific
community and 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.
2004 Award to
Dr. Brian Druker
The 2004 recipient of the Daniel Nathans Memorial Award is Brian Druker, M.D., of the Oregon
Health & Science University Cancer Institute and the Howard Hughes Medical Institute. Dr. Druker’s
research focuses on translating knowledge of the molecular pathogenesis of cancer into specific
therapies, and he is investigating the optimal use of molecularly targeted agents. Dr. Druker will visit
Grand Rapids in September 2005 to deliver two lectures, one directed to a scientific audience and the
second directed to the general public.
Previous Award Recipients
2000 Richard D. Klausner, M.D.
2001 Francis S. Collins, M.D., Ph.D.
2002 Lawrence H. Einhorn, M.D.
2003 Robert A. Weinberg, Ph.D.
71

Postdoctoral Fellowship Program

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 2004 and early 2005 are listed below.
Eduardo Azucena
Wayne State University, Detroit, Michigan
VARI mentor: Sara Courtneidge
Paul Bromann
Northwestern University, Evanston, Illinois
VARI mentor: Sara Courtneidge
Jennifer Bromberg-White
Pennsylvania State University College of
Medicine, Hershey
VARI mentor: Craig Webb
Jun Chen
West China University of Medical Sciences,
Chengdu, China
VARI mentor: Nian Zhang
Yunju Chen
University of Glasgow, U.K.
VARI mentor: Arthur Alberts
Philippe Depeille
University of Montpellier, France
VARI mentor: Nicholas Duesbery
Mathew Edick
University of Tennessee, Memphis
VARI mentor: Cindy Miranti
Kathryn Eisenmann
University of Minnesota, Minneapolis
VARI mentor: Arthur Alberts
Kunihiko Futami
Tokyo University of Fisheries, Japan
VARI mentor: Bin Teh
Chongfeng Gao
Tokyo Medical and Dental University, Japan
VARI mentor: George Vande Woude
Troy Giambernardi
University of Texas Health Science Center, San
Antonio
VARI mentor: Bart Williams
Carrie Graveel
University of Wisconsin – Madison
VARI mentor: George Vande Woude
Holly Holman
University of Glasgow, U.K.
VARI mentor: Arthur Alberts
Hasan Korkaya
International Center for Genetic Engineering
and Biotechnology, New Delhi, India
VARI mentor: Sara Courtneidge
Schoen Kruse
University of Colorado, Boulder
VARI mentor: Eric Xu
Xudong Liang
Qinghai Medical University, Xining, China
VARI mentor: Nicholas Duesbery
Phumzile Loudidi
Cambridge University, England
VARI mentor: Eric Xu
Wei Ma
Chinese Academy of Science, Beijing
VARI mentor: Nian Zhang
75

Donald Pappas, Jr.
Louisiana State University, Baton Rouge
VARI mentor: Michael Weinreich
Ian Pass
University of Dundee, Scotland
VARI mentor: Sara Courtneidge
Michael Shafer
Michigan State University, East Lansing
VARI mentor: Brian Haab
Muthu Shanmugam
National University of Singapore, Singapore
VARI mentor: Brian Haab
Paul Spilotro
St. George University, Grenada
VARI mentor: Nicholas Duesbery
Suganthi Sridhar
Southern Illinois University, Carbondale
VARI mentor: Cindy Miranti
Jun Sugimura
University of Morioka Medical School, Japan
VARI mentor: Bin Teh
Min-Han Tan
National University of Singapore, Singapore
VARI mentor: Bin Teh
Rebecca Uzarski
Michigan State University, East Lansing
VARI mentor: Sara Courtneidge
Pengfei Wang
Fourth Military Medical University, Xian, China
VARI mentor: Bin Teh
Qian Xie
Fudan University, Shanghai, China
VARI mentor: George Vande Woude
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, Birmingham
VARI mentor: Eric Xu
76

Student Programs

Grand Rapids Area Pre-College Engineering Program
The Grand Rapids Pre-College Engineering Program (GRAPCEP) is administered by Davenport
College 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 training in research methods,
the students also learn workplace success skills such as teamwork and leadership. The three 2004
GRAPCEP students were
Lynda Gladding (Resau/Duesbery)
Union High School
Ricky Gonzales (Resau/Duesbery)
Ottawa Hills High School
Mehreteab Mengsteab (Xu)
Creston High School
79

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 May 2004 to March 2005, VARI hosted 44 students from 17 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.
Aquinas College, Grand Rapids, Michigan
Brent Goslin* (Weinreich)
Calvin College, Grand Rapids, Michigan
Arianne Folkema* (Williams)
Jason Koning (Williams)
Cornell University, Ithaca, New York
Susan Kloet (Teh)
Dalhousie University, Nova Scotia
Jasmine Belanger* (Haab)
Grand Rapids Community College, Michigan
Jose Toro (Williams)
Grand Valley State University,
Allendale, Michigan
Timothy Bearup* (Cao)
Jack DeGroot (Vande Woude)
Nicole Evans (Williams)
Erik Freiter (Miranti)
Lisa Orcasitas (Duesbery)
Benjamin Staal* (Vande Woude)
Neil Swanson (Duesbery)
Kelli VanDussen (Weinreich)
Tricia Velting* (Xu)
Grove City College, Pennsylvania
Sarah Feenstra (Vande Woude)
Hope College, Holland, Michigan
Marie Graves (Cavey)
Wendy Johnson (Cavey)
Tom LaRoche (Haab)
Richard Schildhouse (Haab)
Mary VerHeulen (Vande Woude)
Michigan State University, East Lansing
Aaron DeWard (Weinreich/Alberts)
Stephanie Ellison* (Vande Woude)
Mia Hemmes* (Duesbery)
Jennifer Kaufman (Resau)
Yaojian Liu (Alberts)
Charles Miller (Weinreich)
Michigan Technological University, Houghton
Hien Dang (Resau)
Nanjing Medical University, China
Yong-jun Jiao (Cao)
Jin Zhu (Cao)
Xin Wang (Cao)
University of Bath, United Kingdom
William Bond (Zhang)
Katharine Collins (Alberts)
Amber Crampton (Weinreich)
Victoria Hammond (Weinreich)
Amy Percival (Resau)
80

University of Detroit Mercy
Brandon Leeser (Resau)
University of Illinois at Urbana-Champaign
Huang Tran (Resau)
University of Michigan, Ann Arbor
Benjamin Briggs* (Furge)
Michael Wells* (Teh)
Natalie Wolters* (Courtneidge)
University of Richmond, Virginia
Jeremiah NcNamara* (Webb)
Western Michigan University, Kalamazoo
Kenneth Olinger* (Resau)
Nicole Repair* (Miranti)
81

Han-Mo Koo Memorial Seminar Series

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.
2004
January
Karen Vousden, Beatson Institute for Cancer Research, Glasgow, Scotland
“The p53 pathway as a therapeutic target”
Patrick Brophy, University of Michigan, Ann Arbor
“Ureteric budding: controlling two ends of the event”
February
Jeffrey Settleman, Harvard Medical School, Boston, Massachusetts
“Rho GTPase signaling in development”
Josef Prchal, Baylor College of Medicine, Houston, Texas
“Tumors, hypoxia, and polycythemic disorders”
March
Robert Weinberg, Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
The Daniel Nathans Lecture: “Mechanisms of human tumor formation”
“How cancer begins” (lay audience)
Mike Caliguiri, Ohio State University, Columbus
“Natural killer cells: biology and clinical implications”
James Basilion, Massachusetts General Hospital, Charlestown, Massachusetts
“Noninvasive imaging of gene expression: imaging multiple targets simultaneously”
David Frank, Harvard University, Cambridge, Massachusetts
“STAT signal transduction in the pathogenesis and treatment of cancer”
April
Ernst-Robert Lengyel, University of Chicago, Illinois
“Regulation of proteolysis by adhesion receptors”
Kathleen Siminovitch, Mount Sinai Hospital, Toronto, Canada
“The Wiskott-Aldrich syndrome protein: forging the link between actin and T cell activation”
Jose Cibelli, Michigan State University, East Lansing
“Embryonic stem cells by parthenogenesis in mammals”
85

May
Francesco Marincola, National Institutes of Health, Bethesda, Maryland
“Anti-cancer vaccines: from bench to bedside and from bedside to bench”
Robert Gallo, University of Maryland, Baltimore
“HIV in the third decade — some lessons from past experiences and future prospects”
June
Cynthia Wetmore, Mayo Clinic, Rochester, Minnesota
“Implications of Ptc haploinsufficiency for the proliferation and cell fate of neuronal precursors”
July
Arnold Glazier, Drug Innovation and Design, Newton, Massachusetts
“Pattern recognition tumor targeting”
Renata Pasqualini, M.D. Anderson Cancer Center, Houston, Texas
“Translating function protein interactions into targeted therapies for cancer and obesity”
September
Douglas Hanahan, University of California, San Francisco
“Mechanisms of angiogenesis, and anti-angiogenic therapies in mouse models of cancer”
Henry Higgs, Dartmouth Medical School, Hanover, New Hampshire
“Comparative molecular physiology of mammalian formin proteins: potent actin assembly factors”
October
Janice P. Dutcher, Our Lady of Mercy Medical Center, New York
“Clinical features and treatment of metastatic renal cell cancer”
Robert L. Heinrikson, Proteos, Kalamazoo, Michigan
“Innovation in design and production of proteins and peptides — the Proteos approach”
Qing-Xiang Sang, Florida State University, Tallahassee, Florida
“Metalloproteases and nonprotease factors in early stages of tumor invasion: new hypotheses on
the mutated stem cells and cancer drug resistance”
November
John Carpten, Translational Genomics Research Institute, Phoenix, Arizona
“Searching for prostate cancer in tumor suppressor genes”
December
Marcos Dantus, Michigan State University, East Lansing
“The development of future biomedical applications using coherent laser control”
Roland S. Annan, GlaxoSmithKline, King of Prussia, Pennsylvania
“A qualitative and quantitative view of phosphorylation-dependent biological function”
86

2005
January
Ali Shilatifard, St. Louis University, Missouri
“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, Indiana
“Microfluidic technologies for cancer detection and drug delivery”
Paul A. Krieg, University of Arizona, Tucson
“Growth factor regulation of vascular development”
March
Max Wicha, University of Michigan, Ann Arbor
“Stem cells in normal human breast development and cancer”
Judah Folkman, Harvard Medical School, Boston, Massachusetts
“Platelet angiogenic profile as an early biomarker for cancer”
Robert J. Amato, The Methodist Hospital, Houston, Texas
“Therapeutic updates for the management of patients with renal cell carcinoma”
Aaron M. Zorn, Cincinnati Children’s Hospital Research Foundation, Ohio
“Sox17 and β-catenin signaling in development”
April
Martin E. Hemler, Harvard Medical School and Dana-Farber Cancer Institute, Boston,
Massachusetts
“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, Illinois
“Role of c-Met in lung cancer”
87

Organization

Van Andel Research Institute Boards
VARI Board of Trustees
David L. Van Andel, Chairman and CEO
Christian Helmus, M.D.
Fritz M. Rottman, Ph.D.
James B. Wyngaarden, M.D.
David L. Van Andel
Board of Scientific Advisors
The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing
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 Institute’s research programs.
The members are
Alan Bernstein, Ph.D.
Malcolm Brenner, M.D., Ph.D.
Patrick O. Brown, M.D., Ph.D.
Joan Brugge, Ph.D.
Webster Cavenee, Ph.D.
Frank McCormick, Ph.D.
Davor Solter, M.D., Ph.D.
Bruce Stillman, Ph.D.
91

Van Andel Research Institute
Office of the Director
Director
George Vande Woude, Ph.D.
Deputy Director
for Clinical Programs
Deputy Director
for Special Programs
Rick Hay, M.D., Ph.D.
James H. Resau, Ph.D.
Deputy Director
for Research Operations
Director for
Research Administration
Bin T. Teh, M.D., Ph.D.
Roberta Jones
92

Van Andel Research Institute
Administrator to
the Director
Science Editor
Michelle Reed
David E. Nadziejka
Administration Group
From left to right, back row: Dingman, Holman, Stougaard, Ferrell, Carrigan;
front row: Antio, Koo, McGrail, Novakowski
93

Van Andel Institute Administrative Organization
The organizational units listed below provide administrative support to both the Van Andel
Research Institute and the Van Andel Education Institute.
Executive
Steven R. Heacock, Chief Administrative
Officer and General Counsel
R. Jack Frick, Chief Financial Officer
Ann Schoen, Executive Assistant
Communications and Development
Patrick Kelly, Vice President
John Van Fossen
Dianna Davidson
Andrea Nielsen
Margo Pratt
Information Technology
Bryon Campbell, Ph.D.,
Chief Information Officer
David Drolett, Manager
Michael Roe, Manager
Tom Barney
Phil Bott
Kenneth Hoekman
Kimberlee Jeffries
Theo Pretorius
Russell Vander Mey
Candy Wilkerson
Human Resources
Linda Zarzecki, Director
Margie Hoving
Pamela Murray
Angela Plutschouw
Grants and Contracts
Carolyn W. Witt, Director
Rob Junge
Sara O’Neal
David Ross
Finance
Timothy Myers, Controller
Heather Ly, Supervisor
Richard Herrick
Keri Jackson
Angela Lawrence
Susan Raymond
Kevin Tefelsky
Jamie VanPortfleet
Purchasing
Richard Disbrow, Manager
Chris Kutchinski
Amy Poplaski
John Waldon
Facilities
Samuel Pinto, Manager
Jason Dawes
Richard Sal
Richard Ulrich
Security
Kevin Denhof, CPP, Chief
Christen Dingman
Sandra Folino
Emily Young
Glass Washing/ Media Preparation
Heather Frazee
Marlene Sal
Contract Support
Valeria Long, Librarian
(Grand Valley State University)
Jim Kidder, Safety Manager
(Michigan State University)
Raymond Rupp
Patty Sund
Al Troupe
94

95

Van Andel Institute
Van Andel Research Institute
96

Recent VARI Photos

99

100

Back cover photo: Spectral karyotyping of a tumor cell line
Fluorescence microscopy for spectral karyotyping (SKY) analysis of a human tumor cell line. The red light
helps reduce fading in the fluorescent slides, which are light sensitive.
(Julie Koeman)
The Van Andel Institute and/or its affiliated organizations (VARI and VAEI), through its responsible managers,
recruits, hires, upgrades, trains, and promotes in all job titles without regard to race, color, religion, sex,
national origin, age, height, weight, marital status, disability, pregnancy, or veteran status, except where an
accommodation is unavailable and/or it is a bone fide occupational qualification.
Typesetting and printing by RiverRun Press, Parchment, Michigan.