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Leading Edge Research in Cancer Biology, Protein Structure &Bioinformatics

Graduate studies in Medical Biophysics at the University of Toronto


Cell, Molecular
& Structural Biology

Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
Table of Contents
Chairmans Message p. 1
Our Graduate Program p. 1
Cell &Molecular Biology Stream p. 1
The Current Faculty p. 2
Structural Biology Stream p. 3
Research Themes p. 4
Landmark Publications p. 5
Major Funding p. 6
Graduate Admissions p. 6
Where are they now? Recent Ph.D. Graduates p. 7
Faculty &Project Descriptions - Table of Contents p. 8
Faculty &Project Descriptions p. 9
Department of Medical Biophysics
Cell, Molecular &Structural Biology
Our Graduate Student Programme
The Department of Medical Biophysics graduate student
training programme is a vital and dynamic component of our
research environment and dates from the Departments earliest
origins. All of the core scientists hold their primary academic
appointment in the Department of Medical Biophysics, which has
no undergraduate programme; thus, faculty teaching is devoted
entirely to graduate courses and thesis supervision.
Graduate students are selected primarily on the basis of
their academic record and for their potential to become research
scientists. The M.Sc. program is a common entry point but the
emphasis is on reclassification into the Ph.D. programme. Course
work is included in the first two years of graduate study and is
intended to broaden the backgrounds of all students, most of
whom enter with a degree in one of the basic sciences of biomedi-
cine. All Medical Biophysics graduate students are supported by
stipends which are reviewed annually to track inflation, including
that of the graduate tuition fees. The approximately 40% of
students who receive competitive scholarships from granting
agencies receive a top-up which helps offset the increased cost of
living in downtown Toronto. Our graduate students offer an
exemplary record of continued achievement after they leave us: a
partial list of PhD graduates and their current positions can be
found on page 7.
The Cell & Molecular Biology Stream
The University of Toronto is an international leader in
the field of cancer biology. The molecular and cellular biology
division of the Department of Medical Biophysics is at the heart
of this strength. The division derives from the earliest days of the
Ontario Cancer Institute/Princess Margaret Hospital when the
biological research group had a strong focus on studies of
leukemia and normal bone marrow, microbial genetics, oncogenic
viruses and the effects of radiation and chemotherapeutic drugs
on cancers and normal tissues. Studies of the molecular mecha-
nisms responsible for the cellular effects studied in those earlier
days still represent some of the research themes running through
the department and motivating clinical studies within the
hospital. The MCB stream of the department has spread out over
the years to have members
in many of the biomedical
research institutes in
Toronto; in particular there
are major groups of
division members located
at Sunnybrook Health
Sciences Centre and in the
Research Institute of the
Hospital for Sick Children
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Chairman's Message
Welcome to the Department of Medical Biophysics at the University of Toronto. Within these pages you will find information
describing the Department, its research and graduate programmes as well as its history, along with profiles of the Faculty and their
specific interests. This booklet covers our streams in Cell, Molelcular and Structural Biology; another decribes the Imaging Physics
stream. You will find information here on applying to our Department for graduate studies: we encourage you go to our website at
http://medbio.utoronto.ca and to contact the Department Office or the Faculty themselves with questions you may have.
For many undergraduates the first question will be, what is Medical Biophysics? The name reflects our central focus, which is the appli-
cation of research disciplines spanning through biological and physical science to the problems of medicine. Our approach is unique in
many respects and has been driven throughout our 50-year history by our principal research challenge: cancer. In fact, our Department
originated with the Ontario Cancer Institutes original Biology and Physics Divisions and has retained much of these roots: almost all
of our laboratories are in hospital-based institutes and translation of our work into clinical medicine is our shared goal. Our programmes
embrace students from backgrounds in Molecular and Cell Biology, Physiology, Biochemistry and Chemistry as well as Physics,
Mathematics, Engineering, Computer Science and beyond. The diversity of our Faculty and the preponderance of multi-disciplinary
projects reflects these backgrounds. In the descriptions of our laboratories and their projects in this and the companion booklet you will
find projects in tumour biology, radiobiology, membrane function, molecular interactions, gene expression, cell differentiation and
growth control, viral and chemical carcinogenesis, cellular and molecular immunology, hematopoiesis, macromolecular structure, the
physics of radiation therapy and diagnostic imaging, the development of imaging systems involving ultrasound, x-ray, nuclear magnetic
resonance and electron optics, and more. We hope that you will find them as exciting as we do.
Peter N Burns
Chairman of Medical Biophysics
as well as the Ontario Cancer Institute/Princess Margaret
Hospital. The current faculty in the molecular and cellular
biology stream number more than 100, their research spanning a
wide range of cancer biology topics from basic genetics to cell
signaling, to studies in experimental animal models involving
genetically modified mice, to innovative clinical studies of new
drugs and new approaches to assessing treatment efficacy. The
research summaries for the faculty members which follow provide
more information about these topics and illustrate the opportu-
nity for students within the department to benefit from the
breadth of expertise within just one division of the Department.
Over the 50 years since the department was founded in
1958, members of the faculty, postdoctoral fellows and students
within the cell and molecular biology division of the department
have made many seminal contributions to our current under-
standing of the biology of cancer and its treatment including: the
pioneering work in the laboratories of Drs Till and McCulloch on
bone marrow stem cells; the early studies of somatic cell genetics
in the laboratories of Drs Siminovitch, Till and Whitmore that
have subsequently led to the identification of genes involved in
the repair of DNA damage following irradiation or drug treat-
ment; the identification of the first known drug efflux membrane
protein involved in cellular drug resistance (P-glycoprotein) by Dr
Lings laboratory; the identification of the T-cell receptor by Dr
Maks laboratory; the identification of mutations in the Rb
protein responsible for retinoblastoma in the laboratories of Drs
Phillips and Gallie; the first demonstration that exposure to
hypoxia can enhance the metastatic potential of cancer cells in the
laboratory of Dr Hill; the demonstration in clinical studies that a
low fat diet can reduce the onset of breast cancer and that high
breast density on mammograms is a poor prognostic factors for
breast cancer by the group led by Dr Boyd; and the initiation of
studies of metronomic chemotherapy based on studies of the anti-
angiogenic effects of chemotherapeutic drugs in the laboratory of
Dr Kerbel.
Divisional faculty or trainees also play or have played
major roles in the leadership of biomedical research across
Canada. Dr Bernstein is the recently retired president of CIHR,
Drs Branton and Aubin are the current heads of the CIHR
Institutes of Cancer Research and Musculoskeletal Health and
Arthritis respectively; Dr Hudson is the President and Scientific
Director of the new Ontario Institute of Cancer Research and Dr
Philips (a past Chair of Medical Biophysics) its Deputy Director.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Dr. Philip Marsden
Dr. Lisa Martin
Dr. Jane McGlade
Dr. Jeffrey Medin
Dr. Hans Messner
Dr. Mark Minden
Dr. Salomon Minkin
Dr. Benjamin Neel
Dr. Pam Ohashi
Dr. Hitoshi Okada
Dr. Christopher Paige
Dr. Linda Penn
Dr. Mira Puri
Dr. Jonathan Rast
Dr. Robert Rottapel
Dr. Aaron Schimmer
Dr. Andre Schuh
Dr. David Spaner
Dr. Jeremy Squire
Dr. Vuk Stambolic
Dr. Thomas Kislinger
Dr. Emil Pai
Dr. Gil Prive
Dr. Brian Raught
Dr. David Rose
Emeriti
Dr. Arthur Axelrad
Dr. Robert Bruce
Dr. Alistair Cunningham
Dr. Ernest McCulloch
Dr. Richard Miller
Dr. Peter Ottensmeyer
Dr. Robert Phillips
Dr. Mike Rauth
Dr. James Till
Dr. Gordon Whitmore
Our Current Faculty:
Descriptions of current research interests
of individual faculty begin on page 7.
Cellular & Molecular
Biology
Dr. Lee Adamson
Dr. Mike Archer
Dr. Lilliana Attisano
Dr. Jane Aubin
Dr. Dwayne Barber
Dr. Yacov Ben-David
Dr. Neil Berinstein
Dr. Alan Bernstein
Dr. Matthew Bjerknes
Dr. Norman Boyd
Dr. Robert Bristow
Dr. Peter Cheung
Dr. Gregory Czarnota
Dr. Jayne Danska
Dr. Susan Done
Dr. Daniel Dumont
Dr. Jorge Filmus
Dr. Brenda Gallie
Dr. Abhijit Guha
Dr. Razquallah Hakem
Dr. David Hedley
Dr. Richard Hill
Dr. David Hogg
Dr. Thomas Hudson
Dr. Norman Iscove
Dr. Michael Julius
Dr. Igor Jurisica
Dr. Suzanne Kamel-Reid
Dr. Gordon Keller
Dr. Robert Kerbel
Dr. Rama Khokha
Dr. Anne Koch
Dr. Michelle Letarte
Dr. Fei-Fei Liu
Dr. Geoffrey Liu
Dr. Tak Mak
Dr. David Malkin
Dr. Armen Manoukian
Dr. Ian Tannock
Dr. Elisabeth Tillier
Dr. David Tritchler
Dr. Suzanne Trudel
Dr. Ming Tsao
Dr. Derek Van Der Kooy
Dr. Homayoun Vaziri
Dr. Richard Wells
Dr. Shun Wong
Dr. Minna Woo
Dr. James Woodgett
Dr. Eldad Zacksenhaus
Structural Biology &
Bioinformatics
Dr. Cheryl Arrowsmith
Dr. Avijit Chakrabarty
Dr. Aled Edwards
Dr. Paul Fraser
Dr. Jean Garipy
Dr. Mitsuhiko Ikura
Dr Ling has recently
resigned as the Research
Director of the BC
Cancer Agency in
Vancouver to take up a
position as the head of
the new Terry Fox
Cancer Institute; Dr
Carlsen is Vice-President
for research at the
Saskatoon Cancer Agency; Dr Paige is the Vice-President for
research of the University Health Network in Toronto; Dr Julius
is the Vice-President for research at Sunnybrook Health Sciences
Centre; Dr Mak is the head of the Campbell Family Institute for
Breast Cancer Research affiliated with the University Health
Network/Princess Margaret Hospital in Toronto; Dr Siminovitch
was the founding chair of the Department of Medical Genetics at
the University of Toronto; Dr McCulloch was the founding chair
of the Institute of Medical Science at the University of Toronto;
Dr Woodgett is the current head of the Lunenfeld Research
Institute at the Mt Sinai Hospital in Toronto and Dr Wu was
until recently Dean of Science at York University in Toronto.
The Structural Biology Stream
Scientists in The Department of Medical Biophysics at
the University of Toronto are among the pioneers of the study of
the structure and functional analysis of macromolecules. Even
before the field was firmly established by several key recruitments
in the early 1990s, Peter Ottensmeyer, Professor Emeritus and
former Chair of the Department, was a pioneer in the use of the
electron microscope to reconstruct three-dimensional macromole-
cular structures from images of individual particles, a technique
that is at the forefront of Structural Biology in the 21st century.
Between 1990 and 1995, a joint recruitment between the
University and several Research Institutes resulted in the hiring of
a significant core of Structural Biologists using the major
techniques of X-ray crystallography and nuclear magnetic
resonance spectroscopy. These included Medical Biophysics
faculty Drs Arrowsmith, Ikura, Rose and Priv, who joined Drs
Ottensmeyer, Gariepy and,
later, Chakrabartty and Pai,
to form a group with inter-
ests in macromolecular
structures and their role in
diseases. A separate
Departmental Stream,
closely related to Cell and
Molecular Biology but with
a distinct flavour, was
formed in the late 1990s.
This was followed by the
formation of an interde-
partmental graduate
program in Biomolecular
Structure, of which
Medical Biophysics is a
founding member, signi-
fying the close collaboration among members of the Toronto
Structural community.
MBP has also played a founding role in another relevant
interdepartmental graduate program in Proteomics and
Bioinformatics. Paul Fraser, a structural neurobiologist at the Tanz
Institute on campus, and an international authority on the molec-
ular basis of Alzheimer's Disease, is also a MBP Faculty
member.With the recruitment of Aled Edwards and participation
of Cheryl Arrowsmith, Toronto, and Medical Biophysics specifi-
cally, have been at the forefront of the international effort in
Structural Genomics, the high-throughput determination of
macromolecular structures that followed the sequencing of the
human genome. Edwards and Arrowsmith have been instru-
mental in several initiatives in this field, including the founding of
the highly successful Structural Genomics Consortium.
In 2006, most of the Structural Biology group relocated
to the new TMDT/ MaRS complex on College Street, to be
joined by Thomas Kislinger and Brian Raught, with expertise in
mass spectrometric analysis of protein structure, and Elizabeth
Tillier, who uses computational approaches to understand macro-
molecular interactions and evolution. Although Structural Biology
research and training exists in several Departments at the
University of Toronto, Medical Biophysics is unique in three
major respects. The first is that Structural research is embedded in
the Research Institutes (specifically the Ontario Cancer Institute)
and, as such, is within the context of a program emphasizing the
application of research to human health and diseases. Secondly,
and related, is the constant interaction with colleagues in MBP
who are involved in disparate areas of research, from Medical
Imaging and Physics through to Cancer and Cell Biology. Thirdly,
Structural Biology research in MBP spans almost the complete
breadth of modern techniques in this field, including three-
dimensional structural analysis, protein chemistry, mass spectrom-
etry and computational biology.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Research Themes
CANCER BIOLOGY
The molecular basis underlying lung, pancreatic and head &
neck cancer
Models of human leukemias in mice
The role of hypoxia in solid tumour progression
BREAST CANCER
The role of inherited breast cancer susceptibility genes
Tumour immunology as a novel modality in the treatment of
breast cancer
Epidemiology of breast cancer
STEM CELLS
Self-renewal properties of hematopoietic stem cells
Characterization of stem cells in neural and pancreatic devel-
opment
ONCOGENES
Functional role of oncogenes in cancer
TUMOR SUPPRESSORS
The role of PTEN in cancer development
SIGNAL TRANSDUCTION
Activation and regulation of serine/threonine and tyrosine
kinases
Tyrosine phosphatases in signal termination and human
disease
Role of adaptor proteins in mediating intracellular signalling
Signalling pathways involved in stem cell, B cell, T cell and
red cell development
APOPTOSIS
The role of Myc in biology
Intrinsic and extrinsic pathways of apoptosis
DNA STRUCTURE AND REPAIR
Basic investigations into chromatin structure and regulation of
the histone code
Mouse models to examine the role of DNA repair pathways in
mouse development
IMMUNOLOGY
B cell development
T cell tolerance
DEVELOPMENTAL BIOLOGY
Protein Kinase B regulation in Drosophila
Sea Urchin as a model to study aging and immunity
Targeted and random mutagenesis to inactive genes in the
mouse genome
EXPERIMENTAL THERAPEUTICS
Animal models to test novel therapeutic agents
GENE THERAPY
Gne therapy approaches to treat rare metabolic disorders
CHEMICAL BIOLOGY
High throughput screens to identify novel inhibitors that can
be utilized in cancer treatment
Screens to identify off-patent compounds that may have novel
inhibitory functions in cancer
BIOINFORMATICS
Cancer informatics
Novel software to analyze protein-protein interaction
databases
Bioinformatics strategies to characterize domain families
STRUCTURAL BIOLOGY
Three dimensional structural analysis of glycolytic enzymes,
transcription factors and signalling proteins
High throughput proteomics
PEPTIDE CHEMISTRY
Peptides as novel therapeutic agents
Role of peptide biology in neurodegenerative disorders such as
ALS and Alzheimers disease
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Some Landmark MBP Publications ...
DISCOVERY OF THE STEM CELL - Till & McCulloch Labs
Till JE and McCulloch EA. Radiat Res. 13: 115-25, 1960.
Becker AJ et al. Nature. 197: 452-4, 1963.
Siminovitch L et al. J Cell Physiol. 62: 327-36, 1963
THE SEPARATION OF CELLS - Phillips Lab
Miller RG and Phillips RA. J Cell Physiol. 73: 191-201, 1969.
DISCOVERY OF P-GLYCOPROTEIN - Ling Lab
Ling V, Thompson LH. J Cell Physiol. 83: 103-16, 1974
A MODEL FOR GASTRIC CANCER - Archer Lab
Correa P, et al. Lancet. 2: 58-60, 1975.
CLONING OF THE T-CELL RECEPTOR - Mak Lab
Yanagi Y, et al. Nature. 308: 145-9, 1984.
DISCOVERY THAT P53 IS A TUMOUR SUPPRESSOR -
Benchimol Lab
Mowat M et al. Nature. 314: 633-6,1985.
ASSOCIATIONBETWEEN HYPOXIA AND METASTASIS
IN SOLID TUMOURS - Hill Lab
Young, S.D et al. Proc. Natl Acad. Sci. USA, 85: 9533-9537, 1988.
DISCOVERY OF FLI2 - Ben-David Lab
Lu, S.J., et al. Proc. Natl. Acad. Sci. U.S.A., 91: 8398-8402, 1994.
DISCOVERY OF SAP/JNK KINASES - Woodgett Lab
Kyriakis JM, et al. Nature. 369: 156-60, 1994.
INFLUENCE OF PEPTIDE CONCENTRATIONS T-CELL
RECEPTOR FUNCTION - Ohashi Lab
Sebzda E et al. Science. 263: 1615-8, 1994.
DISCOVERY THAT LITHIUM IS AN INHIBITOR OF
GSK-3 - Woodgett Lab
Stambolic V, et al. Curr Biol. 6: 1664-8, 1996
IDENTIFICATION OF TEP1 AS PART OF THE TELOM-
ERASE HOLOENZYME COMPLEX - Harrington Lab
Harrington L, et al. Science. 275: 973-7, 1997.
FUNCTION OF PTEN TUMOUR SUPPRESSOR - Mak Lab
Stambolic V et al. Cell. 95:29-39, 1998.
DELETION OF CASPASE 9 IN MICE - Mak Lab
Hakem R, et al. Cell. 94: 339-52, 1998.
ROLE OF FADD IN REGULATING APOPTOSIS - Mak Lab
Yeh WC, et al. Science. 279: 1954-8, 1998.
IN VIVO FUNCTION OF VEGF RECEPTOR-3
- Dumont Lab
Dumont DJ et al. Science. 282: 946-9, 1998.
RECONSTITUTION OF TELOMERASE - Benchimol Lab
Vaziri H, Benchimol S. Curr Biol. 8: 279-82, 1998.
SUPPRESSOR OF CYTOKINE SIGNALLING BINDS TO
C-KIT RECEPTOR - Rottapel Lab
De Sepulveda P et al. EMBO J. 18: 904-15, 1999.
DISCOVERY THAT THE ADAPTOR PROTEIN GADS
PARTICIPATES IN T CELL SIGNALING - McGlade Lab
Liu SK et al. Curr Biol. 9: 67-75, 1999.
MOUSE MODEL OF SIMPSON-GOLABI-BEHMEL
SYNDROME - Filmus Lab
Cano-Gauci DF, et al. J Cell Biol. 146: 255-64, 1999.
METRONOMIC THERAPY - Kerbel Lab
Klement G, et al. J Clin Invest. 105: R15-24, 2000
STRUCTURAL PROTEOMICS OF ARCHAEBACTERIA,
METHANOBACTERIUM THERMOAUTOTROPHICUM -
Arrowsmith, Edwards & Pai Labs
Christendat D et al. Nature Struct Biol. 7: 903-9, 2000.
GENE THERAPY TO CORRECT FABRY DISEASE IN A
MOUSE MODEL - Medin Lab
Takenaka T, et al. Proc Natl Acad Sci U S A. 97: 7515-20, 2000.
GLYCOGEN SYNTHASE KINASE 3-BETAS ROLE IN
CELL SURVIVAL - Woodgett Lab
Hoeflich KP et al. Nature. 406: 86-90, 2000.
ROLE OF TIMP-3 IN LUNG DEVELOPMENT AND
MAMMARY GLAND APOPTOSIS - Khokha Lab
Leco KJ et al. J Clin Invest. 108: 817-29, 2001.
Fata JE, et al. J Clin Invest. 108: 831-41, 2001.
STRUCTURE OF A GLYCOLYTIC ENZYME - Rose Lab
van den Elsen JM et al. EMBO J. 20: 3008-17, 2001.
THE IMPORTANCE OF DENSITY AS A RISK FACTOR IN
BREAST CANCER - Boyd Lab
Boyd NF et al. N Engl J Med. 347: 886-94, 2002.
ACTIVATED RETINOBLASTOMA IN BREAST CANCER
- Zacksenhaus Lab
Jiang Z, Zacksenhaus E. J Cell Biol. 156: 185-98, 2002.
T CELL PHOSPHATASE SELECTS JAK1 AND JAK3 AS
SUBSTRATES - McGlade & Barber Labs
Simoncic PD et al. Curr Biol. 12: 446-53, 2002.
BCL6-SMRT STRUCTURE - Prive Lab
Ahmad KF et al. Mol Cell. 12, 1551-1564. 2003.
CONDITIONAL INACTIVATION OF CASPASE 8 IN T
CELLS - Hakem Lab
Salmena L et al. Genes Dev. 17: 883-95, 2003.
QUANTITATIVE ANALYSIS OF STEM CELL ENGRAFT-
MENT - Iscove Lab
Benveniste P et al. Nature Immunol. 4: 708-13, 2003.
STAT1 IS A NEGATIVE REGULATOR OF RED BLOOD
CELL PRODUCTION - Barber Lab
Halupa A, et al. Blood. 105: 552-61, 2005.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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CRYSTALLIZATION OF A MEMBRANE PROTEIN
- Pai Lab
Payandeh J, Pai EF. EMBO J. 25: 3762-73, 2006.
RELATIONSHIP OF AGE AND TELOMERE LENGTH IN
LI-FRAUMENI SYNDROME - Malkin Lab
Tabori U et al. Cancer Res. 67: 1415-8, 2007.
ROLE OF GATA-6 IN ASTROCYTOMA - Guha Lab
Kamnasaran, D et al. Proc. Natl. Acad. Sci. 104: 8053-8058, 2007.
ROLE OF INORGANIC PHOSPHATE IN BONE FORMA-
TION - Aubin Lab
Yoshiko Y et al. Mol Cell Biol. 27: 4465-74, 2007.
HOMING OF STEM CELLS AFTER RADIATION
- F-F Liu Lab
Bastianutto C et al. Cancer Res. 67: 10112-6, 2007.
ROLE OF PEPTIDE FOLDING IN AMYOTROPHIC
LATERAL SCLEROSIS - Chakrabartty Lab
Rakhit R et al. Nature Med. 13: 754-9, 2007.
ROLE OF H2A.Z IN CHROMATIN STRUCTURE
- Cheung Lab
Sarcinella E, et al. Mol Cell Biol. 27: 6457-68, 2007.
ROLE OF SIRPA IN STEM CELL ENGRAFTMENT
- Danska Lab
Takenaka K, et al. Nature Immunol. 8: 1313-23, 2007.
Major Funding
Research in the Department of Medical Biophysics is supported
through numerous peer-reviewed grants held by Faculty from
many agencies, including:
Canadian Institutes of Health Research
National Cancer Institute of Canada
Ontario Instutute of Cancer Research
Natural Sciences and Engineering Research Council of Canada
National Institutes of Health, US
Cancer Research Society
Canadian Breast Cancer Research Alliance
Prostate Cancer Research Foundation of Canada
Leukemia and Lymphoma Society
Leukemia and Lymphoma Society of Canada
Kidney Foundation of Canada
Foundation Fighting Blindness
Heart and Stroke Foundation
Research infrastructure support for the Department of Medical
Biophysics is provided by our host Research Institutes: the
Ontario Cancer Institute and Princess Margaret Hospital;
Sunnybrook Health Sciences Centre; the Hospital for Sick
Children; the Lunenfeld Research Institute at the Mount Sinai
Hospital. Further funding is derived from:
Canadian Foundation for Innovation
Ontario Research Foundation
Ontario Innovation Trust
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Graduate Admission: How to apply
The Department welcomes applications from graduates in any of the biological or physical sciences including chemistry, biology,
genetics, immunology and biochemistry, or from medicine, engineering, computer science or related sciences. Applicants are
evaluated on both their academic record and potential for creative research.
To be considered for admission, the minimum requirement is an appropriate four-year bachelor's degree from a recognized univer-
sity in Canada, or its equivalent from another institution. Most students must have at least an A- average in the final two years,
but this condition is flexible, especially for applicants who have demonstrated exceptional aptitude for research. To have the best
chances for acceptance and for receiving University of Toronto Fellowships or Connaught Scholarships, applications should be
received by February 1.
In general, there is no direct entry into the Ph.D. programme except for an applicant who already holds a Master's degree from
a Canadian university. Students who are accepted into our Master's programme may reclassify, if eligible, into the Ph.D.
programme within 18 months of their registration. Students who have been accepted directly into the Ph.D. programme must
pass a qualifying examination within 15 months of registration.
For more information on graduate admissions please contact Justin Thielman in the Medical Biophysics Office at (416) 946-2819.
Application packages are downloadable from our website at http://medbio.utoronto.ca.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Where are they now? Ph.D. Graduates from Cell, Molecular & Structural Biology
Dr. Veronique Dorval Post-doctoral fellow, McGill University
Dr. Jian Payandeh Post-doctoral fellow, University of Washington, Seattle, WA
Dr. Kenneth Yip Post-doctoral fellow, Burnham Institute, La Jolla, CA
Dr. Li Zhang Post-doctoral fellow, Mass General Hospital, Boston, MA
Dr. Robert Garces Post-doctoral fellow, Harvard Medical School, Boston, MA
Dr. Dominic Falconi Abbott Pharmaceuticals, Montreal, QC
Dr. Ivan Bosanac Post-doctoral fellow, Genentech
Dr. Kyoko Yap Post-doctoral fellow, Mount Sinai Hospital, NY
Dr. Grant Welstead Post-doctoral fellow, MIT, Cambridge, MA
Dr. Carlo Hojilla Medical School, University of Toronto
Dr. Brett Robb Post-doctoral fellow, U Mass, Worcester, MA
Dr. Sean Bevan Research Program Manager, Ontario Genomics Institute
Dr. Cara Maguire Post-doctoral fellow, Cancer Care Ontario
Dr. Lauren Brown Post-doctoral fellow, Harvard Medical School, Boston, MA
Dr. Rusty Jones Post-doctoral fellow, University of Pennsylvania
Dr. Dan Mao Post-doctoral fellow, Samuel Lunenfeld Res Inst, Toronto
Dr. Erinn Soucie Post-doctoral fellow, Marseille, France
Dr. Jing Jin Post-doctoral fellow, Samuel Lunenfeld Res Inst, Toronto
Dr. Paul LaPointe Post-doctoral fellow, Scripps Research Institute
Dr. Cynthia Ho Post-doctoral fellow, Samuel Lunenfeld Res Inst, Toronto
Dr. Victoria Ahn Post-doctoral fellow, Stanford University
Dr. Farid Ahmad Post-doctoral fellow, UCSF
Dr. Rob Cairns Post-doctoral fellow, Stanford University
Dr. Kevin Truong Assistant Professor, Biomedical Engineering, U of Toronto
Dr. Geoffrey Wood Assistant Professor, University of Guelph
Dr. Tim Davison Scientist, Asuragen, Austin, TX
Dr. Valerie Olmstead Assistant Professor, Memorial University
Dr. Shane Green Program Director, Ethics, Ontario Genomics Institute
Dr. Vivian Saridakis Assistant Professor, York University
Dr. Lisa Martin Assistant Professor, MBP, University of Toronto
Dr. Jason Barlow BioStrategies Group, Chicago, IL
Dr. Jacqueline Mason Scientist, Campbell Family Breast Cancer Institute
Dr. Rena Oulton Patent Examiner, Canadian Intellectual Property Office
Dr. Klaus Hoeflich Genentech, San Francisco, CA
Dr. Valerie Notenboom Scientist, Schering-Plough, Netherlands
Dr. Leslie Brail Scientist, Lilly Research Labs, Indianapolis, IN
Dr. Louis Boucher Resident, Diagnostic Radiology, University Health Network
Dr. Jacinth Abraham Patent Examiner, Canadian Intellectual Property Office
Dr. Anthony Brade Radiation Oncologist, Princess Margaret Hospital
Dr. Homayoun Vaziri Assistant Professor, MBP, University of Toronto
Dr. Stanley Liu Resident, Radiation Oncology, Princess Margaret Hospital
Dr. Nina Jones Assistant Professor, University of Guelph
Dr. Kim Kawamura Scientist, Campbell Family Breast Cancer Institute
Dr. Andr White Scientist, Boehringer-Ingelheim, Ridgefield, CT
Dr. Filio Billia Clinician Scientist & Research Fellow, Toronto General Hospital
Dr. Vuk Stambolic Assistant Professor, MBP, University of Toronto
Dr. Sona Vasudevan Research Asst Professor, Georgetown University
Dr. Xiao Qiang Yan Head, Biomed Res, Hutchison Medipharma, Shanghai, China
Dr. Maryanne Trevisan Patent Attorney; Wolf, Greenfield & Sacks, Boston, MA
Dr. Valerie Wallace Associate Professor, Professor, University of Ottawa
Dr. Joyce Slingerland Professor, University of Miami, Miami, FL
Dr. Stuart Berger Associate Professor, Department of Immunology, U of Toronto
Dr. Phil Branton Professor, McGill University
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Faculty & Project Descriptions
Dr. Michael Archer p. 9
Dr. Cheryl Arrowsmith p. 9
Dr. Jane Aubin p. 10
Dr. Dwayne Barber p. 11
Dr. Yaacov Ben-David p. 12
Dr. Norman Boyd p. 13
Dr. Robert Bristow p. 14
Dr. Peter Cheung p. 15
Dr. Gregory Czarnota p. 16
Dr. Jayne Danska p. 17
Dr. Daniel Dumont p. 17
Dr. Jorge Filmus p. 18
Dr. Paul Fraser p. 19
Dr. Jean Garipy p. 19
Dr. Abhijit Guha p. 20
Dr. Razquallah Hakem p. 21
Dr. David Hedley p. 22
Dr. Richard Hill p. 23
Dr. Mitsuhiko Ikura p. 24
Dr. Norman Iscove p. 25
Dr. Michael Julius p. 25
Dr. Igor Jurisica p. 26
Dr. Suzanne Kamel-Reid p. 27
Dr. Gordon Keller p. 28
Dr. Robert Kerbel p. 29
Dr. Rama Khokha p. 30
Dr. Thomas Kislinger p. 31
Dr. Anne Koch p. 31
Dr. Michelle Letarte p. 32
Dr. Fei-Fei Liu p. 33
Dr. Geoffrey Liu p. 34
Dr. David Malkin p. 34
Dr. Armen Manoukian p. 35
Dr. Philip Marsden p. 36
Dr. Lisa Martin p. 37
Dr. Jane McGlade p. 37
Dr. Jeffrey Medin p. 38
Dr. Benjamin Neel p. 39
Dr. Pamela Ohashi p. 39
Dr. Hitoshi Okada p. 40
Dr. Emil Pai p. 40
Dr. Christopher Paige p. 41
Dr. Linda Penn p. 42
Dr. Gilbert Priv p. 42
Dr. Mira Puri p. 43
Dr. Jonathan Rast p. 44
Dr. Brian Raught p. 45
Dr. David Rose p. 45
Dr. Aaron Schimmer p. 46
Dr. David Spaner p. 47
Dr. Vuk Stambolic p. 47
Dr. Ian Tannock p. 48
Dr. Elisabeth Tillier p. 49
Dr. Suzanne Trudel p. 49
Dr. Ming-Sound Tsao p. 50
Dr. Derek van der Kooy p. 50
Dr. Richard Wells p. 51
Dr. Shun Wong p. 52
Dr. Minna Woo p. 52
Dr. James Woodgett p. 53
Dr. Eldad Zacksenhaus p. 54
Michael Archer, Ph.D.
Professor,
Department of Medical Biophysics
m.archer@utoronto.ca
Role of dietary factors and susceptibility
genes in cancer development
One objective of our research program is to identify molecular
targets of dietary factors involved in breast and colon cancer devel-
opment. Current research focuses on HMG-CoA reductase and the
mevalonate pathway, cyclooxygenase-2, fatty acid synthase, and
insulin, IGF-I, and other obesity-related factors. A second objective
is to understand the genetic basis for the differences in susceptibility
of rat strains to breast and liver cancer induction.
Selected Publications:
Duncan, R.E., and Archer, M.C. Farnesol induces thyroid
hormone receptor (THR)1 but inhibits THR-mediated signaling
in MCF-7 human breast cancer cells. Biochem. Biophys. Res.
Commun., 343, 239-243, 2006.
Lu, S., and Archer, M.C. Celecoxib reduces fat accumulation in
rats by decreasing fatty acid synthase expression via down-regula-
tion of c-Jun amino-terminal kinase1 (JNK1). Exp. Biol. Med.
232, 643-653, 2007.
Ealey, K.N., Xuan, W., Lu, S. and Archer, M.C. Colon carcinogen-
esis in liver-specific IGF-I-deficient (LID) mice. Int. J. Cancer,
122, 472-476, 2008.
Ealey, K.N, Lu, S., and Archer, M.C. Development of aberrant
crypt foci in the colons of ob/ob and db/db mice: evidence that
leptin is not a promoter. Molecular Carcinogenesis (in press).
Cheryl Arrowsmith, Ph.D.
Professor,
Department of Medical Biophysics
carrow@uhnres.utoronto.ca
p53 related chromatin and ubiquitylation
pathways
Our research focuses on the structural and biochemical
characterization of proteins involved in cancer pathways, especially
that of the tumor suppressor, p53. The goals of our research are
to understand how p53 and related proteins communicate and
signal in complex processes such as DNA damage recognition and
repair, transcription regulation, and ubiquitin-mediated degrada-
tion. We take a genome-wide approach, studying whole families
of related proteins that may have an impact on these pathways in
order to understand how proteins selectively interact with specific
members of a sequence-related family. We use Nuclear Magnetic
Resonance (NMR) Spectroscopy and x-ray crystallography in
conjunction with other physical and biochemical techniques to
study the three-dimensional (3D) structure, dynamics and
biochemical properties of proteins, protein-DNA and protein-
protein complexes.
p53 is a central integrator of signaling pathways that
guard genomic integrity, thereby preventing tumorigenesis. The
cellular levels of p53 are tightly regulated by several ubiquitin E3
ligases that promote ubiquitylation and target p53 for 26S proteo-
some-dependent protein degradation. We are studying proteins
that both add and remove ubiquitin from p53. We are also inves-
tigating other post-translational modifications of p53 (phosphory-
lation, methylation and acetylation), and how these modifications
may act as signals by altering p53s interactions with other
proteins, DNA and/or chromatin. Other systems under study in
the lab include the breast cancer susceptibility gene, BRCA1, the
human ubiquitylation system, and proteins that read and
write histone marks such as methyl- lysine recognizing proteins
such as MBT repeat proteins, Chromodomains and methyl- and
acetyl-transferases.
Selected Publications:
Min J, Allali-Hassani A, Nady N, Qi C, Ouyang H, Liu Y,
MacKenzie F, Vedadi M and Arrowsmith CH. L3MBTL1 recog-
nition of mono- and dimethylated histones, Nature Struct. Mol.
Biol. 14, 1229-30 (2007)
Kaustov L, Lukin J, Lemak A, Duan S, Doherty R, Penn LZ,
Arrowsmith CH.. The conserved CPH domains of Cul 7 and
PARC are protein-protein interaction modules that bind the
tetramerization domain of P53. J Biol Chem. 282, 11300-7
(2007).
Sheng Y, Saridakis V. Sarkari F, Duan S, Wu T, Arrowsmith CH,
and Frappier L. Molecular recognition of p53 and MDM2 by
USP7/HAUSP, Nat Struct Mol Biol, 13, 285-91. (2006).
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
9
Jane Aubin, Ph.D.
Professor,
Department of Medical Biophysics
jane.aubin@utoronto.ca
Mesenchymal stem cells and skeletal
development
Research in our lab is focused on development and
postnatal activity of the skeleton. Osteogenic, chondrogenic,
adipogenic and myogenic cells develop from a pool of
mesenchymal stem cells and primitive progenitor populations in
bone and bone marrow. One aspect of our work addresses both
how to characterize and how to control self-renewal, fate choice,
proliferation and differentiation of the stem, progenitor and more
mature precursor populations so as to map the developmental
hierarchies underlying mesenchymal lineages. To do this, we are
assessing the ability of endogenously produced or exogenously
supplied hormones, cytokines, and growth factors to influence
progenitor proliferation, self-renewal and differentiation to dissect
deterministic versus stochastic pathways of mesenchymal cell
development. We have acquired evidence for differentiation stage-
specific regulatory pathways and for dose-, and time-dependent
biphasic effects of many regulators of interest. We are also inter-
ested in the regulatory and developmental basis of the phenotypic
heterogeneity we have documented in post-proliferative mature
cell phenotypes in the developing and mature skeleton.
Amongst regulators of the highly dynamic postnatal
skeleton, we are currently studying the family of estrogen
receptor-related orphan nuclear receptors, and have developed
novel gain-of-function and loss-of-function transgenic mice to
delineate the functional role of these transcription factors in
health and disease of bones and joints. We are also using genome-
wide chemical (N-ethyl-N-nitrosourea; ENU) mutagenesis in
mice to identify novel genes and mutations that regulate forma-
tion and turnover of the skeleton and are characterizing their
cellular and molecular modes of action in the developing and
postnatal skeleton.
My laboratory has developed many transgenic and
knockout mouse models with skeletal anomalies, primary cell
culture models for mesenchymal cell differentiation, assays for
enriching stem and progenitor populations and dissecting devel-
opmental transition points, and tools for analysing skeletal cells
and their fate including fluorescence-activated cell sorting (FACS),
colony assays, gene expression profiling, immunocytochemistry
and imunnohistochemistry, in situ hybridization of embryonic
and adult tissues and cells, Western blotting, single cell assays and
other state-of-the-art cell and molecular biology techniques. By
this multi-faceted model organism, developmental, cell and
molecular biological approach, we are advancing understanding of
normal development and diseases of the skeleton.
Selected Publications:
Zhang S, Chan M and Aubin JE. Pleiotropic effects of the steroid
hormone 1,25-dihydroxyvitamin D3 on the recruitment of
mesenchymal lineage progenitors in fetal rat calvaria cell popula-
tions. J. Mol. Endocrinol. 36:425-433, 2006.
Falconi D, Oizumi K and Aubin JE. Leukemia inhibitory factor
influences the fate choice of mesenchymal progenitor cells. Stem
Cells. 25:305-312, 2007.
Bonnelye E, Zirngibl R, Jurdic P and Aubin JE. Estrogen
receptor-related receptor alpha is a regulator of Sox9 and is
involved in cartilage formation and integrity. Endocrinology.
148:1195-1205, 2007.
Yoshiko Y, Candeliere GA, Maeda N and Aubin JE. Osteoblast
autonomous Pi regulation via Pit1 plays a role in bone mineraliza-
tion. Molec Cell Biol 27:4465-4474, 2007.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
10
Developing mouse cartilage cultures double-labelled by immunoflu-
orescence for proliferating cells (BrdU; green) and differentiating
chondrocytes (type II collagen (ColII); red). The image shows a 3D
reconstruction of a stack of confocal microscopy images in which
chondrocytes are seen as spherical Col II-positive cells that deposit
cartilage matrix and form 3D nodular structures. Several optical
sections have been removed from the top to show the interior of the
dense cell layers.
A mouse model for human osteogenesis imperfecta (OI).
Micro-computed tomography (microCT) images through
the distal femur. The image on the left is from a wild type
mouse; the image on the right is from a mutant mouse
with a low bone mass phenotype. The low bone mass
results from a point mutation, created by chemical
mutagenesis, in the gene for collagen type I a chain, which
results in reduced osteoblast differentiation and reduced
bone matrix production.
Dwayne Barber, Ph.D.
Professor & Vice-Chair,
Department of Medical Biophysics
dbarber@uhnres.utoronto.ca
Signalling Mechanisms in Hematopoietic
Cells
Cells within the bone marrow respond to a host of
growth factors that promote their growth, survival and differentia-
tion. Our laboratory studies signal transduction in normal and
leukemogenic hematopoiesis. Current studies are focussed in three
specific areas: understanding the role of Erythropoietin (EPO) in
red blood cell production, characterization of the signal transduc-
tion pathways activated downstream of BCR-ABL and delineation
of the molecular mechanism underlying the poor prognosis of
Chronic Myeloid Leukemia patients harbouring deletions at
Chromosome 9q34.
EPO is the major cytokine regulator of red blood cell
production. This cytokine binds to its cognate receptor (EPO-R)
and initiates signal transduction through activation of the JAK2
cytoplasmic tyrosine kinase. JAK2 subsequently phosphorylates
the EPO-R on several cytoplasmic tyrosine residues, which facili-
tates recruitment of several SH2 domain-containing proteins.
Genetic evidence suggests that deletion of EPO, EPO-R or JAK2
in mice results in embryonic lethality due to a fatal anemia that
develops during embryogenesis. This implies that critical signals
emanate downstream of the EPO-R and/or JAK2.
Our approaches to dissect EPO-mediated signalling are
to utilize knockout mice devoid of expression of critical
downstream players. In addition, we have used microarray to
examine gene regulation mediated by EPO. We are most inter-
ested in characterizing the transcriptome in primary cells under
conditions that support normal and stress erythropoiesis.
Approximately one-half of leukemias arise from chromo-
somal translocations. Novel fusion proteins aregenerated which
deliver a growth advantage to the stem cell in which they reside in
the bone marrow. We are interested in the tyrosine kinase subclass
of chromosomal translocations including TEL-JAK2 and BCR-
ABL. Our goal is to identify the critical downstream signalling
pathways that contribute to leukemogenesis mediated by BCR-
ABL. We are utilizing retroviral transduction/bone marrow trans-
plantation technology to approach this problem.
The causative agent of Chronic Myeloid Leukemia
(CML) is the BCR-ABL chromosomal translocation. Dr. Jeremy
Squire has demonstrated that a subset of CML patients with poor
prognosis harbour deletions centromeric to the ABL gene at
Chromosome 9q34. His laboratory has mapped a minimal
deleted region of 120 MB that contains several genes. Our current
studies are to identify and characterize the expression pattern of
these genes in CML cell lines and primary CML cells. Our long-
term goal is to develop mice devoid of expression of the genes
located in the deleted region to determine whether BCR-ABL
causes a more aggressive disease in a bone marrow transplant
model.
Selected Publications:
Richmond TD, Chohan M, and Barber, D.L. 2005. Turning Cells
Red: Signal Transduction Mediated by Erythropoietin. Trends
Cell Biol. 15:146-55.
Halupa A, Bailey M, Huang K, Iscove NN, Levy DE, Barber DL.
A Novel Role for STAT1 in Regulating Murine Erythropoiesis:
Deletion of STAT1 Results in Overall Reduction of Erythroid
Progenitors and Alters Their Distribution. Blood. 105: 552-61,
2005.
Ho JM, Nguyen MH, Dierov JK, Badger KM, Beattie BK,
Tartaro P, Haq R, Zanke BW, Carroll MP, Barber DL. TEL-JAK2
constitutively activates the extracellular signal regulated kinase
(ERK), stress-activated protein/Jun kinase (SAPK/JNK), and
p38 signaling pathways. Blood. 100:1438-48, 2002.
Kolomietz E, Marrano P, Yee K, Thai B, Braude I, Kolomietz A,
Chun K, Minkin S, Kamel-Reid S, Minden M, Squire JA.
Quantitative PCR identifies a minimal deleted region of 120 kb
extending from the Philadelphia chromosome ABL translocation
breakpoint in chronic myeloid leukemia with poor outcome.
Leukemia. 17: 1313-23, 2003.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
11
Cytospin of cells isolated from Phenylhydrazine-primed spleens
from wild type and Ship1-/- mice
Yaacov Ben-David, Ph.D.
Professor,
Department of Medical Biophysics
bendavid@sri.utoronto.ca
Molecular analysis of genes involved in
Leukemia
It is now widely accepted that cancer is the result of a
multistage process involving the activation of dominant acting
oncogenes and the inactivation of tumor suppressor genes. The
erytholeukemias induced by Friend virus are amongst the most
thoroughly studied experimental models of the multistage nature
of cancer. The distinct early and late stages of Friend leukemia,
the rapid and efficient induction of disease by a single injection of
virus, and the identification of a number of unique host genes
that control susceptibility to leukemia induction, provide a unique
model for the identification and analysis of genes involved in this
multistage malignancy.
The research focus in our laboratory is to understand the
molecular mechanisms underlying the multistage malignancy
induced by Friend virus. This disease is initiated with a
preleukemic and non-tumorigenic stage that is associated with a
marked polyclonal increase in the number of erythroid progeni-
tors. The next stage involves the appearance of clonal leukemia
cells in the spleens of infected mice. With respect to the genes
that are responsible for the transition of polyclonal preleukemic
infected erythroid progenitors to clonal leukemic cells, our work
and that of others, have shown that the evolution of clonal
erytholeukemia by various strains of Friend virus is associated
with the inactivation of either of the tumor suppressor genes p53
and p45 NFE2 as well as the activation of either dominant acting
oncogenes Fli-1 or Spi-1. The latter two genes encode transcrip-
tion factors, which belong to the family of ets oncogenes, and are
activated as a result of proviral integration in the majority of
Friend erytholeukemia cell lines. p53, which is also a transcrip-
tion factor, is inactivated in the majority of the Friend
erytholeukemia cell clones as a result of deletions, proviral inser-
tions and mutations. In addition, we have recently identified
another common site for retroviral integration, named Fli-3, from
DNA of erytholeukemia cell lines and shown that the coding
sequence within this locus is identical to a cluster of microRNA, a
new class of gene that is involved in regulation of other genes.
Recent studies in my laboratory demonstrated that activation of
this gene accelerates the progression of Friend erythroleukemia.
My laboratory is currently investigating the molecular
and cellular function of Fli-1, p45 NFE2 and p53. Since Fli-1,
p45 NFE2, and p53 are shown to be transcription factors, their
function is assumed to regulate the expression of cellular gene(s)
that are involved in cell growth and differentiation. Thus, the
identity of target genes whose expression are regulated by these
proteins may eventually address the broad roles played by these
genes in oncogenesis. For example, target genes for Fli-1 have
been implicated in erythropoietin signal transduction pathway in
erythrocytes. Therefore, study of these genes could increase
important insight into normal process of erythropoiesis and
malignant transformation. Furthermore we are also studying the
possible involvement of Fli-1, Fli-3 and p45 NFE2 in human
hematopoietic malignancies.
Selected Publications:
Jiu-Wei Cui, You-Jun Li, Aloke Sarkar, Jeremy Brown, Ye-Hui
Tan, Marina Premyslova, Crystal Michaud, Norman Iscove,
Guan-Jun Wang and Yaacov Ben-David (2007). Retroviral inser-
tional activation of the Fli-3 locus in erythroleukemias encoding a
cluster of microRNAs that converts Epo-induced differentiation
to proliferation. Blood, 110(7): 2631-40.
Cervi, D., Shaked, Y., Haeri, M., Usenko, T., Lee, C., Hincklin, D.,
Nagy, A., Kerbel, R.S., Yefenof, E., and Ben-David, Y. (2007).
Enhanced Natural Killing and Erythropoietic Activity in VEGF
Overexpressing Mice Delays F-MuLV Induced Leukemogenesis.
Blood, 109(5):2139-46.
Shaked, Y., Cervi, D., Neuman, M., Pak, B., Kerbel, R.S., and Ben-
David, Y. (2005). Splenic microenvironment is a source of angio-
genesis/inflammatory mediators accelerating the extramedullary
expansion of murine erythroleukemic cells, Blood, 105: 4500-
4507.
Truong. A.H.L., Cervi, D., Lee, J., and Ben-David, Y. (2005).
Direct transcriptional regulation of MDM2 by Fli-1: A role for
Fli-1 in p53 modulation, Oncogene, 24: 962-969.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology 12
Norman Boyd, M.D., D.Sc.
Professor,
Department of Medical Biophysics
boyd@uhnres.utoronto.ca
Epidemiology and Prevention of Breast
Cancer
Our research is concerned with the development of
strategies to prevent breast cancer. Breast cancer is the most
common cause of death from any cancer in women in most of the
Western world, and the leading cause of death from all causes
among women aged less than 50. Several factors have been identi-
fied that influence risk of the disease, including the characteristics
of breast tissue on either mammography or histology, the number
of pregnancies, alcohol, and body weight.
Mammographic density has consistently been shown to
be one of the strongest known risk factors for breast cancer.
Research is designed to improve our understanding of this risk
factor, its measurement, its causes, and its significance as a
biomarker of breast cancer risk. Current methods of measuring
mammographic density are based on the 2-dimensional image. In
collaboration with Dr M Yaffe (Medical Biophysics and Imaging
Research, Sunnybrook Health Sciences Centre) we are now evalu-
ating novel methods of measurement that assess the volume of the
tissues in the breast that contribute to radiological density. Data
collection has been completed and final calibration of the
measurements and data analysis is in progress.
The breast is especially susceptible to carcinogenic events
at early ages. In collaboration with Dr M Bronskill (Imaging
Research, Sunnybrook Health Sciences Centre), The goal of this
research is to use magnetic resonance (MR) imaging to generate
quantitative estimates of breast tissue composition in young
women, and to examine factors likely to be associated with varia-
tion in these measurements, in particular the relationship between
lifestyle factors and genes, hormones and growth factors that
influence the growth and development of breast tissue.
Striking differences exist between countries in the
incidence of breast cancer. The causes of these differences are
unknown, but because incidence rates change in migrants, they
are thought to be due to differences in lifestyle. The goal of this
research is to identify the factors responsible for international
differences in breast cancer risk, and to determine whether differ-
ences in breast tissue composition, and the hormonal and growth
factors associated with them contribute to differences in risk.
Previous twin studies have shown that mammographic
density is highly heritable. In collaboration with Drs Rommens
and Paterson at the Hospital for Sick Children, we are now
carrying a large scale multicentre family-based linkage study with
whole genome scanning to identify the genetic variants that influ-
ence density..
In collaboration with Dr L Martin, we have recently
completed a multicentre randomized trial in women with high
risk mammographic changes that tests the hypothesis that a 25%
dietary reduction of calories from fat, with isocaloric replacement
of carbohydrate, will reduce the incidence of breast cancer by
approximately 35% over 10 years. The trial is now under analysis.
Additional studies will examine the effect of this dietary interven-
tion on plasma hormones and growth factors that may mediate
environmental influences on risk of breast cancer.
Selected Publications:
Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, Jong RA,
Hislop G, Chiarelli A, Minkin S, Yaffe MJ.Mammographic density
and the risk and detection of breast cancer. N Engl J Med. 2007
Jan 18;356(3):227-36.
Boyd NF, Martin LJ, Sun L, Guo H, Chiarelli A, Hislop G, Yaffe
M, Minkin S. Body size, mammographic density, and breast
cancer risk. Cancer Epidemiol Biomarkers Prev. 2006
Nov;15(11):2086-92.
Martin LJ, Greenberg CV, Kriukov V, Minkin S, Jenkins DJ, Boyd
NF. Intervention with a low-fat, high-carbohydrate diet does not
influence the timing of menopause. Am J Clin Nutr. 2006
Oct;84(4):920-8.
Boyd NF, Dite GS, Stone J, Gunasekara A, English DR,
McCredie MR, Giles GG, Tritchler D, Chiarelli A, Yaffe MJ,
Hopper JL. Heritability of mammographic density, a risk factor
for breast cancer.. N Engl J Med. 2002 Sep 19;347(12):886-94.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
13
Robert Bristow, M.D., Ph.D.
Associate Professor,
Department of Medical Biophysics
rob.bristow@rmp.uhn.on.ca
DNA Repair and Genetic Instability in Solid
Tumours
Cells have developed a sophisticated approach to the
initial sensing and subsequent repair of DNA damage to preserve
genetic stability. The objective of our clinico-translational labora-
tory is to understand the effect of the tumour microenvironment
on the ATM-p53-53BP1 DNA damage signaling pathway and
DNA double-strand break (DNA-dsb) repair. Our studies suggest
that hypoxic tumour cells can have decreased DNA-dsb repair
(e.g. decreased homologous recombination) and an aggressive
mutator phenotype. We are therefore tracking DNA damage
responses and repair within normal and tumour tissues to develop
novel diagnostics and molecular-targeted therapies.
We interrogate protein-protein interactions during
DNA-dsb repair and cell-cycle checkpoints using: siRNA knock-
downs, DNA-rejoining assays (comet and CFGE assays),
chromatin immunoprecipitation (ChIP), biochemical fractiona-
tion, fluorescently-tagged proteins and quantitative confocal
microscopy with UV-microbeams (www.sttarr.ca).
(i) p53 and DNA repair: Mutations in the p53 tumour
suppressor protein are common in many human cancers. We are
interested in certain MTp53 proteins that have acquired novel
properties or "gain of function" in their ability to detect DNA-
dsbs, but over-ride DNA damage cell cycle checkpoints. This can
lead to therapy resistance. We are tracking the sub-cellular
location and function of ATM-dependent p53 phosphoforms and
53BP1 (a p53-binding protein) in response to DNA breaks and
evaluating new therapies that target MTp53.
(ii) Hypoxia, DNA repair and prostate cancer: Many prostate
cancer patients die each year solely from the failure of radical
radiotherapy to control the primary tumour. We are interested in
developing genomic (SNP, CGH) and proteomic (serum, plasma
or urine) biomarkers to predict cancer therapy cure and toxicity.
This includes the assessment of tissue microarrays (TMAs) for
novel protein expression in patients who fail therapy. For example,
we are investigating the role of hypoxia as a negative prognostic
factor in prostate and other cancers. We believe that novel cancer
therapies can target these resistant hypoxic cells by taking advan-
tage of DNA repair defects. We therefore hope to select the most
effective treatment for individual patients based on individual
biology.
For more information, see:
http://www.radiationatpmh.com/body.php?id=165
Selected Publications:
Al Rashid ST, Dellaire G, Cuddihy A, et al. Evidence for the
Direct Binding of Phosphorylated p53 to Sites of DNA Break In
Vivo. Cancer Research, 65(23):10810-21, 2005.
Choudhury A, Cuddihy A, Bristow RG. Radiation and Other
New Molecular-Targeted Agents, Part I: Targeting ATM-ATR
Checkpoints, DNA Repair and the Proteasome. Seminars in
Radiation Oncology: 16(1): 51-58, 2006.
Chan N, Meng A, Bindra R, et al. Chronic hypoxia decreases
synthesis of homologous recombination proteins and therapeutic
resistance. Cancer Research: 68(2), 2008.
Bristow RG and Hill RP: Hypoxia, DNA Repair and Genetic
Instability: A Driving Force in Cancer Progression and A Basis
for Novel Therapies. Nature Cancer Reviews, In Press, 2008
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
14
Human fibroblast nuclei (blue) in G1 (upper) and S-phase (lower)
stained for DNA damage sensing proteins gammaH2AX(green)
and 53BP-1(red). The S-phase cell shows punctate DNA repair
foci at active DNA replication forks.
Image analysis of single human fibroblast nuclei stained for the
DNA damage biomarker 53BP-1 and quantitated 3-D for
expression before (left) and after (right) 2 Gy of IR. Increased
height and number of 53BP-1 spikes within each
nuclear area shows 53BP-1 binding to sites of DNA breaks.
Peter Cheung, Ph.D.
Assistant Professor,
Department of Medical Biophysics
pcheung@uhnres.utoronto.ca
Signaling and epigenetic regulation of
gene expression through chromatin
modification
Cancer is a disease often caused by dysregulated expres-
sion of oncogenes or tumour suppressor genes. One of the funda-
mental mechanisms that regulate global gene expression is
through post-translational modification of histone proteins. In
the context of the human genome, DNA is complexed with
histone to form nucleosomes and chromatin. This functional
organization of the genome is tightly regulated such that only
appropriate genes are maintained in an open context that is acces-
sible by transcription factors and RNA polymerases. In contrast,
inactive genes are sequestered into compact structures and are
transcriptionally silenced. Histone modifications regulate the
balance between these open and closed chromatin states and
define the gene expression profile of the functional genome.
Our lab utilizes a combination of molecular biology and
biochemistry techniques to dissect the mechanisms of how
histones and histone modifications regulate gene functions. Our
projects are broadly divided into 2 main interests: 1) how signal
transduction pathways converge onto histones to regulate gene
functions, and 2) how histone variants function in the epigenetic
regulation of gene expression.
For the first interest, we focus on the role of histone H3
phosphorylation in gene activation. Upon growth factor stimula-
tion, the activated MAP kinase pathway ultimately converges onto
chromatin and results in the phosphorylation of H3 at two
specific serine resides (S10 and S28). The phosphorylation events
are rapid and transient, and mirror the transcriptional activation
of genes such as c-fos and c-jun. We are currently examining how
H3 phosphorylation leads to transcriptional activation and we are
testing whether these modifications recruit or repel regulatory
factors to the c-fos and c-jun promoters. Our previous work also
showed that H3 phosphorylation promotes acetylation on the
same histone and these modifications function together to activate
gene expression. The idea that histone modifications work in
combinations was formally described as the Histone Code
Hypothesis, and we are currently developing new techniques to
dissect the intricate interplay between histone modifications in
vivo and to test the validity of this hypothesis.
For the second interest, we are studying the functional
role of an H2A variant, H2A.Z, in the regulation of gene expres-
sion. H2A.Z is essential for cell viability in that loss of H2A.Z
function in mammalian cells leads to cell death or senescence.
H2A.Z is localized to the transcription start sites of genes, and
cumulative evidence suggests that this variant has both positive
and negative regulatory functions in the gene expression process.
We have found that a fraction of H2A.Z is modified by the
addition of a single ubiquitin group and such modified H2A.Z is
associated with transcriptionally silenced heterochromatin. We
are currently testing how addition or removal of the ubiquitin
modification on H2A.Z regulates transcription and gene expres-
sion.
Selected Publications:
Sarcinella, E., Zuzarte, P. C., Lau, P. N. I., Draker, R., Cheung, P.
2007. Mono-ubiquitylation of H2A.Z distinguishes its association
with euchromatin or facultative heterochromatin. Mol. Cell. Biol.
27: 6457-6468.
Cheung, P., Lau, P. 2005. Epigenetic regulation by histone methy-
lation and histone variants. Mol. Endocrinol. 19: 563-573.
Boggs, B., Cheung, P., Heard, E., Chinault, A. C., Allis, C. D.
2002. Differentially methylated forms of histone H3 show
unique association patterns with human X chromosomes. Nature
Genetics. 30: 73-76.
Cheung, P., Allis, C. D., Sassone-Corsi, P. 2000. Signaling to
chromatin through histone modifications. Cell. 103: 263-271.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
15
Immunofluorescence staining of histone
H2A.Z on mouse metaphase chromosomes.
Gregory Czarnota, Ph.D.
Assistant Professor,
Department of Medical Biophysics
gregory.czarnota@sunnybrook.ca
Ultrasound in Cancer Treatment: New
Therapies and Methods of Therapy
Evaluation
Our research is centered on understanding how radiation
affects blood vessels and how this contributes to tumour death.
We have developed a number of small animal micro-ultrasound
methods in our laboratory to detect vascular changes and
separately apoptotic cell death in vivo. A number of different
research projects are underway to study the basic science behind
vascular responses to radiation and to therapeutically exploit this
in order to radiosensitize tumours. In that regard our research
interests include the novel use of pharmacologic anti-angiogenic
agents and new ultrasound-activated anti-angiogenic agents.
Other projects include:
(A) Cell and Molecular Biology
Despite the use of medical ultrasound for decades the
features inside cells that contribute to ultrasound backscatter at
conventional- and high-frequencies remain unknown. We are
systematically probing how subcellular constituents such as DNA,
RNA, protein and lipids contribute to backscatter. In particular
we are interested in how nuclear and chromatin structure affects
ultrasound signals since we have found it to be a dominant struc-
ture in the formation of backscatter signals.
(B) Image and Spectroscopic Analysis
We are collaboratively investigating a number of spectro-
scopic parameters for characterizing tumours and tumour
responses to chemotherapy and radiation therapy at conventional
and high-frequencies. We are developing these methods to
generate colour-coded ultrasound parameteric maps to aid in
assessing tumour responses to therapy. Since these spectroscopic
signals are potentially linked to nuclear structure and chromatin
structure which differs between normal and neoplastic tissue there
is potential to develop our spectroscopic methods not only into a
method to track tumour responses but a potentially important
diagnostic tool.
(C) Clinical Evaluation of Ultrasound Imaging & Spectroscopy
We are instituting a number of clinical evaluations of our
spectroscopic detection of cell death. Our main investigational site
is breast cancer patients with large 'locally-advanced" breast
cancers who receive neoadjuvant combined chemotherapy and
radiation therapy. We hope to be able to rapidly ascertain
responding tumours from those that are non-responding so that
the latter may be treated with different chemotherapy regimens or
with radiation sensitizers in order to hopefully improve outcomes.
Selected Publications:
Czarnota GJ, Kolios M, Vaziri H, Benchimol S, Ottensmeyer FP,
Hunt JW: Ultrasound Biomicroscopy of Living, Dead, and
Apoptotic Cells. Ultrasound in Medicine and Biology 23: 961-5,
1997.
Czarnota GJ, Kolios M, Abraham J, Portnoy M, Sherar MD,
Ottensmeyer FP, Hunt JW: Ultrasound Imaging of Apoptosis:
High resolution Non-invasive Monitoring of Programmed Cell
Death in vitro, in situ, and in vivo. British Journal of Cancer
81:520-7, 1999.
Tunis AS, Czarnota GJ, Giles A, Sherar MD, Hunt JW, Kolios
MC: Monitoring structural changes in cells with high?frequency
ultrasound signal statistics. Ultrasound in Medicine and Biology
31, 1041-9, 2005.
Czarnota GJ: Role of ultrasound in the detection of apoptosis.
Eur. J. Nucl. Med. Mol. Imaging 32:622, 2005.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
16
Results of ultrasound imaging of apoptotic cells. Each panel is a representative ultrasound scan of a pellet of acute myeloid leukaemia cells.
The bottom of each ultrasound scan is at the bottom of each frame. Pellets are immersed in buffered saline. From left to right, panels corre-
spond to cells treated with cisplatinum for 0, 6, 12, 24 and 48 h to induce varying degrees of apoptosis. A bar at the bottom right of the
figure indicates the colour map used in this image, the left of the bar indicating the colour that corresponds to pixel values of 0 and the
right giving the colour that corresponds to a pixel value of 256. At 0, 6, 12, 24 and 48 h histological analysis indicated that 1.6, 2, 36,
87 and 93% of all cells showed nuclear fragmentation, respectively. At the 6-h time point, 72% of the cells exhibited prominent nuclear
condensation changing from a nuclear diameter 70% of the cellular diameter before addition of the drug, to a diameter 40% of the
cellular diameter at 6 hours. After the 6-h time point, 95% of all cells exhibited nuclear condensation or fragmentation. The speckle
pattern is characteristic of ultrasound images. The scale bar indicates 1 mm. From Czarnota et al, 1999.
Jayne Danska, Ph.D.
Professor,
Department of Medical Biophysics
jayne.danska@sickkids.ca
Functional Genomics of Type 1 Diabetes
Type 1 diabetes (T1D) is caused by autoimmune
destruction of insulin-producing islet cells and afflicts 0.3-0.6
percent of North Americans. The disease is multifactorial caused
by genetic variation at multiple loci, modified by poorly defined
environmental factors. The objective of our research program is to
1) identify genes that control this process in mouse models and in
human patients, 2) understand the autoimmune response that
results in islet cell death, and 3) to test the idea that early immune
system exposures to microbes significantly modifies genetic risk
for developing T1D. We use positional cloning, gene expression
microarray analysis and immunological analyses to identify the
pathways controlled by T1D risk genes and understand how they
are modified by exposure to intestinal commensal bacteria. We are
also engaged in high-throughput human gene association studies
of T1D.
Molecular pathways of Acute Lymphoblastic Leukemia
Our research program is designed to probe the develop-
mental steps and mechanisms of leukemia in mouse models and
in human patients. The objective of this program is to translate
knowledge gained in mouse models and primary human
leukemia, to improve the diagnosis and treatment of leukemia.
Our specific areas of interest are: 1) signalling pathways that
govern cell survival, differentiation or death fate decisions in
normal and neoplastic lymphoid cells, 2) identification and
analysis of the cells that initiate and sustain the leukemic clone
(cancer stem cells) to understand molecular pathways underlying
leukemia initiation and progression, and 3) how leukemic blasts
expand in the central nervous system (CNS) causing a major
clinical complication of leukemia and lymphomas, and 4) features
of the immune system that determine engraftment of normal
human blood stem cells in settings of clinical transplantation.
Selected Publications:
Ivakine EA, Gulban O, Mortin-Toth SM, Scott C, Surrell D,
Canty A, Danska JS. (2006). Molecular genetic analysis of the
Idd4 locus implicates the interferon response in Type 1 diabetes
in susceptibility of NOD mice. J. Immunol. 176 :2976-90.
Matei IR, Gladdy RA, Nutter L, Guidos CJ, Danska JS (2007)
ATM deficiency disrupts Tcra locus integrity and the maturation
of CD4+CD8+ thymocytes. Blood 109 :1887-96.
Takenaka K, Prasolava TK, Wang JC, Mortin-Toth SM, Khalouei
S, Gan OI, Dick JE, Danska JS. (2007) Polymorphism in Sirpa
modulates engraftment of human hematopoietic stem cells. Nat.
Immunol. 8: 1313-1323. See also News and Views 8: 1287-1289.
Daniel Dumont, Ph.D.
Professor,
Department of Medical Biophysics
dan.dumont@sunnybrook.ca
Endothelial Cell Growth & Signalling
The receptor tyrosine kinase (RTK) family of cell surface
proteins is known to play key roles in cell-cell communication in
multi-cellular metazoan organisms. Genetic and biochemical
studies on this large family of proteins have shown that different
RTKs are responsible for transducing important developmental,
proliferative, cell survival and migratory signals from the outside
to the inside of the cell. The development and proper functioning
of cell systems as diverse as the compound eye in the fly, the vulva
in the nematode and hematopoiesis and endothelial growth in the
mouse all depend on intact signalling pathways that are controlled
by different members of the RTK family.
Our lab is investigating the signal transduction pathways
of different RTKs during vascular development in the mouse and
during tumour formation. Vascular endothelial cells constitute an
unusually quiescent epithelial cell population. The turnover rate
of both large and small vessel endothelium is very low. The
mechanisms that underlie this growth control are not well under-
stood, and the factors which initiate and control subsequent
proliferation are unknown. It is clear, however, that vascular
growth occurs under nonpathological conditions (e.g. wound
healing, corpus luteum formation, and development) and that this
growth is somehow terminated at the correct time. In contrast,
uncontrolled vessel growth (including tumour vascularization,
diabetic retinopathy, and arthritis) are associated with many
different diseased states. The determinants which control these
processes remain unknown; however the study of peptide growth
factors, their receptors and their downstream substrates will
provide both a biochemical and genetic entry point into the eluci-
dation of the underlying controls of these processes.
Our group uses gene-targeting in embryonic stem cells,
transgenic mice, proteomics and receptor biochemistry to attempt
to address the importance of these different RTKs and their
related signal transduction pathways during vascular growth in
development and in disease.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
17
GPC3-deficient mice develop cystic kidneys
Wild- type GPC3 GPC3-deficient
Our research has the potential to impact numerous
diseases where aberrant vessel growth or vessel stability lead to
progression of disease or increased complications, such as diabetes,
heart disease and cancer. Furthermore, his group is also using this
knowledge to in fact also augment vessel growth for applications
in regenerative medicine..
Selected Publications:
Ward, N.L., Putocski, T., Mearo, K., Ivanco, T.L. and Daniel J.
Dumont (2005) Vascular-specific growth factor angiopoietin 1 is
involved in the organization of neuronal processes. The Journal
of Comparative Neurology 482(3):244-256 .
Voskas, D., Jones, N., Van Slyke, P., Sturk, C., Chang, W., Haninec,
A., Babichev, Y.O., Tran, J., Master, Z., Chen, S., Ward, N., Cruz,
M., Jones, J., Kerbel, R.S., Jothy, S., Dagnino, L., Arbiser, J.,
Klement, G., and Daniel J. Dumont (2005) A cyclosporine-sensi-
tive psoriasis-like disease produced in Tie2 transgenic mice.
American Journal of Pathology 166(3):843-855.
Chen, S.H., Babichev, Y. Rodrigues, N., Voskas, D., Ling, L.,
Nguyen V. P., and D.J. Dumont (2005) Gene expression analysis
of Tek/Tie2 signaling. Physiological Genomics 22(2):257-267.
VanSlyke, P., Coll, M.L., Master, Z., Kim, H., Filmus, J. and D.J.
Dumont (2005) Dok-R mediates attenuation of epidermal
growth factor-dependent mitrogen-activated protein kinase and
Akt activation through processive recruitment of c-Src and Csk.
Molecular & Cellular Biology 25(9): 3831-3841.
Haninec, A.L., Voskas, D., Needles, A., Brown, A.S, Foster, S.F.
and D.J. Dumont (2006) Transgenic expression of angiopoietin
1 in the liver leads to changes in lymphatic and blood vessel archi-
tecture Biochemical and Biophysical Research Communications
(BBRC) 345(4):1299-1307.
Bureau, W., Van Slyke, P., Jones, J., Han, R.N., Stewart, D.J. and
D.J. Dumont (2006) Chronic systemic delivery of angiopoietin 2
reveals a possible independent angiogenic effect. AJP: Heart and
Circulatory Physiology 291(2):H948-H956.
Bogdanovic, E., Nguyen, V.P. and D.J. Dumont (2006) Activation
of Tie2 by angiopoietin-1 and angiopoietin-2 results in their
release and receptor internalization. 119(17): 3551-3560 Journal
of Cell Science .
Vicky P.K.H. Nguyen, Stephen H. Chen, Jason Trinh, Harold
Kim, Brenda L. Coomber, Daniel J. Dumont (2007) Differential
Response of Lymphatic, Venous and Arterial Endothelial Cells to
Angiopoietin-1 and Angiopoietin-2. BMC Cell Biology, 8:10.
Jorge Filmus , Ph.D.
Professor,
Department of Medical Biophysics
jorge.filmus@sri.utoronto.ca
The role of glypicans in the regulation of
Wnt and Hedgehog activity in develop-
ment and cancer
The Wnt and Hedgehog families of growth factors play
a critical role in developmental morphogenesis by regulating stem
cell renewal, cell proliferation, cell survival, and cell differentia-
tion. In addition, the signaling pathways driven by these growth
factors become hyperactivated in most human cancers. Glypicans
are cell surface proteins that play an important role in develop-
ment and cancer by regulating the activity of Wnts and
Hedgehogs. Not surprisingly, therefore, mutation of GPC3, one
of the 6 mammalian glypicans, is the cause of the Simpson-
Golabi-Behmel syndrome, which is characterized by overgrowth
and a long list of developmental abnormalities. In addition,
mutations and altered expression of several glypicans have been
reported in various cancer types, including hepatocellular carci-
noma, rhabdomyosarcoma, and breast cancer. Our laboratory is
interested in characterizing the molecular mechanisms by which
the various glypicans regulate Wnt and Hedgehog activity. The
identification of these mechanisms may be useful in the design of
novel therapeutic approaches to treat cancer by inhibiting Wnt
and Hedgehog signaling.
The Wnt signaling pathway as a therapeutic target in hepatocel-
lular carcinoma
Hepatocellular Carcinoma (HCC) is the fifth most frequent
neoplasm in the world, and the third most common cause of
cancer-related death. Because liver function in most HCC
patients is deficient, chemotherapy is not tolerated. There is there-
fore an urgent need for non-cytotoxic therapies. Because the Wnt
signaling pathway is hyperactivated in a large proportion of
HCCs, we are trying to develop therapeutic tools to inhibit this
signaling pathway. In particular, we are testing various inhibitors
of the interaction of Wnts with their signaling receptors in liver
cancer cells.
Selected Publications:
Capurro,M., Wanless,I., Sherman, M., Deboer,G., Shi,W.,
Miyoshi,E., and Filmus,J. (2003). Glypican-3: a novel serum and
histochemical marker for hepatocellular carcinoma.
Gastroenterology 125:89-97.
Song, H., Shi, W., Xiang, Y., and Filmus, J. (2005) The loss of
Glypican-3 induces alteration in Wnt signalling. J. Biol. Chem.
280:2116-2125.
Capurro,M.I., Xiang,Y.Y., Lobe,C., and Filmus, J. (2005) Glypican-
3 promotes the growth of hepatocellular carcinoma by stimu-
lating canonical Wnt signaling. Cancer Res. 65:6245-6254.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
18
Paul Fraser, Ph.D.
Professor,
Department of Medical Biophysics
paul.fraser@utoronto.ca
Role of Amyloid & Presenelin Proteins in
Alzheimers Disease
Research in our laboratory focuses on the biochemistry
and biophysics of amyloid plaques and their relationship to
sporadic and familial forms of Alzheimers disease. Plaques are a
principal pathological feature of Alzheimers disease and appear as
abnormal accumulations of fibrous or thread-like structures
within the brain. These plaques are assembled by the misfolding
and aggregation of the amyloid- (A) protein. We have been
studying its properties with an emphasis on the factors responsible
for the transition from the normal to diseased fibrous state, the
ability of aggregated A to kill nerve cells in culture, and the
mechanism by which this is accomplished. Considerable
advances have been made in this area and we are expanding our
efforts to look for modulators of plaque formation as well as the
cellular receptors which we feel are responsible for amyloids
killer action. Our ultimate goal is to understand the events that
culminate in these abnormal and detrimental proteins and the
development of drugs capable of controlling these processes.
These investigations are relevant to both the sporadic and familial
forms of Alzheimers disease which exhibit identical amyloid
pathology but differ only in their rate of progression.
In conjunction with our amyloid research and drug
development, our group has been concentrating its efforts on
understanding the biochemistry and structural biology of the
presenilin family of proteins. Mutations in the presenilin genes are
the major cause of inherited forms of Alzheimers disease and we
have been examining the location and expression of these proteins
in neuronal cells and their relationship to the pathology of
Alzheimers disease. This biochemical and molecular biological
work is complemented by our examination of the three-dimen-
sional organization of particular regions of the presenilin protein
using a variety of biophysical techniques. Through these two
approaches, we will be able to provide details on presenilin
function and the molecular mechanism by which this is achieved.
The importance of these studies is that they enable us to under-
stand the earliest events in Alzheimers disease and allow us to
develop novel approaches to the treatment of a principal cause of
Alzheimers disease.
Selected Publications:
Chen, F., Hasegawa, H., Schmitt-Ulms, G., Kawarai, T., Bohm, C.,
Katayama, T., Gu, Y. Sanjo, N., Glista, M., Rogaeva, E., Wakutani,
Y., Piquard, R.P., Ruan, X., Tandon, A., Checler, F., Marambaud,
P., Hansen, K., Westaway, D., St George-Hyslop P. and Fraser,
P.E. (2006) TMP21 is a presenilin complex component that
modulates ?- but not e-secretase activity. Nature 440: 1208-12.
Dorval, V. and Fraser, P.E. (2006) SUMO modification of
natively unfolded proteins tau and alpha-synuclein. J. Biol. Chem.
281: 9919-24.
Gu, Y., Sanjo, N., Chen, F., Hasegawa, H., Petit, A., Ruan, X., Li,
W., Shier, C., Kawarai, T., Schmitt-Ulms, G., Westaway, D., St
George-Hyslop, P. and Fraser, P.E. (2004) The presenilin proteins
are components of multiple membrane-bound complexes which
have different biological activities. J. Biol. Chem. 279: 31329-36.
Yang, D-S., Tandon, A., Chen, F., Yu, G., Arawaka, S., Yu, H.,
Hasegawa, H., Duthie, M. Ramabhadran, T.V., Mathews, P.M.,
Gandy, S.E., Mount H.T.J., St George-Hyslop, P. and Fraser,P.E.
(2002) Mature glycosylation and secretory trafficking of nicastrin
modulate its binding to presenilins. J. Biol. Chem. 277:28135-42.
Jean Garipy, Ph.D.
Professor & Biology Admissions Co-ordinator,
Department of Medical Biophysics
gariepy@uhnres.utoronto.ca
Molecular Engineering and the develop-
ment of targeted biological therapies
Our laboratory is interested in developing both in vivo
imaging modalities and targeted therapies directed at epithelial
cancers (breast, colon, prostate, ovarian, lung, pancreas). Our
research program is a fine balance of both basic and applied
research projects aimed at understanding how peptides, proteins
and oligonucleotide templates work and how they can be
engineered or utilized in developing directed therapies against
tumor cells (either through the design of cancer vaccines,
RNA/DNA/protein therapeutics or delivery vectors).
The spectrum of approaches taken by our group to
address our design and discovery programs ranges from peptide
synthesis, combinatorial protein or DNA library design/screening,
cell biology/microscopy techniques to yeast genetics and mouse
models.
We presently are designing and screening protein
libraries as well as SELEX/DNA aptamer libraries with a view to
discover new surface markers on epithelial cancer cells and in
designing tumor-specific agents. We are also closely involved in
constructing new imaging agents for the in vivo detection of
millimeter size tumor masses. Since marker discovery is a major
part of our future work and is based on no a priori knowledge of
such molecules (we are scanning the surface of cancer cells), we
are developing strategies, in collaboration with mass
spectrometrists at UHN to identify such novel markers.
Because of the broad nature of our molecular engineering
efforts, projects in our laboratory are usually tailored to a students
expectations and aptitudes. For more information please visit
http://www.jeangariepy.com
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
19
Selected Publications:
McCluskey AJ, Poon GMK, Garipy J. (2007) A Rapid and
Universal Tandem-Purification Strategy for Recombinant
Proteins. Protein Science. 16, 2726-32.
Poon GM, Garipy J. (2007). Cell-surface proteoglycans as molec-
ular portals for cationic peptide and polymer entry into cells.
Biochem Soc Trans. 35, 788-93.
Kawamaura KS, Sung M, Bolewska-Pedyczak E and Garipy J.
(2006). Probing the impact of valency on the routing of arginine-
rich peptides into eukaryotic cells. Biochemistry 45, 1116-27.
LaPointe P, Wei X and Garipy J. (2005). A role for the protease-
sensitive loop region of Shiga-like toxin 1 in the retrotransloca-
tion of its A1 domain from the endoplasmic reticulum lumen. J
Biol Chem 280, 23310-8.
Brokx RD, Revers L, Zhang Q, Yang S, Mal TK, Ikura M and
Garipy J. (2003). Nuclear magnetic resonance-based dissection of
a glycosyltransferase specificity for the mucin MUC1 tandem
repeat. Biochemistry. 42, 13817-25.
Brokx RD, Bolewska-Pedyczak E and Garipy J. (2003). A stable
human p53 heterotetramer based on constructive charge interac-
tions within the tetramerization domain. J Biol Chem. 278, 2327-
32.
Kawamura KS, Su RC, Nguyen LT, Elford AR, Ohashi PS and
Garipy J. (2002). In vivo generation of cytotoxic T cells from
epitopes displayed on peptide-based delivery vehicles. J Immunol
168, 5709-15.
Abhijit Guha, M.D.
Professor,
Department of Medical Biophysics
abhijit.guha@uhn.on.ca
Biology of Central Nervous System Tumours
Our research interests centre on studying signaling
complexes relevant to nervous system tumours with the aim of
undertaking pre-clinical proof-of-principle experiments, which
may translate into effective clinical therapies. We study two
tumors: Astrocytomas- The most common primary central
nervous system (CNS) tumour (studies described below);
Neurofibromas- The most common peripheral nervous system
(PNS) tumors.
Our interests are on aberrant signaling pathways
involving growth factors, growth factor receptors and downstream
signaling pathways that contribute to growth of these tumors.
Specifically we are interested in mutant EGFR and aberrant p21-
Ras and PI3Kinase mediated signaling complexes. Towards these
studies we are using proteomic and transgenic mouse model
technologies. These mouse models are used to explore genetic
interactions, their role in tumor initiation and progression,
discover novel genetic alterations and potential pre-clinical models
to test therapies.Variations in the molecular profile of these
tumors, as a reflection of the tumor microenvironment and epige-
netic regulation, are another area of interest. Specifically, we are
interested in variations in regulators of angiogenesis (VEGF,
Angiopoietins), invasion (Npn1, Semaphorins, Plexins) and
apoptosis, between the central hypoxic and more peripheral
normoxic regions of these tumors. We believe this heterogeneity
underlies differences in their biological and therapeutic behaviour.
In summary, our research program takes clinical
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology 20
Genetically Engineered Mouse
Astrocytoma Model: Sagittal
section of E16.5 transgenic
mouse embryo expressing
activated 12V-HaRas:IRES-
LacZ under control of astrocyte
specific Glial Fibrillary Acidic
Protein (GFAP) promoter. Blue
LacZ staining, denoting expres-
sion of the transgene, is
restricted to the Central Nervous
System (CNS-brain and spinal
cord). These transgenic mice are
born normally but develop and
die over a period of 3-4 months
from malignant astrocytomas,
which share many of the pathological and molecular similarities
found in the most common of all human CNS tumors, the malig-
nant astrocytoma.
neurooncological problems to the laboratory, where a variety of
molecular investigations are undertaken to decipher the pathogen-
esis and function, with research ultimately aimed at developing
novel therapies to help patients afflicted with these nervous system
tumours.
Selected Publications:
Kamnasaran, D, Hawkins, C, Guha, A: Characterization and trans-
formation potential of synthetic astrocytes differentiated from
murine embryonic stem cells (Glia-in press)
Kamnasaran, D, Qian, B, Hawkins, C, Stanford, W, Guha, A.
GATA6, a Novel Astrocytoma Tumor Suppressor Gene Identified
by Gene Trapping from a Genetically Engineered Mouse Model
of Astrocytoma: PNAS. 2007 May8;104(19):8053-8058
Wei, Q, Clarke, L, Scheidenhelm, DK, Qian, B, Tang, A, Sabha, N,
Karim, Z, Bock, N, Reti, R, Swoboda, R, Purev, E, Lavoie, J-F,
Bajenaru, MJ, Shannon, P, Herlyn, D, Kaplan, D, Henkelman, RM,
Gutmann, DH, Guha, A: High-Grade Glioma Formation Results
from Postnatal Pten Loss or Mutant Epidermal Growth Factor
Receptor Expression in a Transgenic Mouse Glioma Model.
Cancer Research. 2006 Aug1: 66(1): 7429-7437
Zadeh, G, Reti, R, Kouisham, K, Baoping, Q, Shannon, P, Guha,
A: Regulation of the pathological vasculature of malignant astro-
cytomas by angiopoietin-1. Neoplasia 2005 Dec;7(12):1081-90
Shannon, P, Sabha, N, Lau, N, Gutmann, DH, Guha, A:
Pathological and Molecular Progression of Astrocytomas in a
GFAP:12V-Ha-Ras Mouse Astrocytoma Model: Am J. of
Pathology: Sep 167(3): 859-867, 2005
Zadeh, G, Koushman, K, Shannon, P, Guha, A: Interaction of
Angiopoietins and VEGF in Astrocytomas: J. Neuropath & Expt.
Neurology: 63(9): 978-989,2004
Razquallah Hakem, Ph.D.
Associate Professor,
Department of Medical Biophysics
rhakem@uhnres.utoronto.ca
Molecular Mechanisms Of DNA Damage
Repair And Apoptosis And Their Role In
Cancer
The main aspect of our research focuses on studying the
underlying mechanisms behind cancer development, including
the influence of impaired DNA repair responses and/or apoptosis,
on the onset of the disease. Although a tremendous amount of
information has been gathered on the disease, there still exists a
variety of questions that remain unanswered, including the
mechanisms behind cancer initiation, progression and tissue speci-
ficity. Moreover, although several genes have been identified as
either tumor suppressors or oncogenes, there likely exist other
cancer genes that have not been identified; be it a cancer
suppressing or cancer promoting function.
In our ongoing efforts to study the link between defec-
tive DNA damage repair/response and cancer our laboratory has
studied both known, as well as novel tumor suppressors, including
BRCA1 and Mus81, respectively. Mutations of BRCA1 increase
the risk for breast, ovarian and other forms of cancer. Using
mouse models, our laboratory has demonstrated that in the
absence of Brca1, cells accumulate DNA damage that activates the
Chk2-p53 pathway, leading to cell cycle arrest and apoptosis. We
have also demonstrated essential roles for the Chk2-p53 pathway
in suppressing Brca1- associated cancer. Currently we are investi-
gating the role that apoptosis plays in preventing Brca1-associated
cancer.
Mus81, in association with Eme1, constitutes an
endonuclease that cleaves branched DNA structures such as repli-
cation forks (RFs). We demonstrated that cells deficient for
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
21
Increased genomic instability in BRCA1
-/-
CHK2
-/-
cells. b: DNA break; t: Triradial
Mus81 or Eme1, exhibit elevated genomic instability and
enhanced sensitivity to the chemotherapeutic drug, mitomycin
(MMC). Mus81 mutants were susceptible to developing sponta-
neous lymphomas, and drastically modified the tumor spectrum
of p53 mutant mice. Other ongoing studies for Mus81 include
the analysis of its functional interactions with Chk2, Rad54, or
Blm, and the effects of dual mutations of these genes on DNA
damage repair and cancer. We are also currently studying the in
vivo function of Pirh2, an E3 ubiquitin ligase that interacts and
ubiquitinates p53, leading to its degradation. We have generated
Pirh2-deficient mice and are currently investigating the role Pirh2
plays in the regulation of p53, the DNA damage response, and
cancer.
Our laboratory also focuses on identifying the mecha-
nisms behind apoptosis. Two major apoptotic pathways exist in
mammalian cells: the death receptor pathway and the mitochon-
drial mediated apoptotic pathway. We are currently assessing the
effect of dual inhibition of the death receptor pathway of
apoptosis and inhibition of the mitochondrial mediated apoptotic
pathway on apoptotic responses, development and diseases
including cancer.
The overall goal of our lab is to contribute to a better
understanding of the mechanisms behind cancer, which can then
be translated to improving the response of tumors to radio and
chemotherapies.
Selected Publications:
McPherson, J. P., Lemmers, B., Hirao, A., Hakem, A., Abraham, J.,
Migon, E., Matysiak-Zablocki, E., Tamblyn, L., Sanchez-
Sweatman, O., Khokha, R., Squire, J., Hande, M. P., Mak, T. W.,
and Hakem, R. Collaboration of Brca1 and Chk2 in tumorigen-
esis. Genes Dev, 18: 1144-1153, 2004.
McPherson, J. P., Lemmers, B., Chahwan, R., Pamidi, A., Migon,
E., Matysiak-Zablocki, E., Moynahan, M. E., Essers, J., Hanada,
K., Poonepalli, A., Sanchez-Sweatman, O., Khokha, R., Kanaar,
R., Jasin, M., Hande, M. P., and Hakem, R. Involvement of
mammalian Mus81 in genome integrity and tumor suppression.
Science, 304: 1822-1826, 2004
Ashwin Pamidi, Renato Cardoso, Anne Hakem, Elzbieta
Matysiak-Zablocki, Anuradha Poonepalli, Laura Tamblyn,
Bayardo Perez-Ordonez, M. Prakash Hande, Otto Sanchez and
Razqallah Hakem. 2007. Functional Interplay Of p53 And Mus81
In DNA Damage Responses And Cancer. Cancer Research.
67:8527-8535, 2007.
Salmena, L., Lemmers, B., Hakem, A., Matysiak-Zablocki, E.,
Murakami, K., Au, P. Y., Berry, D. M., Tamblyn, L., Shehabeldin,
A., Migon, E., Wakeham, A., Bouchard, D., Yeh, W. C., McGlade,
J. C., Ohashi, P. S., and Hakem, R. Essential role for caspase 8 in
T-cell homeostasis and T-cell-mediated immunity. Genes Dev, 17:
883-895, 2003.
David Hedley, M.D., Ph.D.
Professor,
Department of Medical Biophysics
david.hedley@uhn.on.ca
Understanding Molecular Cancer
Therapeutics
The development of molecular cancer therapeutics is a
complex process that starts with the identification and validation
of potential drug targets based on understanding of the molecular
mechanisms of human cancers, and then proceeds through the
development of novel agents that are tested initially in the labora-
tory, and eventually in human clinical trials. In many instances
the results treating cancer patients are less dramatic than those
seen in experimental models. This likely comes about due to a
combination of circumstances including low drug target expres-
sion in some patients, the presence of additional oncogenic
mutations that render the target non-critical for cancer growth,
and suboptimal drug dosing. Our laboratory aims to understand
the mechanisms of molecular cancer therapeutics action using
xenograft models, linked to the analysis of samples obtained
during early clinical trials. The eventual goal is a science-driven
process that can identify optimum treatment schedules of molec-
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology 22
Primary pancreatic cancer xenograft grown orthotopically, showing
typical morphological features including dense fibrovascular stroma,
increased EGFR and HER2, and activation of PKB/Akt.
ular targeted agents for individual cancer patients.
The laboratory has a particular interest in pancreatic
cancers, which are highly resistant to standard cancer treatments.
These cancers have a distinctive set of molecular features
including K-ras mutations and overexpression of multiple growth
factor receptors that explain their aggressive biology, and provide
clues for novel drug targets. Currently we are working on novel
drugs targeting PI3-kinase, MEK, Src, FAK, and GSK3. As well
as standard laboratory models, we use primary xenografts derived
from pancreas cancer patients. As shown in the figure, when
grown in the pancreas of SCID mice these closely simulate the
actual clinical situation, and allow detailed study of the effects of
novel agents, linked to the underlying genetics of each tumour, in
a realistic clinical setting. For monitoring in vivo drug effects, we
make extensive use of sophisticated microscopy and flow cytom-
etry methods to study complex cellular processes including effects
of microenvironmental factors on tumour response, as well as the
microCT, MRI, and PET imaging facilities in the STTARR
Program.
Important features of our laboratory are its extensive
network of interactions with the clinical trials program as well as
basic and translational scientists in the University of Toronto, the
application of a wide range of analytical methods, and access to
exciting new drugs that are just entering human clinical trials.
Selected Publications
Yau,C.Y.F., Wheeler,J., Sutton,K., and Hedley,D.W. Inhibition of
Integrin-Linked Kinase (ILK) by QLT0254 Inhibits Akt-
Dependent Signaling Pathways and is Growth Inhibitory in
Orthotopic Primary Pancreatic Cancer Xenografts. Cancer
Research 65:1497-1504, 2005
Birle DC and Hedley DW. Signaling interactions of rapamycin
combined with erlotinib in cervical carcinoma xenografts.
Molecular Cancer Therapeutics 2006;5:2494-2502.
Birle, D. and Hedley DW. Suppression of the Hypoxia-Inducible
Factor-1 Response in Cervical Carcinoma Xenografts by
Proteasome Inhibitors. Cancer Research 2007;67:1735-43
Pham, N-A., Tsao, M-S, Cao, P. and Hedley, DW. Dissociation of
gemcitabine sensitivity and protein kinase B signaling in pancre-
atic ductal adenocarcinoma models. Pancreas 2007; 35:e16-26
Richard Hill, Ph.D.
Professor,
Department of Medical Biophysics
hill@uhnres.utoronto.ca
Role of Hypoxia in Solid Tumour Progression
& Metastasis
Radiation is one of the primary modalities for the treat-
ment of localized cancer and a number of factors can influence
the response of tumours and surrounding normal tissues to such
treatment. These factors, which can be specific to the individual
tumour or normal tissue and to their environment, can vary from
patient to patient. One part of the research in our laboratory
focuses on understanding how these factors influence tumour and
normal tissue response to radiation treatment in individual
patients. Our current work involves:
1) Examination of hypoxia and high interstitial fluid pressure
(IFP) in animal models of human tumours with a focus on cancer
of the cervix. In these studies we are collaborating with the clinical
groups at the Princess Margaret Hospital in examining methods
to exploit these factors to predict and improve treatment
outcome.
2) Studies of the radiosensitivity of normal dermal fibroblasts in
vivo. These studies are focusing on measuring DNA damage as a
biological dosimeter for potential use in individuals who have
been accidentally exposed to irradiation or for assessment of
differences between in vivo and in vitro radioresponsiveness of
fibroblasts from patients with soft tissue sarcoma who demon-
strate significant complications following combined radiation
therapy and surgery.
3) Examination of the sensitivity of lung tissue to different
volumes of irradiation. These studies in rat and mouse lung are
investigating mechanisms associated with the response of the lung
to radiation damage and drugs that may be useful to mitigate the
long term effects of this damage on lung function, when applied
after the radiation exposure.
The second major focus of our research is the spread of
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
23
SiHa tumours: EF5 staining in red (hypoxia); CD31 staining in
green (blood vessels); Hoechst (blood perfusion) in blue
cancer from its initial site of growth to other locations in the body
(metastasis), which is a major factor influencing the likelihood of
successful treatment. The formation of metastasis by tumour cells
is thought to be dependent on the expression of specific pheno-
types by individual tumour cells. Our research is examining
metastatic phenotypes that are expressed only transiently and that
may be induced by exposure of tumour cells to conditions, such
as hypoxia, which occur in the tumour microenvironment. Recent
clinical results have suggested that tumours that contain substan-
tial hypoxic regions may be more likely to form metastases. We
have found in animal model systems that exposure to hypoxia,
both in vitro and in vivo, can cause transient increases in the
metastatic potential of tumour cells and that exposure to transient
hypoxic episodes may be particularly important for this increased
metastatic potential. We are examining the effect of different levels
of hypoxic exposure and of exposure to intermittent hypoxia in
modifying the expression of genes likely to be associated with
metastasis and tumour progression in xenograft models of human
cervix carcinoma and soft tissue sarcoma. We are also initiating
studies to examine the role of hypoxia in modifying or
maintaining the aggressive phenotype of tumour stem cells.
Selected Publications:
Langan AR, Khan MA, Yeung IWT, VanDyk J, Hill RP. The
protective effects of Eukarion-189, a superoxide dismutase-
catalase mimetic, on volume effects in radiation-induced lung
damage. Radioth Oncol:79(2):231-8, 2006.
Chaudary N and Hill RP. Hypoxia and metastasis. Clin Cancer
Res. 2007;13(7):1947-9.
Fyles A, Milosevic M, Pintilie M, Syed A, Levin W, Manchul L,
Hill RP. Long-term Performance of IFP and Hypoxia as
Prognostic Factors in Cervix Cancer. Radiother Oncol. 2006; 80
(2):132-137.
R. Hill, P. Kaspler, A. Griffin, B. OSullivan, C. Catton, H. Alasti,
A. Abbas, M. Heydarian, P. Ferguson, J. Wunder. Studies of the in
vivo radiosensitivity of human skin fibroblasts. Radiother Oncol.
2007; 84(1):75-83.
Zhang L and Hill RP. Hypoxia Enhances Metastatic Efficiency in
HT1080 Fibrosarcoma Cells by Increasing Cell Survival in Lungs
not Cell Adhesion and Invasion. Cancer Res. 2007; 67(16):7789-
97.
Hill RP and Perris R. Destemming cancer stem cells. J Natl
Cancer Inst 2007;99:1435 40.
Mitsuhiko Ikura, Ph.D.
Professor,
Department of Medical Biophysics
mikura@uhnres.utoronto.ca
Structural Biology and Cell Signalling
Creating and maintaining tissue organization requires
strict control of three processes governed by cellular signalling: cell
division, differentiation and growth. Cells which escape from
normal controls, and proceed along a path of uncontrolled
growth and migration, often lead to cancer. To this end, we are
investigating the structure-function relationships of key signalling
proteins by various biochemical and biophysical method,
including nuclear magnetic resonance (NMR) spectroscopy, X-ray
crystallography, and fluorescence resonance energy transfer
(FRET) imaging microscopy. NMR and X-ray crystallography
enable us to determine the three-dimensional atomic structures of
proteins and protein complexes. FRET allows for visualization of
the dynamic behavior of signalling proteins in living cells.
Currently our research focuses on proteins involved in
calcium signalling, cell adhesion, transcriptional regulation and
bacterial signal transduction. We are part of the newly funded
research program on Genomic Instability and Cancer Survival at
the Advanced Medical Discovery Institute within OCI. This
program will enhance our research activities in the area of cancer.
In 2006 a state-of-art Untra-Shielded 800-MHz NMR instru-
ment has been installed, which is equipped with a high-sensitivity
triple-resonance cryogenic probe. This instrument is crucial for
the investigation of larger proteins which are soluble only at low
protein concentrations. Many cancer-related proteins fall into
this category of targets, including transcriptional regulators,
metabolic enzymes and regulators, and various cancer signaling
proteins. Linking detailed structural analysis with molecular and
cellular functional analyses of cancer proteins, we hope to make
marked contributions to the fight against cancer.
3D structural information of proteins can also provide
useful clues in designing new therapeutic compounds to specifi-
cally inhibit certain protein activities. In the coming years, we
hope to determine 3D structures of more protein-protein
complexes by NMR or X-ray crystallography to visualize, via
FRET imaging microscopy, specific protein-protein interactions
in various cells including tumour cells.
For further information, visit our laboratory website at:
http://nmr.uhnres.utoronto.ca/ikura
Selected Publications:
Plevin, M.J., Zhang J., Guo C., Roeder R.G., Ikura M. The
acute myeloid leukemia fusion protein AML1-ETO targets E
proteins via a paired amphipathic helix-like TBP-associated
factor homology domain (2006). Proc Natl Acad Sci USA.
103(27):10242-7.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
24
Fluorescent imaging of
tumour growth and spread.
Fluorescent cells can be
used to study the spread of
disease to organs, such as
lung (left). Cells that emit
light of different colours
can be used to track specific
populations of cells. The
image shows a mouse lung
bearing tumour colonies 2 weeks after an intravenous injection of
red and green cells. Hoechst (blood perfusion) is in blue.
Bosanac I, Yamazaki H, Matsu-Ura T, Michikawa T,
Mikoshiba K, Ikura M. Crystal structure of the ligand binding
suppressor domain of type 1 inositol 1,4,5-trisphosphate
receptor. (2005) Mol Cell. Jan. 21;17(2): 193-203.
Bosanac I, Alattia JR, Mal TK, Chan J, Talarico S, Tong FK,
Tong KI, Yoshikawa F, Furuichi T, Iwai M, Michikawa T,
Mikoshiba K, Ikura M. (2002) Structure of the inositol 1,4,5-
trisphosphate receptor binding core in complex with its ligand.
Nature 420 , 696-700.
Hoeflich KP, Ikura M. (2002) Calmodulin in action: diversity
in target recognition and activation mechanisms. Cell 108: 739-
742.
Norman Iscove, M.D., Ph.D.
Professor,
Department of Medical Biophysics
iscove@uhnres.utoronto.ca
Specification of Hematopoietic Stem Cell
Identity
Adult stem cells maintain proliferating cellular systems
for life. They do this by generating not only cells primed to enter
differentiation pathways, but also cells that instead retain the
undifferentiated stem cell characteristics of the parent.
Generation of new stem cells is called "self-renewal". We
are interested in two aspects of self-renewal. The first concerns the
genes that come into play when stem cells undergo self-renewal
divisions. The second concerns the means by which some intially
renewing daughter cells lose self-renewal capacity while others
continue to retain it through multiple cell divisions. We use the
hemopoietic system to model these processes. Our findings have
led us to focus increasingly on particular homeobox genes as
central effectors of self-renewal divisions, and on the genes that
control their expression as specifiers of stable stem cell identity in
this system.
Selected Publications:
NN Iscove, M Barbara, M Gu, M Gibson, C Modi, N
Winegarden. Representation is faithfully preserved in global
cDNA amplified exponentially from sub-picogram quantities of
mRNA.. Nat Biotechnol 20:940-943, 2002.
P Benveniste, C Cantin, D Hyam, NN Iscove. Hematopoietic
stem cells engraft in mice with absolute efficiency. Nature
Immunology 4:708-713, 2003.
G Sauvageau, NN Iscove, RK Humphries. In vitro and in vivo
expansion of hematopoietic stem cells. Oncogene 23:7223-7232,
2004.
Michael Julius, Ph.D.
Professor,
Department of Medical Biophysics
michael.julius@sri.utoronto.ca
Signal Transduction in Lymphocyte
Activation
Our work over the last decade has demonstrated that the
modifications of signals emanating from the T cell antigen
receptor complex (TcR/CD3) mediated by accessory activation
molecules (AAM) including CD4/8, CD45, CD28 and glyco-
sylphosphatidylinositol anchored proteins (GPI-AP) can be
extreme, resulting in perturbation of cell growth control, anergy,
or death. Our global objectives are to: characterize the ordered
interaction of AAM with TcR/CD3; define the sequence and
temporal engagement of the second messenger generating systems
engaged; and to characterize the TcR/CD3/AAM induced spatial
re-distribution of second messenger generating systems supporting
cellular activation and growth. Our approach utilizes primary T
cells; T cell clonal variants and transgenesis: enabling a genetic
analysis and facilitating the biochemical characterization under-
pinning the signaling phenotype.
The AAMs CD4 and CD8 play fundamental roles in
the initiation of the earliest signals induced upon TcR/CD3
engagement through their regulation of activity and delivery of
function of Src family PTK. We have discovered the interde-
pendent temporal and spatial regulation of Lck and Fyn during
proximal TcR signaling. Specifically, in primary CD4+ peripheral
lymph node T cells lipid rafts (LR) function to segregate Lck and
Fyn. Upon antigen receptor engagement, Lck is activated within
seconds outside LR, followed by its translocation into LR and the
ensuing activation of co-localized Fyn.
Genetic models and structure function analyses have
enabled the formal demonstration that Fyn activation is predi-
cated by its physical interaction with kinase active Lck within LR,
and revealed a c-terminal sequence common to all members of the
Lck subfamily of Src family PTK that predicates their partitioning
to LR.
Our current working model proposes a biological frame-
work based on structural distinctions amongst Src family subfami-
lies that rationalizes the involvement and regulation of function of
multiple Src-family PTK reported to be critically involved in a
variety of receptor-mediated signaling pathways.
Selected Publications:
Filipp, D., Zhang, J., Leung, B. L., Shaw, A., Levin, S. D., Veillette,
A. and Julius, M. 2003. Regulation of Fyn Trough Translocation
of Activated Lck into LR. J. Exp. Medicine 197: 1221.
Filipp, D., Leung, B. L., Zhang, J., Veillette, A. and Julius, M. 2004.
Enrichment of Lck in LR Regulates Co-localized Fyn Activation
and the Initiation of Proximal Signals Through TcRab. The
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
25
Journal of Immunology 172:4266-4274.
Filipp, D. and Julius, M. LR: Resolution of Fyn problem? 2004.
Molecular Immunology 41: 645-656.
Dominik Filipp, Behrouz Moemeni, Alessandra Ferzoco,
Kirishanthy Kathirkamathamby, Jenny Zhang, Domenique
Davidson, Andr Veillette, and Michael Julius. 2007. Lck
Dependent Fyn Activation requires C-terminus dependent
targeting of kinase active Lck to lipid rafts. Journal of Biological
Chemistry (in press)
Igor Jurisica, Ph.D.
Assistant Professor,
Department of Medical Biophysics
juris@ai.utoronto.ca
Integrative Computational Biology
Merely coping with the deluge of data is no longer an
option; their systematic analysis is a necessity in biomedical
research.
Computational biology is concerned with developing
and using techniques from computer science, informatics, mathe-
matics, and statistics to solve biological problems. Analyzing
biomedical data requires robust approaches that deal with high
dimensionality, multi-modal and rapidly evolving representations,
missing information, ambiguity and uncertainty, noise, and
incompleteness of domain theories.
Our research is focussed on integrative computational
biology, and representation, analysis and visualization of high
dimensional data generated by high-throughput biology experi-
ments, in the context of cancer informatics. Of particular interest
is the use of comparative analysis for the mining of integrated
different datasets such as protein-protein interaction, gene expres-
sion profiling, and high-throughput screens for protein crystalliza-
tion. We integrate multiple data sources and database, develop
and apply combination of diverse algorithms in a systematic
manner. Besides performance, our focus is on scalable algorithms
with easy use by non-experts.
Truly understanding biological systems requires the
integration of data across multiple high-throughput platforms. It
has been established that despite inherent noise present in protein-
protein interaction data sets, systematic analysis of resulting
networks uncovers biologically relevant information, such as
lethality, functional organization, hierarchical structure, dynamic
modularity and network-building motifs. These results suggest
that protein interaction networks have a strong structure-function
relationship, which we use to help interpret integrated cancer
profile data. Focusing on network analysis and modeling,
integrated with cancer profiles will enable us to identify diagnostic
and prognostic biomarkers, understand disease initiation and
progression, which will lead to improving cancer treatment. Tools,
such as BTSVQ, I2D (http://ophid.utoronto.ca/i2d), and
NAViGaTOR (http://ophid.utoronto.ca/navigator) enable users to
interpret integrated cancer profiles, and create relevant models
dynamically.Many targets discovered using approaches described
above will lead to uncharacterized proteins. To further our under-
standing of their function, we may use X-ray crystallography or
NMR to determine their 3D structure.
Protein crystallization is a major bottleneck in high-
throughput structure determination, partially due to many param-
eters affecting the crystallization outcome (e.g., purity of proteins,
intrinsic physico-chemical, biochemical, biophysical and biolog-
ical parameters), and the unknown correlations between the
parameter and the propensity for a given protein to crystallize.
Protein crystallization has two phases: search and optimization.
High-throughput screening (HTS) can speed up the search phase,
and has the potential to increase process quality. To achieve these
benefits, we work on developing a sophisticated automated image
classification algorithms, data mining and reasoning approaches to
optimize protein crystallization plans, improve our understanding
of the crystallization process, and increase a number of deter-
mined disease-related protein structures
(http://www.cs.toronto.edu/~juris/WCG/).
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
26
NAViGaTOR (Network Analysis, Visualization, & Graphing
TORonto) is a software package for visualizing and analyzing
protein-protein interaction networks. NAViGaTOR can query
OPHID / I2D - online databases of interaction data - and display
networks in 2D or 3D. To improve scalability and performance,
NAViGaTOR combines Java with OpenGL to provide a 2D/3D
visualization system on multiple hardware platforms.
NAViGaTOR also provides analytical capabilities and supports
standard import and export formats such as GO and the
Proteomics Standards Initiative (PSI).
Selected Publications:
Gortzak-Uzan, L., Ignatchenko, A., Evangelou, A., Agochiya, M.,
Brown, K., St.Onge, P., Kireeva, I., Schmitt-Ulms, G., Brown, T.,
Murphy, J., Rosen, B., Shaw, P., Jurisica, I., Kislinger, T. A
Proteome Resource of Ovarian Cancer Ascites: Integrated
Proteomic and Bioinformatic Analyses To Identify Putative
Biomarkers. J Proteome Res (2007).
Brown, K. and I. Jurisica. Unequal evolutionary conservation of
human protein interactions in interologous networks. Genome
Biology,8(5), 2007.
Przulj, N, D. G. Corneil, I. Jurisica. Efficient estimation of
graphlet frequency distributions in protein-protein interaction
networks. Bioinformatics, 22(8):974-980, 2006.
Cumbaa, C. A. and I. Jurisica. Automatic classification and pattern
discovery in high-throughput protein crystallization trials, Journal
of Structural and Functional Genomics,6(2-3):195-202, 2005.
Suzanne Kamel-Reid, Ph.D.
Professor,
Department of Medical Biophysics
Suzanne.Kamel-Reid@uhn.on.ca
Identifying Biomarkers of Cancer Initiation,
Progression and Recurrence
Work in our laboratory focuses on the application of high
throughput technologies for example, gene expression microar-
rays, tissue arrays, ChIP-on-chip, microRNA arrays and protein
arrays to enhance our understanding of cancer biology.
Specifically, we are interested in identifying biomarkers of cancer
initiation, progression and recurrence in two types of human
cancer: Head and Neck Squamous Cell Carcinoma (HNSCC)
and Acute Promyelocytic Leukemia (APL).
Head and Neck Squamous Cell Carcinoma
In this arm of our research laboratory, we are specifically inter-
ested in the etiology of oral cancers (OSCCs). The molecular
genetic changes involved in oral cancer development are poorly
understood. Our work focuses on three major questions:
1.) What are the gene(s) involved in recurrence of oral cancer?
Approximately 50% of OSCCs recur after surgery. Our current
approaches involve analyzing OSCC tumour samples, as well as
samples taken from the regions immediately surrounding the
tumours, at time of surgery (surgical resection margins), in order
to identify genes deregulated in the tumour and in the
surrounding margin(s), which may be predictive of tumour recur-
rence.
2) .What are the steps involved in the development of oral cancer?
A significant fraction of OSCCs arise from precursor lesions called
leukoplakias. In these studies, we are interested in profiling the
genetic changes specifically, changes in microRNA expression
in leukoplakias and comparing them to genetic changes found in
tumours.
3.) What are the underlying genetic differences between OSCCs in
young patients (< 45 yrs of age) and older patients (> 45 yrs of age)?
OSCCs are strongly associated with the risk factors of alcohol
consumption and smoking tobacco. However, younger patients
who do not exhibit exposure to either of these two risk factors are
still diagnosed with OSCC. We have observed defective DNA
mismatch repair and differential gene expression in young patients
compared to older patients. This line of study is aimed at further
understanding the genetic differences between young and older
patients, and at understanding the role of defective DNA repair in
OSCCs.
Acute Promyelocytic Leukemia
Leukemias are often associated with chromosomal
translocations that give rise to fusion proteins which may deregu-
late cellular signaling or transcription. Our group cloned and
characterized two variant fusion proteins involved in APL, NPM-
RAR and NuMA-RAR, which are both aberrant transcription
factors; we are especially interested in understanding the roles of
these fusion proteins in the cell. Specifically, we use cell lines,
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
27
Matrigel Invasion Assay for Cells with Over-Expressed and
Silenced CLDN1
NPM Localization in U937-NuMA-RAR Cells
mouse models and human patient samples in order to understand
leukemia biology. The questions we are most interested in include:
1.) What are the common downstream genetic targets of the APL fusion
proteins?
APL is associated with seven known fusion proteins, all of which
involve the RAR transcription factor. Yet, all fusion proteins give
rise to the same disease. We hypothesize that this is because all
fusion proteins have a common set of direct transcriptional
targets, and are utilizing gene expression arrays and ChIP-on-chip
technology to address this issue.
2.) What are the cell biology effects of the APL fusion proteins?
While most studies have focused on the APL fusions as transcrip-
tion factors, more recent evidence has suggested that these
proteins behave distinctly and have effects through protein-
protein interactions on other signaling pathways within the
leukemic cell. We are applying the same concept as above, and
hypothesizing that there may be a common set of interactions
and/or deregulated pathways shared by all APL fusion proteins,
and are characterizing the protein-protein interactions of the
fusions in order to find this out.
3.) What are the necessary secondary events in leukemia development?
Previous studies have indicated that the APL fusions are necessary,
but insufficient, for the development of leukemia in mice,
suggested that additional genetic events may cooperate with the
fusions in leukemia. We are addressing this question through the
above studies, and also through genetic studies of the hCG-
NuMA-RAR mouse model that we previously developed and
characterized.
Selected Publications:
Hummel JL, Zhang T, Wells RA, Kamel-Reis S. The Retinoic acid
receptor alpha (RARalpha) chimeric proteins PML-, PLZF-,
NPM-, and NuMA-RARalpha have distinct intracellular localiza-
tion patterns. Cell Growth Differ. 2002;13:173-183.
Kamel-Reid S, Zhang T, Wells RA. Expression of the NPM-RAR
fusion gene in hematopoietic cells confers sensitivity to troglita-
zone-induced apoptosis. Oncogene, 2003;22:6424-6435.
Sukhai M, Wu X, Xuan Y, Zhang T, Reis PP, Dub K, Rego E,
Bhaumik M, Wells RA, Kamel-Reid S, Pandolfi PP. Myeloid
Leukemia with Promyelocytic Features in Transgenic Mice
Expressing hCG-NuMA-RAR?. Oncogene 2004;23:665-678.
Warner, GC, Reis, PP, Jurisica, I, Sultan, M, Arora, S, Macmillan,
C, Makitie, AA, Grenman, R, Reid, N, Sukhai, M, Freeman, J,
Gullane, P, Irish, J, Kamel-Reid, S. (2004) Molecular classification
of oral cancer by cDNA microarrays identifies overexpressed
genes correlated with nodal metastasis. Int J Cancer
2004;110:857-868.
Tremblay S, Pintor Dos Reis P, Bradley G, Galloni NN, Perez-
Ordonez B, Freeman J, Brown D, Gilbert R, Gullane P, Irish J,
Kamel-Reid S. Young patients with oral squamous cell carcinoma:
study of the involvement of GSTP1 and deregulation of the
Fanconi anemia genes. Arch Otolaryngol Head Neck Surg.
2006;132:958-966.
Gordon Keller, Ph.D.
Professor,
Department of Medical Biophysics
gkeller@uhnres.utoronto.ca
Lineage Specific Differentiation of
Embryonic Stem Cells
The overall goals of our research program are to under-
stand the mechanisms that control mesoderm and endoderm
induction and specification in mouse and human embryonic stem
(ES) cell cultures. Our studies focus specifically on the generation
of the following derivative lineages: hematopoietic, vascular,
cardiac, hepatic and pancreatic. Research projects include:
1.) characterization of the earliest developmental stages of lineage
commitment
2.) identification of the signaling pathways regulating lineage
specification and maturation, and
3.) generation of functional cell populations for transplantation in
preclinical models of human disease.
For the mesoderm-derived lineages we have demon-
strated that the earliest stages of hematopoietic and cardiac devel-
opment in ES cell cultures are defined by the appearance of
progenitors that display both tissue specific and vascular potential.
Within the hematopoietic system, these progenitors are known as
hemangioblasts whereas those that define the onset of cardiac
development are known as cardiovascular progenitors. Our
current studies are aimed at defining the mechanisms that control
the proliferation and differentiation of these progenitor popula-
tions. The long-term goal of these transplantation studies is to
develop new therapies for the treatment of hematopoietic and
cardiovascular diseases. With respect to endoderm derivatives, we
have shown that the combination of activin A, BMP-4 and bFGF
will lead to the efficient generation of cells with characteristics of
immature hepatocytes. Modification of this induction protocol
results in the development of pancreatic progenitors. Our current
research projects in this area are focused on understanding the
pathways that regulate the maturation of these populations into
functional hepatocytes or insulin-secreting pancreatic beta cells.
Access to ES cell-derived hepatocytes and beta cells will provide a
novel source of these cells for cell based therapy approaches for the
treatment of diabetes and certain types of liver disease.
Selected Publications:
Gouon-Evans V, Boussemart L, Gadue P, Nierhoff D, Koehler
CI, Kubo A, Shafritz DA and Keller G. 2006. BMP-4 is required
for hepatic specification of mouse embryonic stem cell-derived
definitive endoderm. Nat Biotech. 24:1402-1411
Kattman SJ, Huber TL and Keller G. 2006. Multipotent flk-1+
cardiovascular progenitor cells give rise to the cardiomyocyte,
endothelial, and vascular smooth muscle lineages. Dev Cell. 2006
11: 723-732
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
28
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
29
Robert Kerbel, Ph.D.
Professor,
Department of Medical Biophysics
robert.kerbel@sri.utoronto.ca
Perturbing Angiogenic Pathways as a
Novel Therapeutic Modality
Our research is focussed in the area of tumor angiogen-
esis and antiangiogenic therapy. In this regard, one of the most
significant developments in medical oncology over the last five
years has been the approval of three different antiangiogenic drugs
for the treatment of a number of human cancers, e.g. breast,
colorectal, renal, and hepatocellular carcinoma. This includes
bevacizumab (Avastin), the humanized anti-VEGF monoclonal
antibody which is now undergoing evaluation in almost 400
clinical trials around the world. Despite the successes of antiangio-
genic drugs, including bevacizumab, remarkably little is known
about how these drugs actually work as anti-cancer agents. The
reason for the counterintuitive property of an antiangiogenic drug
enhancing the efficacy of chemotherapy is unknown and is a
subject of investigation in our laboratory as well as a number of
others around the world. In addition, the best way to administer
chemotherapy with an antiangiogenic drug remains to be deter-
mined. Another major question in the field is how to determine
the optimal biologic dose for antiangiogenic drugs and to monitor
their activity in vivo.
The Kerbel group is studying the hypothesis that
conventional chemotherapy can induce remarkably rapid and
marked mobilizations of a proangiogenic cell population from the
bone marrow compartment, known as endothelial progenitor
cells, which can subsequently home to drug treated tumors and
accelerate their recovery from the initial chemotherapy treatment.
This reactive host response can be blocked using certain antian-
giogenic drugs. The molecular pathways by which these processes
occur is under intensive investigation in Dr. Kerbels laboratory.
Dr. Kerbel is also pioneering a concept known as metro-
nomic chemotherapy (where chemotherapy is given at close
regular intervals at low doses with no prolonged drug-free breaks)
as an exciting and novel new way to combine chemotherapy with
a targeted antiangiogenic drug such as bevacizumab, for the treat-
ment of advanced metastatic disease. We have developed a
number of new models of advanced metastatic disease in immune
deficient mice involving human tumor cell lines, including breast,
ovarian, and hepatocellular carcinoma, as well as malignant
melanoma. These studies have evolved in a new research initiative,
namely, the development of models of spontaneous brain metas-
tases and their use to study the biology and treatment of such
lesions. Finally, our group is at the forefront of studying circu-
lating cellular and molecular surrogate biomarkers in peripheral
blood for antiangiogenic drugs and/or metronomic low-dose
chemotherapy which can help determine the optimal biologic
dose to be used for such drugs/treatments.
Selected Publications
Ebos JM, Lee CR, Christensen JG, Mutsaers AJ, Kerbel RS.
Multiple circulating proangiogenic factors induced by sunitinib
malate are tumor-independent and correlate with antitumor
efficacy. Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):17069-
74. Epub 2007 Oct 17.
Kerbel RS. Antiangiogenic therapy: a universal chemosensitization
strategy for cancer? Science. 2006 May 26;312(5777):1171-5.
Shaked Y, Ciarrocchi A, Franco M, Lee CR, Man S, Cheung AM,
Hicklin DJ, Chaplin D, Foster FS, Benezra R, Kerbel RS.
Therapy-induced acute recruitment of circulating endothelial
progenitor cells to tumors. Science. 2006 Sep 22;313(5794):1785-
7.
Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ,
Bohlen P, Kerbel RS. Continuous low-dose therapy with vinblas-
tine and VEGF receptor-2 antibody induces sustained tumor
regression without overt toxicity. J Clin Invest. 2000
Apr;105(8):R15-24. Erratum in: J Clin Invest.
2006Nov;116(11):3084. J Clin Invest. 2006 Oct;116(10):2827.
Rama Khokha, Ph.D.
Professor,
Department of Medical Biophysics
rkhokha@uhnres.utoronto.ca
Tissue Homeostasis and Cancer
Development
The complex microenvironment contains distinct
entities including the structural extracellular matrix, anchored and
soluble growth factors and cytokines, and a variety of immune
and inflammatory cells. Signals generated within this space dictate
cell fate by influencing cell proliferation, differentiation, motility,
and cell death. We aim to understand how proteolysis
(MMP/TIMP/ADAM), specific growth factors (IGF-II, HGF,
TNF), and tumor suppressors and oncogenes impact these signals
in tissue homeostasis and tumorigenesis. We use genetic mouse
models of human cancers and disease to study the cellular and
molecular basis of breast, liver, lung, and bone cancers, and also
investigate the role of proteolytic systems in tissue homeostasis in
models of heart disease, inflammation and tissue regeneration. A
central hypothesis for our work is that cellular microenvironment
converges at the cell surface to trigger intracellular signaling
pathways and influence cell fate.
In the past two years, we identified TIMP3 as an innate
negative regulator of systemic inflammation. Its loss not only
increases the proteolytic activity of ADAM17, but also of MT1-
MMP known to be central to cell motility and invasion. Multiple
organs of timp3-/- mice exhibit greater metastatic dissemination
to several cancer cell types. We have also found that Rho C and
RANKL are critical for metastasis. Using primary cultures derived
from timp3 deficient mice we have shown dysregulated -catenin
signaling with specific effects on its target genes (cyclin D1,
MMP7) in mammary epithelial and embryonic fibroblasts; and
with primary cultures of neonatal cardiac myocyte and fibroblast
we have identified a novel concurrent amplification of TGF and
TNF signaling due to increased proteolytic activity.
Our ongoing work on TIMP4 in heart disease reveals a
differential function for this TIMP following myocardial infarc-
tion and pressure overload. Some of our recent discoveries in
mouse models are particularly exciting as they hold a high poten-
tial for clinical impact, both in breast and bone tumors. A new
avenue of our research involves the development of innovative
imaging techniques to probe and visualize collagen changes in
vivo in mouse models of human diseases.
Selected Publications:
Murphy G, Murthy A, Khokha R. Clipping, shedding and
RIPping keep immunity on cue. Trends Immunology, in press.
Hojilla CV, Kim I, Kassiri Z, Fata JE, Fang H, Khokha R.
Metalloproteinase axes increase beta-catenin signaling in primary
mouse mammary epithelial cells lacking TIMP3. J Cell Sci,
120:1050-60, 2007
Cruz-Munoz W, Sanchez OH, Grappa M, English JL, Hill RP,
Khokha R. Enhanced metastatic dissemination to multiple organs
by melanoma and lymphoma cells in timp-3(-/-) mice. Oncogene,
25:6489-96. 2006
English JL, Kassiri Z, Koskivirta I, Atkinson SJ, Di Grappa M,
Soloway PD, Nagase H, Vuorio E, Murphy G, Khokha R.
Individual Timp deficiencies differentially impact pro-MMP-2
activation. J Biol Chem, 281(15):10337-462005, 2006.
Smookler DS, Mohammed FF, Kassiri Z, Duncan GS, Mak TW,
Khokha R. TIMP3 regulates TNF-dependent systemic inflam-
mation. J Immunol. 176(2):721-5, 2006.
Jones DH, Nakashima T, Sanchez OH, Kozieradzki I, Komarva
SV, Sarosi I, Morony S, Rubin E, Sarao R, Hojilla CV,
Komnenovic V, Kong YY, Dixon SJ, Sims SM, Khokha R, Wada
T, Penninger JM. Regulation of cancer cell migration and bone
metastasis by RANKL. Nature 440(7084):692-6, 2006.
Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G,
Wakeham A, Khokha R, Mak T. RhoC is Dispensable for
Embryogenesis and Tumor Initiation but is Essential for
Metastasis. Genes Dev, 19:1974-1979, 2005.
Kassiri Z, Oudit GY, Mohammed FF, Sanchez O, Dawood F,
Nuttall RK, Edwards DR, Liu PP, Backx PB, Khokha R.
Combination of tumor necrosis factor-alpha ablation and matrix
metalloproteinase inhibition prevents heart failure after pressure
overload in tissue inhibitor of metalloproteinase-3 knock-out
mice. Circ Res, 97(4):380-90, 2005.
Mohammed FF, Smookler DS, Taylor SEM, Fingleton B, Kassiri
Z, Sanchez OH, English JL, Matrisian LM, Au B, Yeh W-C,
Khokha R. Abnormal TNF activity in timp-3-/- mice leads to
chronic hepatic inflammation and failure of liver regeneration.
Nature Genet. 36:969-977, 2004.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
30
Multiphoton imaging of collagen in the mammary gland and
liver. Normal tissues (left) are contrasted with small mammary
tumors (top right) or liver micrometastases of osteosarcoma
(bottom right).
Green = collagen; red = cellular autofluorescence.
Thomas Kislinger, Ph.D.
Assistant Professor,
Department of Medical Biophysics
thomas.kislinger@utoronto.ca
Proteome Research
The human genome has been sequenced and now
sophisticated technologies are used to characterize the proteome,
the set of proteins generated by that genome, as a function of
developmental stage, environment, tissue-type, and pathology.
The goal of our research is to understand, on a systems level, the
complex biological processes that are fundamental to physiology
and disease. A particular focus of our laboratory is cancer research
(ovarian and breast cancer) and vascular/cardiovascular biology.
Our laboratory develops and applies powerful tools of
proteomics and bioinformatics to analyze the protein complement
in protein complexes, cells, tissues and organisms. Our goal is not
only to determine the level of proteins but also their localization,
dynamics, and interaction partners. In recent years we have devel-
oped sensitive methodologies to isolate specific subcellular
fractions from mammalian cell culture or tissues.
Currently our focus is on optimizing technologies that
allow for the sensitive and selective isolation and characterization
of surface, plasma membrane proteins from cell culture (in vitro)
or directly from the surface of microvascular endothelial cells (in
vivo). The ultimate goal of these studies is to investigate the
proteome dynamics of surface membrane proteins in the vascula-
ture of disease models or in cell culture in response to specific
treatments. Additionally, we are interested in dynamics of protein-
protein interactions. To accomplish this goal we are applying
tandem affinity purifications (TAP-tagging) in combination with
mass spectrometry. Briefly, the protein of interest (bait) is
expressed as a fusion protein with a dual affinity tag. This allows
for mild and efficient purification of this protein and most of its
interacting partners.
Our laboratory is well equipped and currently has full-
time access to two latest generation mass spectrometers, a linear
ion-trap (Thermo Scientific LTQ) and the new Thermo Scientific
LTQ-Orbitrap XL, a high resolution, high mass accuracy hybrid
mass spectrometer. These mass spectrometers are currently the
standard for global, high-throughput proteomics. Additionally, we
have access to a new triple quadrupole mass spectrometer for
target driven proteomics and accurate quantification.
Selected Publications:
Gortzak-Uzan L, Ignatchenko A, Evangelou A, Agochiya M,
Brown KA, St.Onge P, Kireeva I, Schmitt-Ulms G, Brown TJ,
Rosen B, Shaw P, Jurisica I, & Kislinger T. A Proteome resource
of ovarian cancer ascites: integrated proteomic and bioinformatic
analyses to identify putative biomarkers. J Proteome Research
2007 (in press)
Kislinger T, Cox B, Kannan A, Chung C, Hu P, Ignatchenko A,
Scott SS, Gramolini AO, Morris Q, Hallett MT, Rossant J,
Hughes TR, Frey B, & Emili A. Global Survey of Organ and
Organelle Protein Expression in Mouse: Combined Proteomic
and Transcriptomic Profiling. Cell 2006, 125: 173-186
Kislinger T, Gramolini AO, Pan Y, Rahman K, MacLennan DH,
& Emili A. Proteome dynamics during C2C12 myoblast differen-
tiation. Mol Cell Proteomics 2005; 7: 887-901
Kislinger T, Rahman K, Radulovic D, Cox B, Rossant J, & Emili
A. PRISM: A generic large-scale proteomics investigation strategy
for mammals. Mol Cell Proteomics 2003; 2:96-106
Anne Koch, M.D., Ph.D.
Assistant Professor,
Department of Medical Biophysics
akoch@uhnres.utoronto.ca
DNA damage signaling, repair and
cancer
DNA double-strand breaks (DSBs) represent the most
lethal form of DNA damage. DSBs can result from exposure to
exogenous genotoxins, such as ionizing radiation (IR) and
chemotherapeutic drugs, or as a consequence of intrinsic cellular
processes such as DNA replication and metabolic processes that
generate DNA-damaging free radicals. If left unrepaired, DSBs
can result in the loss of genetic material and DSBs are an obligate
intermediate of numerous types of gross genetic aberrations such
as chromosome translocations. Given the critical importance of
DSBs in cancer etiology and therapy, the study of the cellular
response to DSBs is of utmost importance to cancer biology. In
vertebrates, there are two principal DSB repair pathways: nonho-
mologous end joining (NHEJ) and homologous recombination
(HR). HR acts primarily after DNA replication where it
accurately repairs the DSB using the undamaged sister-chromatid
as a DNA template. On the other hand, NHEJ is active
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
31
High-throughput immunofluorescence
confocal microscopy examining IR-induced
Double Strand Breaks.
throughout the cell cycle, predominating during G0 and G1, is
error-prone and uses little or no sequence homology to repair
DSBs. NHEJ is the major pathway involved in the repair of DSBs
induced by DNA damaging agents, such as IR or radio-mimetic
drugs. By virtue of its importance in promoting repair of DSBs
caused by therapeutic IR and anti-cancer drugs, the study of
NHEJ is germane for the development novel anti-cancer thera-
pies.
Our laboratory is focused on studies examining the
complex regulation of the DNA damage response and how its
deregulation relates to carcinogenesis. Some of our specific
research interests include the identification and characterization of
novel DNA damage signalling pathways and repair factors
involved in the cellular response to IR-induced DNA damage. We
are also interested in the detailed examination of these pathways,
and how they may be perturbed, in the context of human breast
cancer.
Selected Publications:
Macrae, CJ, RD McCulloch, J Ylanko, D Durocher and CA Koch.
APLF (C2orf13) facilitates nonhomologous end-joining and
undergoes ATM-dependent hyperphosphorylation following
ionizing radiation. DNA Repair, 2007. In press
Bernstein, NK, RS Williams, ML Rakovszky, D Cui, R Green, F
Karimi-Busheri, RS Mani, S Galicia, CA Koch, CE Cass, D
Durocher, M Weinfeld, JNM Glover. The molecular architecture
of the mammalian DNA repair enzyme, polynucleotide kinase.
Mol Cell 17(5):657-70 (2005)
Koch, CA, R Agyei, S Galicia, P Metalnikov, P ODonnell, A
Starostine, M Weinfeld, D Durocher. Xrcc4 physically links DNA
end processing by polynucleotide kinase to DNA ligation by
DNA ligase IV. EMBO J 23(19):3874-85 (2004)
Michelle Letarte, Ph.D.
Professor,
Department of Medical Biophysics
mablab@sickkids.on.ca
Pathways and Diseases Associated with
Receptors of the TGF- Superfamily
Our research concentrates on vascular diseases caused by
mutations in genes coding for receptors of the transforming
growth factor beta (TGF-) superfamily. We focus primarily on
endoglin and ALK1, which are specifically expressed on endothe-
lial cells. These cells line the blood vessels and are essential for
providing oxygen and nutrients to all tissues as well as serving a
barrier function and sensing changes in blood flow and pressure.
Mutations in endoglin and ALK1 genes are responsible for
Hereditary Hemorrhagic Telangiectasia (HHT) and in some cases
pulmonary arterial hypertension. Our recent studies have demon-
strated that a soluble form of endoglin is associated with pre-
eclampsia, a pregnancy disorder characterized by increased blood
pressure in the mother. We have shown that TGF- can cause
vasorelaxation and stimulate endothelial nitric oxide synthase
(eNOS) activity. Furthermore endoglin interacts with eNOS and
in fact the TGF- receptor complex associates with the eNOS
activation complex, to regulate blood vasomotor function. To
delineate TGF- signaling pathways mediated by endothelial
specific receptors, we have performed robotic screens of protein-
protein interactions in transfected mammalian cells. We have
identified several novel interacting partners of endoglin and ALK1
receptors.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
32
Wild-type Eng-/-
Decreased vascular network in the lungs of the Endoglin heterozy-
gous (Eng+/-) mouse. X-ray micro-CT imaging was performed at
the Mouse Imaging Centre, with Dr M. Henkelman. The perfused
control Eng+/+ mouse lungs reveal a normal vascular network
including the main pulmonary arteries (PA), extensive branching
into arterioles and capillaries and return through the pulmonary
veins (PV). In the Eng+/- mouse, pulmonary arteries are enlarged
and there is a reduced number of peripheral vessels and no perfu-
sion of the venous side. This abnormal remodeling of the lung
vasculature likely contributes to the increased pulmonary vascular
resistance seen in this mouse model of vascular disease.
Our current aim is to determine how these novel inter-
acting proteins interact with the receptors, mediate signaling and
engage in cross-talk with known pathways. We have generated
mouse models of HHT and pulmonary arterial hypertension,
which are useful in determine the protein interactions and
pathways affected in these diseases.Since TGF- is a critical
contributor to several cancers, we are studying the possible role of
some of the novel receptor interacting proteins in ovarian cancer.
Our primary focus is on molecular changes associated with early
events in the disease. We are comparing ovarian epithelial cells
derived from women with BRCA mutations to those of normal
women in an attempt to delineate early changes caused by these
mutations and potentially contributing to cell transformation.
Our work may lead to the identification of early markers needed
for the early diagnostic of this devastating cancer.
Selected Publications:
Toporsian M, Gros R, Kabir MG, Vera S, Govindaraju K,
Eidelman DH, Husain M and Letarte, M. (2005) A role for
endoglin in coupling eNOS activity and regulating vascular tone
revealed in hereditary hemorrhagic telangiectasia. Circ. Res. 96:
684-92.
Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim
YM, Bdolah Y, Lim KH, Yuan HT, Libermann TA, Stillman IE,
Roberts D, D'Amore PA, Epstein FH, Sellke FW, Romero R,
Sukhatme VP, Letarte M and Karumanchi SA. (2006) Soluble
endoglin contributes to the pathogenesis of preeclampsia. Nat.
Med. 12: 642-649.
Abdalla SA and Letarte M. (2006) Hereditary haemorrhagic
telangiectasia: current views on genetics and mechanisms of
disease. J Med Genet 43: 97-110.
Motamed-Khorasani A, Jurisica I, Letarte M, Shaw PA, Parkes
RK, Zhang X, Evangelou, A, Rosen B, Murphy KJ and Brown
TJ. (2007) Differentially androgen-modulated genes in ovarian
epithelial cells from BRCA mutation carriers and control patients
predict ovarian cancer survival and disease progression.
Oncogene 26:198-214.
Fei-Fei Liu, M.D.
Professor,
Department of Medical Biophysics
Fei-Fei.Liu@rmp.uhn.on.ca
Translational Research on Breast & Head &
Neck Cancer
Our lab is focussed on several different aspects of translational
molecular oncology, ranging from high-throughput screens to
identify novel anti-cancer therapeutics, to developing predictive
gene sets for human breast and head/neck cancers, to cancer stem
cell investigations. Using robotic high-throughput screens, we
have identified novel anti-cancer activities of existing antimicro-
bials. We are now pursuing the identification of novel radiosensi-
tizers to enhance the anti-cancer properties of ionizing radiation.
Using diagnostic human cancer samples, and extracting RNA, we
are in the process of conducting global expression analysis of
micro-RNA, a novel genetic species, which appears to be a
powerful modulator of gene and protein expression in biological
systems. Preliminary data are very promising, and in addition to
identifying predictive signatures for improving cancer patient
management, we are also studying their biological function in pre-
clinical models of human cancers. Finally, we are also interested
in understanding how hematopoietic stem cells traffic in response
to ionizing radiation, in order to better understand how local
radiation therapy contributes to secondary acute myeloid
leukemia, a fatal iatrogenic complication in cancer patients.
Selected Publications:
Yip KW, Mao X, Au PYB, Hedley DW, Chow S, Dalili, Mocanu
JD, Bastianutto C, Schimmer A, Liu F-F: Benzethonium chloride:
A novel anticancer agent identified utilizing a cell-based small
molecule screen. Cl Cancer Res 12:5557-5569, 2006
Bachtiary B, Boutros PC, Pintilie M, Shi W, Bastianutto C, Li J-H,
Schwock J, Zhang W, Penn LZ, Jurisica I, Fyles A, Liu F-F. Gene
expression profiling in cervical cancer An exploration of intra-
tumour heterogeneity. Cl Cancer Res 12:5632-5640, 2006
Shi W, Bastianutto C, Li A, Perez-Ordonez B, Ng R, Chow K-Y,
Zhang W, Jurisica I, Bayley A, Kim J, OSullivan B, Siu L, Chen E,
Liu F-F. Multiple dysregulated pathways in nasopharyngeal carci-
noma revealed by gene expression profiling. Int J Cancer
119:2467-2475, 2006
Bastianutto C, Mian A, Symes J, Mocanu J, Alajez N, Sleep G, Shi
W, Keating A, Crump M, Gospodarowicz M, Medin J Minden M,
Liu F-F: Local radiotherapy induces homing of hematopoietic
stem cells to the irradiated bone marrow. Cancer Res 67:10112,
2007
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
33
Geoffrey Liu, M.D.
Assistant Professor,
Department of Medical Biophysics
geoffliu@uhnres.utoronto.ca
Epidemiology of Solid Tumour Progression
Our laboratory is studying the behaviour of cancers of
the aerodigestive tract (esophageal cancer, head and neck cancer,
lung cancer) as well as orphan thoracic tumours such as thymoma
and mesothelioma. As a unique translational epidemiologic
laboratory within MBP, we are focused on discovering novel
molecularly-based methods for risk stratification, prognostication,
and treatment of patients.
Genetic and environmental influences are interwoven
into the fabric of cancer prognosis. Our laboratory performs
human population-based research, using candidate gene
approaches, pathway-based approaches, and high density SNP
technology to identify the potential biologic pathways involved in
esophageal cancer development. Discovery of putative pathways
becomes the first step in narrowing the list of potential environ-
mental carcinogens. We are currently developing customized high
density SNP-chips for assessment in aerodigestive cancers, and
collaborating with computational biologists on their analysis.
These same genetic and environmental factors can play
critical roles in cancer behaviour after diagnosis,and in response to
therapy. Our laboratory is studying pharmacogenomic factors in
cancer treatment, both in terms of traditional chemotherapeutic
agents as well as molecular targeted agents. We recently assessed
the role of several functional polymorphic variants of EGFR in
lung cancer patients treated with an EGFR-tyrosine kinase
inhibitor, and found that these polymorphic variants predict
survival and treatment response. Another focus in our laboratory
is the assessment of putative serum biomarkers to help monitor
patient therapy and response. In addition to human population
studies, pre-clinical models of esophageal and gastric cancers are
being developed in our laboratory. These resources are being used
to assess genotype-phenotype correlations, testing of molecular
targeted drugs and novel therapeutics, and for pre-clinical testing
of biomarkers.
Currently, we are assessing biomarkers and genetic varia-
tions in biologic samples obtained from the following large scale
studies: Lung Cancer Screening Study; Mesothelioma Screening
Study; Molecular Epidemiology of Lung, Esophageal Cancer,
Thymoma and Mesothelioma, a series of US-based studies of
esophageal, lung and pancreatic cancer), and Head and Neck
Cancers from PMH. In addition, our laboratory is involved in the
biologic assessments of a number of Phase I/II/III cancer treat-
ment studies. We are and have been CIHR, NCI (US), NCIC-
CTG, and Doris Duke funded, with close collaborations with
Harvard, Laval, and Seattle.
Selected Publications:
Heist RS, Zhou W, Chirieac LR, Cogan-Drew T, Liu G, Su L,
Neuberg D, Lynch TJ, Wain JC, Christiani DC. MDM2 polymor-
phism, survival, and histology in early-stage non-small-cell lung
cancer. J Clin Oncol. 2007 Jun 1;25(16):2243-7.
Liu G, Gurubhagavatula S, Zhou W, Wang Z, Yeap BY,
Asomaning K, Su L, Heist R, Lynch TJ, Christiani DC.
Epidermal growth factor receptor polymorphisms and clinical
outcomes in non-small-cell lung cancer patients treated with
gefitinib. Pharmacogenomics J. 2007
Liu G, Zhou W, Yeap BY, Su L, Wain JC, Poneros JM, Nishioka
NS, Lynch TJ, Christiani DC. XRCC1 and XPD polymorphisms
and esophageal adenocarcinoma risk. Carcinogenesis. 2007
Jun;28(6):1254-8.
Liu G, Zhou W, Christiani DC. Molecular epidemiology of non-
small cell lung cancer. Semin Respir Crit Care Med. 2005
Jun;26(3):265-72.
David Malkin, M.D.
Professor,
Department of Medical Biophysics
david.malkin@sickkids.ca
Genetic Determinants of Childhood
Cancer Predisposition
Almost every type of cancer has been reported to occur
in a familial form. Li-Fraumeni syndrome (LFS) is a paradigm
cancer predisposition syndrome in which affected family members
develop a wide spectrum of cancers including sarcomas, breast
cancer, brain tumors and adrenocortical carciomas at very young
ages. Many affected individuals develop multiple cancers during
their lifetime and these cancers frequently occur at ever younger
ages with each subsequent generation. We have previously found
that constitutional (inherited or de novo) mutations of the p53
tumour suppressor gene are associated with cancer predisposition
in many of these families, as well as in patients with multiple
primary cancers, and children with osteosarcoma, rhabdomyosar-
coma, choroid plexus carcinoma or adrenocortical carcinoma in
the absence of a family history of cancer.
Our research focuses on the genetic basis of LFS, and
particularly on the epigenetic and molecular cellular events that
modify the underlying constitutional mutant p53 genotype in
LFS families. We have recently demonstrated that accelerated
telomere attrition measured in peripheral blood lymphocytes is
associated with accelerated tumor onset in successive generations,
and that this early onset is further influenced by the existence of a
variable single nucleotide polymorphism (SNP) in the hMDM2
gene. We are now examining the role of telomere dysfunction and
telomerase function in the context of constitutional p53
genotypes, as well as in specific subsets of LFS tumors. We are also
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
34
utilizing high-throughput genome-wide array analyses to explore
the interaction of other elements of the human genome in
modulating the phenotypic variations found in LFS in the context
of distinct underlying p53 alterations. Our goal is to develop an
understanding of the underlying genetic basis of cancer predispo-
sition in these cancer-prone individuals, and to eventually develop
a molecular metric that may inform clinical surveillance opportu-
nities for early tumor detection.
To complement our genetic approach to LFS, we are
exploring the cell signaling mechanisms involved in the initiation
and progression of rhabdomyosarcoma (RMS) a tumor derived
from immature skeletal muscle elements that represents the most
common childhood cancer in LFS, and the most common
sporadic soft tissue sarcoma of childhood. We have previously
demonstrated that RMS cells express vascular endothelial growth
factor receptor (VEGFR) and are responsive to VEGF ligand.
Inhibition of this ligand-receptor interaction leads to growth
inhibition of RMS cells. We are interested in determining the
functional interaction of this kinase pathway with other p53-
dependent and independent signaling mechanisms in RMS.
Characterization of these functional pathways may lead to the
identification of novel molecular therapeutic targets for these
aggressive malignancies.
Selected Publications:
Tabori U, Nanda S, Druker H, Lees J, Malkin D. Younger age of
cancer inititation is associated with shorter telomere length in Li-
Fraumeni syndrome. Cancer Res 67(4): 1415-18, 2007.
Strahm D, Durbin AD, Sexsmith E, Malkin D. The CXCR4-
SDF1 axis is a critical mediator of rhabdomyosarcoma metastatic
signaling induced by bone marrow stroma. Clin Exp Metastasis
Sep 1, 2007. [epub ahead of print]
Tabori U, Vokovic B, Zielenska M, Hawkins C, Braude I, Rutka J,
Bouffet E, Squire J, Malkin D. The role of telomere maintenance
in the spontaneous growth arrest of pediatric low-grade gliomas.
Neoplasia 8(2): 136-142, 2006.
West NA, Neale GA, Pounds S, Figueredo BC, Rodriguez-
Galindo C, Pianovski MA, Oliveira Fiho AG, Malkin D, Lalli E,
Ribeiro R, Zambetti GP. Gene expression profiling of childhood
adrenocortical tumors. Cancer Res 67(2): 600-8, 2007.
Armen Manoukian, Ph.D.
Associate Professor,
Department of Medical Biophysics
armenm@uhnres.utoronto.ca
Understanding PKB Function via Drosophila
Genetics
Our laboratory is interested in signal transduction pathways
which regulate Protein Kinase B (PKB) and Glycogen Synthase
Kinase-3 (GSK-3). These serine/threonine kinases are involved in
multiple signaling pathways and have been shown to be linked to
a variety of human diseases. For example, the hyperactivation of
PKB has been shown to be involved in all stages of tumorigenesis.
We use a combination of genetic, biochemical, cell biological and
pharmacological approaches to the study of the cellular processes
which require the function of these kinases. In order to gain
insight into the mechanism of action of these enzymes, we use
Drosophila melanogaster as a genetic model system. Our labora-
tory initiated these studies via identification of loss of function
alleles of Drosophila PKB . We have used this allele as a tool for
second site genetic modifier screens to identify many novel
components of PKB function as well as identifying novel roles for
PKB during development. Recently, we have used Drosophila as
a drug discovery model in the identification and design of novel
small molecule therapeutics using genetic tools in Drosophila.
Our goal is to combine the powerful genetics of Drosophila with
pharmacology to fast track the process of drug discovery. Using
our approach, we have also linked GSK-3 to the regulation of the
circadian clock in Drosophila and in mammalian cells. This work
has provided provocative links between GSK-3 function and
bipolar therapeutics including lithium. We will be using our
genetic and pharmacogenetic tools in Drosophila to further
explore the mechanism of action of these therapeutics in the
design and identification of novel therapeutics.
Selected Publications:
Staveley BE, Ruel L, Jin J, Stambolic V, Mastronardi FG, Heitzler
P, Woodgett JR, Manoukian AS (1998) Genetic analysis of PKB
in Drosophila. Current Biology 8, 599-602.
Jin J, Anthopoulos N, Wetsch B, Binari RC, Isaac DD, Andrew
DJ, Woodgett JR, Manoukian AS (2001) Regulation of
Drosophila tracheal system development by PKB.
Developmental Cell 1, 817-827.
Martinek S, Inonog S, Manoukian AS, Young MW (2001) A role
for the segment polarity gene shaggy/GSK-3 in the Drosophila
circadian clock. Cell 105, 769-779.
Kaladchibachi SA, Doble B, Anthopoulos N, Woodgett JR,
Manoukian AS (2007) GSK-3, circadian rhythms, and bipolar
disorder: a molecular link in the therapeutic action of lithium.
Journal of Circadian Rhythms 5, 3.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
35
Philip Marsden, M.D.
Professor,
Department of Medical Biophysics
p.marsden@utoronto.ca
Regulation of gene expression in endothe-
lial cells
Current work in our lab focuses upon newer aspects of the
transcriptional and post-transcriptional regulation of endothelial
gene expression in health and disease. Exciting new information
indicates that chromatin structure and DNA methylation play a
key role in the cell-restricted expression of important endothelial
genes. Furthermore, RNA binding proteins and antisense RNA
interactions also play an unexpected role in regulation of endothe-
lial gene expression, in part, through modulation of RNA stability
and translational efficiency. We suspect that these pathways may
involve RNA interference. Epigenetic pathways and RNA inter-
ference are newer concepts in the regulation of genes in the
cardiovascular system.
Models of Endothelial Activation
The study of endothelial cells has provided unique
insight into important cardiovascular diseases and the control of
angiogenesis during tumour development. The control of new
blood vessel formation, or angiogenesis, is orchestrated by vascular
endothelium and endothelial cells respond to unique signals in
their environment with a repertoire of cellular and molecular
responses. Studies directed towards dissecting the molecular
mechanisms underlying alterations in genotype and phenotype are
underway using prototypic endothelial cell gene products (e.g.
endothelin-1, eNOS, CXCR4, adhesion molecules such as
VCAM-1 or ICAM-1) and exciting models of cellular activation
(hypoxia, shear stress and epigenetic modifiers). An excellent
example of the applicability of this approach is our recent finding
that verotoxins, bacterial-derived exotoxins that cause severe
inflammation of capillary beds in patients with E coli 0157:H7,
regulate the expression of genes in vascular endothelium at the
post-transcriptional level through AUF1 and proteasomal
pathways.
Regulated expression of nitric oxide (NO) in vascular tissue - role
of epigenetics.
Release of NO from the vascular endothelium represents
a sensitive and highly effective local system for maintaining local
blood flow to an organ. This research program played a major role
in the cloning and functional characterization of endothelial NO
synthase (eNOS) cDNAs and in the demonstratiion that expres-
sion of this endothelial gene is perturbed in atherosclerosis. Recent
studies seek to define the structure and organization of the eNOS
gene and examine mechanisms of regulation that are relevant to
the pathobiology of endothelial cells. Current work focuses upon
newer aspects of the transcriptional and post-transcriptional
regulation of eNOS mRNA in development and disease.
Selected Publications:
Mawji, I.A., Robb, G.B., Tai, S.C., Marsden, P.A. Role of the 3-
untranslated region of human endothelin-1 in vascular endothelial
cells: Contribution to transcript lability and the cellular heat shock
response. J Biol Chem, 279: 8655-67, 2004
Chan, Y., Fish, J.E., DAbreo, C., Lin, S., Robb, G.B., Teichert,
A.M., Karantzoulis-Fegaras, F., Keightley, A., Steer, B.M.,
Marsden, P.A. The cell-specific expression of endothelial nitric
oxide synthase: a role for DNA methylation. J. Biol. Chem, 279:
35087-100, 2004
Robb. G.B., Carson, A.R., Tai, S.C., Fish, J.E., Singh, S., Yamada,
T., Scherer, S.W., Nakabayashi, K., Marsden, P.A. Post-transcrip-
tional regulation of endothelial nitric oxide synthase by an
overlapping antisense mRNA transcript. J. Biol. Chem,. 279:
37982- 96, 2004
Fish, J.E., Matouk, C.C., Rachlis, A., Lin, S., Tai, S.C., DAbreo,
C., Marsden, P.A. The expression of endothelial nitric oxide
synthase is controlled by a cell-specific histone code. J Biol Chem.
280:24824-38, 2005
Chan, G.C., Fish, J.E., Mawji, I.A., Leung, D.D., Rachlis, A.C.,
Marsden, P.A. Epigenetic basis for the transcriptional hypore-
sponsiveness of the human inducible nitric oxide synthase gene
in vascular endothelial cells. J Immunol. 175: 3846-3861, 2005.
Ward, M.E., Toporsian, M., Scott, J.A., Teoh, H., Govindaraju, V.,
Wener, A., Bevan, S.C., Newton, D.C., Marsden, P.A. Hypoxia
induces a functionally significant and transitionally efficient
neuronal nitric oxide synthase mRNA variant. J Clin Invest. 115:
3128-3139, 2005.
Fish, J.E., Matouk, C.C., Yeboah, E., Bevan, S.C., Khan, M., Patil,
K., Ohh, M, Marsden, P.A. Hypoxia-inducible expression of a
natural cis-antisense transcript inhibits endothelial nitric-oxide
synthase. J Biol Chem, 282:15652-66, 2007
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
36
Lisa Martin, Ph.D.
Assistant Professor,
Department of Medical Biophysics
lmartin@uhnres.utoronto.ca
Etiology and Prevention of Breast Cancer
Our research focuses on the etiology and prevention of
breast cancer with an emphasis on mammographic density.
Mammographic density refers to a pattern of breast tissue compo-
sition (as seen on a mammogram) that is strongly associated with
the risk of developing breast cancer, is common in the population,
and is highly heritable. An understanding of the etiology of
mammographic density, and how it is related to breast cancer risk,
is likely to lead to effective preventative interventions for breast
cancer. Our research uses an epidemiological approach that
includes the study of diet and lifestyle factors, as well as biochem-
ical, genetic and molecular factors measured in a variety of biolog-
ical samples.
We have recently completed a long-term, multi-centre
clinical trial to determine if a low-fat diet will reduce the
incidence of breast cancer in women with extensive mammo-
graphic density (in collaboration with Dr. Boyd, Principal
Investigator). The analysis of the data from this trial is currently
underway. Future work will include an examination of the effects
of diet on mammographic density, blood hormones and growth
factors as well as breast tumour characteristics.
Additional studies are investigating the relationship of
mammographic density with markers of oxidative stress/inflam-
mation and with blood telomere length. We have previously
shown that increased urinary malondialdehyde, a marker of lipid
peroxidation, is associated with increased mammographic density.
We will extend this observation by studying more specific
measures of lipid peroxidation (eg. urinary isoprostanes) and
markers of inflammation produced from arachidonic acid via the
COX-2 pathway (eg. urinary prostaglandin E2).
Telomeres, which cap the ends of chromosomes and
promote their stability, become shorter with cell replication and
exposure to oxidative stress. Shorter blood telomere length has
been associated with higher risk of some types of cancer.
Currently we are examining whether blood telomere length is
associated with mammographic density and other risk factors for
breast cancer. We have set up a quantitative PCR technique to
facilitate measurement of blood telomere length in large epidemi-
ological studies, This work may suggest a novel way in which
mammographic density is related to breast cancer and motivate
future studies to examine whether blood telomere length is a
marker of breast cancer risk.
Selected Publications:
Martin LJ, and Boyd NF. Potential mechanisms of breast cancer
risk associated with mammographic density: hypotheses based on
epidemiological evidence. Breast Cancer Research. Accepted
November 2007.
Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, Jong R,
Hislop G, Chiarelli A, Minkin S, Yaffe M. Mammographic
density, and risk and detection of breast cancer in screened
populations. N Eng J Med. 356:11-20, 2007.
Boyd NF, Martin LJ, Sun L, Guo H, Chiarelli A, Hislop G, Yaffe
M, Minkin S. Body size, mammographic density and breast
cancer risk. Cancer Epidemiol Biomarkers Prev. 15:2086-2092,
2006.
Martin LJ, Greenberg C, Kriukov V, Minkin S, Jenkins DJA, Boyd
N. Intervention with a low-fat, high carbohydrate diet does not
influence the timing of menopause. Am J Clin Nutr. 84:920-928.
2006
Jane McGlade, Ph.D.
Professor & Graduate Co-ordinator,
Department of Medical Biophysics
jmcglade@sickkids.on.ca
Signal Transduction in Normal
Development and Disease.
Our research focus is on the protein networks and
signal-transduction pathways that control normal cellular function
and human disease. Through identification and functional charac-
terization of intracellular adapter proteins we study the molecular
mechanisms that control how signal transduction pathways are
integrated, localized, and down regulated. We have discovered and
characterized new adapters such as Gads and SLAP/SLAP2 that
function in growth factor and antigen-receptor signaling, as well
as the adaptors NUMB, EHD and Lulu/Ymo1, which regulate
the activity of trans-membrane receptors that determine cell fate
and polarity during development. Our lab is also studying the role
of ubiquitin dependent regulation of signaling pathways. We
have identified a family of novel E3 ubiquitin ligases, including
LNX, and our current focus is to discover the role of LNX activity
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
37
Variations in mammographic density
in cell fate determination and the establishment of cell polarity
during embryonic development. As part of both of these projects
we are developing high throughput assays of signal transduction
and protein degradation pathways using robotics, protein arrays
and high throughput imaging systems (www.sickkids.ca/sidnet).
More information can be found at http://www.sickkids.ca/mcglade
Selected Publications:
Simoncic, D.P, Bourdeau, A., Lee-Loy, A., Rohrschneider, L.R.,
Tremblay, M.L., Stanley, E.R. and C.J. McGlade (2006) T-cell
Protein Tyrosine Phosphatase (Tcptp) is a negative regulator of
CSF-1 signaling and mononuclear phagocyte development. Mol.
Cell Biol. 26:4149-4160.
Wolting, C., McGlade, CJ., and D. Tritchler (2006) Cluster analysis
of protein array results via similarity of gene ontology annota-
tion. BMC Bioinformatics, 7:338.
Smith, CA., Lau, K., Rahmani, Z., Dho, SE, Brothers, G., She,
Y., Bonneil, E., Thibault, P., Berry, DM, Le Borgne. R.,
Shweisguth, F., and C.J. McGlade (2007) aPKC-mediated
phosphorylation regulates asymmetric membrane localization of
the cell fate determinant Numb. EMBO J. 26:468-80.
Pakuts B, Debonneville C, Liontos LM, Loreto MP, McGlade CJ.
(2007) The Src-like adaptor protein 2 regulates colony-stimulating
factor-1 receptor signaling and down-regulation. J. Biol. Chem.
282:17953-63
Jeffrey Medin, Ph.D.
Associate Professor & Curriculum Co-ordinator,
Department of Medical Biophysics
jmedin@uhnres.utoronto.ca
Gene Transfer/Therapy projects to
generate specific immune responses
against cancers and to correct inherited /
acquired diseases
We employ the latest-generation of recombinant
lentiviral gene transfer vectors to deliver important transgenes into
target hematopoietic cells and other tissues. We also have gener-
ated novel vectors that incorporate advanced suicide safety
systems based on modified human enzymes and unique prodrug
combinations that can be used to control the fate of any vector-
transduced cell in order to maximize safety after transplantation
into both usual and innovative niches. Our work thus involves the
full-spectrum from biochemistry to subcloning/site-directed
mutation of interesting factors to construction of novel recombi-
nant gene transfer vectors to testing outcomes in vitro to testing
outcomes in vivo in small and large animal models. In addition,
we are licensing vectors we have generated to a company for
preparation of clinical-grade supernatants for Phase I trials.
Selected Publications:
Sato T, Neschadim A, Konrad M, Fowler DH, Lavie A, Medin
JA. Engineered human tmpk/AZT as a novel enzyme/prodrug
axis for suicide gene therapy. Mol Ther (2007) 15:962-70.
Silvertown JD, Symes JC, Neschadim A, Nonaka T, Kao JC,
Summerlee AJ, Medin JA. Analog of H2 relaxin exhibits antago-
nistic properties and impairs prostate tumor growth. FASEB J
(2007) 21:754-65.
Yoshimitsu M, Higuchi K, Ramsubir S, Nonaka T, Rasaiah VI,
Siatskas C, Liang SB, Murray GJ, Brady RO, Medin JA.
Efficient correction of Fabry mice and patient cells mediated by
lentiviral transduction of hematopoietic stem/progenitor cells.
Gene Ther (2007) 14:256-65.
Neschadim A, McCart JA, Keating A, Medin JA. A roadmap to
safe, efficient, and stable lentivirus-mediated gene therapy with
hematopoietic cell transplantation. Biol Blood Marrow Transplant
(2007) 13:1407-16.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
38
Phosphorylation controls asymmetric localization of the cell
fate determinant Numb. Numb protein (green)lacking
two protein kinase C phosphorylation sites (Numb2A) is
symmetrically rather than asymmetrically localized in
polarized epithelial cells as shown above in optical sections
taken in the z-axis. Polarized MDCK cells were fixed and
co-stained with anti-aPKC (red) and anti-ZO-1 (blue)
which mark the sub-apical region and the tight junction
respectively. Confocal x-y sections show (I) the sub-apical
localization of aPKC and (II) the basal-lateral accumula-
tion of Numb. Size bar indicate 10 microns.
Benjamin Neel, M.D., Ph.D.
Professor,
Department of Medical Biophysics
bneel@uhnres.utoronto.ca
Signal transduction in stems cells and
disease
Our laboratory studies cell signaling, with a particular
emphasis on protein-tyrosine phosphatases (PTPs). We also have a
developing interest in normal and tumor stem cell signaling. The
roles of the SH2 domain-containing phosphatase Shp2 and its
binding proteins in several human diseases are a major focus.
Shp2 is expressed ubiquitously and is a positive signal transducer,
required for Ras/Erk activation downstream of most receptor
tyrosine kinases, cyotkine receptors, and integrins. Shp2 is
required for a variety of developmental processes, including the
survival of trophoblast stem cells, As a consequence of loss of the
latter function, Shp2-null embryos die peri-implantation. In the
absence of an appropriate phosphotyrosyl peptide, Shp2 is
inactive, because the N-SH2 domain is inserted into the catalytic
cleft of the phosphatase (PTP) domain. We showed earlier that
mutations in the SH2/PTP interface can yield "activated
mutants". Analogous mutations in humans cause 50% of cases of
the autosomal dominant disorder Noonan Syndrome (NS), while
somatic mutations cause some leukemias/myeloproliferative disor-
ders (MPD). Interestingly, another autosomal dominant disorder,
LEOPARD syndrome, also is caused by Shp2 mutations, but
surprisingly, we showed recently that such mutations are catalyti-
cally inactive/impaired and act as dominant negative mutants in
transfection assays. We have generated an allelic series of inducible
and stable NS and leukemia mutant knock-in mutants, as well as
inducible Shp2 knockout mice and cell lines.
Current work is aimed at elucidating key Shp2 substrates
using both candidate and unbiased proteomic approaches, delin-
eating its role in specific tissues using the inducible knockouts,
determining the cellular and molecular basis of how Shp2
mutations cause NS and hematopoietic disease, and generating
mouse models of NS. We have also recently identified another
major NS gene, and are studying the biochemical and cellular
effects of these mutations, as well as the potential involvement of
this gene in human tumors. Finally, we are testing the effects of
specific oncogene/tumor suppressor gene mutations on prospec-
tively purified mammary stem and progenitor cell populations,
and attempting to identify/purify and culture cancer stem cells
from several types of solid tumors.
Selected Publications:
Neel BG, Hayward WS, Robinson HS, Fang J, Astrin SM. Avian
leukosis virus-induced tumors have common proviral integration
sites and synthesize discrete new RNAs: oncogenesis by promoter
insertion. Cell, 1981; 23:323-334.
O'Reilly AM, Pluskey S, Shoelson SE, Neel BG. Activated
mutantsof SHP-2 preferentially induce elongation of Xenopus
animal caps. Mol. Cell. Biol., 2000; 20:299-311.
Araki T, Mohi MG, Ismat FA, Bronson RT, Williams IR, Kutok
JL, Pao LI, Gilliland DG, Epstein JA and Neel BG.Murine Model
of Noonan Syndrome Reveals Cell Type- and Gene Dosage-
Dependent Effects of PTPN11 Mutation Nature Medicine 2004;
10:849-57.
Mohi MG, Williams IR, Dearolf CR, Chan G, Kutok JL, Cohen
S, Morgan K, Boulton C, Shigematsu H, Keilhack H, Akashi K,
Gilliland DG and Neel BG. Prognostic, therapeutic and mecha-
nistic implications of mouse model of leukemia evoked by Shp2
(PTPN11) mutations. Cancer Cell, 2005; 7:179-91
Pamela Ohashi, Ph.D.
Professor,
Department of Medical Biophysics
pohashi@uhnresearch.ca
T Cell Tolerance
The focus of our lab is to investigate the mechanisms that
maintain tolerance or promote T cell activation, leading to the
induction of immunity, autoimmunity or potentially tumor
immunity. We have used a combination of transgenic models that
allow us to follow a responding T cell population specific for a
defined self antigen or tumor associated antigen. In many studies
we have also combined these models with various gene deficient
mice to examine the importance of different molecules on the
induction of T cell tolerance or activation. Our lab pursues three
basic directions:
1.) Investigating the role of survival versus apoptosis on tolerance
and autoimmunity.
2.) Investigating signaling pathways that control T cell tolerance,
activation, immunity, autoimmunity or tumor immunity.
3.) Examining the potential for immune surveillance and tumor
immune therapy.
Selected Publications:
Calzascia, T., Pellegrini, M., Hall, H., Sabbagh, L., Ono, N.,
Elford, A.R., Ohashi, P.S. (2007) TNF-alpha is critical for anti-
tumor but not anti-viral T cell immunity. J. Clin. Invest. (in press).
Gronski, M.A., Boulter, J.M., Moskophidis, D., Nguyen, L.T.,
Holmberg, K., Elford, A.R., Deenick, E.K., Kim, H.O.,
Penninger, J.M., Odermatt, B., Gallimore, A., Gascoigne, N.R.J.
and Ohashi, P.S. (2004) TCR affinity and negative regulation limit
autoimmunity. Nature Med. 10:1234-1239.
Millar, D.G., Garza, K.M., Odermatt, B., Elford, A.R., Ono, N.,
Li, Z. and Ohashi, P.S. (2003) Hsp70 promotes antigen-presenting
cell function and converts T-cell tolerance to autoimmunity in
vivo. Nat. Med. 9:1469-1476.
Nguyen, L.T., Elford, A.R., Murakami, K., Garza, K.M.,
Schoenberger, S.P., Odermatt, B., Speiser, D. and Ohashi, P.S.
(2002) Tumor growth enhances cross-presentation leading to
limited T cell activation without tolerance. J Exp Med 195:423-35.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
39
Hitoshi Okada, M.D., Ph.D.
Assistant Professor,
Department of Medical Biophysics
hokada@uhnres.utoronto.ca
Regulation of Cell Cycle Progression
Our laboratory primarily is interested in the signaling
pathways regulating the entry into and the progression of the cell
cycle - factors which are crucial to the maintenance of genomic
stability. The pathways regulating such processes also have a direct
link to cellular proliferation, differentiation and survival. In fact,
dysregulation of such function results in numerous adverse patho-
logical conditions, including cancer.
By developing comprehensive data sets, we endeavor to
understand how normal signaling pathways are regulated and how
such functional characteristics differ in the presence of disease. In
order to achieve this goal, we are currently investigating several
aspects of the regulatory pathways of proliferation and survival.
Firstly, we are studying the p53 signaling pathway which is essen-
tial for cell cycle arrest and apoptotic induction which occurs
during numerous stresses. We have recently identified Bat3 as a
novel positive regulator of p53 transactivation in DNA damage
response and we are now in the process of investigating the
biological activity, tissue specific function and interacting
molecules of Bat3. Incidentally, the existence of bi-allelic inacti-
vating mutation of Bat3 has recently been identified in a colon
cancer cell line. Secondly, we are focusing on one of the heat
shock proteins, HSP70-2. HSP70-2 is essential for meiotic cell
division in male germ cells and cell proliferation of advanced
human cancers, but is not required for proliferation of primary
cells. Further investigation has suggested that HSP70-2 may
regulate histone modifications. However, the precise molecular
mechanism of just how HSP70-2 regulates cell survival and prolif-
eration remains unclear and we therefore continue to study the
signaling pathways and interacting proteins relevant to its
function. Of significance, HSP70-2 was previously identified as a
functional tumor antigen. We firmly believe that dissection of the
mechanisms of action of cell signaling pathways holds great
promise for identification of new potential therapeutic targets.
Selected Publications:
Sasaki, T., Gan, E., Wakeham, A., Kornbluth, S., Mak, TW.,
Okada, H. (2007): HLA-B-associated transcript 3(Bat3)/Scythe is
Essential for p300-mediated Acetylation of p53. GenesDev.
21(7):848-61.
Aronzo, L., Okada, H., Pasolli, HA., Wakeham, A., Itie, You-Ten
A., Mak, TW., Fuchs, E. (2005): Sgk3 Links Growth Factor
Signaling to Maintenance of Progenitor Cells in the Hair Follicle.
J. Cell Biol. 170 (4):559-570
Okada, H. and Mak, T.W. (2004): Pathways of Apoptotic and
Non-Apoptotic Death in Cancer Nat. Rev. Cancer 4(8):592-603.

Emil Pai, Dr. rer. nat.


Professor,
Department of Medical Biophysics
pai@hera.med.utoronto.ca
Structure and Function of Proteins
Knowledge of the three-dimen-
sional structure of a given protein
is an absolutely essential prerequi-
site for the full understanding of
the chemical basis of an enzymes
catalytic mechanism, for inter-
preting the way structural proteins
like e.g. actin interact with each
other or how lead compounds have to be modified to improve
affinity and specificity of drug candidates. In my lab, we use X-ray
crystallographic techniques to establish the molecular architectures
of proteins. We integrate our structural results with biochemical
and molecular-biological as well as computational data, either
collected in our laboratory or available through collaborations
with specialists in the respective fields.
Orotidine 5'-phosphate decarboxylase from archaea, plasmodia
and man
ODCase is one of the most proficient enzymes known. It
catalyzes the conversion of orotidine-5'-mono-phosphate (OMP)
to UMP, the last step in the de novo biosynthesis of pyrimidine
nucleotides enhancing the decarboxylation rate by 17 orders of
magnitude without the help of any cofactors or metal ions. We
have determined more than 50 structures of the various enzymes,
apo-forms as well as complexes with substrate, product and a large
number of inhibitors synthesized and tested in animals as anti-
malaria and anti-cancer agents by our collaborators in medicinal
chemistry, parasitology and cancer research.
CorA -- a Mg
2+
transporter from T. maritima
Represents the labs first membrane protein structure, a
pentameric protein that imports Mg2+-ions into the cell. While
our lab pursues the structures of the
open state of the molecule and
structurally characterizes a large
number of mutants, we also collabo-
rate with colleagues specializing in
electrophysiology, AFM and compu-
tational approaches to elucidate the
details of the proteins workings.
Antibodies as tools in structure
research
Although the three-dimensional structure of antibodies has been
know for a long time, they still represent interesting research
targets as well as unique tools for stabilizing transitional states. We
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
40
are interested in the structural basis of antibody maturation, cross-
reactivity, and the recognition of epitopes on HIV proteins. We
also make use of antibodies in trapping intermediates of the
PrPC-PrPSc transition of prion proteins for structural characteri-
zation.
Selected Publications:
K. Okamoto, K. Matsumoto, R. Hille, B.T. Eger, E.F. Pai & T.
Nishino (2004) Crystal Structure of Xanthine Oxidoreductase in
Catalysis: Hydroxylation Mechanism and Inhibition. Proc. Natl.
Acad. Sci. USA, 101:7931-7936.
M. Fujihashi, A.M. Bello, E. Poduch, L. Wei, S.C. Annedi, E.F. Pai
& L.P. Kotra (2005) An Unprecedented Twist to ODCase
Catalytic Activity. J. Amer. Chem. Soc. 127:15048-15050.
J. Payandeh & E.F. Pai (2006) A Structural Basis for Mg2+
Homeostasis and the CorA Translocation Cycle. EMBO J.
25:3762-3773.
A.M. Bello, E. Poduch, M. Fujihashi, M. Amani, Y. Li, I. Crandall,
R. Hui, P.I. Lee, K.C. Kain, E.F. Pai & L.P. Kotra (2007) A
Potent, Covalent Inhibitor of Orotidine 5-Monophosphate
Decarboxylase with Antimalarial Activity. J. Med. Chem. 50:915-
921.
Christopher Paige, Ph.D.
Professor,
Department of Medical Biophysics
paige@uhnres.utoronto.ca
Cellular Interactions and Biochemical
Mediators that Promote Development
B lymphocytes produce antibodies and present antigen.
Failure to regulate the growth, development, and response of B
cells can lead to malignancy, immunodeficiency and autoimmu-
nity. Our research efforts are directed towards a better under-
standing of the process of B cell development and their role in
disease.
B cell development is dependent on growth and differen-
tiation factors. In some cases, such factors are produced by
stromal cells. We are determining the biochemical nature of the
cell-bound and secreted molecules that mediate these interactions.
We have also identified interactions that occur between devel-
oping B cells themselves. These homotypic interactions appear to
be critical for progression from the pre -B to the B cell stage of
development.. Finally, we are undertaking signal transduction
experiments that seek to determine the interactions between
molecular pathways regulated by critical cell surface receptors such
as the pre B Cell Receptor and the IL-7 Receptor as well as
growth and differentiation factors such as IL-7 and IL 21. We
have cloned and characterized a novel bioactive peptide,
Hemokinin -1, that may also play a role in the early development
of B cell progenitors.
Immune System and Disease
We also study B cell malignancies in both mouse and
human models. Of specific interest is determining the mecha-
nisms of action of the immune system in recognizing and
destroying tumor cells.We have found that IL-12 can play a key
role in promoting the ability of the immune system to recognize
and eliminate the leukemia cell.We have also found that there are
important differences between T cell mediated immune responses
elicited by systemic administration of IL-12 compared to IL-12
gene therapy in which the leukemia cells are infected with
lentiviral vectors that produce IL-12.
Selected Publications:
Labbe, A., A. Tran and C.J. Paige 2006. Murine model of
immune-mediated rejection of the acute lymphoblastic leukemia
70Z/3. J. Immunol. 176(9):5354-61.
Zhang, Y., A. Berger, C.D. Milne and C.J. Paige. 2006.
Tachykinins in the immune system. Curr. Drug Targets.
7(8):1011-20.
Konforte, D. and C.J. Paige. 2006. Identification of cellular inter-
mediates and molecular pathways induced by IL-21 in human B
cells. J. Immunol. 177(12)8381-92.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
41
Linda Penn, Ph.D.
Professor,
Department of Medical Biophysics
lpenn@uhnres.utoronto.ca
Exploiting Molecular Oncology To Trigger
Tumor Cell Death
Cancer occurs when a single cell acquires multiple
genetic mutations that result in uncontrolled growth and survival.
The tumor cell becomes addicted to these mutations and their
activated signaling pathways. Our goal is to understand, exploit
and target these tumor-specific vulnerabilities. To this end we
have two working groups in the lab, TumorsnTargets and
DeathnDrugs. Our ideas are simple and relevant, and our
approach is novel and achievable. We focus on two major areas.
The first area of focus is the regulation and function of
the Myc oncogene. Myc is deregulated in >50% of human
cancers and functions as a regulator of gene transcription. We are
identifying the target genes regulated by Myc, the cofactors
recruited by Myc and the post-translational modifications
regulating these processes. Targeting Mycs potent transforming
activity through the development of novel inhibitors is a major
area of investigation. Interestingly, for a non-transformed cell
with deregulated Myc expression to develop into a fully malignant
cancer, it is essential that Mycs ability to potentiate apoptosis be
overcome. We aim to understand the molecular mechanism of
Myc-induced apoptosis and identify genetic events that can block
this programmed cell death, to cooperate with Myc in tumour
progression. To achieve these goals we use state of the art
technologies, including ChIP-chip, high-throughput FRET/FLIM
as well as functional cloning, and have established several tissue
culture and animal models for our work, including leukemia,
breast, neuroblastoma, and lung cancers.
The other major area of focus is in the use of statins as
anti-cancer agents. Statins are a family of drugs used routinely in
the control of hypercholesterolemia that we and others, have
shown can trigger tumor-specific apoptosis. Statins block HMG-
CoA reductase, the rate-limiting enzyme of the mevalonate
pathway. This is a basic biochemical pathway essential for cellular
metabolism. Because statins are an approved drug for use in
humans, we can immediately fast-track statins to impact patient
care. To this end, our research goals are to understand why
certain tumor-types are highly sensitive to statin-induced
apoptosis. Moreover, we aim to determine how best to use statins
in treatment regimes in combination with traditional and molec-
ular targeted therapeutic options. Recent evidence in the lab
suggests that statins can also sensitize tumor cells to undergo
apoptosis in a mevalonate-independent mechanism. Biomarkers
of sensitivity and response are also a major focus. The role of
statins and the sensitivity of the tumor stem cell is also a key area
of investigation. Tumor types under study include leukemia,
multiple myeloma and ovarian cancer, however, several others
neoplasias are also potential targets.
Selected Publications:
Meyer N, Kim SS, Penn LZ. The Oscar-worthy role of Myc in
apoptosis. Semin Cancer Biol. 2006 Aug;16(4):275-87. Review
Kim SS, Shago M, Kaustov L, Boutros PC, Clendening JW, Sheng
Y, Trentin GA, Barsyte-Lovejoy D, Mao DY, Kay R, Jurisica I,
Arrowsmith CH, Penn LZ. CUL7 is a novel antiapoptotic
oncogene. Cancer Res. 2007 Oct 15;67(20):9616-22.
Wong WW, Dimitroulakos J, Minden MD, Penn LZ. HMG-CoA
reductase inhibitors and the malignant cell: the statin family of
drugs as triggers of tumor-specific apoptosis. Leukemia. 2002
Apr;16(4):508-19. Review
Wong WW, Clendening JW, Martirosyan A, Boutros PC, Bros C,
Khosravi F, Jurisica I, Stewart AK, Bergsagel PL, Penn LZ.
Determinants of sensitivity to lovastatin-induced apoptosis in
multiple myeloma. Mol Cancer Ther. 2007 Jun;6(6):1886-97.
Gilbert Priv, Ph.D.
Professor,
Department of Medical Biophysics
prive@uhnres.utoronto.ca
Transcriptional repressor complexes and
membrane proteins
Our research centers on the study of protein structure and molec-
ular recognition, with an emphasis on understanding protein-
protein, protein-peptide and protein-lipid interactions. We use a
variety of biophysical, bioinformatic and biochemical techniques
to address these questions, including x-ray crystallography,
spectroscopy and thermodynamic methods.
Structure and function of the BTB domain
Several human BTB-zinc finger proteins, including PLZF and
BCL6, are transcriptional repressors that are implicated in devel-
opment and/or in cancer. The BTB domain in these proteins
represses the expression of target genes by the BTB-mediated
recruitment of HDAC/corepressor complexes to promoter sites
recognized by the C-terminal zinc-finger regions. We are studying
the interactions of BTB domains with corepressor and cullin-
based ubiquitin ligase complexes with the objective of developing
protein-protein interaction inhibitors to reverse the biological
activities of these oncogene products.
Structural biology of membrane proteins
It is widely recognized that new methods are needed for the struc-
tural study of membrane proteins, and we are addressing the
problem of membrane protein crystallization by developing
lipopeptide detergents (LPDs), a fundamentally different class of
amphiphile designed specifically for the crystallization of
membrane proteins. We are also studying the thermodynamics of
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
42
membrane protein folding, and the fundamental properties of
protein-lipid and protein detergent interactions. We have inter-
ests in several specific membrane and membrane-associated
proteins, including transporters, the PagP acyl transferase, and the
saposin proteins.
More information can be found at
http://xtal.uhnres.utoronto.ca/prive.
Selected Publications:
Parekh S, Priv GG and Melnick A. Therapeutic targeting of the
BCL6 oncogene for diffuse large B-cell lymphomas. Leukemia
and Lymphoma (2008).
Ghetu AF, Corcoran CM, Cerchietti L, Bardwell VJ, Melnick A,
Priv GG. Structure of a BCOR corepressor peptide in complex
with the BCL6 BTB domain dimer. Molecular Cell (2008).
Michaux C, Pomroy NC, Priv GG. Refolding SDS-Denatured
Proteins by the Addition of Amphipathic Cosolvents. J. Mol.
Biol. (2008).
Alattia JR, Shaw JE, Yip CM, Priv GG. Molecular imaging of
membrane interfaces reveals mode of beta-glucosidase activation
by saposin C. Proc. Natl. Acad. Sci. 104, 17388-17393 (2007).
Mira Puri, Ph.D.
Assistant Professor,
Department of Medical Biophysics
mira.puri@sri.utoronto.ca
Cardiovascular and Hematopoietic
Development
Research in my laboratory focuses on the cellular and
molecular mechanisms that lead to the formation and stability of
the cardiovascular and hematopoietic systems in mammals.
During development, this process is tightly coordinated, involving
the differentiation and interaction of several cell lineages to form
the heart, blood vessels and circulating blood cells, and thus is
dependent on signals exchanged by these cell types. Our research
concentrates on two families of signalling molecules, TIE1 and
TIE2 receptor tyrosine kinases, and the TGF/BMP receptor,
Endoglin. These cell surface receptors are expressed in both
endothelial and hematopoietic cells in the embryo and the adult.
Consistent with this restricted
expression patterns, these
molecules have been implicated
in a number of human disease
states involving the unchecked
growth or differentiation of
vascular and hematopoietic cells.
Using the mouse as a model
system, we take a genetic
approach both in vivo and in
embryonic stem cells to define
the cellular processes mediated
by these pathways in establish-
ment of the hematopoietic stem
cell microenvironment, as well
as the patterning of both blood
and lymphatic circulatory
system during embryogenesis
and early postnatal life.
Selected Publications:
Tachibana, K., Jones, N. Dumont, D.J. Puri, M.C., Bernstein A.
(2005) Selective role of a distinct tyrosine residue on Tie2 in heart
development and early hematopoiesis. Mol Cell Biol. 25(11),
4693-702.
Puri, M.C. and Bernstein, A. (2003) Requirement for the TIE
family of receptor tyrosine kinases in adult but not fetal
hematopoiesis. Proc. Natl. Acad. Sci. 100(22); p. 12753
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
43
Developing blood vessels of mid-
gestation mouse embryo visual-
ized by Tie1-LacZ reporter gene.
Crystal structure of a complex between the BCL6 BTB domain
and a peptide from the BCOR transcriptional corepressor
Jonathan Rast, Ph.D.
Assistant Professor,
Department of Medical Biophysics
jrast@sri.utoronto.ca
Gene Regulatory Programmes that Control
Animal Immunity
Research in our laboratory is concerned with character-
izing the gene expression programs that underlie animal
immunity. These gene regulatory networks encompass transcrip-
tion regulatory proteins, the DNA control sequences to which
they bind and the signaling systems which convey nuclear states
among cells. We are specifically interested in understanding
regulatory networks that direct immunocyte specification and
thereby set up programs for innate immune function. We employ
the embryo and larva of the purple sea urchin (Strongylocentrotus
purpuratus) as an uncomplicated experimental system in which to
carry out these investigations. These animals offer advantages for
this work including the availability of enormous quantities of
staged embryos, highly efficient transgenesis and the ability to
specifically perturb gene function using antisense and transgenic
technologies. Importantly, sea urchins are echinoderms, a sister-
group of the chordates, and they thus share a genetic heritage with
the vertebrates critical for our comparative immune investigations.
The purple sea urchin genome sequence reveals an innate
immune system of unprecedented diversity that is rich with exper-
imental opportunities to better understand our own immune
system. We approach these issues from two directions:
1.) We are characterizing expression of sea urchin homologs of
transcription factors that are well known as regulators of
hematopoiesis and immunity in vertebrates. Genes currently
being characterized include homologs of vertebrate GATA-1/2/3,
PU.1/Spi-B/Spi-C, E2A/HEB/ITF-2, SCL/Tal-2/Lyl-, and Id-
1/2/3/4. All of these genes are important in the function of adult
immune cells and in the development of larval immune cells.
2.) In a second approach, we are characterizing genes that are
responsible for immune recognition, regulation and effector
functions. Genes in this category include those encoding immense
superfamilies of recognition proteins such as toll-like receptors
(TLRs), Nod/Nalp-like proteins and SRCR domain proteins,
genes that encode immune signaling molecules such as TNF
family members and IL17 homologs, gene families with character-
istics of antimicrobial peptides, and perforins. We are also looking
at a variety of genes with homologs in the vertebrate adaptive
immune system.
Work in this simple model reveals conserved gene regula-
tory circuitry that can then be characterized in more complicated
vertebrate systems, but the sea urchin also provides an invaluable
set of natural experiments that illuminate the workings of our
own immune system and present us with entirely novel solutions
to the immune challenges all animals face.
Selected Publications:
Rast JP, Smith LC, Loza-Coll M, Hibino T, Litman GW. 2006.
Genomic insights into the immune system of the sea urchin.
Science 314:952-6.
Sea Urchin Genome Sequencing Consortium. 2006. The genome
of the sea urchin Strongylocentrotus purpuratus. Science. 314:
941-52.
Hibino T, Loza-Coll M, Messier C, Majeske AJ, Cohen AH,
Terwilliger DP, Buckley KM, Brockton V, Nair SV, Berney K,
Fugmann SD, Anderson MK, Pancer Z, Cameron RA, Smith LC,
Rast JP. 2006. The immune gene repertoire encoded in the purple
sea urchin genome. Dev Biol. 300:349-65.
Fugmann SD, Messier C, Novack LA, Cameron RA, Rast JP.
2006. An ancient evolutionary origin of the Rag1/2 gene locus.
Proc Natl Acad Sci U S A. 103:3728-33.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
44
A sea urchin
pluteus larva.
The sea urchin is
a relatively close
relative to the
vertebrates. They
can be used as a
model system to
learn how genes
interact to
coordinate
immune
function.
A larval pigment cell that has phagocytosed a labeled
bacterium. The bacterium (green rod) is seen among
the numerous red vesicles of the cell that contain
antibacterial compounds. The colorless cells in the
background are ectodermal cells that form the exterior
of the larva.
Brian Raught, Ph.D.
Assistant Professor,
Department of Medical Biophysics
brian.raught@uhnres.utoronto.ca
Looking for trUbl: Proteomics of ubiquitin
and the ubiquitin-like proteins
We are using a combination of biochemical, genetic, and
proteomics techniques to study a very interesting, but poorly
understood, class of small regulatory proteins - the ubiquitin-like
modifiers (Ubls). The Ubls are post-translationally conjugated to
other biomolecules to regulate their functions: interestingly, the
various Ubls (a family of 12 different isoforms in mammalian
cells) appear to be conjugated to several different types of
molecules, and possess very different functions. The Ubls have
been implicated in a variety of critical cellular functions, such as
cell cycle control, the DNA damage response, and intracellular
localization. Most importantly, several of the Ubls (e.g. NEDD8
and SUMO) have also been implicated in neurological disorders
such as Parkinsons and Alzheimers disease, as well as cancer.
Recently, we developed a novel mass spectrometry-based
pattern recognition tool that can identify previously invisible
Ubl conjugation sites. Using this tool, we were able to demon-
strate that, like ubiquitin, the Ubl SUMO can form several
different types of SUMO-SUMO multimeric chains. We are in
the process of determining the physiological function of these
chains using genetic and biochemical assays in mammalian cells
and the model organism S. cereviseae. More information can be
found at http://www.raughtlab.ca.
Selected Publications:
Pedrioli, P., B. Raught, X.D. Zhang, R. Rogers, J. Aitchison, M.
Matunis, and R. Aebersold (2006) Automated identification of
SUMOylation sites using mass spectrometry and SUMmOn
pattern recognition software. Nature Methods 3:533
King, N.L., E.W. Deutsch, J.A. Ranish, A.I Nesvizhskii, J.S. Eddes,
P. Mallick, J. Eng, F. Deseire, M. Flory, D.B. Martin, B. Kim, H.
Lee, B. Raught and R. Aebersold (2006) Analysis of the
Saccharomyces cerevisiae proteome with PeptideAtlas. Genome
Biol 7:R106
Gingras, A.C., M. Gstaiger, B. Raught and R. Aebersold (2007)
Analysis of protein complexes using mass spectrometry. Nature
Rev Mol Cell Biol 8:645
Gingras, A.C., R. Aebersold and B. Raught (2005) Advances in
protein complex analysis using mass spectrometry. J. Physiol.
563:11
David Rose, D. Phil.
Professor,
Department of Medical Biophysics
drose@uhnres.utoronto.ca
Glycosidases in human health and
disease
Many of the most important macromolecules in physio-
logical processes are neither proteins nor nucleic acids, but carbo-
hydrates (sugars and polysaccharides). Our focus is to investigate
the enzymes that process (assemble or break down) complicated
polysaccharide structures, and the role of these enzymes in health
and disease processes. We emphasize glycosidases because they are
particularly suited for inhibitor development. Glycosidase
inhibitors have already shown promise clinically as anti-cancer,
anti-diabetic and anti-viral compounds.
1. GH38 enzymes and cancer
Several years ago, we first derived the atomic structure of
the Golgi enzyme -mannosidase II (GMII). A participant in the
N-glycosylation pathway of glycoprotein synthesis, GMII is classi-
fied in a family of enzymes, glycosyl hydrolase (GH) 38. Other
members of this family are related to HMII structurally and
mechanistically, but have very different substrate specificities and
localizations in the cell. Inhibition of GMII has been shown to
have clinical effectiveness against the progression of late-stage
cancer, but with side-effects that are attributed to broad inhibition
of other GH38 enzymes. Our approach is to examine the struc-
tural characteristics of this family that give rise to cross-inhibition,
and to design modified inhibitors with more specificity to GMII.
In a recent,
exciting result, we have
derived the structure of
GMII complexed with its
intact polysaccharide
substrate (see Figure 1).
This has allowed us to
identify the precise charac-
teristics of several separate
sugar binding sites. By
linking together these sites
with branched inhibitors,
we hypothesize that higher
specificity for GMII will
result.
2. GH31 enzymes and Diabetes
Starch is the major source of energy from the diet. A
series of intestinal enzymes degrade starch, ultimately into glucose.
Inhibiting some of these enzymes has been shown to be effective
in controlling blood sugar levels. In addition, understanding the
specificity of these enzymes for different forms of starch will allow
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
45
Figure 1
us to understand what nutritional sources are most effective in
generating glucose. This could have applicability in the develop-
ment of less expensive but more effective nutritional sources for
underdeveloped regions.
It turns out that the two main enzymes in the later stages
of starch processing, maltase glucoamylase, MGAM, and sucrase
isomaltase, SIM, are both made up of dual glycosidase domains,
which are all members of the same GH31 family. We are devel-
oping a program to study all four of these domains in order to
understand how they work in concert to degrade different forms
of starch. Recently, we have determined the first MGAM domain
structure (Figure 2) and we are studying how it interacts with
novel glycosidase inhibitors.
Selected Publications:
Sim L, Quezada-Calvillo R, Sterchi E, Nichols BL, Rose DR
(2007) Human Intestinal MaltaseGlucoamylase: Crystal
Structure of the N- Terminal Catalytic Subunit and Basis of
Inhibition and Substrate Specificity J Mol Biol, in press.
Kumar NS, Kuntz DA, Wen X, Pinto BM, Rose DR (2007)
Binding of Sulfonium-Ion Analogues of Di-epi-Swainsonine and
8-epi-Lentiginosine to Drosophila Golgi -Mannosidase II: The
Role of Water in Inhibitor Binding Proteins: Structure, Function
and Bioinformatics, in press.
Aaron Schimmer, M.D., Ph.D.
Assistant Professor,
Department of Medical Biophysics
aaron.schimmer@utoronto.ca
Chemical Biology and Drug discovery
Our lab is interested in chemical biology and drug
discovery with a focus on the apoptosis pathway. Using automated
and robotic equipment we screen chemical and siRNA libraries to
identify chemical and genetic probes and use them as tools to
better understand biological pathways with a focus on apoptosis
and the pathogenesis of leukemia.
For example, to identify small molecule inhibitors of the
anti-apoptotic protein XIAP, a million compound small molecule
library was screened. From this screen, small molecule XIAP
inhibitors were identified and subsequently used as tools to
validate XIAP as a therapeutic target in acute leukemia. Based
partly on this work, a clinical trial of XIAP antisense oligonu-
cleotides in combination with reinduction chemotherapy was
launched in patients with refractory acute myeloid leukemia.
Similar chemical biology approaches have been used to identify
small molecules that sensitize resistant cells to death receptor
ligands by reducing expression of the caspase-8 inhibitor FLIP.
Our work on small molecules that decrease FLIP and activate the
death receptor pathway of caspase activation also led to a clinical
trial of the synthetic triterpenoid CDDO in patients with refrac-
tory leukemia.
Finally, efforts are underway to advance novel molecules
from the lab to the bedside. Here, off-patent drugs are screened
to identify compounds that impact targets important in the
pathogenesis of malignancy and thus have previously unrecog-
nized anti-cancer activity. Through this approach, new insights
into molecular pathways are gained. In addition, these old drugs
can be repurposed and moved rapidly into clinical trial for the
treatment of malignancy.
Selected Publications:
BZ Carter, M Gronda, Z Wang, K Welsh, C Pinilla, M Andreeff,
WSchober, A Nefzi, GP Pond, IA Mawji, RA Houghten, J
Brandwein, MD Minden, A Schuh, RA Wells, H Messner, K
Chun, JC Reed, AD Schimmer. Small-molecule XIAP inhibitors
derepress downstream effector caspases and induce apoptosis of
leukemia cell lines and patient samples. Blood, 2005 105: 4043-50
IA Mawji, CD Simpson, R Hurren, M Gronda, MA Williams, J
Filmus, J Jonkman, R Da Costa, B Wilson, MP Thomas, JC Reed,
GV Glinsky, AD Schimmer. Critical role for Fas-associated death
domain-like interleukin-1-converting enzyme-like inhibitory
protein in anoikis resistance and distant tumor formation. Journal
of the National Cancer Institute, 2007, 99: 811-822
IM Mawji, CD Simpson, M Gronda, MA Williams, R Hurren, C
Henderson, A Datti, JL Wrana, AD Schimmer. A chemical
screen identifies anisomycin that sensitizes resistant cells to
anoikis by decreasing FLIP protein synthesis. Cancer Research.
2007, 67:8307-15
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
46
Figure 2
David Spaner, Ph.D.
Assistant Professor,
Department of Medical Biophysics
david.spaner@sri.utoronto.ca
T cell-mediated Cancer immunotherapy
The goal of our lab is to develop methods to allow more
cancer patients to benefit from the therapeutic potential of tumor-
reactive T cells. The two major projects are to learn how best to
activate tumor-reactive T cells and how to make tumor cells
immunogenic, or able to interact with, and be killed by, T cells.
Malignant Melanoma and Chronic Lymphocytic Leukemia
(CLL) are used as clinical models.
Tumor-reactive T cells are often in a suppressed state.
We have shown that infusing anergic T cells into
lymphopenic hosts reawakens them, and are exploring this
phenomenon as a strategy to amplify T cell responses after treat-
ment with cytotoxic chemotherapy. We are also investigating the
use of antibodies against CTLA-4 (an important negative
regulator of T cell activation) as another strategy to recover
suppressed tumor-reactive T cell function.
Cancer vaccines can also activate tumor-reactive T cells.
We have developed methods to make leukemia cells
differentiate into dendritic cells (the most potent T cell stimula-
tors), and are currently designing clinical trials of these vaccines.
We have extended these methods to generate large numbers of
pseudo-dendritic cells from normal B cells, which can be
infected with antigen-expressing viruses to become effective
vaccine platforms in immunologically susceptible cancers. The
biology and manipulation of these activated B cells in mice and
humans is a major project.
To improve the immunogenicity of tumor cells, we are
exploring the use of immunomodulators such as Toll-like receptor
agonists and type 1 interferons (IFNs). We have shown that
infusions of IFN following melanoma vaccines significantly
improved clinical efficacy and that TLR-7 agonists sensitized CLL
cells to lysis by both T cells, and chemotherapies. However, the
oncogenic events that drive cancers can also corrupt
immunomodulator signaling pathways. A central hallmark of
aggressive cancer cells is their strong dependence on glucose
(rather than other fuels) to meet their metabolic needs. A current
hypothesis in the lab is that this aberrant glucose metabolism
results in glycosylation of intracellular signaling molecules and
inhibition of signals from immunomodulators. The molecular
explanations for these phenomena are being investigated using cell
lines, primary tumor cells, and transgenic mice as tools, and the
identification and development of small molecules that can restore
proper immunomodulator signaling are being actively pursued.
Recently, we identified an extracellular calcium-sensing
receptor on B cells that prepares them to engage in immune
responses and appears to be important in the pathogenesis of
CLL. Cloning this receptor, developing inhibitors, and estab-
lishing its role in other B cell cancers are also current projects in
the lab.
Selected Publications:
Spaner, D., X. Sheng-Tanner, A. Schuh. Aberrant regulation of
superantigen responses during T-cell reconstitution and graft-vs-
host disease in immunodeficient mice. Blood 100:2216-24 (2002).
Astsaturov I, T. Petrella, U. Bagriacik, M. de Benedette, G.
Lumber, N. Berinstein, N. Iscoe, C. Hammond, P. Hamilton, and
D. Spaner. High dose Interferon-?2b amplifies pox-virus induced
anti-tumor T cell responses: implications for cancer vaccines.
Clinical Cancer Research 9: 4347-4355 (2003).
Shi Y, White D, He L, Miller RL, Spaner DE. Toll-like receptor-7
tolerizes malignant B cells and enhances killing by cytotoxic
agents. Cancer Res. 67:1823-31 (2007).
Hammond CM, White D, Tomic J, Shi Y, Spaner DE.
Extracellular calcium sensing promotes human B cell activation
and function. Blood. 110:3985-95 (2007)
Vuk Stambolic, Ph.D.
Assistant Professor,
Department of Medical Biophysics
vuks@uhnres.utoronto.ca
Signal Transduction and Cancer:
Regulation of Cellular Proliferation, Survival
and Apoptosis
Cellular transformation, an early step in tumorigenesis, is
almost exclusively caused by malfunction of signal transduction
pathways. Immediate cellular environment, including metabolic,
mitogenic and positional clues, activity of various modifier genes,
as well as selective pressure for tumor growth, profoundly affects
cellular signaling throughput and plays key roles in cancer initia-
tion, progression and metastasis. Our laboratory is interested in
studying cancer-related signal transduction and its effects on
cellular growth, survival and apoptosis. Our primary interest is the
PI3K signaling pathway, which has been implicated in the
etiology of multiple human malignancies. For instance, PI3K
(PIK3A) is a potent oncogene whose mutations are often found in
colon, breast, brain and lung cancers, whereas PTEN, a gene
whose protein product directly counteracts PI3K activity, is one of
the most frequently mutated tumor suppressor genes in a variety
of human cancers.
Our previous research in this area included characteriza-
tion of the physiological function of PTEN, as well as develop-
ment of several animal model systems that were highly informa-
tive in characterizing the role of 3PI signaling in tumorigenesis
and regulation of cell growth and size. More recently, we have
become interested in the relevance of subcellular localization of
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
47
pathway components as an additional regulatory input into 3PI
signaling and have described the importance of compartmental-
ization of certain pathway components for proper signal propaga-
tion.Our current interests include examination of modes of
PTEN regulation, prioritization of PKB/Akt substrates relevant to
tumorigenesis and characterization of Rheb, a small GTPase and a
downstream target of this pathway.Our work aims to provide a
thorough understanding of molecular mechanisms of signal trans-
duction and may lead to the development of effective therapeutic
strategies for treatment of cancer.
Selected Publications:
Stambolic V. and Woodgett, JW Functional Distinctions of
Protein Kinase B/Akt Isoforms Defined by Their Influence on
Cell Migration. Trends in Cell Biology 2006, 16(9):461-6
Buerger C, Devries B, Stambolic V. Localization of Rheb to the
endomembrane is critical for its signaling function. Biochem
Biophys Res Commun. 2006 J344(3):869-80
Backman SA, Ghazarian D, So K, Sanchez O, Wagner KU,
Hennighausen L, Suzuki A, Tsao MS, Chapman WB, Stambolic V,
Mak TW. Early onset of neoplasia in the prostate and skin of
mice with tissue-specific deletion of Pten. Proc Natl Acad Sci U
S A. 2004 Feb 10;101(6):1725-30.
Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C,
Sasaki T, Ruland J, Penninger JM, Siderovski DP, Mak TW.
Negative regulation of PKB/Akt-dependent cell survival by the
tumor suppressor PTEN. Cell 1998, Vol. 95:29-39.
Ian Tannock M.D., Ph.D.
Professor,
Department of Medical Biophysics
ian.tannock@uhn.on.ca
Microenvironmental causes of resistance
to anticancer drugs
Mechanistic studies of drug resistance in tumours have
concentrated on genetic changes in cancer cells. We are studying
equally important, but neglected mechanisms that depend on
tumour microenvironment. Anti-cancer drugs access solid
tumours via the blood, and must penetrate multiple cell layers to
reach all cancer cells. We study drug penetration using multilay-
ered cell cultures ex vivo, and quantitative immunohistochemistry
to assess drug distribution in tumours of animals. We have shown
limited perivascular distribution of the clinically-used anticancer
drugs doxorubicin, mitoxantrone, and topotecan in solid
tumours, but uniform distribution in normal tissues other than
brain. We are investigating strategies to overcome clinical resist-
ance that include:
i.) Use of the inactive pro-drug AQ4N, which penetrates tissue to
reach hypoxic regions where it is activated to AQ4, an analogue of
mitoxantrone. We have shown complementary special distribu-
tions and synergistic activity of mitoxantrone and AQ4N in
human tumour xenografts and are initiating a phase 1 trial, to be
followed by a phase 2 trial in patients with hormone-refractory
prostate cancer.
ii.) Use of the anti-ulcer agent pantoprazole to inhibit sequestra-
tion of the weak bases doxorubicin and mitoxantrone in acidic
endosomes of tumour cells (by inhibiting the endosomal proton
pump), thereby allowing improved tissue penetration. We expect
to initiate a second clinical trial of pantoprazole and doxorubicin
for treatment of solid tumours. For drugs that are not themselves
fluorescent, we are studying drug distribution in vivo by using
antibodies to the drugs, or that recognize downstream targets of
drug activity.
Repopulation of bone marrow allows recovery after chemotherapy
Repopulation of surviving tumour cells also occurs, and increases
in the rate of repopulation between successive cycles of
chemotherapy may lead to shrinkage and regrowth of tumours
without change in intrinsic drug sensitivity. Another goal of our
program is selective inhibition of repopulation of tumour cells
using molecular targeted cytostatic agents: giving such agents
between courses of chemotherapy (and stopping them prior to the
next cycle) is probably the optimal strategy to inhibit repopulation
and improve outcome of chemotherapy, whereas concurrent
administration, as often used clinically, is likely to be less effective.
Selected Publications:
Kim JJ, Tannock IF. Repopulation of cancer cells during therapy:
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
48
an important cause of treatment failure. Nature Reviews Cancer
5:516-25, 2005.
Primeau AJ, Rendon A, Hedley D, Lilge L, Tannock IF. The
distribution of the anti-cancer drug doxorubicin in relation to
blood vessels in solid tumors. Clin Cancer Res 11:8782-8, 2005.
Minchinton AI, Tannock IF. Drug penetration in solid tumours.
Nature Reviews Cancer 6:583-592; 2006.
Trdan O, Galmarini CM, Patel K, Tannock IF. Drug Resistance
and the solid tumour microenvironment. J Natl Cancer Inst
99:1441-54, 2007.
Elisabeth Tillier, Ph.D.
Assistant Professor,
Department of Medical Biophysics
e.tillier@utoronto.ca
Molecular Evolution, Computational
Biology, Bioinformatics, and Genomics
Recent advances in technology have made possible the rapid
accumulation of massive amounts of molecular biological data.
Namely, rapid sequencing has made possible the complete
sequencing of entire genomes, and DNA microchip technology is
advancing such that the study of the expression patterns of all
genes in a genome can be analyzed rapidly. Our interest is in
developing and improving analysis tools that are needed to
consider such large amounts of varied data in light of studying
biology and evolution on a genomic scale.
Projects in the lab include:
Developing methods of protein sequence analysis for predic-
tion of aspects of their structure and protein interactions.
Developing methods of DNA and RNA sequence analysis to
predict aspects of their function and regulation.
Developing new bioinformatics tools for metagenomic applica-
tions.
Selected Publications:
A Fast and Flexible Approach to Oligonucleotide Probe Design
for Genomes and Gene Families. Feng, S. and E.R.M. Tillier,
Bioinformatics (2007) 23 :1195-202
Maximizing Co-Evolutionary dependencies to discover interacting
proteins. Tillier, E. R. M., L. Biro, G. Li and D. Tillo,
PROTEINS: Structure, Function and Bioinformatics (2006) 63:
822-31
Using multiple interdependency to separate functional from
phylogenetic correlations in protein alignments. Elisabeth R. M.
Tillier and Thomas W. H. Lui, Bioinformatics (2003) 19: 750-755
Genome Rearrangement by Replication-Directed Translocation.
Tillier, E. R. M. and R. A. Collins, Nature Genetics (2000) 26:
195-197
Suzanne Trudel, M.D.
Assistant Professor,
Department of Medical Biophysics
strudel@uhnres.utoronto.ca
Preclinical Validation of Novel Agents for
Treatment of Multiple Myeloma
Our research focus is on novel drug development for the treat-
ment of mature B cell malignancies based on molecular targets.
This encompasses drug discovery and pharmacological profiling of
candidate compounds, pre-clinical studies to validate molecular
and cellular targets, the establishment of appropriate animal
models for drug testing, and the development of Phase I/II
clinical trials.
Specifically, we have recently validated the tyrosine
kinase, Fibroblast Growth Factor Receptor 3 (FGFR3) as thera-
peutic target for a subgroup of multiple myeloma patients that
express this receptor on tumor surface. We in pre-clinical studies
have further identified the small molecule inhibitor, CHIR-258
and a neutralizing anti-FGFR3 antibody, PRO-001 as active
agents against FGFR3 expressing myeloma cells. As a direct result
the first ever, clinical trial of FGFR3 inhibition in myeloma has
been initiated.
Our efforts are now focused on the development and
implementation of relevant biological endpoints to study this
novel class of anti-tumor drugs in the context of clinical trials. In
addition experiments to validate additional molecular targets in
myeloma such as MMSET and the maf and cyclin family of genes
are ongoing or currently development.
Selected Publications:
Trudel, S., Ely, S., Farooqui, Y., Affer, M., Robbiani, D.F., Chesi,
M., Bergsagel, P.L. Inhibition of FGFR3 induces terminal differ-
entiation and apoptosis in t(4;14) myeloma. Blood, 103: 3521-
3528, 2004.
Trudel, S., Li, Z., Wek, E., Wiesmann, M., Chang, H., Chen, C.,
Reece, D., Heise, C. and Stewart, A.K. CIHR-258, a novel,
multi-targeted tyrosine kinase inhibitor for the treatment of
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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CHIR-258 inhibits KMS11 myeloma growth. Real time
monitoring in vivo using luciferase labeled cells
t(4;14) multiple myeloma. Blood, 105:2941-2948, 2005.
Chang, H., Stewart, A.K., Li, Z.H., Yi, Q.L., and Trudel, S.
Immunohistochemistry accurately predicts FGFR3 overexpres-
sion and t(4;14) in multiple myeloma. Blood, 106:353-355, 2005.
Trudel, S., Li, Z.H., Rauw, J., Tiedeman, R.E., Wen. X.Y., Stewart,
A.K. Pre-clinical studies of the pan-Bcl inhibitor obatoclax
(GX015-070) in multiple myeloma. Blood, 109:5430-8, 2007.
Ming-Sound Tsao, M.D.
Professor,
Department of Medical Biophysics
ming.tsao@uhn.on.ca
Molecular Basis of Lung & Pancreatic
Cancer
The laboratory is focused on translational research
projects in lung and pancreatic cancer, two of the most deadly
cancers with overall five-year survival rates of 15% and 2-5%,
respectively. Greater understanding of the molecular basis of their
malignancy will provide insights for improving the early diagnosis
and treatment against these diseases.
The primary goals of our lung cancer projects are to
identify novel genes or proteins that are better than clinical
predictors alone in predicting clinical outcome or response to
therapies. We profile the gene expression and genomic aberrations
of a large number of human lung cancer samples using microarray
techniques, apply computational bioinformatic algorithms to
identify the predictive genes or gene signatures, and validate them
by realtime quantitative PCR technique. The biological functions
of the predictive genes are then studied using in vitro lung cancer
cell line model, primary lung cancer xenograft models and ortho-
topic rodent models of human lung cancers.
The primary goal of our pancreatic cancer project is to
dissect the molecular basis of human pancreatic cancer, which
mostly arises from the duct epithelium. Our laboratory was the
first to establish primary cultures of normal human pancreatic
duct cells. Immortalized cell lines derived from these primary
cultures are used to study the activities of oncogenes and tumor
suppressors that are commonly aberrant in pancreatic cancer. The
approach provides insights into key molecular events that may be
targeted to prevent and treat pancreatic cancer.. Special interest
and emphasis involve the signaling of epidermal growth factor
receptor (EGFR) and Met/hepatocyte growth factor receptor.
These two growth factor signaling loops play important roles in
the growth and metastasis of lung, pancreatic and colorectal
cancer. We are also investigating the role of mutations and overex-
pression of these genes in the biology of these cancers.
Selected Publications:
Qian J, Niu J, Li M, Chiao PJ, Tsao M-S. In vitro Modeling of
Human Pancreatic Duct Epithelial Cell Transformation Defines
Gene Expression Changes Induced by K-ras Oncogenic
Activation in Pancreatic Carcinogenesis. Cancer Res
2005;65:5045-53.
Tsao M-S, Sakurada A, Cutz J-C, Zhu C-Q, et al. Molecular and
clinical predictors of response and prognostic markers of survival
in patients treated with erlotinib in National Cancer Institute of
Canada Clinical Trials Group Trial BR.21. New Engl J Med
2006;353:133-44.
Zhu CQ, Popova S,Brown ERS, Barsyte-Lovejoy D, et al. Integrin
11 regulates IGF-2 expression in fibroblasts to enhance tumori-
genicity of human non-small cell lung cancer cells. Proc Natl
Acad Sci USA 2007;104:11754-9
Derek van der Kooy, Ph.D.
Professor,
Department of Medical Biophysics
derek.van.der.kooy@utoronto.ca
Current Research Interests
The research in the lab is focused on Neurobiology, but is
separated into three distinct areas: Neural Development & Stem
Cell Biology, Neurobiology of Motivation, and Learning and
Memory Genes.
Our Neural Development and Stem Cell Biology project
involves the development of the mammalian brain, eye, and
pancreas. Neural stem cells
also are present throughout
the lifetime of the animal, and
are being localized character-
ized. We have also discovered
a surprising capacity of the
adult mammalian eye to re-
grow. Our experimental
approach involves culturing
mouse retinal stem cells from
normal and genetically
modified mice, in order to understand the factors that control
retinal stem cell activity. Finally, we have isolated a rare cell from
the adult mouse pancreas that can show extensive proliferation
under defined conditions in vitro (Seaberg et al, 2004). These
cells may comprise a population of adult mammalian pancreatic
stem cells, which might in the future be employed in treating type
1 diabetics.
The primary objective of the Neurobiology of
Motivation project is to characterize how the brain processes and
distinguishes different types of rewards, e.g. nicotine, food, opiates
(morphine). Our overall hypothesis is that two discrete neural
mechanisms underlie the rewarding effects of opiates in drug
naive animals (processed by TPP) versus drug-dependent and
deprived animals (utilizing dopamine). Proposed experiments will
further test and reveal the structure of motivational systems in the
mammalian brain.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
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Our Learning and Memory Genes project has resulted in
the development of associative and non-associative learning
paradigms using olfactory and taste stimuli in the worm C.
elegans. Mutational screens in progress have identified new genes
that code for critical components of associative and non-associa-
tive learning; revealing the separable neuronal and molecular
substrates underlying associative learning and habituation. Our
goal is to use the power and specificity of modern molecular
genetics to reveal the component processes of learning and
memory by using the C.elegan's genes.
Selected Publications
Laviolette, S.R., Gallegos, R.A., Henriksen, S.J., and van der Kooy,
D. Opiate state controls bi-directional reward signaling via
GABAA receptors in the ventral tegmental area. Nature
Neuroscience, 7 (2004) 160-169.
Smukler, S.R., Runciman, S.B., Xu, S., Mak, T.W., and van der
Kooy, D. A default mechanism underlies the direct acquisition of
a primitive neural stem cell identity by embryonic stem cells.
Journal of Cell Biology, 172 (2006):79-90.
Richard Wells M.D., D.Phil.
Assistant Professor,
Department of Medical Biophysics
richard.wells@sunnybrook.ca
The Nature of the Haematopoietic Stem
Cell in Myelodysplastic Syndrome
The myelodysplastic syndromes (MDS) are a group of clonal
bone marrow stem cell disorders characterized by low blood cell
counts, an increased rate of apoptosis of haematopoietic progen-
itor cells, and a high risk of transformation to acute leukaemia.
The goals of our research program are to gain insights into the
biology of MDS, and to use such insights to design rational and
effective MDS therapies.
The MDS stem cell advantage EAR-2, MDS, and AML
A central paradox in MDS biology is how the myelodysplastic
stem cell out-competes normal stem cells and comes to dominate
the bone marrow. We hypothesized that the competitive advan-
tage enjoyed by the MDS stem cell consists in an enhanced
capacity for self-renewal, and identified gene that mediate this
property in leukaemia cells. One of the genes we identified, EAR-
2, is also more highly expressed in MDS and leukaemia than in
normal bone marrow. We have found that EAR-2 expression
blocks differentiation of leukaemia cells in culture and leads to the
development of leukaemia when overexpressed in mouse bone
marrow. We are now studying the mechanism by which EAR-2
alters stem cell behaviour, and its role in the multistep pathogen-
esis of MDS and AML.
When less is more Candidate genes in del(5q) and del(7q) MDS
Clonal cytogenetic abnormalities are present in more than 50% of
cases of MDS. Two of the most frequently seen abnormalities
involve involve deletions of large tracts of the long arms of
chromosomes 5 or 7. These deletions are thought to contribute
to the development of MDS by resulting in the loss of tumour
suppressor genes located on these chromosomes; however, the
identity of such tumour suppressor genes remains a mystery. We
are investigating two candidate MDS tumour suppressor genes:
SPARC (chromosome 5q), a mediator of the interactions between
the cell surface and the extracellular matrix, and HIPK2 (chromo-
some 7q), a serine/threonine kinase that has roles in the regulation
of proliferation and apoptosis.
Iron, oxidative stress, and the stem cell
Owing to the failure of normal haematopoiesis, patients with
myelodysplastic syndrome commonly require regular blood trans-
fusions in order to survive. This results in accumulation of iron,
which results in cellular damage via the generation of reactive
oxygen species (ROS). Since accumulation of ROS leads to HSC
senescence in mice, we have hypothesized that iron overload in
MDS patients creates a vicious cycle, in which iron deposition
results in ROS generation, leading to further impairment in
haematopoiesis, leading to even greater requirement for blood
transfusion. Furthermore, we believe the DNA-damaging effects
of ROS may contribute to the progression of MDS to AML. We
are conducting experiments to measure this phenomenon, and to
explore how it may be reversed by iron chelation and anti-oxidant
agents.
Selected Publications:
Chan LS, Wells RA. Manipulation of reciprocal salt bridges at the
heterodimerization interface alters the dimerization properties of
mouse RXRalpha and PPARgamma1. Biochem Biophys Res
Commun. 2007 Jul13;358(4):1080-5.
Ichim CV and Wells RA. First among equals: The cancer cell
hierarchy. Leukemia and Lymphoma, 2006;47 (10):2017-27.
Gu C, Teng T, and Wells RA. Synergistic effects of troglitazone in
combination with cytotoxic agents in acute myelogenous
leukaemia cells. Leukemia Research, 2006; 30; 1447-1451.
Disperati P, Ichim CV, Tkachuk D, Chun K, Schuh AC, and Wells
RA. Progression of myelodysplastic syndrome to acute
lymphoblastic leukaemia: Implications for disease biology.
Leukemia Research. 2006;30:233-9.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
51
Shun Wong, M.D.
Professor,
Departments of Medical Biophysics
shun.wong@sunnybrook.ca
Effects of ionizing radiation on the central
nervous system
Radiation therapy is a major cancer treatment modality.
Radiation injury of the brain and spinal cord has devastating and
sometimes fatal consequences. The overall goal of my laboratory
research program is to understand the mechanisms of radiation
injury in the central nervous system (CNS), and to develop
neuroprotective strategies against this injury.
The underlying mechanisms of this injury remain
unclear. There is an increasing body of data including those from
our laboratory that suggests that the radiation response in the
CNS is a continuous, dynamic, and interacting process. It is
recognized that clonogenic cell death is not the only mode of
radiation-induced cell death. Certain glial, neuronal and endothe-
lial cells including neural progenitor cells in the CNS undergo
apoptosis within a few hours after irradiation. There is also
component of secondary injury and cell death that is mediated by
neuro-inflammation, oxidative stress and microenvironmental
alterations. Disruption of the vasculature and microenvironment
after irradiation may influence cell fate and lead to inhibition of
neurogenesis and neurocognitive deficits.
We are currently studying these effects at the tissue, cell
and molecular level. Our work is focused on potentially reversible
components of cell death and damage since targeting these
damage pathways provide the best opportunities for neuroprotec-
tion. Current work includes characterizing apoptosis, neuro-
inflammation and perturbations of the vascular/neural progenitor
cell niche and cell fate determination after irradiation. Our studies
are performed in vivo using transgenic mouse models of radia-
tion-induced neurobehavioral damage and myelopathy, and in
vitro using glial and neural progenitors cultured from the CNS of
these animals.
Selected Publications:
Li YQ, Chen P, Haimovitz-Friedman A, Reilly RM, Wong CS:
Endothelial apoptosis initiates acute blood-brain barrier disrup-
tion after ionizing radiation. Cancer Res 2003; 63:5950-5956.
Nordal RA, Nagy A, Pintilie M, Wong CS. Hypoxia and hypoxia-
inducible Factor-1 target genes in central nervous system radia-
tion injury: A role for vascular endothelial growth factor. Clin
Cancer Res 10(10):3342-3353, 2004.
Lu F, Wong CS. Time-dependent neurosphere-forming ability of
adult rat spinal cord after irradiation. Radiat Res 168: 453-461,
2007.
Minna Woo, M.D., Ph.D.
Assistant Professor,
Department of Medical Biophysics
mwoo@uhnres.utoronto.ca
Tissue-specific Molecular Determinants of
Survival, Apoptosis and Metabolism
Our laboratory is focusing in elucidating tissue-and
context-specific roles of molecules that determine cellular survival
and function. Interestingly, many of the molecules involved in
cancer formation are also involved in obesity and metabolism.
Our goal is therefore to understand tissue-specific roles of these
complex molecules. Our focus is in caspases, as well as tumour
suppressors and oncogenes such as PTEN and myc. Several
projects in our lab involve using genetically engineered mice and
taking biological, biochemical and molecular approaches to define
pathogenic mechanisms of disease. These approaches to clarify
tissue-specific molecular mechanisms have wide implications for
treatment of type 1 and type 2 diabetes, as well as islet transplan-
tation.
One main area of research in my laboratory is to eluci-
date genetic determinants of insulin producing pancreatic beta cell
growth and apoptosis in healthy states and in diabetes. In type 1
diabetes, beta cells undergo apoptosis from autoimmune mediated
islet destruction. In type 2 diabetes, obesity and inflammation
are the underlying cause that ultimately lead to gluco- and lipo-
toxicity of the beta cells. While these two types of diabetes have
distinct pathogenesis, beta cell defect and deficiency are features
that may utilize common cellular machinery. We use tissue-
specific genetic approaches such as the cre-loxP system to analyze
the in vivo functions of the critical genes.
PI3K signaling pathway induces a complex pleiotropic
biological outcome that is highly context dependent. PTEN is a
phosphatase that is a major negative regulator of this important
signaling pathway. PTEN, although first discovered as a tumour
suppressor, also appears to have critical effects on metabolism and
therefore likely plays a key role in the pathogenesis of insulin
resistance and type 2 diabetes. Our goal is to understand the
molecular mechanism of PTEN type 2 diabetes models.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
52
Immunofluorescent image of a
pancreatic islet from a healthy
non-diabetic mouse stained for
insulin in red depicting beta
cells at the core of the islet and
for glucagon in green showing
alpha cells at the rim of the
islet.
Selected Publications:
N. Liadis., L. Salmena, E. Kwan., P. Tajmir, A. Radziszewska, X.
Li, L. Sheu, M. Eweida, S. Xu, H.Y. Gaisano, R. Hakem, and M.
Woo. Distinct in vivo roles of Caspase-8 in cells in physiological
and diabetes models Diabetes 2007 56(9):2302-11
Nguyen KT, Tajmir P, Lin CH, Liadis N, Zhu XD, Eweida M,
Tolasa-Karaman G, Cai F, Wang R, Kitamura T, Belsham DD,
Wheeler MB, Suzuki A, Mak TW, Woo. M. Essential role of Pten
in body size determination and pancreatic beta-cell homeostasis
in vivo. Molecular and Cellular Biology 2006 26(12):4511-8.
N. Liadis, M. Eweida, E. Elford, R. Hakem, P. Ohashi, M. Woo.
Caspase-3-Dependent beta-Cell Apoptosis in the Initiation of
Autoimmune Diabetes Mellitus Molecular and Cellular Biology
2005 25(9):3620-9.
Wijesekara, D. Konrad, M. Eweida, N. Liadis, A. Giacca, C. R.
Kahn, A. Klip, M. Woo. Ablation of muscle PTEN protects
mice from high fat and age-induced diabetes Molecular and
Cellular Biology 25(3):1135-45
James Woodgett, Ph.D.
Professor,
Department of Medical Biophysics
woodgett@mshri.on.ca
Functional Characterization of the Wnt &
PI3 Kinase Pathways
Our laboratory is focused on the role of specific signal
transduction pathways in development and disease. Specifically,
we are interested in the Wnt and phosphatidylinositol 3 kinase
(PI3K) pathways as these are frequently activated in human
cancers as well as other disorders. We employ molecular and
cellular biological approaches to probe the components of these
pathways and we are particularly interested in breast and colon
cancer as well as diabetes and neurological processes. These
signaling pathways share several common elements including
regulation of a protein-serine kinase termed GSK-3, which acts as
a global suppressor of the functions of a variety of regulatory
proteins. Using knockout mice that harbour conditional alleles
for the two mammalian isoforms of this kinase, we are investi-
gating the consequences of inactivating this regulatory protein in
specific tissues. In embryonic stem cells, GSK-3 inhibition
contributes to a block to differentiation and we find a similar
effect in progenitor cells in the developing mouse as well as the
adult.
To understand how the PI3K and Wnt pathways exert
their effects in cancer, we are examining the state of activation of
specific downstream components of these systems in transgenic
mice as well as cell lines guided by surveys of human cancer
specimens. To fill in the missing connections in the signaling
pathways, we are also using mass spectrometry to identify key
targets of the protein kinases in these pathways in a systematic
approach.
More information can be found at http://www.mshri.on.ca/woodgett/.
Selected Publications:
MacAulay, K., Doble, B.W., Patel, S., Hansotia, T., Sinclair, E.M.,
Drucker, D.J., Nagy, A. and Woodgett, J.R. (2007) Glycogen
synthase kinase-3-specific regulation of hepatic glycogen metabo-
lism. Cell Metabolism 6, 329-337.
Doble, B., Patel, S., Wood, G.A., Kockeritz, L.K. and Woodgett,
J.R. (2007) Functional redundancy of GSK-3? and in Wnt/-
catenin signaling in an allelic series of embryonic stem cell lines.
Developmental Cell 12, 957-971.
Linding, R. et al. (2007) Systematic discovery of in vivo phospho-
rylation networks. Cell 129, 1415-1426.
Tessier, M. and Woodgett, J.R. (2006) Role of the PX domain and
phosphorylation in activation of serum and glucocorticoid-
regulated kinase-3. J. Biol. Chem. 281, 23978-23989.
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
53
Eldad Zacksenhaus, Ph.D.
Associate Professor,
Department of Medical Biophysics
eldad.zacksenhaus@utoronto.ca
Breast Cancer Stem Cells; Tumour
Suppressor pRb
There is growing but incomplete evidence that cancer
may be organized in a hierarchy in which only a fraction of cells,
termed Tumor Initiating Cells (TICs; also referred to as Cancer
Stem Cells, CSC), is capable of instigating cancer and metastatic
disease. In contrast, the majority of tumor cells represents progen-
itor and partially differentiated cells that have lost their tumori-
genic potential. Thus, targeted killing of TICs may be curative.
Our laboratory is using several mouse models of breast
cancer to analyze TICs, their biology, the pathways by which they
divide and their unique properties relative to mammary stem cells
(MSCs). The use of mouse models provides ample supply of
primary tumors with defined genetic background to study these
rare tumorigenic cells. In addition, powerful genetic manipula-
tions in the mouse allow us to test the basic paradigms of the
CSC model in vivo in isogenic mice.
We recently reported on the first identification of TICs
in a mouse model of Her2/Neu, one of the most aggressive forms
of breast cancer in human. We showed that TICs are functionally
indistinguishable from tumorsphere initiating cells, which give rise
to spheres in non-adherent conditions. These tumorspheres
provide a means by which to screen small molecule libraries for
TIC specific therapeutic targets. We are also attempting to purify
Her2/Neu TICs to near homogeneity so we can test whether a
single TIC can induce Her2/Neu tumors following transplanta-
tion into the mammary gland of a recipient mouse, as well as
establish a gene expression signature for the Her2/Neu TIC. Our
long-term goals are to develop TIC specific inhibitors for the
major breast cancer subtypes.
One novel model that we have recently developed
involves the targeted inactivation of the tumor suppressor pRb in
the mammary gland. Rb is mutated in nearly 30% of human
breast tumors and the mice we created produce similar spectrum
of breast tumors as in human. We will next attempt to identify
TICs and therapeutic targets in these Rb mutant tumors as well as
define the effect of Rb status on the response of tumor cells to
chemo- and hormonal therapies in vivo.
In addition to mutations that disrupt the Rb gene, pRb
is often inactivated in cancer by phosphorylation induced by
cyclin dependent kinases. To study the effect of pRb phosphoryla-
tion in vivo, we created mutant mice in which key Ser/Thr
phospho-acceptor sites are substituted to Ala residues by homolo-
gous re-combination. Analysis of these phospho-mutant Rb
knock-in mice reveals unanticipated roles for this tumor
suppressor in genomic stability and aging.
Selected Publications:
Jeff C. Liu, Tao Deng, Rajwinder S. Lehal , Jinny Kim and Eldad
Zacksenhaus. Identification of tumorsphere- and tumor-initiating
cells in Her2/Neu mammary tumors. Cancer Research (2007)
67:8671-8681.
Jiang Z. and E. Zacksenhaus. Activation of retinoblastoma
protein in mammary gland leads to ductal growth suppression,
precocious differentiation and adenocarcinoma. J. Cell Biology
(2002) 156:185-198.
Ho, A., H. Li,, R. Hakem, T. W. Mak and E. Zacksenhaus.
Coupling of Caspase-9 to Apaf-1 in response to loss of pRb or
cytotoxic drugs is cell type dependent. EMBO J (2004) 23:460-
472
Ho, T. A., H. Li, Okada, H., T. W. Mak and E. Zacksenhaus.
XIAP dictates Apaf-1 dependency for caspase-9 activation. Mol.
Cell. Biol. (2007) 27:5673-5685
Dept Medical Biophysics, University of Toronto Cell, Molecular and Structural Biology
54
Cell, Molecular and Structural Biology Dept Medical Biophysics, University of Toronto
Research Laboratories
Sunnybrook Health Sciences Centre
Depar t ment of Medi cal Bi ophysi cs
610 University Avenue Rm 7-411, Toronto ON, Canada M5G 2M9 Tel 416-946-2819 Fax 416-946-2050
medbio@uhnres.utoronto.ca
http://medbio.utoronto.ca
Research Atrium, Princess Margaret Hospital