The Annual EMS Meeting: April 8 - 13, 2000 New Orleans, LA

James Tucker, Program Chair

Meeting Program

Saturday, April 8, 2000

8:00am -
10:00am Strategic Planning Committee Meeting

1:00pm -
5:00pm EMS Council Meeting Poydras B

6:00pm -
9:30pm Student Poster Session and Reception
{Sponsored by the Genetic Toxicology Association} Cabildo C
6:00pm The Poster Session

7:30pm Introduction
Jennifer Sasaki, Lawrence Livermore National Lab

7:35pm Welcome Address
David DeMarini, U.S.E.P.A.

7:45pm Presentation
Carlos Sierra-Torres, University of Texas Medical Branch at Galveston

8:00pm Presentation
Krista Dobo and Bob Mauthe, Pfizer Central Research

8:30pm Presentation of the EMS Education Committee Award and Best Student / New Investigator Poster Award

Sunday, April 9, 2000

7:00am -
8:30am Breakfast Meetings
Students Group

New Technologies Group

Program Committee (First Meeting)

Nominating Committee

8:30am -
12:00pm Ames Test Workshop
Presiding: Judy Mayo, Pharmacia & Upjohn Inc., and Jenness Majeska, Boehringer Ingelheim Pharmaceuticals Regency Ballroom E
Bacterial mutation assays remain a cornerstone in worldwide regulatory risk assessment today, hence it is timely that the EMS review the "State of the Ames Test" as it is currently perfomed. This workshop will provide a forum for discussion of new information on the Ames Assay including: a) Survey results from 23 labs on details of spontaneous revertant rates, new strains, culture/control conditions, etc., b) Method modifications being developed to reduce test article requirement, c) Regulatory requirements, d) Artefacts, e) Panel discussion of problems/solutions/helpful hints. A summary of the survey information will be presented in a poster later in the meeting.

8:30am Welcome
Jenness Majeska and Judy Mayo

8:40am Overview of Regulatory Requirements
Leonard Schectman, USFDA

8:55am Ames Survey Results
Jenness Majeska

9:20am Artefacts in the Ames Test
Elmar Gocke, Roche Ltd

Method Modifications for Reduced Test Article Requirement

9:40am Spot Test Update
Skip Wagner, Bioreliance

10:00am Coffee

10:30am Spiral Assay
Larry Claxton, USEPA

10:50am MiniAmes Using 6-Well Plates
Marilyn Diehl, Abbott Laboratories and Judy Mayo, Pharmacia & Upjohn

11:10am Testing Combinatorial Libraries
Leon Stankowski, Chrysalis, and Marque Todd, Chiron Corporation

11:30am Discussion Panel: Audience and Survey Questions

8:30am -
12:00pm Workshop on Suitable Follow-up Testing and Risk Evaluation for In Vitro Positive Pharmaceuticals
{Sponsored by Merck, The Procter & Gamble Co., Dow Corning Company, Astra-Zeneca, and Eli Lily}
Presiding: Sheila Galloway, Merck Research Laboratories, and Lutz Müller, Federal Institute for Drugs & Medical Devices, Berlin Regency Ballroom FGH
The workshop will address pharmaceutical candidate compounds that gave only positive results in mammalian cells in vitro in the initial test battery. Speakers from governmental agencies and industry will present scientific rationales to investigate the significance of such findings. Examples will be discussed.

8:30am Mechanistic studies on thresholds and the involvement of cytotoxicity in induction of chromosome aberrations with drug candidates including angiotensin II receptor antagonists
Sheila Galloway

9:05am Evaluation of in vitro clastogens: Examples including catecholamines and acetylcholinesterase inhibitors
Lutz Müller

9:40am Japanese regulatory approaches to clarify the significance of positive results in in vitro mammalian cell genotoxicity assays
Makoto Hayashi, National Institute of Health Sciences, Tokyo

10:15am Coffee

10:45am Examples from current experience with in vitro genotoxins, with discussion of how transgenic mouse tumorigenicity models are considered in clarifying genotoxicity issues
Joe Contrera, US Food and Drug Administration

11:20am Useful and scientifically sound follow-up test approaches for compounds that initially were only positive in mammalian cell tests in vitro
James MacGregor, US Food and Drug Administration

1:00pm -
2:00pm Plenary Talk 1: Mechanism of Trinucleotide Repeat Instability in Human Neurological Disease
Speaker: Cynthia T. McMurray, Mayo Clinic and Foundation Regency Ballroom FGH

2:00pm -
5:00pm Genomics and Evolution
Chair: Mark A. Batzer, Louisiana State University Health Sciences Center; Co-Chair: Prescott L. Deininger, Tulane University Cancer Center Regency Ballroom FGH
The fields of genomics and evolution are expanding rapidly as a greater proportion of human and model organism genomes are sequenced. The symposium will provide an overview of different classes of mutations that occur within single nucleotides, trinucleotide repeats, mobile elements and larger chromosomal segments. In addition, the natural history of combinations of mutations into haplotypes will be presented. The single nucleotide polymorphisms (SNPs) are the most common form of genetic variation and are of great interest to a variety of researchers. Trinucleotide repeat based mutations display genetic anticipation and have been implicated in a number of human neuromuscular disorders. Mobile element based mutations may occur as a result of insertional mutagenesis or by enhancing genomic fluidity subsequent to genomic integration by facilitating recombination events between elements. The evolution of large multigene families through the duplication of loci has also occurred throughout evolution and influenced the structure of many eukaryotic genomes. The advent of parallel processing and array technology for re-sequencing individual genes and genotyping multiple loci has changed the framework for evolutionary biology and genomics from the single gene format to genome wide surveys of genetic variation. This shift in emphasis will provide many new opportunities to investigate differences in disease susceptibility between individuals and analyze mutational processes on a genome wide basis.

2:00pm Human DNA Variation in Noncoding Regions in Worldwide Populations
Wen-Hsiung Li, University of Chicago

2:25pm Genetic Structure of Human Populations: Role of Founder Effect and Admixture
Damian Labuda, University of Montreal

2:50pm Trinucleotide Repeat Expansions and Evolution
Bronya J. B. Keats, Louisiana State University Health Sciences Center

3:15pm Coffee

3:45pm Contribution of Interspersed Elements to Human Genomic Diversity
Mark A. Batzer

4:10pm The Complex Genomic Organization of the Olfactory Receptor Gene Family
Barbara J. Trask, University of Washington

4:35pm Overview and Summary
Prescott L. Deininger

5:00pm -
7:30pm Poster Session I
{Sponsored by Dupont Pharmaceuticals Company} French Market Exhibit Hall

8:00pm -
10:00pm EMS Awards and Reception
{Sponsored by BioReliance, Inc. and Covance Laboratories} Regency Ballroom FGH and French Market Exhibit Hall

Monday, April 10, 2000 Theme: Human Genetic Variability and Susceptibility

7:00am -
8:30am Breakfast Meetings
Germ Cell Mutagenesis/Human Genetics/Aneuploidy Group

Organization Committee

Public Relations & Communications Committee

Finance Committee

Education and Student Members Committee

Future Meetings Committee

8:30am -
11:30am Molecular Epidemiology
Chair: Fred Kadlubar, National Center for Toxicological Research Regency Ballroom FGH
The emergence of Molecular Epidemiology has enabled researchers to apply findings from the laboratory to population-based epidemiological studies. Recent advances in molecular techniques and an understanding of disease etiology at the molecular level have also enabled researchers to study determinants of disease risk from a new and different multidisciplinary perspective. The result is a rapidly growing area of research that presents extraordinary opportunities and challenges in that it brings together efforts of epidemiologists, molecular biologists, biochemists, toxicologists, nutritionists, geneticists, clinicians, pathologists, biostatisticians, ethicists, and researchers from other scientific disciplines. By working together, it should be possible to develop and implement new strategies to address current and future issues in the study of complex gene-environment interactions. In this symposium, we will present the latest developments from laboratory and epidemiological studies in relation to the etiology of several human cancers.

8:30am Introduction: Exposure and Susceptibility Biomarkers for Colon and Pancreas Cancer
Fred Kadlubar

9:00am Genotypes of Hormone and Drug Metabolism in Cancer Predisposition
Tim Rebbeck, University of Pennsylvania School of Medicine

9:30am Role of Heterocyclic Amines and Breast Cancer
Rashmi Sinha, National Cancer Institute

10:00am Coffee {Sponsored by Taconic Farms, Inc.}

10:30am Environmental Exposures in the Molecular Epidemiology of Breast Cancer
Mary Wolff, Mt. Sinai School of Medicine

11:00am Molecular Epidemiology of Lung Cancer: A Model for Environmental-Genetic Interactions
David Christiani, Harvard School of Medicine and Public Health

11:30am -
1:00pm EMS Annual Business Meeting
Lunch provided! Regency Ballroom FGH

1:00pm -
4:00pm Pharmacogenomics
{Sponsored by Pharmacia & Upjohn}
Chair: Sid Aaron, Pharmacia & Upjohn Inc. Regency Ballroom FGH
Pharmacogenomics is a cornerstone in the modern process of drug discovery and development. Clearly, heritable changes in a variety of genes can lead to increased (and decreased) susceptibility to drugs and this factor has played a role in the past. The proactive stance being taken by pharmaceutical researchers in identifying these populations of altered susceptibility has resulted in rapid development and deployment of molecular methods of determining the factors responsible for the differences. Ultimately, this will lead to drug prescriptions written for a person based on high likelihood of a successful treatment outcome. This symposium will explore the state of the art and understanding with respect to pharmacogenetic principles and the application of those principles and new technologies to serious human disease susceptibility.

1:00pm Pharmacogenomics: A Brave New/Old World
Sid Aaron

1:30pm The Impact of Pharmacogenetics on Medicine
Wendel Weber, University of Michigan

2:00pm Genetic Susceptibility to Infectious Diseases
Emil Skamene, McGill University

2:30pm Coffee

3:00pm Sequencing Entire Genomes of Free Living Organisms: Implications for Pharmacology and Toxicology
Sam Broder, Celera Genomics

3:30pm Application of Pharmacogenomics to Drug Discovery and Development
Michael Silber, Pfizer Central Research

4:00pm -
6:30pm Poster Session II
{Sponsored by The R.W. Johnson Pharmaceutical Research Institute} French Market Exhibit Hall

6:30pm -
8:30pm Legal and Ethical Issues in DNA Sampling and Testing
{Sponsored by the National Institute of Environmental Health Sciences}
Chairs: Errol Zeiger, OECD and NIEHS, and Ami Jaeger, Biolaw Group Regency Ballroom FGH
This symposium will provide an overview of legal and ethical issues associated with the use of DNA testing of humans. Such testing may be done for health or diagnostic reasons, or for reasons of policy, i.e., the requirement that DNA samples be taken from newborns, members of the military, arrested people, or convicted felons. Issues to be addressed will include the use of DNA-derived information for the purposes for which it was intended, and for uses not foreseen by the DNA donor, and issues of databases and medical information privacy. Members of the EMS are familiar with the scientific and technical issues involved in DNA sampling. However, it is important that there be an open dialogue between the people who create and practice the science and those who have to apply or interpret it in a societal context. This Symposium will provide an opportunity to hear the views of people who have approached these issues from the social science and legal cultures. The presenters will include lawyers and ethicists who have studied the social and legal issues associated with this technology, and will offer insights into the future of DNA databases and the uses of this information. The session will allow time for questions after each of the speakers, and will provide time during the panel discussion for participation by the audience.

6:30pm Introduction
Errol Zeiger

6:35pm General Overview of Legal and Ethical Issues in the Use of Genetic Information
Ami Jaeger

7:10pm Health Care: Legal Considerations in the Use of Genetic Information
Robert Schwartz, UNM School of Law

7:35pm Forensics: Ethical, Legal and Social Implications
Jean McEwen, National Human Genome Research Institute

8:00pm Panel Discussion: Guidelines for Genetic Testing and the Use of Genetic Samples
Errol Zeiger

Tuesday, April 11, 2000 Theme: Mechanisms of Mutation and Cancer

7:00am -
8:30am Breakfast Meetings
Risk Assessment Group

DNA Repair Group

Molecular Epidemiology Group

Awards and Honors Committee

Exhibitor's Breakfast

8:30am -
11:30am DNA Mismatch Repair
Chair: Tom Kunkel, National Institute of Environmental Health Services Regency Ballroom FGH
This symposium will focus on the multiple roles of DNA mismatch repair proteins, including repair DNA replication errors, meiotic recombination, cellular responses to DNA damage and carcinogenesis. Presentations will include studies on the genetics and biochemistry of these processes in bacterial, yeast, mouse and human systems.

8:30am Functional Studies of DNA Mismatch Repair Proteins in Yeast
Tom Kunkel

9:00am DNA Mismatch Repair: From Structure to Mechanism
Wei Yang, Laboratory of Molecular Biology

9:30am Implications of DNA Mismatch Repair Inactivation for Colon Cancer Development and Treatment
Martina Veigl, Case Western Reserve University

10:00am Coffee {Sponsored by Chrysalis Preclinical Services}

10:30am Role of Mismatch Repair Genes in Mitosis and Meiosis
Raju Kucherlapati, Albert Einstein College of Medicine

11:00am The Many Faces of Mismatch Repair in Meiosis
Rhona Borts, University of Oxford

8:30am -
11:30am Dietary Supplementation (Nutriceuticals)
{Sponsored by The Dow Chemical Company}
Chair: Lynn Ferguson, Auckland Cancer Society Research Centre Regency Ballroom E
Alternative medicine and diet supplements are one of the most significant growth areas in the health industry. A great variety of supplements and functional foods are being marketed as anti-carcinogenic agents based on epidemiological and/or experimental evidence (e.g. fiber and antioxidants). In most cases evidence for protective effects from prospective, placebo-controlled trials in humans is not yet available. The actual benefits of these supplements to individuals have not been quantified or verified and little is known of the impact of genetic background to ultimate outcome although it is evident that individuals with specific metabolic defects may require different nutrient intakes. While in the past there has been great focus on the genotoxic impact of exposure to potential carcinogens it is increasingly becoming evident that deficiencies in specific micronutrients (e.g. vitamin C, folic acid, vitamin B12, Mg, Zn, Fe, etc.) as well as non-nutrients (e.g. flavonoids) may just as effectively damage DNA and thus facilitate the carcinogenic process. Natural products are also efficient inducers of Phase I and Phase II enzymes that modulate endogenous anti-oxidant defense mechanisms and alter the impact of carcinogen exposure. With the emergence of more reliable and practical biomarkers of DNA damage in human cells it has become possible to seriously consider a concerted research effort to define the recommended dietary intakes of micronutrients for the prevention of genomic instability. Preliminary evidence already suggests that current recommended daily intake levels for folate and vitamin B12, while adequate to prevent anaemia may not be sufficient for the prevention of chromosome damage. Natural compounds may also intervene in later stages of cancer development by interfering with critical events that control cell proliferation such as synthesis of polyamines and the processing of oncogene products. Some of these products, such as limonene, are undergoing clinical trials for the prevention of breast and other cancers. The aim of the symposium is to review the current state of play in this exciting area of research and to explore in greater detail the interface of mutagenesis, carcinogenesis and nutrition.

8:30am Overview: Nutriceuticals and their Role in Cancer Prevention
Lynn Ferguson

9:00am Vitamin and Mineral Supplementation: Effects on Genomic Stability
Michael Fenech, CSIRO Division of Human Nutrition

9:30am Monoterpenes with Protective Effects Against Breast Cancer
Michael Gould, Dept of Human Oncology, University of Wisconsin, Dept of Human Oncology, University of Wisconsin

10:00am Coffee {Sponsored by Chrysalis Preclinical Services}

10:30am Diet/gene Interactions with Emphasis on Cell Cycle Control and Apoptosis
Michael Wargovich, South Carolina Cancer Center

11:00am beta-Catenin Mutation in Colon Tumors Promoted by Dietary Phytochemicals
Roderick Dashwood, Linus Pauling Institute

11:30am -
12:30pm Plenary Talk 2: Risk Characterization of Dioxin
Linda S. Birnbaum, US Environmental Protection Agency Regency Ballroom FGH

12:30pm -
6:30pm Free Afternoon
New Orleans Visitors' Guide

6:30pm -
10:00pm Social Event: Mardi Gras Evening

Wednesday, April 12, 2000 Theme: Disease Models

8:30am -
12:00pm Hot Topics and Late-Breaking Ideas
Chair: John Heddle, York University Regency Ballroom FGH
This symposium consists of talks selected on a competitive basis by members of the Program Committee. Click on the title of a talks that are hyperlinked to see their abstracts.

8:30am Chromosomal Instability in Preneoplastic Ulcerative Colitis: a Mutator Phenotype?
Peter Rabinovitch, University of Washington

9:00am The Generation and Utilization of Tissue- and Chromosome Region-Specific Gene Expression Libraries
Allen T. Christian, Lawrence Livermore National Laboratory

9:30am Analysis of Sequence Alterations in a Defined DNA Region Using Temperature-Modulated Heteroduplex Analysis (TMHA"): Efficient Separation of Mutant hprt Sequences
Thomas R. Skopek, Merck Research Laboratories

10:00am A Novel Fidelity System Enabled by the dnaX Gene
Roel M. Schaaper, National Institute of Environmental Health Sciences

10:30am Coffee {Sponsored by Covance Laboratories, Inc.}

11:00amMutator Alleles Affecting Polymerase Fidelity and Cancer in Mice
Robert E. Goldsby, University of Utah

11:30am RNA Polymerase II as a Sensor for DNA Damage
Mats Ljungman, University of Michigan Comprehensive Cancer Center

8:30am -
12:00pm Apoptosis
Chairs: Mathieu Noteborn, Leadd BV and Leiden University Medical Center, and Prof. van der Eb, Leiden University Regency Ballroom E
Overview: Eukaryotic cells, when stimulated by certain signals, have the capacity to enter a defined program leading to cell death. This process, called apoptosis, is a gene-directed, physiologically programmed form of cell death. Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, and condensation and cleavage of DNA. The apoptotic remnants are enveloped by membranes and, in vivo, undergo rapid phagocytosis by neighboring cells. In contrast to necrosis, which constitutes a non-physiological process of cell death, apoptosis does not lead to the release of cellular content, so that inflammation reactions are avoided. Apoptosis plays an important role in many natural processes, e.g., the formation of tissues and organs during embryogenesis as well as in certain processes in the adult organism such as tissue renewal, regulation of the immune system, and elimination of neoplastic cells that threaten to become tumors. Apoptosis is also involved in a variety of diseases, such as AIDS (increased apoptosis) and cancer (decreased apoptosis). Dr J. John Cohen, University of Colorado Medical School, Denver, USA will present an overview of apoptosis and its relevance in health and disease. Originally, it was thought that the development of tumors is caused mainly by enhanced cell proliferation. Subsequent research showed, however, that a decreased level of apoptosis also contributes to tumor formation. Cells that have incurred damage in their DNA must repair this damage before they divide. If they are not able to repair the damage, mutations may arise which lead to oncogenic transformation. However, if damaged cells have incurred too much damage, they may instead undergo apoptosis, and by doing so, prevent tumor growth. The tumor suppressor protein p53 plays an important role in the prevention of oncogenic transformation. More than 90% of individuals with a hereditary p53 defect develop sarcomas, breast tumors, adrenal carcinomas, etc. It is generally assumed that tumor suppression is due, at least in part, to p53-induced apoptosis and that during tumor formation, a selection for loss of wild-type p53 function takes place. Another gene that plays a role in apoptosis is the proto-oncogene c-myc. Expression of c-myc results in either net cell proliferation or apoptosis, depending on the cellular background or environment. For example, over-expression or deregulated expression of c-myc results in apoptosis in the presence of p53, whereas in its absence, enhanced cell proliferation occurs. Many tumors lack functional p53 and/or over-express c-myc. Dr George Prendergast, Dupont Pharmaceuticals, Philadelphia, USA, will discuss the regulation of apoptosis by c-myc and c-myc-related genes. The anti-apoptotic proto-oncogene Bcl-2, and a number of related proteins, e.g. Bcl-xL, BAG-1, and Bak, are also involved in tumor development. Bcl-2 is over-expressed as a result of chromosomal translocations in various tumors, e.g. leukemia, lymphoma, and breast cancer. Most of the DNA tumor viruses that are known to date not only harbor transforming genes, but also have genes that prevent or inhibit apoptosis, including Bcl-2-like proteins or proteins up-regulating Bcl-2 synthesis. Dr Amy Kronenberg, Lawrence Berkeley National Laboratory, Berkeley, USA, will present her studies on the relationship of radiation, mutation and apoptosis. Her presentation will describe the role of over-expression of Bcl-2 proteins in high allelic recombination caused by radiation or mutagenic agents and its implications for neoplastic progression. Dr J. Marie Hardwick, John Hopkins University, Baltimore, USA will present data on the regulation of virus-induced neuronal apoptosis by the Bcl-2 proteins. Apoptosis is essential not only for tumor growth, but can also be exploited for the treatment of tumors. Many tumor cells have defects in the decision machinery for apoptosis, but retain an intact execution system. Thus such tumor cells may still die if they are provided with an effective apoptotic signal. Chemotherapeutic agents normally induce apoptosis via activation of p53. However, more than 50% of human tumors, including melanoma, lung cancer, or colon carcinoma, do not contain functional p53. Patients with such tumors have a low chance of responding to chemo- and/or radiation therapy. Likewise, over-expression of the anti-apoptotic proteins Bcl-2 or BCR-ABL also negatively influences the success rate of chemotherapeutic treatment of a large number of leukemias and lymphomas. Based on this information, considerable efforts are currently being invested in the development of new anti-tumor therapies. Dr Noteborn, Leadd BV and Leiden University Medical Center, Leiden, The Netherlands, will discuss the viral protein, Apoptin, which induces apoptosis specifically in tumor cells.

8:30am The Biology of Apoptosis: Why Does it Occur so Readily?
J. John Cohen, University of Colorado Medical School

9:05am Regulation of Virus-Induced Neuronal Apoptosis by Bcl-2 Proteins
J Marie Hardwick, John Hopkins University

9:40am Loss of the Myc-Bin1 Cell Death Pathway in Cancer
George Prendergast, Dupont Pharmaceuticals

10:15am Coffee {Sponsored by Covance Laboratories, Inc.}

10:50am Programmed Cell Death and Allelic Recombination
Amy Kronenburg, Lawrence Berkeley National Laboratory

11:25am Apoptin Induces Tumor-Specific Apoptosis
Mathieu H.M. Noteborn

1:00pm -
3:30pm Poster Session III
{Sponsored by Chrysalis} French Market Exhibit Hall

3:30pm -
6:30pm Hypermutation and Disease
{Sponsored by Merck}
Chair: Lawrence A. Loeb, University of Washington Regency Ballroom FGH
It is becoming increasingly evident that mutation rates in organisms are not fixed. Instead, they are finely tuned to changes in the environment. This variation facilitates adaption to environmental stresses, including changes in nutrition, decreased oxygen, nutrients, exposure to toxic drugs, and other adverse conditions. However, increased mutagenesis can exceed the error threshold for survival. This symposium will consider first the evidence that HIV-1 exists as a genetically heterogeneous population, a "quasispecies", that is ever-changing in response to its environment. Secondly, we will follow the consequences of increasing the mutation frequency of HIV. Thirdly, we analyze the ability of the malaria parasite to obtain drug resistance by altering the expression of dihydrofolate reductase, and the feasibility of analyzing these mutations in yeast. Fourthly, we will focus on strategies utilized by E. coli to adapt to environmental challenges. "Mutases" can target specific genes that evade host immune systems. Lastly, we will consider the host's response to this ever-changing environment and the mechanisms for hypermutations that occur in immunoglobulin genes. This process of somatic hypermutation is linked both to transcription initiation and presumably involves specific mutator factors.

3:30pm Lethal Mutagenesis of HIV: Pushing the Virus Over the Cliff
Lawrence A. Loeb

4:00pm Mechanisms Driving HIV-1 Variation
Bradley Preston, University of Utah

4:30pm Analysis of Yeast Anti-Folate Resistance in the Malarium Parasite, P. falciparum
Carol Sibley, University of Washington

5:00pm Coffee

5:30pm Somatic Hypermutation of Ig Genes Causes Mutations at Many Thousand-fold Higher Than Spontaneous Mutation Rate
Ursula Storb, University of Chicago

6:00pm Fine-Tuning of Mutagenesis: Genetic Gambling While Climbing the Slopes of Evolutionary Landscapes
Miroslav Radman, Inst. Jacques Monod

3:30pm -
6:30pm Transgenics
Chairs: Barry Glickman, University of Victoria, and Peter Stambrook, University of Cincinnati College of Medicine Regency Ballroom E
Cells in culture have been invaluable in establishing a base-line for understanding cellular mechanisms that contribute to the genesis of mutations. However, extrapolation to in vivo mechanisms in intact animals requires the generation of model mammalian systems. The first generation of mouse models has used bacterial transgenes as in vivo reporters of mutagenesis producing models such as Big Blue and Muta Mouse. These have been followed by a model in which the reporter transgene is of mammalian origin and by another in which the reporter gene is an endogenous gene. The first half of this symposium deals with advances using mouse models with bacterial reporters and the second half describes data from two of the newer models using mammalian genes as reporters of mutagenesis. The first speaker, Dr. Johan de Boer, will present an overview of in vivo mutagenicity data derived with Big Blue. He will be followed by Dr. Elizabeth Snyderwine who will describe how genetic background (in this case the overexpression of a c-myc transgene in the liver) can affect the frequency of mutation following administration of a food derived carcinogen. Dr. Takehiko Nohmi will close the first half of this session by discussing data from large-scale studies in which the transgenic mouse gpt delta has been used. The two talks in the second half focus on in vivo data derived from mammalian reporter genes. Dr. Stringer will describe a mouse model in which the reporter gene in a human placental alkaline phosphatase (PLAP) gene whose product can be detected in situ in all tissues. By using reversion strategy, he will describe the distribution of frameshift and missense mutations in different tissues and the consequences of mismatch repair deficiency. The last speaker, Dr. Harry Vrieling, has used the endogenous Aprt gene as a reporter of mutagenic activity in mice heterozygous at that locus. A major class of mutagenic events that is not detected by the other models is loss of heterozygosity due to mitotic recombination. This class of event represents a mutagenic mechanism that previously has been under appreciated, but is identified in this model as an important and prominent event.

3:30pm The Big Blue World: an Overview
Johan de Boer, University of Victoria Centre for Environmental Health

4:00pm Mutagenicity and Carcinogenicity of a Food-Derived Heterocyclic Amine in c-myc/lacZ Bitransgenic Mice
Elizabeth Snyderwine, Laboratory of Experimental Carcinogenesis

4:30pm Transgenic Mouse gpt delta: Development, Recent Progress and Future Directions
Takehiko Nohmi, National Institute of Health Sciences

5:00pm Coffee

5:30pm Mismatch Repair and Somatic Mutation in Individual Cells of Diverse Mouse Tissues
Jim Stringer, University of Cincinnati, College of Medicine

6:00pm Acute and Long Term Effects of DNA Damage in NER Deficient Transgenic Mice
Harry Vrieling, Leiden University Medical Center

6:30pm -
7:30pm Plenary Talk 3: Mutation Research Award Winner Talk
(Speaker and title to be announced at the meeting.) Regency Ballroom FGH

Thursday, April 13, 2000 Theme: Mechanisms of DNA Repair & Cancer

7:00am -
8:30am Breakfast Meetings
Transgenics Group

Program Committee (Second Meeting)

Membership & Professional Development Committee

Hollaender Committee

8:30am -
10:30am Molecular Cytogenetics
Chair: David Ward, Yale University Regency Ballroom FGH
Molecular cytogenetic techniques have become powerful tools for the detection of genetic alterations in chromosomes, cells and tissues. With increasing regularity they are being applied to problems in both clinical diagnostics and molecular biology. In this symposium several of the recent advances in molecular cytogenetics will be highlighted. These include: The application of comparative genome hybridization (CGH) using both chromosome and DNA array based formats to define DNA gains and losses in solid tumors that subsequently lead to the identification of specific genes implicated in tumor progression; Molecular beacon probes that rapidly and quantitatively detect point mutations in genomic DNA using homogeneous solution assays; Rolling circle DNA amplification for the detection of point mutations or polymorphisms in genomic DNA in situ or in arrays; and Enhancements in fluorescence in situ hybridization techniques (M-FISH and SKY) for the karyotype analysis of cancer cells and the multi-parametric imaging of interphase nuclei.

8:30am High resolution multicolor analysis in 2- and 3-dimensions
Michael Speicher, Institut fur Anthropologie und Human Genetik

9:00am Multicolor Molecular Beacons for the Identification of Single-nucleotide Polymorphisms
Fred Kramer, Public Health Research Institute

9:30am Two Molecular Cytogenetic Views of Cancer: An Instability Paradox
Joe Gray, University of California. San Francisco

10:00am Rolling Circle DNA Amplification: A New Diagnostic Paradigm
Dave Ward

10:30am Coffee

11:00am -
1:00pm Homologous Recombinational Repair, BRCA Proteins, and Cancer
Chair: Larry Thompson, Lawrence Livermore National Laboratory Regency Ballroom FGH
From studies of mutant cell lines of chicken, rodent, and human origin, it has become clear that the pathway of homologous recombinational repair (HRR) is a major pathway that acts on double-strand breaks and other DNA damages. Moreover, there is now substantial evidence that perturbations in this pathway are causally involved in human cancer. Several genetic and biochemical links between the BRCA1 and BRCA2 familial breast cancer genes and the HRR protein machinery have been established. Moreover, several types of human tumors were recently found to have mutations in the HsRad54 and HsRad51B genes. This symposium will discuss the genes identified so far in HRR, phenotypes of available mutants, biochemical mechanisms, interactions between the BRCA proteins and those mediating homologous repair, and new ways of measuring repair of damage (i.e. double-strand breaks) by HRR. A divergent family of five RAD51-like proteins appear to have arisen during the evolution of vertebrates from lower eukaryotes, resulting in much greater biochemical complexity than exists in the historically studied Saccharomyces cerevisiae.

11:00am Overview of Double-Strand Breaks in Mammalian Cells
Larry Thompson

11:10am Homologous Recombination is Not a Minor DNA Repair Pathway in Vertebrates
Larry Thompson

11:30am Effects Of ERCC1 Gene Knock-Out On Homologous Recombination In CHO Cells
Gerald Adair, University of Texas Cancer Center

12:00pm Roles of BRCA1 and BRCA2 in DNA Repair and Cancer
Wen-Hwa Lee, University of Texas Health Science Center at San Antonio

12:30pm Dsb Repair, Homologous Recombination, and Genomic Integrity: A Role for BRCA1
Maria Jasin, Sloan Kettering Institute

1:00pm -
5:00pm EMS Council Meeting Poydras B

Hot Topic Abstracts

The generation and utilization of tissue- and chromosome region-specific gene expression libraries.

Allen T. Christian1, Matthew A. Coleman1, Elizabeth G.Snyderwine2, and James D. Tucker1 1. Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, P.O. Box 808, L-448, Livermore, CA, 94551. 2. Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, National Cancer Institute, Building 37, Room 3C28, Bethesda, MD, 20892.

We have developed a process to make gene expression libraries from specific regions of chromosomes, using any tissue, normal or cancerous, for any species of animal. Using this technique, the involvement of known genes in various diseases can be determined, as well as the potential involvement of expressed novel genes. This technology can also be used to map genes in species whose genome is less well-known than the human genome by isolating cDNA from a particular region and comparing those sequences against the human expressed sequence tag database.

We have combined this technique with comparative genomic hybridization (CGH) to identify and map genes to chromosomal regions that are ubiquitously deleted in rat tumors. In rat mammary carcinomas induced by the heterocyclic amine PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), CGH results show that five regions of the genome are consistently lost. Using the microdissection gene recovery technique to isolate cDNA from the coextant regions in normal rat metaphase spreads, we have sequenced numerous genes of interest from two of these regions, including DNA repair and tumor suppressor genes that were not previously mapped in the rat. We have also sequenced novel genes whose function is as yet unknown.

This technique represents a powerful new way to analyze the response of both known and novel genes to a specific chemical or physical agent. Once the expressed sequences from a region have been isolated, the libraries can be placed on 'chip arrays' and used to assay the presence, absence and differential expression levels of genes from specific chromosomal regions. As an added benefit, gene location on a chromosome can be mapped easily. cDNA from one species can also be hybridized to the chromosomes of another species, allowing synteny to be established between them. This provides a valuable means of linking data from animal models to the human genome. When coupled with the ever-growing database of genomic sequence information, this technique will have a large impact on the use of expressed sequence data in the fields of carcinogenesis and genetic toxicology.

This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 with support from NIH grant CA55861.

Analysis of sequence alterations in a defined DNA region using temperature-modulated heteroduplex analysis (TMHA™): efficient separation of mutant hprt sequences.

Thomas R. Skopek, Merck Research Laboratories, West Point, PA 19486.

Temperature-modulated heteroduplex analysis (TMHA™) is a novel approach for investigating DNA sequence heterogeneity. Mutant DNA sequences are first denatured and allowed to reanneal with wild type (wt) so that mismatched heteroduplexes are formed. Reversed-phase high-performance liquid chromatography is then used to resolve the mutant-containing heteroduplexes from homoduplex species. The TMHA™ chromatography system consists of hydrophobic particles as the stationary phase and triethylammonium acetate (TEAA) and acetonitrile as the mobile phase. TEAA adsorbs to the surface of the particles and presents a positive charge that attracts negatively-charged DNA. While on the column DNA is exposed to denaturing conditions produced by both heat and the denaturing properties of acetonitrile. When denaturing conditions are increased to the point where the least stable portion of the DNA duplex is denatured, the association of the DNA with the beads is reduced, and the partially-melted molecule moves more quickly through the column. Heteroduplexes with mismatches in this region will denature and move faster on the column at lower denaturing conditions than perfectly base-paired homoduplexes, and thus can be resolved.

We have assessed the ability of TMHA™ to separate mutant sequence from wt using a collection of twenty mutations in a 279-base fragment from exon 8 of the human hprt gene. All mutant/wt heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment (including transitions, transversions, and frameshifts) were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved with DGGE, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA™ and DGGE are measuring similar melting characteristics in heteroduplex molecules.

TMHA™ has two technical advantages. First, the DNA under analysis remains in solution and can be easily collected and manipulated, unlike gel-based techniques. Secondly, heteroduplexes run ahead of the homoduplexes rather than behind in the homoduplex peak "tail". Both characteristics offer significant advantages when attempting to enrich rare mutant sequences from wt. For example, collection of a single fraction preceding the wt homoduplex peak would capture the majority of mutations in the low temperature melting domain. This fraction could be diluted into a number of PCR reactions, amplified, and subsequently analyzed at the sequence level. An approach for accomplishing this and progress to date will be presented.

A novel fidelity system enabled by the dnaX gene.

Roel M. Schaaper, Laboratory of Molecular Genetics, E3-01, National Institute of Environmental Health Sciences, PO Box 12233, 111 TW Alexander Drive, Research Triangle Park, NC 27709.

A novel DNA replication error prevention system enabled by the dnaX geneAccessory factors of DNA replication complexes may be important fidelity determinants. We have started a systematic investigation of the replication complex of E. coli. We have discovered that mutants in the tau subunit (dnaX gene product), display a mutator activity of unusual specificity. The data, combined with in vitro studies on the replication fidelity of pol III holoenzyme, suggest a role for this subunit in a hitherto undescribed fidelity mechanism. Tau dimerizes the two core polymerases that conjointly replicate the leading and lagging strand; it is also the scaffold for the gamma complex responsible for repeated clamp loading in the lagging strand. It also coordinates the HE with the DNA helicase that unwinds the fork ahead of the complex. The mutator activity of our dnaX mutants is unique, as we observe specific enhancement of only two types of mutations: transversions and (-1) frameshifts. All affected dnaX mutants contain a substitution in the C-terminal tip of tau responsible for interaction with the polymerase subunit. Our data support a model in which tau plays a central, coordinating role within the HE, facilitating, among others, communication between the leading and lagging strand polymerases. Such communication is essential when one of the two polymerases runs into a block, and stoppage of the other polymerase is desirable. In vitro replication studies suggest that single-base mismatches created by a polymerase misincorporation already constitute such a (temporary) block (1,3).We propose that, following a misincorporation, (i) the responsible polymerase may be temporarily halted (this depends on the type of error), that (ii) tau senses this halting, (iii) stops the other polymerase, and (iv) initiates removal of the offending error (by proofreading or polymerase dissociation). In the absence of the sensing mechanism, coordination of leading and lagging strands is lost (accounting for certain replication defects of the mutants) and many errors are not removed (mutator effect). The specificity of mutagenesis is consistent with this model, as transversion mismatches (pur.pur) are most likely to stall polymerases. If not removed, they lead to transversions or, by slippage, frameshifts (1,3). Our dnaX mutants have also been investigated for the replication fidelity bias between leading and lagging strains, as described previously for wild-type strains (2). The bias is altered in dnaX mutants in a manner consistent with the above model.

(1) Pham, P.T., Olson, M.W., McHenry, C.S., and Schaaper, R.M. (1998) The base substitution and frameshift fidelity of E. coli DNA polymerase III holoenzyme in vitro. J. Biol. Chem. 273:23576-23584.
(2) Fijalkowska, I.J., Jonczyk, P., Tkaczyk, M.M., Bialoskorska, M., and Schaaper, R.M. (1998) Unequal fidelity of leading- and lagging-strand DNA replication on the E. coli chromosome. Proc. Natl. Acad. Sci. USA 95:10020-10025.
(3) Pham, P.T., Olson, M.W., McHenry, C.S., and Schaaper, R.M. (1999) Mismatch extension by E. coli DNA polymerase III holoenzyme. J. Biol. Chem. 274:3705-3710.

Mutator alleles affecting polymerase fidelity and cancer in mice.

Robert E. Goldsby, Mallika Singh, Elise Olmsted, Nicole A. Lawrence, Xin Chen, Peggy A. O'Neil, Geraldine Phear, Mark Meuth, and Bradley D. Preston Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA and Institute for Cancer Studies, University of Sheffield, Sheffield, UK.

Accurate DNA synthesis is contingent on multiple factors (dNTP pools, polymerase fidelity, proofreading and repair), yet only repair defects are clearly associated with human cancers. To assess the potential role of other mutators in tumorigenesis, we generated mice with defects in either polymerase proofreading or dNTP metabolism and examined these animals for cancer predisposition.

Using a genetic screening system in yeast, we first identified several alleles of DNA polymerase delta (pol delta) that disrupt exonucleolytic proofreading and confer a strong mutator phenotype (predominantly base substitutions but no microsatellite instability). To determine the significance of defective proofreading in mammals, we introduced one of these mutator alleles into mice. The mouse Pold1 gene (which encodes the catalytic subunit of pol delta) was cloned, a point mutation in the exonuclease-encoding domain (D400A) was created, and the mutant Pold1 gene was reintroduced into mouse ES cells using targeted homologous recombination. RT-PCR confirmed that the mutant allele is expressed at normal levels. Chimeric mice were generated from these mutant ES cells, and germline transmission of the pol delta exonuclease (exo) mutation was established. Exo +/- and -/- mice are viable and fertile. Moreover, the exo -/- animals are developing tumors at an early age. With the oldest mouse being ~26 weeks old, 8 of the 44 exo -/- mice have developed thymic lymphomas, while none of the wild type or heterozygous mice (N=165) have developed tumors. The phenotypes of the exo +/- and -/- mice are under further investigation, but these initial findings strongly indicate that defective pol delta proofreading results in cancer susceptibility (p<0.00001; Fisher's Exact Test).

To examine the impact of altered dNTP pools on tumorigenesis, we expressed a mutator allele of CTP synthetase (CTPS) in transgenic mice. CTPS catalyzes the amination of UTP to CTP in an essential, rate-limiting step during de novo dCTP synthesis. A dominant mutation in CTPS (I568T) that eliminates feedback inhibition by CTP was identified in Chinese hamster ovary cells (Whelan et al., Nat. Genet. 3:317, 1993). The mutant cells have increased pools of CTP and dCTP and a mutator phenotype characteristic of dCMP misincorporation by DNA polymerases (N-to-C base substitutions; next nucleotide effect; no microsatellite instability). Five C57BL/6J transgenic mouse lines were created that express this CTPS mutator allele under control of either an SV40 or CMV promoter. Among 29 transgenic mice examined, 10 animals developed tumors at 7 to 27 months of age; only one control mouse developed a tumor (liver) among 34 age-matched, non-transgenic littermates (p<0.005). Two transgenic mice developed primary tumors in more than one tissue. The CTPS-induced tumors occurred in diverse tissues (4 liver, 4 hematopoietic, 2 lung, 1 uterus, 1 ovary) and were of both epithelial and mesenchymal origin. Most of the tumors were malignant (80% based on histopathology), but metastases were rare (one animal). This study identifies a novel mutator and cancer susceptibility pathway involving dysregulation of dNTP pools.

Together these experiments show that microsatellite-stable mutators increase cancer susceptibility. Moreover, these data point to DNA polymerase fidelity as an important determinant of tumorigenesis.

RNA polymerase II as a sensor for DNA damage.

Mats Ljungman, Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109-0936.

Many environmental mutagens induce lesions in DNA that impedes the translocation of RNA polymerases. These types of agents pose two different kinds of problems for a cell. First, the cell has to deal with the immediate effects of the DNA damage on transcription and second, the cell needs to avoid the formation of mutations. Since the transcription machinery is constantly "scanning" the DNA of active genes, it is an ideal sensing apparatus for the cell to use to monitor the integrity of DNA. Blockage of RNA polymerase II has been shown to induce transcription-coupled repair (TCR) and to trigger the induction of both p53 and apoptosis. Cells defective in TCR induce p53 and apoptosis at much lower doses of UV light or cisplatin than cells with proficient TCR (Oncogene 13:823-831, 1996; Oncogene 18:583-592, 1999). Thus, proficiency in TCR confers a survival advantage for cells but by reducing apoptosis may actually increase mutagenesis.

It has been shown that following UV-irradiation, RNA polymerase II is actively ubiquitylated and subsequently degraded by the 26S proteasome. We have found that following a dose of 10 J/m2 of UVC light, the cellular level of the large subunit of RNA polymerase II decreases within three hours but returns to baseline levels within 6 hours which co-incides with the recovery of poly(A)RNA synthesis. Interestingly, UV-irradiated Cockayne's syndrome (CS) cells do not degrade their RNA polymerase II. Since CS cells are defective in TCR and the recovery of RNA synthesis following UV-irradiation, the results suggest that degradation of RNA pol II may contribute to these processes. In fact, we have found that inhibition of the 26S proteasome in repair-proficient cells interferes with the recovery of RNA synthesis following UV-irradiation. Our results further suggest that the degradation of RNA pol II is linked to elongation (and presumably blockage at UV-induced lesions) since pretreatment with drugs that inhibits the RNA polymerase from escaping from the promoter blocks RNA pol II degradation.

How does blockage of RNA polymerase signal p53? One potential signaling protein that is part of the transcription machinery is the CDK-activating kinase (CAK). CAK has been shown to be able to modify p53 either directly or indirectly via Cdk2 or Cdc2 which phosphorylates p53 on ser315. We have recently identified a pair of protein phosphatases, Cdc14A and Cdc14B that can interact with p53 and specifically dephosphorylate the ser315 site of p53 (JBC, in press). We are currently investigating the role of these kinases and phosphatases in the regulation of p53 following blockage of RNA polymerase II.

We have found that UV-induced apoptosis is not dependent on p53 in human fibroblast cells. In fact, we have found that cells with compromised p53 function induce apoptosis at lower doses than cells harboring wtp53 (Neoplasia 1:276-284, 1999). Furthermore, expression of p53 prior to but not following UV-irradiation confers resistance to UV light-induced apoptosis (manuscript submitted). The p53-mediated resistance correlates to enhanced recovery of both of RNA polymerase II protein levels and mRNA synthesis. It is possible that the degradation of RNA polymerase II following UV-irradiation is linked to apoptosis in cells unable to efficiently recover RNA synthesis. This would be a fail-proof mechanism to ensure that severely damaged cells will not survive since they would deplete themselves of the essential RNA polymerase II protein.