I. Bernard Weinstein, MD - US grants

The objectives of the Columbia University Comprehensive Cancer Center are to: 1) support and facilitate basic laboratory and clinical research related to cancer and foster collaborative research projects between epidemiologists, laboratory scientists and clinical investigators; 2) to promote a targeted research program in human cancer causation and prevention, which interfaces with other research activities of the Center; 3) to provide education and training in cancer research for scientists, clinical investigators and health professionals at all levels; and 4) to promote the dissemination of cancer-related clinical and technical expertise and to foster cancer control activities in the community. These objectives are facilitated by an organizational structure that has 4 major Divisions; 1) Basic Research; 2) Clinical Research; 3) Etiology, Prevention and Control; and 4) Research Education. Essential support is also provided by the Office of Administration, an External Advisory Committee and the Office of Communications and Development. Fields of research are organized into a total of 21 Programs that facilitate research within a given discipline and foster interdisciplinary-collaboration. A major function of this grant is to support a series of basic science and clinical shared research Facilities that provide state of the art expertise, technology and resources; with considerable efficiency and cost savings. A major emphasis of this Cancer Center, and a unifying theme that joins together this interdisciplinary research approach, is the use of concepts and methods in cellular and molecular biology to enhance our understanding of the etiology and pathophysiology of human cancer, and to develop more effective strategies for cancer prevention, diagnosis and treatment.

The purpose is to continue our studies on the biochemical and molecular mechanisms that underlie the process of tumor promotion. Because of the evidence that the enzyme protein kinase C (PKC) plays a central role in growth control and mediates the action of the phorbol ester tumor promoters, we will concentrate on this enzyme system. Studies from several laboratories, including our own, indicate that the mammalian genome encodes several isoforms of this enzyme. Our major strategy will be to use genetic techniques to develop derivatives of the Rat 6 fibroblast cell line that stably overexpress the beta 1, gamma, or epsilon isoforms of PKC. These cell lines will be used to determine possible biochemical differences between these isoforms, to assess their effects on growth control and synergy with oncogenes, and to develop more specific inhibitors of PKC. Using 32P-labelling of intact cells, coupled with two dimensional gel electrophoresis and immunoprecipitation methods, we will attempt to identify the major protein substrates phosphorylated in these cells and the possible roles of these phosphoproteins in mediating the effects of various isoforms of PKC. Region-specific mutations will be introduced into different domains of PKC beta 1 to assess the biochemical functions of these domains and their roles in growth control. A specific project will explore the role of PKC in activation of human T cells and in replication of the human immunodeficiency virus. We will also isolate a full length cDNA sequence for the gene TPA-R1, whose expression is inhibited by TPA, and determine whether overexpression of this sequence suppresses the transformed phenotype. A long term goal is to develop transgenic strains of mice that overexpress specific isoforms and mutant forms of PKC, to define the role of this enzyme system in the multistage carcinogenic process in the intact animal. We are hopeful that the insights obtained from these studies, and the model systems that are developed, will provide a more rational basis for detecting potential tumor promoters in our environment and lead to new strategies of cancer prevention.

We propose to conduct a series of related in vivo pilot studies that take the first steps toward validating a recently developed immunoassay for benzo[a]pyrene (BaP) covalently bound to DNA as a dosimeter for "biologically effective dose" of the carcinogen. A secondary goal is to preliminarily evaluate the potential of the assay as an indicator of increased risk of lung cancer. Specifically we will investigate relatively unknown aspects of in vivo binding, including the sensitivity of the assay, "dose-response" relationships, the persistence of the adduct, the extent of interindividual variation, the correlation between concentrations of adducts in lung and white blood cell DNA in laboratory animals, and the possibility that lung cancer patients have a greater tendency to bind BaP to DNA than controls. The research will consist of (1) experimental studies of limited numbers of laboratory animals and highly exposed cigarette smoking volunteers in which exposure to BaP will be controlled and (2) observational and clinical studies of coke oven workers and lung cancer cases and controls. DNA extracted from lung and blood samples of animals and blood samples of humans will be assayed by the same enzyme linked immunosorbent assay (ELISA). For the human subjects, results in the ELISA will be analysed in conjunction with data obtained by questionnaire concerning exposure to BaP and to a limited number of agents believed to modify BaP metabolism in humans. This work is intended to verify and extend results of an earlier pilot project involving laboratory animals and hospital patients in order to develop meaningful data on the potential usefulness of the BaP-DNA assay for use as a dosimeter in future molecular epidemiological studies in cancer causation, and as a means of screening individuals who might be at elevated risk of lung cancer.

The objective continues to be the elucidation of the molecular mechanisms of action of chemical carcinogens. This grant will facilitate an interdisciplinary approach, which is required in view of the complexity of the multistage process of carcinogenesis. The proposed studies will follow a logical continuum that includes the following major topics: 1) the chemistry and stereochemistry underlying the covalent binding of chemical carcinogens to DNA, 2) elucidation of the changes in DNA conformation that result from these chemical modifications, as well as studies on their functional significance, 3) the development of highly specific and sensitive immunoassays to facilitate molecular studies on chemical carcinogenesis and 4) molecular genetic studies designed to assess the possibility that the structural changes produced in DNA, as a result of modification by chemical carcinogens, can induce, not only point mutations, but also complex genomic effects, including gene amplification, stable alterations in gene expression and gene transposition (Project E). This integrated approach should provide fundamental insights into basic mechanisms of action of chemical carcinogens and thus provide rational approaches to carcinogen detection, risk extrapolation and cancer prevention.

The long-term objective of the proposed research is to elucidate the mechanisms and role of genetic toxicity in chemical carcinogenesis. In multidisciplinary integrated studies, various complementary markers of genetic toxicity will be investigated: carcinogen-DNA and protein adducts using immunological methods, sister chromatid exchange (SCE) and micronuclei in lymphocytes (MNL), and products of activated oncogenes. The overall approach will be to validate these markers in parallel experimental animal and molecular epidemiological studies by assaying biological samples following in vivo exposure to a number of carcinogens. In the first phase, quantifiable and well-characterized model compounds (chemotherapy agents, ethylene oxide) will be studied. In the second phase, following development of antibodies to the specific DNA and protein adducts of interest, the same battery of immunological and cytogenetic assays will be applied to two environmentally relevant chemicals (vinyl chloride and styrene). The feasibility of extending this approach to benzene and butadiene will also be evaluated. This approach will provide greater understanding of the relationship between DNA binding and measurable chromosomal damage and will permit assessment of the relative usefulness of each as a marker of biologically effective dose of carcinogen in future biomonitoring and molecular epidemiology studies. The proposed research will be carried out by developing new antibodies to carcinogen-DNA and protein adducts, modifying existing immunological methods to increase their sensitivity, implementing the MNL assay, and applying innovative methods (cytofluorography, fluorescence line narrowing, and image intensification microscopy) to quantification of DNA adducts.

The objectives of this Cancer Center are to: 1) support and facilitate basic laboratory and clinical research related to cancer and foster collaborative research projects between epidemiologists, laboratory scientists and clinical investigators; 2) to promote a targeted research program in human cancer causation and prevention, which interfaces with other research activities of the Center; 3) to provide education and training in cancer research for scientists, clinical investigators and health professionals at all levels; and 4) to promote the dissemination of cancer-related clinical and technical expertise and to foster cancer control activities in the community. These objectives are facilitated by an organizational structure that has 4 major Divisions; 1) Basic Research; 2) clinical Research; 3) Cancer Causation and Prevention, and 4) Research Education. Essential support is also provided by the Office of Administration and, a Visiting Scientific Advisor Committee. Fields of research are organized into a total of 9 Programs that facilitate research within a given discipline and foster interdisciplinary collaboration. A major function of this grant is to support a series of 14 basic science and clinically-related shared research facilities that provide state-of- the-art expertise, technology and resources with considerable efficiency and cost savings. A specific emphasis of this Cancer Center, and a unifying theme that joins together this interdisciplinary research approach, is the use of concepts and methods in cellular and molecular biology to enhance our understanding of the etiology and pathophysiology of human cancer, and to develop more effective strategies for cancer prevention, diagnosis and treatment.

DESCRIPTION: The purpose of this proposal is to examine the role of the cyclin D1 gene, and related genes, in multistage carcinogenesis, emphasizing cancers of the esophagus. The rationale for these studies is that the chromosome locus 11q13 which contains this gene is frequently amplified in esophageal and certain other types of human cancer. Furthermore, in recent studies we found that about 30% of human esophageal tumors display not only amplification of cyclin D1 but also increased expression of this gene. A second rationale is that mutations in cyclin D1, and in other cyclins or cyclin-related genes, could play a critical role in carcinogenesis by perturbing cell cycle control, and thereby enhancing cell proliferation and genomic instability. To determine the effects of cyclin D1 overexpression on the phenotype of cells a series of cell culture lines that overexpress cyclin D1 will be compared to control cell lines for possible differences in growth properties, cell cycle progression, transformation, gene expression, differences in responses to chemical carcinogens, and susceptibility to gene amplification. To determine whether cyclin D1 exerts its effects by interacting with the p110 Rb protein, or other specific cellular proteins, we will utilize the yeast two-hybrid system and also carry out protein phosphorylation assays. Taken together, these studies will provide insights into the role of cyclin genes in multistage carcinogenesis and may suggest novel strategies for cancer chemoprevention and treatment.

oncogenic virus; radiobiology; carcinogenesis; neoplasm /cancer radiation therapy; neoplasm /cancer epidemiology; urinary tract neoplasms; nervous system neoplasms; brain neoplasms; neoplasm /cancer immunology; neoplasm /cancer genetics; molecular oncology; developmental genetics; hematology;

phorbols; protein kinase C; isozymes; cell growth regulation; regulatory gene; transfection /expression vector; neoplasm /cancer genetics; cellular oncology; point mutation; enzyme mechanism; gene expression; oncogenes; transcription factor; leukocyte activation /transformation; molecular oncology; apoptosis; tissue /cell culture; genetically modified animals; laboratory mouse;