The goal is to define on a molecular level the multifactor interactions involved in carcinogenesis. The role of specific viral genes in regulating carcinogen-enhancement of viral transformation (CET) will be studied using a highly transformable rat embryo fibroblast (CREF) cell line, well characterized type 5 adenovirus (Ad5) mutants and various molecular biological techniques. Experiments designed to determine the role of specific viral genes in regulating the CET phenotype will focus on a cold-sensitive host-range mutant of Ad5, H5hr1, which transforms CREF cells at a higher frequency than wild type Ad5 (H5wt) at 37 or 39.5 degrees C, but is defective for transformation at 32 degrees C. The H5hr1 mutant also displays a unique CET phenotype not observed in carcinogen-treated CREF cells infected with H5wt or other cold-sensitive host-range Ad5 mutants. Experiments will be designed to determine if carcinogens alter the proportion of cells in an infected population which initially contain viral DNA and ultimately become transformed. The effect of carcinogens on Ad5-gene expression in cryptically transformed CREF cells, i.e., transformed cells containing integrated Ad5 DNA but not expressing Ad5-genetic information, will also be evaluated. To define directly the role of specific regions of the H5hr1 genome in regulating the CET effect, CREF cells will be coinfected with H5hr1 and various genetically engineered Ad5 mutants and the H5hr1 genome will be reconstructed by genetic recombination techniques. To identify potentially relevant changes in cellular gene expression induced by carcinogen treatment and H5hr1 infection high resolution 2-D gel protein electrophoresis will be employed. Experiments using 2-D gel protein electrophoresis analysis will be designed to identify: (a) the temporal sequence of cellular protein changes induced by carcinogen treatment and infection with H5hr1; (b) protein alterations which are unique to specific DNA damaging and carcinogenic agents which induce a CET phenotype; and (c) by using CREF cells resistant to the CET phenotype, specific protein changes which may be crucial for induction of a CET phenotype. These studies should provide important insights into the function of specific viral genes in mediating chemical-viral carcinogenesis and should permit a further molecular definition of the multiple factors involved in the carcinogenic process.
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