MUTATION AND DEREPRESSION OF GENES IN CARCINOGENESIS

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The fundamental working hypothesis of this proposal is that carcinogenesis is a multi-staged process, consisting of an irreversible conversion of the normal cell to a premalignant cell (i.e., initiation phase) and the clonal amplification of the initiated cell to a "critical mass" allowing the metastatic phenotype to appear (i.e., promotion and progression phases). Our aim on these two distinctly different but interdependent processes during carcinogenesis continues to be the study of the biochemical and cellular mechanisms of initiation and promotion using in vitro techniques with various mammalial, including human, cells. Specifically, we will study: a) mammalian mutagenesis and its possible role in the initiation phase of carcinogenesis and b) gap-junctional intercellular communication and its potential role in tumor promotion. We will use our Chinese hamster V79 Alpha DNA polymerase "mutator" (aphidicolin-resistant) mutant to study its role in spontaneous and induced mutations by characterizing various types of revertant mutants, some with presumptive qualitative or quantitative changes in the Alpha-DNA polymerase, and others with altered nucleotide pools. We will also attempt to isolate aphidicolin-sensitive Chinese hamster cells to test if certain mutant Alpha DNA polymerases could be "anti-mutators". In addition, we will extend our studies on the role of Alpha DNA polymerase on gene and chromosomal amplification. The role of gap-junctional communication in tumor promotion will be studied by extending our observations that certain oncogenes (i.e., src, ras) inhibit intercellular communication. Plasmids containing the src and ras genes, respectively, will be transfected into the gap-junction proficient Chinese hamster cells, and into a communicating and diethylstibesterol-differentiation-inducible rat liver "oval" cell line. In addition, we will study the possible role of ras and myc oncogenes, alone and in combination, on cell communication and transformation. We will be using, along with traditional techniques to measure gap-junction function, two of our newly developed techniques, "Fluorescence Redistribution After Photobleaching" (FRAP analysis) and "scrape-loading" to perform our studies on gap-junction function.