IN VITRO MUTAGENESIS STUDIES USING MAMMALIAN CELL LINES

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We study the mechanisms by which mutations arise in mammalian cells using both human and hamster cell lines in tissue culture. The goal of these studies is to define how various environmental mutagens/carcinogens perturb normal cellular responses leading to an increased probability of a genomic error. Our studies with human cancer lines focus on the role of specific mismatch repair (MMR) gene products on the control of genomic stability. Using cell lines known to be defective in any one of several genes involved in MMR, including the hMSH2, GTBP, hMLH1 and hPMS2 genes, we are quantitating mutation rates at the hprt locus. These cell lines, designated RER+, show instability at genomic microsatellite sequences; however, more subtle differences in the effect of a specific MMR gene defect on genomic stability requires the use of a more complex mutational target sequence such as hprt. Mutation rates determined thus far range from 5.9 x 10e-7 for a cell line in which the MMR defect has been corrected to a high of 5.9 x 10e-5 for an hPMS2 defective cell line. We are currently isolating hprt mutant clones for use in generating a mutational sequence spectrum in these lines. Such studies define both the type and site of a mutation and can be expected to provide mechanistic insights regarding the role of specific MMR genes the process of spontaneous mutation. In addition, studies evaluating the differential response of any of these MMR deficient cell lines to various types of DNA damage is in progress. Finally, we have completed our studies with the AS52 CHO cell line into which the human CYP2A6 gene was transfected. This CYP450 activity metabolizes the tobacco specific nitrosamine, NNK, to yield mutagenic DNA methylation damage. In addition, we have nearly completed sequencing the site of gpt integration in AS52 cells. Both the plasmid integration as well as flanking CHO genomic sequences provide motifs that might explain the sensitive mutational response for this cell line for mutation induction by radiomimetic agents.