IN VITRO MUTAGENESIS STUDIES USING MAMMALIAN CELL LINES

我们研究了哺乳动物细胞中突变产生的机制， 组织培养中的人和仓鼠细胞系。 这些目标 研究的目的是确定各种环境诱变剂/致癌物 干扰正常的细胞反应，导致 一个基因错误我们对人类癌症细胞系的研究集中在 特异性错配修复（MMR）基因产物对基因组调控的作用 稳定使用已知在几种基因中的任何一种有缺陷的细胞系 参与MMR的基因，包括hMSH 2、GTBP、hMLH 1和hPMS 2基因， 我们正在量化HPRT基因座的突变率。 这些细胞系， 命名为RER+，在基因组微卫星序列上显示不稳定性; 然而，一个特定的MMR基因的作用更微妙的差异， 基因组稳定性的缺陷需要使用更复杂的突变 靶序列如HPRT。目前确定的突变率 从5.9 x 10 e-7的细胞系，其中MMR缺陷已经 对于hPMS 2缺陷细胞系，校正为5.9 × 10 e-5的上限。 我们 目前正在分离HPRT突变克隆用于产生 突变序列谱。 这些研究定义了 突变的类型和位点，并且可以预期提供 关于特定MMR基因的作用的机制见解， 自发突变的可能性 此外，评估 这些MMR缺陷细胞系中的任何一种对各种 DNA损伤正在发生。 最后，我们完成了我们的 使用人CYP 2A 6基因转染的AS 52 CHO细胞系进行的研究 被感染了 这种CYP 450活性代谢烟草特异性 亚硝胺，NNK，以产生致突变的DNA甲基化损伤。 在 此外，我们已基本完成了gpt整合位点的测序。 在AS 52细胞中。 质粒整合以及侧翼CHO 基因组序列提供了可能解释敏感的 该细胞系对突变诱导的突变反应， 拟辐射剂。

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.