of Work: We continue to use mammalian cell lines in culture to address specific questions related to the cellular processes that regulate genome stability and to study how those processes can be perturbed by specific gene mutations or by environmental mutagens/carcinogens. Mostly, we have focused on mismatch repair deficient human cancer lines. Genomic stability in cell lines known to be affected by defects in any one of the five known human MMR genes. The specific genes implicated in the human MMR pathway include homologues of the bacterial MMR proteins MutS and MutL, including MSH2, MSH3, MSH6 (GTBP), MLH1 and PMS2. Several of these proteins have been shown to function in MMR as heterodimers. For example, the mismatch recognition heterodimer, hMutS-alpha is comprised of the MutS homologues, MSH2 and MSH6 and the hMutLa heterodimer is comprised of the MutL homologues, MLH1 and PMS2. An additional mismatch recognition heterodimer, hMutS-beta, has also been proposed, consisting of the MutS homologues, MSH2 and MSH3. The mutation rate and mutational specificity at the HPRT locus were determined in an endometrial carcinoma cell line defective in the MMR genes, MSH6 and MSH3. This cell line, HHUA, exhibited a substantially elevated mutation rate at the HPRT locus, and the resulting mutational spectrum consisted of an approximately equal incidence of base substitution and frameshift mutations. Restoration of either defective gene by chromosome transfer resulted in HHUA cell lines with substantially reduced mutation rates. The specificity of mutation at the HPRT locus, combined with in vitro studies of substrate specificity using cell extracts and analyses of microsatellite stability in the parent HHUA line suggest that both single-base mismatches and frameshift intermediates can be repaired by either MSH3 or MSH6, thereby supporting the notion of a redundancy for substrate recognition and repair by hMutSa or hMutSb. In addition, we have evaluated the cytotoxic and mutagenic response at the HPRT locus following exposure to the chemotherapeutic agent, 6-thioguanine (6-TG) or to the alkylating agent MMS in human cancer cell lines defective in mismatch repair (MMR) and in cell lines in which the MMR defects were complemented by chromosome transfer. Our findings suggest that cytotoxicity to MMS is mediated through MMR, as indicated by an increased resistance to either 6-TG or MMS in MMR-deficient cells and that resistance to such damage in MMR-defective cells correlates with an increase in genomic mutations.
