MISMATCH REPAIR DEFECTS AND O-6-METHYLGUANINE-DNA METHYLTRANSFERASE EXPRESSION IN ACQUIRED-RESISTANCE TO METHYLATING AGENTS IN HUMAN-CELLS

Fifteen variants with greater than or equal to 30-fold resistance to N -methyl-N-nitrosourea were isolated from the Burkitt's lymphoma Raji c ell line. Eight had received a single treatment with a highly cytotoxi c dose. The remainder, including the previously described RajiF12 cell line, arose following multiple exposures to initially moderate but es calating doses. Surprisingly, methylation resistance arose in three cl ones by reactivation of a previously silent O-6-methylguanine-DNA meth yltransferase gene, Five clones, including RajiF12, displayed the micr osatellite instability and increased spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyltransferase locus, consistent with deficiencies in mismatch repair. Defects in either the hMutS alpha or hMutL alpha mismatch repair complexes were identified in extracts of these resistant clones by in vitro complementation using extracts from colorectal carcinoma cell lines. Defects in hMutL alpha were confirme d by Western blot analysis. Remarkably, five methylation-resistant clo nes in which mismatch repair defects were demonstrated by biochemical assays did not exhibit significant microsatellite instability.