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 cell line. Eight had received a single treatment with a highly cytotoxic dose. The remainder, including the previously described RajiF12 cell line, arose following multiple exposures to initially moderate but escalating doses. Surprisingly, methylation resistance arose in three clones by reactivation of a previously silent O-6-methylguanine-DNA methyltransferase gene, Five clones, including RajiF12, displayed the microsatellite 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 confirmed by Western blot analysis. Remarkably, five methylation-resistant clones in which mismatch repair defects were demonstrated by biochemical assays did not exhibit significant microsatellite instability.