HUMAN-CHROMOSOME-3 CORRECTS MISMATCH REPAIR DEFICIENCY AND MICROSATELLITE INSTABILITY AND REDUCES N-METHYL-N'-NITRO-N-NITROSOGUANIDINE TOLERANCE IN COLON-TUMOR CELLS WITH HOMOZYGOUS HMLH1 MUTATION

The human colon tumor cell line HCT 116 is known to have a homozygous mutation in the mismatch repair gene hMLH1 on human chromosome 3, to e xhibit microsatellite instability, and to be defective in mismatch rep air. In order to determine whether the introduction of a normal copy o f hMLH1 gene restores mismatch repair activity and corrects microsatel lite instability, a single human chromosome 3 from normal fibroblasts was transferred to HCT 116 cells via microcell fusion. As a control, h uman chromosome 2 was also transferred to HCT 116 cells. Two HCT 116 m icrocell hybrid clones that received a single copy of chromosome 2 (HC T 116+ch2) and two that received a single copy of chromosome 3 (HCT 11 6+ch3) were isolated and characterized. A G G mismatch in MW-derived h eteroduplex DNA was efficiently repaired in cell extracts from HCT 116 +ch3 cells, but not in those of parent HCT 116 cells or HCT 116+ch2 ce lls. Microsatellite alterations at the D5S107 locus containing CA repe ats were seen in 8 of 80 subclones from HCT 116 cells, and in 13 of 15 0 subclones from HCT 116+ch2 cells. In contrast, none of the 225 subcl ones derived from mismatch repair-proficient HCT 116+ch3 cells showed alterations in the microsatellite at the same locus. The effect of int roducing chromosome 3 on the sensitivity of HCT 116 cells to N-methyl- N' nitro-N nitrosoguanidine (MNNG) was examined, since enhanced tolera nce to MNNG is accompanied by loss of mismatch repair activity in seve ral cell lines. Within 3 days after treatment with 5 mu 3 MNNG, HCT 11 6+ch3 cells became morphologically flat and stopped growing. Their col ony-forming ability, determined 10 days after treatment, was reduced 2 00-fold when compared to MNNG-treated parental HCT 116 and HCT 116+ch2 cells. These results support the hypothesis that mutations in both al leles of the hMLH1 gene are necessary for the manifestation of defecti ve mismatch repair and microsatellite instability and for enhanced MNN G tolerance. The results also suggest that the mismatch repair system contributes to the process that causes growth arrest in response to DN A damage by alkylating agents.