Evidence for a Connection between the Mismatch Repair System and the G2 Cell Cycle Checkpoint1

The human colon tumor cell line HCT116 is deficient in wild-type liMIJII. is defective in mismatch repair (MMR), exhibits microsatellite instability, and is tolerant to JV-methylWV'-nitro-JV-nitrosoguanidine (MNNG). Transferring a normal copy of li.MI.III on chromosome 3 into the cell line restores MMR activity, stabilizes microsatellite loci, and increases the sensitivity of the cell to MNNG. Previous studies in other cell lines tolerant to alkylating agents such as MNNG or JV-methylnitrosourea have shown cross-tolerance to 6-thioguanine (6TG), leading to a hypoth esis that tolerance to MNNG or 6TG may be the result of MMR deficiency. To test this hypothesis, we studied the effects of 6TG on the MNNGtolerant, MMR-deficient IK I IK. cell line and its MNNG-sensitive, MMR-proficient, MNNG-tolerant, and MMR-deficient derivatives. Con tinuous exposure to low doses of 6TG (0.31-1.25 ug/ml) had no apparent effect on colony-forming ability (CFA) in MNNG-tolerant, MMR-deficient cells, whereas MNNG-sensitive, MMR-proficient cells exhibited a dosedependent decrease in CFA. Growth kinetics and cell cycle analysis re vealed that the growth of 6TG-treated HCT116+chr3 cells was arrested at G2 after exposure to low dose of 6TG. In contrast, the same exposure to 6TG did not induce G2 arrest but rather a G, delay in IK"l'I 16 and HCT116+chr2. To obtain further evidence for the role of MMR on 6TG and MNNG toxicity, we isolated an MNNG-resistant revertan! clone, M2, from the MNNG-sensitive, MMR-proficient HCT116+chr3 cell line and characterized the MMR activity, hMLHl status, and 6TG response. The results showed that M2 cells lost MMR activity as well as the previously introduced normal hMLHl gene. Restoration of the CFA of M2 and an absence of G2 arrest were observed after treatment with low doses of 6TG. These results suggest that the mismatch repair system interacts with the G2 checkpoint in response to 6TG or MNNG-induced DNA lesions. The results further suggest that any agent that induces DNA mispairs will cause G2 arrest in MMR-proficient cells but not in MMR-deficient cells.