Loss of DNA mismatch repair imparts defective cdc2 signaling and G₂ arrest responses without altering survival after ionizing radiation

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G₂ arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch repair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G₂ arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G₂ arrest in response to IR (6 Gy). These differences in IR-induced G₂ arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G₀/G₁ or S phase, indicating that MMR indeed dramatically affects the G₂-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G₂ arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15 -cdc2), a key regulator of the G₂-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G₂ arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G₂ arrest, suggesting that MMR proteins may exert their effect on IR-induced G₂ arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G₂-M checkpoint and might affect overall mutation rates.