Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation

Our previous data demonstrated that cells deficient in MutL homologue-1 (ML H1) expression had a reduced and shorter G(2) arrest after high-dose-rate i onizing radiation (IR), suggesting that the mismatch re pair (MMR) system m ediates this cell cycle checkpoint. We confirmed this observation using two additional isogenctically matched human MLH1 (hMLH1)-deficient and -profic ient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cel ls, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-defleient and -proficient human e ndometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR i n general, is involved in IR-related G(2) arrest responses. As in MLH1-defi cient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arre st in response to IR (6 Gy). These differences in IR-induced G(2) arrest be tween MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unl ike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cell s 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 G(2) arrest, we examined the levels of tyrosine 15 phosphorylatio n of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. I ncreased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-c dc2 rapidly decreased in MMR (hMILH1 or hMSH2) -deficient cell lines at tim es coincident with progress from the IR-induced G(2) arrest through M phase . Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rat e IR correspond temporally with the observed differences in the IR-induced G(2), arrest, suggesting that INJINIR proteins may exert their effect on IR -induced G(2) arrest by signaling the cdc2 pathway. Although MMR status doe s not significantly affect the survival of cells after high-dose-rate IR, i t seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.