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

• Published in: Cancer research (Chicago, Ill.) Ill.), 2001-11, Vol.61 (22), p.8290-8297
• Main Authors: Yan, T, Schupp, J E, Hwang, H S, Wagner, M W, Berry, S E, Strickfaden, S, Veigl, M L, Sedwick, W D, Boothman, D A, Kinsella, T J
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Base Pair Mismatch; Carrier Proteins; CDC2 Protein Kinase - metabolism; CDC2 Protein Kinase - physiology; Cell Survival - drug effects; Cell Survival - physiology; Cell Survival - radiation effects; Colorectal Neoplasms - metabolism; Colorectal Neoplasms - pathology; DNA Repair - physiology; Endometrial Neoplasms - metabolism; Endometrial Neoplasms - pathology; Female; G2 Phase - drug effects; G2 Phase - physiology; G2 Phase - radiation effects; Humans; MutL Protein Homolog 1; Neoplasm Proteins - deficiency; Nuclear Proteins; Ovarian Neoplasms - metabolism; Ovarian Neoplasms - pathology; Phosphorylation; S Phase - drug effects; S Phase - physiology; S Phase - radiation effects; Signal Transduction - physiology; Signal Transduction - radiation effects; Thioguanine - pharmacology; Tumor Cells, Cultured
• ISSN: 0008-5472

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (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 G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between 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, 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 G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-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 G(2) 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 G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) 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 G(2)-M checkpoint and might affect overall mutation rates.