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 (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.
Yan
T
T
Department of Radiation Oncology, Ireland Comprehensive Cancer Center/University Hospitals of Cleveland, Cleveland, OH 44106-4942, USA.
Schupp
J E
JE
Hwang
H S
HS
Wagner
M W
MW
Berry
S E
SE
Strickfaden
S
S
Veigl
M L
ML
Sedwick
W D
WD
Boothman
D A
DA
Kinsella
T J
TJ
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D016203:CDC2 Protein Kinase / Q000378:metabolism / Q000502:physiology*; D002352:Carrier Proteins; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology; D004260:DNA Repair / Q000502:physiology*; D016889:Endometrial Neoplasms / Q000378:metabolism / Q000473:pathology; D005260:Female; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D006801:Humans; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000172:deficiency; D009687:Nuclear Proteins; D010051:Ovarian Neoplasms / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D016196:S Phase / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D015398:Signal Transduction / Q000502:physiology / Q000528:radiation effects; D013866:Thioguanine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016203:CDC2 Protein Kinase; D000070957:MutL Protein Homolog 1; D013866:Thioguanine