Defective Expression of the DNA Mismatch Repair Protein, MLH1, Alters G sub(2)-M Cell Cycle Checkpoint Arrest following Ionizing Radiation

A role for the Mut L homologue-1 (MLH1) protein, a necessary component of DNA mismatch repair (MMR), in G sub(2)-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G sub(1) arrest and/or G sub(1)-S transition after damage was, however, not discounted. We report that MLH1-deficient human colon carcinoma (HCT116) cells showed decreased survival and a concomitant deficiency in G sub(2)-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected human colon carcinoma (HCT116 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embryonic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from MLH1 knockout mice also demonstrated classic DNA damage tolerance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLH1 super(+)) compared with HCT116 (MLH1 super(-)) cells after IR or 6-TG. Retroviral vector-mediated expression of the E6 protein did not, however, affect the enhanced G sub(2)-M cell cycle arrest observed in HCT116 3-6 compared with MLH1-deficient HCT116 cells. A role for MLH1 in G sub(2)-M cell cycle checkpoint control, without alteration in G sub(1), after IR was also suggested by similar S-phase progression between irradiated MLH1-deficient and MLH1-proficient human or murine cells. Introduction of a nocodazoleinduced G sub(2)-M block, which corrected the MLH1-mediated G sub(2)-M arrest deficiency in HCT116 cells, clearly demonstrated that HCT116 and HCT116 3-6 cells did not differ in G sub(1) arrest or G sub(1)-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G sub(1) cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLH1-mediated G sub(2)-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells.