Role of the hMLH1 DNA mismatch repair protein in fluoropyrimidine-mediatedcell death and cell cycle responses

DNA mismatch repair (MMR) is an efficient system for the detection and repa ir of mismatched and unpaired bases in DNA, Deficiencies in MMR are commonl y found in both hereditary and sporadic colorectal cancers, as well as in c ancers of other tissues. Because fluorinated thymidine analogues (which thr ough their actions might generate lesions recognizable by MMR) are widely u sed in the treatment of colorectal cancer, we investigated the role of MMR in cellular responses to 5-fluorouracil and 5-fluoro-2 ' -deoxyuridine (FdU rd), Human MLH1(-) and MMR-deficient HCT116 colon cancer cells were Ib-fold more resistant to 7.5 muM 9-fluorouracil (continuous treatment) and 17-fol d more resistant to 7.5 muM FdUrd in clonogenic survival assays compared wi th genetically matched, MLN1(+) and MMR-proficient HCT116 3-6 cells. Likewi se, murine MLH1(-) and MMR-deficient CT-5 cells were 3-fold more resistant to a 2-h pulse of 10 muM FdUrd than their MLH1(+) and MMR-proficient ME-10 counterparts, Decreased cytotoxicity in MMR-deficient cells after treatment with various methylating agents and other base analogues has been well rep orted and is believed to reflect a tolerance to DNA damage. Synchronized HC T116 3-6 cells treated with a low dose of FdUrd had a 2-fold greater G(2) c ell cycle arrest compared with MMR-deficient HCT116 cells, and asynchronous ME-10 cells demonstrated a 4-fold greater G(2) arrest after FdUrd treatmen t compared with CT-5 cells. Enhanced G(2) arrest in MMR-proficient cells in response to other agents has been reported and is believed to allow time f or DNA repair. G(2) cell cycle arrest as determined by propidium iodide sta ining was not a result of mitotic arrest, but rather a true G(2) arrest, as indicated by elevated cyclin B1 levels and a lack of staining with mitotic protein monoclonal antibody 2, Additionally, p53 and GADD45 levels were in duced in FdUrd-treated HCT116 3-6 cells, DNA double-strand break (DSB) form ation was 2-fold higher in MMR-proficient HCT116 3-6 cells after FdUrd trea tment, as determined by pulsed-field gel electrophoresis, The formation of DSBs was not the result of enhanced apoptosis in MMR-proficient cells. FdUr d-mediated cytotoxicity was caused by DNA-directed and not RNA-directed eff ects, because administration of excess thymidine (and not uridine) prevente d cytotoxicity, cell cycle arrest, and DSB formation. hMLH1-dependent respo nses to fluoropyrimidine treatment, which may involve the action of p53 and the formation of DSBs, clearly have clinical relevance for the use of this class of drugs in the treatment of tumors with MMR deficiencies.