The mismatch repair protein, hMLH1, mediates 5-substituted halogenated thymidine analogue cytotoxicity, DNA incorporation, and radiosensitization in human colon cancer cells

Deficiency in DNA mismatch repair (MMR) is found in some hereditary (hereditary nonpolyposis colorectal cancer) and sporadic colon cancers as well as other common solid cancers. MMR deficiency has recently been shown to impart cellular resistance to multiple chemical agents, many of which are commonly used in cancer chemotherapy. It is therefore of interest to find an approach that selectively targets cells that have lost the ability to perform MMR. In this study, we examine the response of MMR-proficient (hMLH1⁺) and MMR-deficient (hMLH1^-) colon carcinoma cell lines to the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) before and after irradiation. These dThd analogues are used clinically as experimental sensitizing agents in radioresistant human cancers, and there is a direct correlation between the levels of dThd analogue DNA incorporation and tumor radiosensitization. In contrast to the well-characterized, marked increase in cytotoxicity (> 1 log cell kill) found with 6-thioguanine exposures in HCT116/3-6 (hMLH1⁺) cells compared to HCT116 (hMLH1^-) cells, we found only modest cytotoxicity (10-20% cell kill) in both cell lines when treated with IdUrd or BrdUrd for 1 population doubling. Upon further analysis, the levels of halogenated dThd analogues in DNA were significantly lower (two to three times lower) in HCT116/3-6 cells than in HCT116 cells, and similar results were found in Mlh1(+/+) spontaneously immortalized murine embryonic fibroblasts and fibroblasts from Mlh1 knockout mice. As a result of the higher levels of the dThd analogue in DNA, there was an increase in radiation sensitivity in HCT116 cells but not in HCT116/3-6 cells after pretreatment with IdUrd or BrdUrd when compared to treatment with radiation alone. Additionally, we found no differences in the cellular metabolic pathways for dThd analogue DNA incorporation because the enzyme activities of dThd kinase and thymidylate synthase, as well as the levels of triphosphate pools, were similar in HCT116 and HCT116/3-6 cells. These data suggest that the hMLH1 protein may participate in the recognition and subsequent removal of halogenated dThd analogues from DNA. Consequently, whereas MMR-deficient cells and tumor xenografts have shown intrinsic resistance to a large number of chemotherapeutic agents, the 5-halogenated dThd analogues appear to selectively target such cells for potential enhanced radiation sensitivity.