Previously we hypothesized and then proved that 2',2'-difluoro-2'-deoxycytidine (dFdCyd, gemcitabine) is a potent radiation sensitizer. During the current grant period we demonstrated that radiosensitization correlated strongly with inhibition of ribonucleotide reductase by the 5'-diphosphate of dFdCyd, resulting in greater than 80 percent depletion of dATP, whereas the 5'-triphosphate contributed primarily to the cytotoxic effect. Cells that were radiosensitized by dFdCyd accumulated in the less radiosensitive S-phase during drug exposure. Radiosensitization did not require apoptotic cell death, and it could occur in p53 wild-type as well as mutant p53 cell lines. Gemcitabine did not increase DNA double strand breaks prior to or following irradiation, nor did it inhibit their repair. Thus, the accumulated data suggest that radiosensitization with dFdCyd is determined by the nature of the DNA damage sustained prior to irradiation rather than the response to DNA damage after irradiation. We now hypothesize that the critical lesion for radiosensitization with dFdCyd is the incorporation in DNA of an incorrect nucleotide caused by the depleted dATP and, if this lesion is not repaired prior to S-phase, irradiation will prevent repair of the misincorporation events resulting in permanent mutation and enhanced cell death. This hypothesis would predict that cells defective in repairing misincorporated nucleotides, such as mismatch repair deficient cells, would be better radiosensitized than cells proficient in this pathway. Preliminary data indicate that mismatch repair deficient HCT-116 cells are radiosensitized by dFdCyd whereas the mismatch repair proficient counterpart was not, although it was more sensitive to cytotoxicity with dFdCyd. We propose to evaluate the factors that determine dFdCyd cytotoxicity and radiosensitivity in isogenic cell lines defective or proficient in mismatch repair. Using a shuttle vector assay, we will determine whether the hypothesized radiosensitizing lesions, misincorporated nucleotides, are more prevalent in cells that are radiosensitized by dFdCyd. In addition to defining the mechanism of radiosensitization for dFdCyd, we will also determine the mechanism of synergy when it is combined with docetaxel, based on high antitumor efficacy reported recently in patients. Preliminary data demonstrate that this synergy is not related to the ability of docetaxel to block cells in G2/M, but rather we hypothesize docetaxel commits dFdCyd-treated cells to S-phase specific cell death.
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