| Gemcitabine (dFdCyd) is a nucleoside analog that has demonstrated synergistic cell killing with ionizing radiation (termed "radiosensitization") in a variety of solid tumor cells. The diphosphate derivative, dFdCDP, inhibits ribonucleotide reductase, which primarily produces a decrease in dATP levels in solid tumor cells, resulting in decreased DNA synthesis. The triphosphate derivative, dFdCTP, competes with dCTP for incorporation into DNA, which correlates with cytotoxicity, and inhibits DNA synthesis. Although the potent radiosensitizing ability of dFdCyd has been well documented, the mechanism has not been elucidated. This dissertation examines the roles of p53, mismatch repair (MMR), and mismatches in DNA on the mechanism of dFdCyd radiosensitization.; Radiosensitization with dFdCyd has correlated with dATP depletion and S-phase accumulation, without apparent alterations in DNA double strand breaks or their repair. The role of p53 and whether its expression would prevent S-phase accumulation due to a G1 block was examined in matched MCF-7 breast cancer cells with either wild-type or mutant p53. Both cell lines exhibited depleted dATP pools, accumulated in S-phase prior to irradiation, and were radiosensitized. Results demonstrated that radiosensitization was not prevented by p53.; To account for the strong correlation between dATP depletion and radiosensitization by dFdCyd, we hypothesized that DNA mismatches from the dATP pool imbalance lead to radiosensitization. By comparing cells that differed in their ability to repair DNA mismatches, we demonstrated that HCT116 MMR-deficient cells were better radiosensitized by dFdCyd than the corresponding HCT116 MMR-proficient cells.; Using an episomal supF gene as a marker in both MMR-deficient and proficient cells, we determined that dFdCyd and hydroxyurea produced increased nucleotide changes only at radiosensitizing concentrations. We hypothesize that the dFdCyd-induced imbalance in dNTP pools leads to errors in DNA replication, which are normally corrected by MMR. The data suggest that when the MMR capacity is exceeded, the nucleotide misincorporations caused by depleted dATP pools are the lesions responsible for dFdCyd radiosensitization. |
