Radiation sensitization by 2 ¶,2 ¶-difluoro-2 ¶-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDPmediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2-to 3-fold increase; P V 0.01) after radiosensitizing concentrations of dFdCyd F 5 Gy radiation, which persisted for at least 96 h. dFdCyd (10 nmol/L) did not radiosensitize MMR-proficient HCT116 or A549 cells, but following small interfering RNA -mediated suppression of hMLH1, this concentration produced excellent radiosensitization (radiation enhancement ratios = 1.6 F 0.1 and 1.5 F 0.1, respectively; P < 0.05) and a 2.5-fold increase in mutation frequency in A549 cells. Cytosine arabinoside (1-B-D-arabinofuranosylcytosine), which can be incorporated into DNA but does not inhibit RR, failed to radiosensitize MMR-deficient cells or increase mutation frequency in the MMR-deficient and MMR-proficient cells. However, the RR inhibitor hydroxyurea radiosensitized MMR-deficient cells and increased nucleotide misincorporations (z5-fold increase; P < 0.05), thus further implicating the inhibition of RR as the mechanism underlying radiosensitization by dFdCyd. These data showed that the presence and persistence of mismatched nucleotides is integral to radiosensitization by dFdCyd and suggest a role for hMLH1 deficiency in eliciting the radiosensitizing effect.
The antitumor drug 5-fluoro-2Ј-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC 50 ϭ 3.5 M) versus HCT116 1-2 cells (IC 50 ϭ 0.75 M), when irradiation followed FdUrd (IC 50 ) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) ϭ 1.8 Ϯ 0.28 versus 1.1 Ϯ 0.1, respectively] and an increase (Ն8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wildtype cells, FdUrd (IC 50 ) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER ϭ 1.6 Ϯ 0.10 and 1.5 Ϯ 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC 90 ) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER ϭ 1.8 Ϯ 0.03 and 1.7 Ϯ 0.13, respectively) and nucleotide misincorporations (Ͼ10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.
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