Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a nucleoside analogue and a potent radiosensitizer in tumor cells in vitro and in vivo. The active metabolite dFdCDP inhibits ribonucleotide reductase, decreasing synthesis of dNTPs for DNA synthesis. This inhibition primarily results in a decrease in dATP and this depletion is strongly correlated to radiosensitization. We hypothesize that radiosensitization by dFdCyd is the result of incorrect nucleotide incorporation into DNA, as a result of DNA replication in the presence of imbalanced dNTP pools, which, if not repaired, augments cell death following irradiation. This hypothesis was supported by the finding that mismatch repair (MMR)- deficient HCT116 cells, which lack hMLH1, were radiosensitized by dFdCyd at ≤IC50 while the isogenic MMR-proficient cell line, HCT116 1-2, which contains full-length wild-type hMLH1 cDNA to correct the hMLH1 deficiency, was not. The plasmid shuttle vector pSP189, which encodes an amber suppressor tRNA sequence (supF gene) to allow expression of beta-galactosidase in MBM7070 E. coli cells, was used to directly examine the ability of dFdCyd to produce nucleotide misincorporation by measuring plasmid mutation rates in the MMR-deficient cell line compared to the MMR-proficient cell line. In the absence of drug, mutation rates were similar between cell lines. Following dFdCyd treatment at concentrations ≤ IC50 only the MMR- deficient cell line showed significant nucleotide misincorporation with a ≥ 2-fold increase in mutation rate (p-value ≤ 0.01). Similar to MMR-proficient HCT116 1-2 cells, D-54 glioblastoma cells cannot be radiosensitized with dFdCyd at concentrations ≤ IC50. However, when HCT116 1-2 cells or D-54 cells were incubated with the IC96 for dFdCyd, plasmid mutation rates were ≥ 3-fold greater than in the control condition (p-value ≤ 0.01). This concentration of dFdCyd also produced excellent radiosensitization in both cell lines (radiation enhancement ratio = 1.5 ± 0.1). To determine whether mismatched nucleotides were repaired after irradiation, plasmid mutation rates were determined in MMR-deficient HCT116 cells for 4 days following dFdCyd treatment at ≤ IC50, 5 Gy radiation, and ≤ IC50 dFdCyd followed by 5 Gy radiation. Significant incorrect nucleotide incorporation was observed at 24 hr following treatment with a ≥ 2-fold increase in plasmid mutation rate for each condition. At 72 hr the increase in mutations persisted only in the group that received both dFdCyd and radiation where a frequency of 0.20 ± 0.02 % was determined compared to 0.04 ± 0.01 % for the control group. Studies in HCT116 and D-54 cells indicate that only concentrations of dFdCyd that produce radiosensitization result in mismatched nucleotides in DNA. Furthermore, these data strongly suggest that the presence and persistence of the mismatched nucleotide lesion is integral to radiosensitization by dFdCyd.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]