Received for publication November 12, 2007.
Revised June 5, 2008.
Accepted for publication June 5, 2008.

MLH1 Deficiency Enhances Radiosensitization with 5-Fluorodeoxyuridine by Increasing DNA Mismatches

Abstract

The antitumor drug 5-fluoro-2'-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. While 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 which, if not repaired prior to irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells which lack the expression of the required MMR protein MLH1 (inactive MLH1), and MMR-proficient (wildtype MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC₅₀= 3.5 µM) versus HCT116 1-2 cells (IC₅₀ = 0.75 µM), when irradiation followed FdUrd (IC₅₀) the MLH1-inactivated cells exhibited greater radiosensitization compared to MMR- wildtype cells (radiation enhancement ratio (RER) = 1.8 ± 0.28 vs. 1.1 ± 0.1, respectively) and an increase ( 8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wildtype cells, FdUrd (IC₅₀) did not produce radiosensitization nor did it increase the mutation frequency, but following shRNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 ± 0.10, and 1.5 ± 0.06) and an increase in nucleotide misincorporations (8-fold and 6-fold respectively). Incubation with higher concentrations of FdUrd (IC₉₀) after suppression of MLH1 produced a further increase in IR sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 ± 0.03, and RER = 1.7 ± 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.

Key words: Mechanisms of cell killing/apoptosis, Nucleoside/Nucleotide derivatives, Oncogenes