Defining DNA Double Strand Breaks and the Role of Homologous Recombination in the Mechanism of Radiosensitization by Gemcitabine.

Abstract

Gemcitabine (2’,2’-difluoro-2’-deoxycytidine; dFdCyd) is an analog of deoxycytidine used in the treatment of a variety of solid malignancies. When tumor cells in vitro are exposed to dFdCyd prior to ionizing radiation (IR), a synergistic enhancement in cell killing, termed radiosensitization (RS), is observed. While RS with gemcitabine is correlated with depletion of dATP, mediated by gemcitabine diphosphate inhibition of ribonucleotide reductase, and the consequent mismatched nucleotides in DNA, the effect of these events on DNA damage was unknown. This dissertation seeks to elucidate the mechanism underlying this synergy by examining the role of homologous recombination (HR) in DNA double strand break (DSB) formation and resolution.

Radiosensitization by other nucleoside analogs is attributed to increased DSBs, or inhibition in their repair. This dissertation shows that the pattern of initial DSB formation and resolution (0-4h post-IR) did not correlate with radiosensitization by gemcitabine (RS-dFdCyd), consistent with earlier reports, however, a second increase was observed at 24 and 48h time points that greatly exceeded the initial DSBs and positively correlated with RS-dFdCyd. I hypothesized that gemcitabine-mediated mismatches in DNA interrupted HR-mediated repair of the late DSBs observed with dFdCyd+IR. The data show that shRNA knockdown of XRCC3, a required HR protein, in human MCF-7 breast cancer cells, completely inhibited RS-dFdCyd. Furthermore, XRCC3 knockdown reduced formation of late DSBs as evidenced by gamma-H2AX foci and phosphorylated ATM evaluation. These data demonstrate for the first time that RS-dFdCyd produces late increases in DSBs, and formation of these DSBs requires HR. I suggest that HR is blocked by the persistent mismatches in DNA produced by gemcitabine. Thus, failed HR-mediated repair of late DSBs is crucial for RS-dFdCyd, suggesting that HR-deficient tumors will be most effectively treated by this approach.