Abstract 1316: Late-occurring DNA damage is responsible for radiosensitization with gemcitabine

Gemcitabine (2’,2’-difluoro-2’-deoxycytidine; dFdCyd) has excellent activity in the treatment of a variety of solid malignancies. After transport into the cell, dFdCyd is phosphorylated to two active metabolites, dFdCDP and dFdCTP. Incorporation of dFdCTP into DNA correlates with cytotoxicity. dFdCDP is a mechanism-based inhibitor of ribonucleotide reductase (RR) primarily causing a depletion of dATP in solid tumor cells, which inhibits DNA synthesis and contributes to cell death. When tumor cells are exposed to dFdCyd prior to ionizing radiation (IR), a synergistic enhancement in cell killing, termed radiosensitization (RS), is observed. This RS is independent of analog incorporation into DNA, but correlates with dATP depletion and the resulting S-phase accumulation/inhibition of DNA synthesis. Evidence now demonstrates that dFdCDP-mediated dATP depletion results in misincorporation of deoxynucleotides into DNA, which occurs only under radiosensitizing conditions, and these mismatches persist for at least 96 hr post-IR. While we have identified mismatches in DNA as the initial lesions necessary for RS, the mechanism by which these mismatches result in RS is not clear. Evaluation of DNA damage by pulsed field gel electrophoresis demonstrated that dFdCyd did not increase the number of DNA double strand breaks (dsbs) prior to or combined with IR, nor did dFdCyd decrease the rate of dsb repair 4 hr after IR when the majority of the dsbs were repaired. In view of our findings that misincorporated deoxynucleotides persisted in DNA for >96 hr, we hypothesized that there was a disruption of DNA repair at late times following dFdCyd + IR. To test this hypothesis, MCF-7 breast cancer cells were treated with dFdCyd alone (24 hr) or with IR. We identified DNA dsbs using the sensitive technique of γ-H2AX immunostaining and measured the number of foci that formed for 72 hr following dFdCyd + IR. MCF-7 cells exhibited excellent RS with IC 10 , IC 50 and IC 90 dFdCyd (radiation enhancement ratios of 1.9, 1.7 and 2.7, respectively). Compared to controls, γ-H2AX foci increased 2 − 5-fold at 0.5 hr after a 24 hr incubation with dFdCyd or treatment with IR alone, but the combination of dFdCyd + IR did not produce a further increase in foci. Under all conditions, the foci resolved to near baseline levels by 4h after drug + IR. Interestingly, a second increase in γ-H2AX foci was observed 24 − 48 hr after drug alone treatment, which exceeded the level at 0.5 hr by 3 − 5-fold. Furthermore, the number of γ-H2AX foci observed at 24 − 48 hr after dFdCyd + IR was increased by a factor of >2-fold compared to dFdCyd alone. This second increase in foci was dFdCyd concentration dependent, did not occur with IR alone, and corresponded to the timing of persistent DNA mismatches that occurred only under radiosensitizing conditions. Taken together, these data demonstrate that mismatches in DNA result in a late disruption of repair of DNA damage, resulting in excellent RS with dFdCyd. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1316.