Studies directed toward the mechanism of radiosensitization by 2',2'-difluoro-2'-deoxycytidine (gemcitabine).

Abstract

2<super>'</super>,2<super>'</super>-Difluoro-2<super>' </super>-deoxycytidine (dFdCyd) is a nucleoside analog that has shown clinical activity against a variety of solid tumor types. There are two pathways through which dFdCyd can exert cytotoxicity: (1) the diphosphate derivative, dFdCDP, inhibits ribonucleotide reductase resulting primarily in the depletion of dATP in solid tumor cells; and (2) the triphosphate derivative, dFdCTP, can be incorporated into DNA and interfere with DNA replication. In addition to its inherent cytotoxicity, when combined with ionizing radiation, dFdCyd produces a synergistic effect in cell killing. This dissertation examines the roles of dATP depletion and cell cycle distribution in the mechanism of dFdCyd mediated radiosensitization. In order to study the mechanism of dFdCyd mediated radiosensitization, U251 human glioblastoma cells, which express a mutant p53 and were readily radiosensitized, were compared to D54 human glioblastoma cells, which express wild-type p53 and could not be radiosensitized. Previous studies correlated radiosensitization by dFdCyd with its ability to deplete dATP. The lack of radiosensitization of D54 cells was not due to a lack of dATP depletion. dATP depletion alone was not sufficient to induce radiosensitization; however, dATP depletion did parallel DNA synthesis inhibition. dATP depletion and the subsequent inhibition of DNA synthesis did not contribute significantly to cytotoxicity. Other studies have demonstrated that S-phase cells are more susceptible to radiosensitization by dFdCyd. Before examining the role of the cell cycle in radiosensitization by dFdCyd, an extensive review of dual parameter flow cytometry measuring BrdUrd incorporation and DNA content was performed. Both acid/heat and DNase denaturation of DNA were equally effective; however, indirect labeling of BrdUrd was more sensitive than direct labeling. Following dFdCyd treatment, U251 cells readily accumulated in S-phase, whereas D54 cells did not. However, treatment of D54 cells with hydroxyurea resulted in S-phase accumulation without subsequent radiosensitization. Removal of p53 controlled cell cycle checkpoints by the introduction of HPV-16 E6 into D54 cells promoted S-phase accumulation following dFdCyd treatment and increased the incidence radiosensitization. These studies suggest that dATP depletion and S-phase accumulation are not sufficient for, but may induce the critical DNA lesion of radiosensitization by dFdCyd.