Gemzar: Mech. of Cytotoxicity and Radiosensitization

描述(由申请人提供)： 之前我们假设并证明了2‘，2’-二氟-2‘-脱氧胞苷(dFdCyd，吉西他滨)是一种有效的辐射增敏剂。在当前的授权期内，我们证明了放射增敏与dFdCyd的5‘-二磷酸抑制核苷酸还原酶密切相关，导致dATP的耗竭超过80%，而5’-三磷酸主要起到细胞毒作用。经dFdCyd辐射增敏的细胞在药物暴露过程中积累在辐射不敏感的S时相。放射增敏不需要细胞凋亡性死亡，野生型和突变型p53细胞均可发生放射增敏。吉西他滨在照射前或照射后不会增加DNA双链断裂，也不会抑制其修复。因此，积累的数据表明，dFdCyd的辐射增敏作用取决于辐射前DNA损伤的性质，而不是辐射后对DNA损伤的反应。我们现在假设，dFdCyd放射增敏的关键损伤是dATP耗尽引起的错误核苷酸在DNA中的掺入，如果这种损伤在S期之前没有修复，辐射将阻止误掺入事件的修复，从而导致永久性突变和细胞死亡增加。这一假说将预测，在修复错误结合的核苷酸方面存在缺陷的细胞，如错配修复缺陷细胞，将比精通这一途径的细胞对辐射更敏感。初步数据表明，错配修复缺陷的HCT-116细胞对dFdCyd具有放射敏感性，而错配修复熟练的细胞则不敏感，尽管它对dFdCyd的细胞毒性更敏感。我们建议评估决定dFdCyd细胞毒性和辐射敏感性的因素在错配修复缺陷或熟练的同基因细胞系中。使用穿梭载体分析，我们将确定假想的放射增敏损伤，错配的核苷酸，是否在dFdCyd辐射增敏的细胞中更普遍。除了确定dFdCyd的放射增敏机制外，我们还将根据最近在患者中报道的高抗肿瘤疗效，确定它与多西紫杉醇联合使用时的协同机制。初步数据表明，这种协同作用与多西紫杉醇阻断G2/M期细胞的能力无关，而是我们假设多西紫杉醇使dFdCyd处理的细胞发生S期特异性细胞死亡。

DESCRIPTION (provided by applicant): Previously we hypothesized and then proved that 2',2'-difluoro-2'-deoxycytidine (dFdCyd, gemcitabine) is a potent radiation sensitizer. During the current grant period we demonstrated that radiosensitization correlated strongly with inhibition of ribonucleotide reductase by the 5'-diphosphate of dFdCyd, resulting in greater than 80 percent depletion of dATP, whereas the 5'-triphosphate contributed primarily to the cytotoxic effect. Cells that were radiosensitized by dFdCyd accumulated in the less radiosensitive S-phase during drug exposure. Radiosensitization did not require apoptotic cell death, and it could occur in p53 wild-type as well as mutant p53 cell lines. Gemcitabine did not increase DNA double strand breaks prior to or following irradiation, nor did it inhibit their repair. Thus, the accumulated data suggest that radiosensitization with dFdCyd is determined by the nature of the DNA damage sustained prior to irradiation rather than the response to DNA damage after irradiation. We now hypothesize that the critical lesion for radiosensitization with dFdCyd is the incorporation in DNA of an incorrect nucleotide caused by the depleted dATP and, if this lesion is not repaired prior to S-phase, irradiation will prevent repair of the misincorporation events resulting in permanent mutation and enhanced cell death. This hypothesis would predict that cells defective in repairing misincorporated nucleotides, such as mismatch repair deficient cells, would be better radiosensitized than cells proficient in this pathway. Preliminary data indicate that mismatch repair deficient HCT-116 cells are radiosensitized by dFdCyd whereas the mismatch repair proficient counterpart was not, although it was more sensitive to cytotoxicity with dFdCyd. We propose to evaluate the factors that determine dFdCyd cytotoxicity and radiosensitivity in isogenic cell lines defective or proficient in mismatch repair. Using a shuttle vector assay, we will determine whether the hypothesized radiosensitizing lesions, misincorporated nucleotides, are more prevalent in cells that are radiosensitized by dFdCyd. In addition to defining the mechanism of radiosensitization for dFdCyd, we will also determine the mechanism of synergy when it is combined with docetaxel, based on high antitumor efficacy reported recently in patients. Preliminary data demonstrate that this synergy is not related to the ability of docetaxel to block cells in G2/M, but rather we hypothesize docetaxel commits dFdCyd-treated cells to S-phase specific cell death.