Gemzar: Mech. of Cytotoxicity and Radiosensitization

格姆扎尔：机甲。

• 批准号: 6748578
• 负责人: DONNA S. SHEWACH
• 金额: $ 26.55万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-08 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6748578
• 关键词: DNA damage; DNA repair; apoptosis; cell cycle; cell line; combination cancer therapy; cytotoxicity; drug interactions; drug metabolism; flow cytometry; gemcitabine; gene mutation; ionizing radiation; neoplasm /cancer chemotherapy; neoplasm /cancer radiation therapy; neoplastic cell; p53 gene /protein; paclitaxel; plasmids; radiosensitizer

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.

描述（由申请人提供）： 以前我们假设并证明2 '，2'-二氟-2 '-脱氧胞苷（dFdCyd，吉西他滨）是一种有效的放射增敏剂。在当前的资助期间，我们证明了放射增敏与dFdCyd的5 '-二磷酸对核糖核苷酸还原酶的抑制密切相关，导致超过80%的dATP消耗，而5'-三磷酸主要对细胞毒性作用起作用。由dFdCyd放射致敏的细胞在药物暴露期间在放射敏感性较低的S期累积。 放射增敏不需要凋亡细胞死亡，它可以发生在p53野生型以及突变型p53细胞系。吉西他滨在照射前或照射后均未增加DNA双链断裂，也未抑制其修复。因此，累积的数据表明，dFdCyd的放射增敏作用是由照射前持续的DNA损伤的性质决定的，而不是由照射后对DNA损伤的反应决定的。我们现在假设dFdCyd放射增敏的关键病变是由耗尽的dATP引起的不正确核苷酸掺入DNA中，如果该病变在S期之前未修复，则照射将阻止错误掺入事件的修复，从而导致永久性突变和增强的细胞死亡。这一假说将预测，在修复错误掺入的核苷酸方面有缺陷的细胞，如错配修复缺陷细胞，将比在这一途径中有专长的细胞更好地放射增敏。初步数据表明，错配修复缺陷的HCT-116细胞是放射增敏的dFdCyd，而错配修复熟练的对应物不是，虽然它是更敏感的细胞毒性与dFdCyd。我们建议评估的因素，确定dFdCyd的细胞毒性和放射敏感性的同基因细胞系缺陷或精通错配修复。使用穿梭载体测定，我们将确定是否假设的放射增敏病变，错误掺入的核苷酸，是放射增敏的dFdCyd的细胞中更普遍。除了定义dFdCyd的放射增敏机制外，我们还将根据最近在患者中报告的高抗肿瘤疗效，确定其与多西他赛联合使用时的协同作用机制。初步数据表明，这种协同作用与多西他赛阻断G2/M期细胞的能力无关，而是我们假设多西他赛使dFdCyd处理的细胞发生S期特异性细胞死亡。