dUTPase and uracil-DNA glycosylase are central modulators of antifolate toxicity in Saccharomyces cerevisiae.
dUTPase and uracil-DNA glycosylase are central modulators of antifolate toxicity in Saccharomyces cerevisiae.
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发表时间：
2002-09
期刊：
影响因子：
11.2
通讯作者：
Beverly A Tinkelenberg;M. Hansbury;R. Ladner
中科院分区：
文献类型：
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作者：
Beverly A Tinkelenberg;M. Hansbury;R. Ladner
胸苷酸合成酶反应仍然是广泛使用的抗癌药物的重要靶点;然而，这些药物的临床应用受到细胞耐药性发生的限制。尽管有大量关于药物作用机制的信息，但导致致死性的下游事件的相对重要性仍不清楚。在这项研究中，我们已经开发了一个模型系统，使用芽殖酵母酿酒酵母解剖dUMP错误掺入DNA的影响，作为抗叶酸剂诱导的细胞毒性的贡献机制。尿嘧啶-DNA代谢中的关键酶dUTR和尿嘧啶-DNA糖基化酶的活性被减弱或增强，并分析了操纵菌株的毒性生化终点。过表达dUTP的细胞通过其防止dUTP池扩增的能力而免受细胞毒性，并且能够从早期S期检查点停滞中恢复。相反，dUTR活性的耗尽导致dUTP池的积累和对抗叶酸剂的敏感性增强。这些细胞也被阻滞在早期S期，停药后无法完成DNA复制，导致死亡。尿嘧啶碱基切除修复的失活诱导了对早期细胞毒性的部分抗性（10 h内）;然而，最终在较晚的时间点（12-24 h）导致了致死性，可能是由于稳定的尿嘧啶错误掺入的有害影响。虽然这些细胞能够用尿嘧啶取代的DNA完成复制，但它们在G（2）-M期停滞。这一发现可能代表了一种新的机制，通过该机制，尿嘧啶取代的DNA的存在发出G（2）-M检查点的信号。总之，这些数据提供了遗传和生化证据，证明酵母中叶酸拮抗剂的致死性主要取决于尿嘧啶错误掺入DNA，与dTTP耗竭相关的尿嘧啶非依赖性机制起次要作用。我们的研究结果表明，dUTR和尿嘧啶-DNA糖基化酶的相对表达水平可以有很大的影响胸苷酸转移酶导向化疗的疗效，从而提高这些蛋白质作为预后标志物和治疗发展的替代目标的候选资格。
The thymidylate synthase reaction remains an important target for widely used anticancer agents; however, the clinical utility of these drugs is limited by the occurrence of cellular resistance. Despite the considerable amount of information available regarding mechanisms of drug action, the relative significance of downstream events that result in lethality remains unclear. In this study, we have developed a model system using the budding yeast Saccharomyces cerevisiae to dissect the influence of dUMP misincorporation into DNA as a contributing mechanism of cytotoxicity induced by antifolate agents. The activities of dUTPase and uracil-DNA glycosylase, key enzymes in uracil-DNA metabolism, were diminished or augmented, and the manipulated strains were analyzed for biochemical endpoints of toxicity. Cells overexpressing dUTPase were protected from cytotoxicity by their ability to prevent dUTP pool expansion and were able to recover from an early S-phase checkpoint arrest. In contrast, depletion of dUTPase activity leads to the accumulation of dUTP pools and enhanced sensitivity to antifolates. These cells were also arrested in early S-phase and were unable to complete DNA replication after drug withdrawal, resulting in lethality. Inactivation of uracil base excision repair induced partial resistance to early cytotoxicity (within 10 h); however, lethality ultimately resulted at later time points (12-24 h), presumably because of the detrimental effects of stable uracil misincorporation. Although these cells were able to complete replication with uracil-substituted DNA, they arrested at the G(2)-M phase. This finding may represent a novel mechanism by which the G(2)-M checkpoint is signaled by the presence of uracil-substituted DNA. Together these data provide both genetic and biochemical evidence demonstrating that lethality from antifolates in yeast is primarily dependent on uracil misincorporation into DNA, and that uracil-independent mechanisms associated with dTTP depletion play a minor role. Our findings indicate that the relative expression levels of both dUTPase and uracil-DNA glycosylase can have great influence over the efficacy of thymidylate synthase-directed chemotherapy, thereby enhancing the candidacy of these proteins as prognostic markers and alternative targets for therapeutic development.