Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA.
Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA.
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DOI：
10.1021/bi102046h
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发表时间：
2011-02-08
期刊：
影响因子：
2.9
通讯作者：
Stivers JT
中科院分区：
文献类型：
--
作者：
Grogan BC;Parker JB;Guminski AF;Stivers JT
5-氟尿嘧啶（5-FU）、5-氟脱氧尿苷（5-dUrd）和雷替曲塞（RTX）是靶向胸苷酸合成酶（TS）从而阻断dUMP转化为dTMP的抗癌剂。在芽殖酵母中，5-FU促进dUMP/dTMP比率大幅增加，导致尿嘧啶（U）大量聚合酶催化掺入基因组DNA中，5-FU在较小程度上促进dUMP/dTMP比率大幅增加，两者均被酵母尿嘧啶DNA糖基化酶（UNG）切除，导致DNA片段化和细胞死亡。相比之下，5-FU和RTX在人和小鼠细胞系中的毒性不涉及UNG，而是涉及可以切除尿嘧啶衍生物的其他DNA糖基化酶。为了阐明酵母和人类细胞中这些不同结果的基础，我们研究了这些药物如何干扰细胞dUTP和TTP池水平以及三种人类DNA糖基化酶（hUNG 2，hSMUG 1和hTDG）切除各种TS药物诱导的DNA损伤的相对能力。我们发现，当在标准培养基中生长时，5-dUrd仅适度增加异步MEF、HeLa和HT-29人类细胞系中的dUTP/dTTP池水平。相反，用5-dUrd或RTX处理鸡DT 40 B细胞导致dUTP/TTP比率大幅增加。令人惊讶的是，即使UNG是DT 40细胞中唯一可以作用于来自dUTP掺入的U/A碱基对的DNA糖基化酶，但与对照细胞相比，同基因UNG−/− DT 40细胞系对RTX的敏感性几乎没有变化。纯化的人酶的体外动力学分析表明，hUNG 2是切除5-FU和U的最强大的催化剂，无论它是否与A，G或存在于ssDNA中的碱基对中。与体外活性测定完全一致，从人和鸡细胞培养物中分离的核提取物显示，hUNG 2对U和5-FU的清除具有压倒性活性，尽管其在这些细胞系中的药物毒性方面处于旁观者状态。TS抑制相对于核苷酸池水平，所产生的DNA损伤的性质，和DNA修复反应的不同结果进行了讨论。
5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd) and raltitrixed (RTX), are anticancer agents that target thymidylate synthase (TS) thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG) leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated the how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1 and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP/dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth is in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U/A base pairs derived from dUTP incorporation, an isogenic ung−/− DT40 cell line showed little change its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A, G or present in ssDNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, nature of the resulting DNA lesion, and the DNA repair response are discussed.
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