Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression
Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression
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DOI：
10.1371/journal.pone.0047318
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发表时间：
2012-10-09
期刊：
影响因子：
3.7
通讯作者：
Myllykallio, Hannu
中科院分区：
文献类型：
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作者：
Ligabue, Alessio;Marverti, Gaetano;Myllykallio, Hannu
背景：5-氟尿嘧啶，一种常用的化疗药物，上调人胸苷酸合成酶（hTS）的表达。已经提出了几种不同的调节机制来介导不同细胞系中的这种上调，但迄今为止尚未研究它们在单个细胞系中的具体贡献。我们已经建立了这些先前提出的监管机制在卵巢癌细胞系2008和相应的顺铂耐药和5-FU交叉耐药的亚系C13* 的相对贡献。方法/主要发现：使用RNA聚合酶II抑制剂DRB处理的细胞培养物，我们表明，70-80%的上调的HTS结果从TYMS mRNA的转录激活。此外，我们报告说，5-FU通过阻断2008和C13* 细胞系的S期而损害细胞周期。由于先前的工作已经确定TYMS mRNA在S和G（1）期合成，并且hTS在S和G（2）-M期定位于细胞核中，因此观察到的细胞周期变化也预期影响hTS的细胞内调节。我们的数据还表明hTS催化活性的抑制和hTS蛋白水平的上调没有因果关系，因为在5-FU暴露后3小时已经检测到由hTS、脱氧尿苷一磷酸和亚甲基四氢叶酸形成的失活三元复合物，而仅在24小时后才检测到总体TS水平的大幅增加。总之，我们的数据表明，组成型TYMS mRNA转录，细胞周期诱导的hTS调节和hTS酶的稳定性是负责5-氟尿嘧啶诱导的上调人胸苷酸合成酶表达在两个卵巢癌细胞系研究的三个关键机制。由于这三种独立的调节现象以精确的顺序发生，我们的工作为早期观察到的5-FU与其他药物的协同组合提供了可行的理论基础，并可能提出新的治疗策略。
Background: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*.Methodology/Principal Findings: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G(1) phase and hTS is localized in the nuclei during S and G(2)-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours.Conclusions/Significance: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies.