The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV– and chemotherapeutic drug–induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU–treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU–based chemotherapy.

中文翻译：

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ATR 激酶的抑制增强了 5-FU 敏感性，独立于非同源末端连接和同源重组修复途径。

抗癌剂 5-氟尿嘧啶 (5-FU) 具有细胞毒性，常用于治疗各种癌症。5-FU 被认为可抑制胸苷酸合酶，胸苷酸合酶在核苷酸合成中发挥作用，并已被发现可诱导单链和双链 DNA 断裂。ATR Ser/Thr 激酶 (ATR) 是 DNA 损伤反应中的主要激酶，在紫外线和化疗药物诱导的 DNA 复制应激反应中被激活，但其在细胞对 5-FU 反应中的作用尚不清楚。在这项研究中，我们检查了 ATR 抑制对哺乳动物细胞 5-FU 敏感性的影响。使用免疫印迹，我们发现 5-FU 治疗剂量依赖性地诱导 ATR 在自身磷酸化位点 Thr-1989 的磷酸化，从而激活其激酶。5-FU 与特定的 ATR 抑制剂一起给药显着降低细胞存活率，与 5-FU 联合其他主要 DNA 修复激酶抑制剂治疗相比。值得注意的是，ATR 抑制增强了 5-FU 处理细胞中 DNA 双链断裂和细胞凋亡的诱导。使用基因表达分析，我们发现 5-FU 诱导了 S 内细胞周期检查点的激活。在 ATR 抑制剂存在的情况下，缺乏 BRCA2 的细胞对 5-FU 敏感。此外，ATR 抑制增强了 5-FU 治疗的功效，独立于非同源末端连接和同源重组修复途径。这些发现表明 ATR 可能是基于 5-FU 的化疗的潜在治疗靶点。使用基因表达分析，我们发现 5-FU 诱导了 S 内细胞周期检查点的激活。在 ATR 抑制剂存在的情况下，缺乏 BRCA2 的细胞对 5-FU 敏感。此外，ATR 抑制增强了 5-FU 治疗的功效，独立于非同源末端连接和同源重组修复途径。这些发现表明 ATR 可能是基于 5-FU 的化疗的潜在治疗靶点。使用基因表达分析，我们发现 5-FU 诱导了 S 内细胞周期检查点的激活。在 ATR 抑制剂存在的情况下，缺乏 BRCA2 的细胞对 5-FU 敏感。此外，ATR 抑制增强了 5-FU 治疗的功效，独立于非同源末端连接和同源重组修复途径。这些发现表明 ATR 可能是基于 5-FU 的化疗的潜在治疗靶点。