Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways
Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways
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DOI：
10.1074/jbc.ra120.013726
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发表时间：
2020-09-11
影响因子：
4.8
通讯作者：
Kirita, Tadaaki
中科院分区：
文献类型：
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作者：
Ito, Soichiro S.;Nakagawa, Yosuke;Kirita, Tadaaki
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被认为抑制胸苷酸合成酶，该酶在核苷酸合成中起作用，并已发现诱导单链和双链DNA断裂。ATR Ser/Thr激酶（ATR）是DNA损伤反应中的主要激酶，并且响应于UV和化疗药物诱导的DNA复制应激而被激活，但其在细胞对5-FU的反应中的作用尚不清楚。在这项研究中，我们研究了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的磷酸化在自磷酸化位点Thr-1989，从而激活其激酶。与5-FU与其他主要DNA修复激酶抑制剂联合治疗相比，5-FU与特异性ATR抑制剂联合治疗显著降低了细胞存活率。值得注意的是，ATR抑制增强了5-FU处理的细胞中DNA双链断裂和凋亡的诱导。利用基因表达分析，我们发现5-FU诱导了S细胞周期内检查点的激活。BRCA 2缺失的细胞在ATR抑制剂存在下对5-FU敏感。此外，ATR抑制增强了5-FU治疗的疗效，独立于非同源末端连接和同源重组修复途径。这些结果表明，ATR可能是一个潜在的治疗目标，在5-FU为基础的化疗。
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 lackingBRCA2were 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.