Zhang Yong-Wei, Jones Tamara L, Martin Scott E, Caplen Natasha J, Pommier Yves
Laboratory of Molecular Pharmacology, Genetics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2009 Jul 3;284(27):18085-95. doi: 10.1074/jbc.M109.003020. Epub 2009 May 5.
To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we conducted synthetic small interfering RNA (siRNA)-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. Silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase complex, respectively, markedly enhanced the cytotoxicity of the topoisomerase I inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by histone gamma-H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels and induces the nuclear translocation of RRM2. The checkpoint kinase 1 (Chk1) was up-regulated and activated in response to CPT, and CHEK1 down-regulation by siRNA and small molecule inhibitors of Chk1 blocked RRM2 induction by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs and DNA repair. Specifically, we propose that upon DNA damage, Chk1 activation, mediated by ATM and ATR, up-regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 expression levels coupled with its nuclear recruitment suggests an active role for ribonucleotide reductase in the cellular response to CPT-mediated DNA damage that could potentially be exploited as a strategy for enhancing the efficacy of topoisomerase I inhibitors.
为了研究药物作用机制并确定合理药物组合开发的分子靶点，我们进行了基于合成小干扰RNA（siRNA）的RNA干扰筛选，以鉴定其沉默会影响抗癌药物反应的基因。分别编码人核糖核苷酸还原酶复合物大亚基和小亚基的RRM1和RRM2的沉默，显著增强了拓扑异构酶I抑制剂喜树碱（CPT）的细胞毒性。还发现RRM2的沉默会增强通过组蛋白γ-H2AX测量的DNA损伤。进一步研究表明，CPT上调RRM1和RRM2的mRNA和蛋白水平，并诱导RRM2的核转位。检查点激酶1（Chk1）在CPT作用下被上调并激活，通过siRNA下调CHEK1以及使用Chk1小分子抑制剂可阻断CPT诱导的RRM2上调。CHEK1 siRNA也抑制了CPT诱导的E2F1上调，而E2F1的沉默则抑制了RRM2的上调。ATR或ATM的沉默以及KU-55933对ATM活性的抑制阻断了Chk1激活和RRM2上调。本研究将CPT诱导的DNA损伤反应的已知成分与dNTP合成和DNA修复所需的蛋白质联系起来。具体而言，我们提出在DNA损伤时，由ATM和ATR介导的Chk1激活通过E2F1转录因子上调RRM2表达。RRM2表达水平的上调及其核募集表明核糖核苷酸还原酶在细胞对CPT介导的DNA损伤的反应中发挥积极作用，这有可能被用作提高拓扑异构酶I抑制剂疗效的策略。
