Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208, United States.
Biochem Pharmacol. 2012 Mar 15;83(6):741-6. doi: 10.1016/j.bcp.2011.12.022. Epub 2011 Dec 24.
There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (∼6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.
有证据表明，RAD51 的表达与对常用化疗药物的耐药性有关。我们之前的工作表明，胸苷酸合成酶 (TS) 的抑制剂诱导 RAD51 依赖性同源重组 (HR)，并且耗尽 RAD51 重组酶使细胞对 TS 抑制剂敏感。在这项研究中，研究了 RAD51 过表达的后果。野生型 RAD51 的过表达（比内源性 RAD51 高约 6 倍）赋予对 TS 抑制剂的抗性。相比之下，RAD51 突变体（T309A）的过表达使细胞对化疗药物更敏感，因为该突变体不能被磷酸化。此外，RAD51 突变体过表达以显性负性方式作用于内源性 RAD51，导致在用 TS 抑制剂处理后 RAD51 焦点的定位减少。为了测量突变型 RAD51 对其他 DNA 损伤化疗药物的细胞反应的影响，使用拓扑异构酶抑制剂依托泊苷。过表达野生型 RAD51 的细胞显示 DNA 链断裂减少，而过表达突变型 RAD51 的细胞则显示出两倍以上的链断裂，这表明突变型 RAD51 积极抑制链断裂的解决。为了直接证明对 HR 的影响，将野生型 RAD51 和 T309A 突变型 RAD51 瞬时表达在含有 HR 报告构建体的 HeLa 细胞中。由 I-SceI 内切酶诱导的 DNA 断裂引发的 HR 事件在表达野生型 RAD51 的细胞中增加，在表达 T309A 突变型 RAD51 的细胞中减少。总之，这些数据表明，干扰 RAD51 介导的 HR 的激活代表了联合治疗的潜在有用的抗癌靶标。
