Department of Toxicology, School of Public Health, Central South University, 110 Xiangya Road, Changsha 410078, Hunan, China.
Toxicol Lett. 2012 Oct 17;214(2):209-17. doi: 10.1016/j.toxlet.2012.08.025. Epub 2012 Sep 5.
DNA polymerase eta (Polη), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Polη and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Polη in the response of L-02 cells to HQ, we employed RNA interference to silence Polη expression in L-02 cells and examined the susceptibility of these Polη-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Polη were determined by real-time PCR, and the protein expression levels of Polη and γ-H2AX were determined by Western blot, γ-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24h in vitro. The results showed that stable Polη-knockdown cells were successfully constructed and more than 80% inhibition of Polη expression was confirmed. The results also showed that down-regulation of Polη led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Polη-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Polη-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of γ-H2AX. In addition, knockdown of Polη resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Polη plays an important role in the response of L-02 cells to HQ-induced DNA damage.
DNA 聚合酶 eta（Polη）是 Xeroderma pigmentosum 变体基因的产物，是跨损伤 DNA 合成所必需的，在预防遗传毒性药物诱导的 DNA 损伤后基因组不稳定性方面发挥着关键作用。先前的研究表明，Polη 与对氢醌（HQ）诱导的毒性的易感性之间存在关联。为了进一步探讨 Polη 在 L-02 细胞对 HQ 反应中的作用，我们采用 RNA 干扰沉默 L-02 细胞中的 Polη 表达，并研究了这些 Polη 缺陷细胞对 HQ 毒性作用的敏感性。在这项研究中，通过 MTT 测定法确定细胞存活率，通过彗星试验测定 DNA 损伤，通过流式细胞术测定细胞凋亡和细胞周期分布，通过实时 PCR 测定 Polη 的 mRNA 表达水平，通过 Western blot 测定 Polη 和 γ-H2AX 的蛋白表达水平，并用共聚焦激光扫描荧光显微镜观察暴露于 HQ 后 24 小时细胞内 γ-H2AX 焦点的形成。结果表明，成功构建了稳定的 Polη 敲低细胞，并证实了 Polη 表达的抑制率超过 80%。结果还表明，下调 Polη 导致细胞增殖减少和对 HQ 诱导的细胞毒性的敏感性增加。与非特异性 siRNA 对照细胞相比，Polη 缺陷细胞对 HQ 的敏感性增加了 2 倍。此外，用 HQ 处理的 Polη 沉默的 L-02 细胞显示出 DNA 双链断裂水平升高，如橄榄尾矩所测，并且 DNA 损伤反应增强，如 γ-H2AX 的诱导所指示。此外，Polη 敲低导致 HQ 处理后细胞凋亡增强，S 期阻滞更为明显。总之，这些结果表明 Polη 在 L-02 细胞对 HQ 诱导的 DNA 损伤的反应中发挥重要作用。
