PO.MCB09.06 · 分子与细胞生物学
线粒体动力学的破坏改变结肠癌的DNA修复能力
Disruption of mitochondrial dynamics alters DNA repair capacity in colon cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
线粒体动力学是指一系列线粒体运动，包括分裂、融合和运输。已知癌细胞会调整线粒体动力学，以提供细胞生长、增殖和迁移所需的代谢可塑性。我们先前已表明，线粒体分裂的关键调节因子Drp1在介导脂肪酸诱导的Wnt/beta-catenin信号激活中在结肠癌里发挥重要作用。在本研究中，我们确定了线粒体动力学与结肠癌细胞中DNA损伤反应之间的功能性相互作用。为破坏线粒体分裂，使用慢病毒介导的RNAi构建了诱导型Drp1敲低结肠癌细胞系。对照细胞和Drp1敲低细胞用放射线或化疗药物伊立替康处理以诱导DNA损伤反应。通过Western blot和RT-qPCR分析DNA损伤程度以及DNA修复信号的激活。我们发现Drp1在伊立替康或放射线处理后被激活，表现为S616位点磷酸化增加。有趣的是，Drp1敲低细胞中由伊立替康或放射线处理诱导的DNA损伤标志物gammaH2AX的表达增加，而DNA损伤感应蛋白（如ATM和ATR）的表达和激活保持不变。为确定脂肪酸摄取增加是否会改变DNA损伤，我们表明外源性脂肪酸的存在减弱了对照细胞中放射线诱导的gammaH2AX表达，而Drp1的敲低则削弱了这一反应。在功能上，通过集落形成试验测定，添加脂肪酸增加了放射后细胞存活，而沉默Drp1则降低了细胞存活。然而，脂肪酸的促存活作用在Drp1敲低细胞中基本被消除。综上所述，我们的结果提示脂肪酸摄取可能通过线粒体分裂依赖性机制增强DNA修复。本研究在结肠癌细胞中建立了脂肪酸代谢、线粒体动力学与DNA损伤反应之间的功能性联系。
查看英文原文 English abstract
Mitochondrial dynamics refers to a collection of mitochondrial movements, including fission, fusion, and transport. Cancer cells are known to adjust mitochondrial dynamics to provide the metabolic plasticity needed for cell growth, proliferation and migration. We have shown previously that Drp1, a key regulator of mitochondrial fission, plays an important role in mediating fatty acid-induced activation of Wnt/beta-catenin signaling in colon cancer. In this study, we determined the functional interaction between mitochondrial dynamics and DNA damage response in colon cancer cells. To disrupt mitochondrial fission, inducible Drp1 knockdown colon cancer cell lines were generated using lentivirus-mediated RNAi. Control and Drp1 knockdown cells were treated with radiation or chemotherapy drug irinotecan to induce DNA damage response. The extent of DNA damage as well as the activation of DNA repair signaling were analyzed by western blot and RT-qPCR. We found that Drp1 was activated upon irinotecan or radiation treatment as shown by increased phosphorylation at S616 site. Interestingly, the expression of gammaH2AX, a DNA damage marker, induced by irinotecan or radiation treatment was increased in Drp1 knockdown cells, whereas the expression and activation of DNA damage sensing proteins (e.g., ATM and ATR) remained unchanged. To determine if increased fatty acid uptake alters DNA damage, we showed that the presence of exogenous fatty acids attenuated gammaH2AX expression induced by radiation in control cells whereas knockdown of Drp1 blunted this response. Functionally, the addition of fatty acids increased, while silencing Drp1 decreased, cell survival post radiation as measured by colony formation assays. However, the pro-survival effect of fatty acids was largely abolished in Drp1 knockdown cells. Taken together, our results suggest that fatty acid uptake may enhance DNA repair via a mitochondrial fission-dependent mechanism. This study establishes a functional link between fatty acid metabolism, mitochondrial dynamics, and DNA damage response in colon cancer cells.
利益披露 Disclosure
M. D. Creech, None..
E. M. Wolf Horrell, None..
T. Gao, None.