Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Free Radic Biol Med. 2012 Nov 1;53(9):1680-8. doi: 10.1016/j.freeradbiomed.2012.08.579. Epub 2012 Aug 27.
Cigarette smoking can contribute to the development of many human diseases such as cardiovascular disease, lung cancer, asthma, and chronic obstructive pulmonary disease. Thousands of compounds are present in cigarette smoke, including a large number of reactive oxygen species that can cause DNA damage, leading to the activation of poly(ADP-ribose) polymerase (PARP) enzymes. The PAR polymer is degraded by poly(ADP-ribose) glycohydrolase (PARG). Here we have investigated the effects of cigarette smoke extract (CSE) on A549 human lung epithelial cells. CSE induced DNA damage (comet assay), PAR accumulation (immunofluorescence and immunoblotting), impaired proliferation (clonogenic survival assay and electric cell-substrate impedance sensing measurement), and cell death (MTT reduction, propidium iodide uptake, lactate dehydrogenase release). CSE-induced cell death was also characterized by mitochondrial depolarization but massive translocation of apoptosis-inducing factor could not be observed. To investigate the role of PARylation in CSE-induced oxidative stress, PARP-1- and PARG-silenced A549 cells were used. Silencing of both PARP-1 and PARG sensitized cells to CSE-induced toxicity: PARP-1- and PARG-silenced cell lines exhibited reduced clonogenic survival, displayed a delayed repair of DNA breaks, and showed higher levels of cytotoxicity. CSE triggered the production of mitochondrial superoxide and hydrogen peroxide. Addition of superoxide dismutase increased, whereas catalase abolished, CSE-induced PAR formation. In summary, our data show that the superoxide-hydrogen peroxide-DNA breakage pathway activates the PAR cycle by PARP-1 and PARG, which serves as a survival mechanism in CSE-exposed cells. Our data also raise the possibility that the PARP-1/PARG status of smokers may be an important determinant of the efficiency of DNA repair in their lungs and of their susceptibility to CS-induced carcinogenesis.
吸烟会导致许多人类疾病的发生，如心血管疾病、肺癌、哮喘和慢性阻塞性肺疾病。香烟烟雾中存在数千种化合物，包括大量的活性氧物质，这些物质会导致 DNA 损伤，从而激活聚（ADP-核糖）聚合酶（PARP）酶。PAR 聚合物被聚（ADP-核糖）糖水解酶（PARG）降解。在这里，我们研究了香烟烟雾提取物（CSE）对 A549 人肺上皮细胞的影响。CSE 诱导 DNA 损伤（彗星试验）、PAR 积累（免疫荧光和免疫印迹）、增殖受损（集落形成存活试验和电动细胞-底物阻抗感应测量）和细胞死亡（MTT 减少、碘化丙啶摄取、乳酸脱氢酶释放）。CSE 诱导的细胞死亡还表现为线粒体去极化，但未观察到凋亡诱导因子的大量易位。为了研究 PAR 化在 CSE 诱导的氧化应激中的作用，我们使用了 PARP-1 和 PARG 沉默的 A549 细胞。PARP-1 和 PARG 的沉默使细胞对 CSE 诱导的毒性敏感：PARP-1 和 PARG 沉默的细胞系显示出较低的集落形成存活率，显示出 DNA 断裂修复延迟，并表现出更高水平的细胞毒性。CSE 触发了线粒体超氧阴离子和过氧化氢的产生。添加超氧化物歧化酶增加，而添加过氧化氢酶则消除了 CSE 诱导的 PAR 形成。总之，我们的数据表明，超氧阴离子-过氧化氢-DNA 断裂途径通过 PARP-1 和 PARG 激活 PAR 循环，这是 CSE 暴露细胞的一种存活机制。我们的数据还提出了一种可能性，即吸烟者的 PARP-1/PARG 状态可能是其肺部 DNA 修复效率和对 CS 诱导致癌作用易感性的重要决定因素。
