PARP－1在甲醛诱导的支气管上皮细胞DNA损伤修复及凋亡中的作用

Role of PARP-1 in formaldehyde-induced DNA damage repair and apoptosis in human bronchial epithelial ceils

目的探讨多聚腺苷二磷酸核糖基聚合酶-1（poly（ADP-ribose）polymerase-1，PARP-1）蛋白在不同浓度甲醛诱导的支气管上皮（HBE）细胞DNA损伤反应中的作用，探索甲醛致癌的机制。方法用免疫印记（Westernblot）法检测蛋白的表达；用免疫共沉淀法检测不同蛋白间的相互结合；采用化学抑制剂验证PARP-1蛋白与DNA损伤修复的关系。结果细胞毒性检测结果显示，HBE细胞经不同浓度甲醛染毒4h后，各浓度组与对照组相比细胞存活率没有显著差异，染毒24h后，80和160μmol／L浓度组与对照组相比细胞存活率差异显著（P〈0．05）。10μmol／L甲醛可诱导PARP-1和XRCC-1蛋白水平显著增高。80μmol／L甲醛可诱导124KD分子量的PARP－1蛋白水平显著减少，而89KD分子量的PARP-1蛋白水平显著增加，XRCC-1蛋白水平无明显改变。免疫共沉淀结果显示10μmol／L甲醛可引起PARP-1和XRCC-1蛋白结合增加，而80μmol／L甲醛染毒后PARP-1和XRCC-1蛋白结合没有明显改变。彗星试验结果显示10μmol／L甲醛染毒4h可以诱导HBE细胞尾部DNA百分含量显著增高，撤除甲醛2h后HBE细胞尾部DNA百分含量与对照组相比没有显著差异。抑制PARP-1后，与HBE组相比，甲醛诱导的尾部DNA百分含量显著增高，DNA修复能力明显减低。结论PARP—1通过招募XRCC-1蛋白到DNA损伤点来介导低浓度甲醛诱导的DNA损伤的修复，并可能参与调控高浓度甲醛诱导的细胞凋亡。

Objective To study the role of poly （ADP-ribose） polymerase-1 （PARP-1） in formaldehyde- induced DNA damage response in human bronchial epithelial （HBE） cells and to investigate the mechanism of formaldehyde carcinogenicity. Methods The protein levels were measured by Western blot. The interaction between different proteins was determined by co-immunoprecipitation assay. The chemical inhibitor was used to confirm the relationship between PARP-1 and DNA damage repair. Results After being exposed to different concentrations of formaldehyde for 4 h, HBE cells showed no significant changes in cell viability. Cell viability was significantly reduced after 24-h exposure to 80 and 160 μmol/L formaldehyde （P〈0.05）. The 10 μmol/L formaldehyde resulted in significant increases in the protein levels of PARP-1 and XRCC-1. However, 80 μmol/ L formaldehyde led to a significant decrease in the protein level of PARP-1 of 124 KD molecular weight but a significant increase in the protein level of PARP-1 of 89 KD molecular weight; there was no significant change in the protein level of XRCC-1. The co-immunoprecipitation assay showed that 10 μmol/L formaldehyde induced increased binding between PARP-1 and XRCC-1, but 80 μmol/L formaldehyde led to no significant change in binding between PARP-1 and XRCC-1. Here, we confirmed the role of 10 μmol/L formaldehyde in strand breaks by comet assay which showed an increase in the tail DNA content of HBE cells after 4-h formaldehyde exposure. No significant difference was observed in tail DNA content between treated HBE cells and control cells at 2 h after formaldehyde was removed. Moreover, compared with control, inhibition of PARP-1 induced a significant increase in tail DNA content, and a significant difference was observed in tail DNA content between inhibited HBE cells and control cells at 2 h after formaldehyde was removed. Inhibition of PARP-1 significantly reduced DNA repair capacity. Conclusion PARP-1 mediated the repair of DNA damage induced by low-concentration formaldehyde through recruiting XRCC-1 protein, and may be involved in the regulation of cell apoptosis induced by high-concentration formaldehyde.