Apoptin诱导内源性DNA损伤的电离辐射增敏作用机制研究

γH2AX is a reactive indicator of DNA damage, we accidentally discovered in the experimental process that apoptin-transfected tumor cells also appeared γ-H2AX levels increased, even without ionizing radiation, suggesting that apoptin could induce cell endogenous DNA damage. The process of DNA replication in the S phase of mitosis. Replication fork is the basic structure of DNA replication. It affects genomic stability. Our study showed that Apoptin induced DNA damage occurs during S phase cells. Previous studies showed that inhibition of DNA replication forks turn into, leading to the DNA replication fork collapse, the cellular DNA damage accumulation, resulting in cell apoptosis. Thus we speculate that apoptin could by interfering with the DNA replication process, affect DNA replication fork stability, so that S phase cell endogenous DNA damage, activation of a series of related signaling pathway, and ultimately increase the cells to ionizing radiation sensitivity? This project intends to human cervical carcinoma cell line as the research object, application of recombinant lentivirus, comet assay and mass spectrometry analysis, RNA interference technology, study on apoptin on DNA replication process effect and explore its lead to the specific mechanisms of endogenous DNA damage, and screening activation key molecules involved in, and to explore its mechanism, to further improve the apoptin research of increasing the sensitivity of cells to ionizing radiation, provide a theoretical basis for the development of new ionizing radiation sensitizer.

γH2AX是直接反应DNA损伤的指标。我们发现表达Apoptin的肿瘤细胞未受电离辐射时也出现γH2AX升高，提示Apoptin可能自身引起细胞内源性DNA损伤。DNA 复制在有丝分裂S 期进行，复制叉是DNA 复制的基本结构，它影响基因组稳定性，进一步研究证实Apoptin导致DNA损伤在S期细胞中发生。既往报道Segall AM等合成两个携带DNA复制叉结合域的蛋白肽段，与DNA复制叉结合导致复制叉垮塌，细胞DNA损伤积累，发生细胞凋亡。而这些肽段结构与Apoptin具有相似之处。由此我们推测：Apoptin可能通过干扰DNA复制过程影响DNA复制叉稳定性，从而使S时相细胞发生内源性DNA损伤，激活一系列相关信号通路，最终增加细胞电离辐射敏感性？本课题拟探讨Apoptin导致细胞内源性DNA损伤的机制，完善Apoptin增加细胞电离辐射敏感性的研究，为开发新电离辐射增敏剂提供理论基础。

本课题研究了病毒蛋白Apoptin的体内外放射增敏效果及其作用机制。通过细胞水平体内实验及裸鼠移植瘤体外实验，分别证实病毒蛋白Apoptin可明显增加肿瘤的放射敏感性作用，并完善Apoptin对肿瘤细胞放射增敏的机制研究，进一步探讨Apoptin对DNA损伤修复多个相关步骤及修复蛋白的影响，初步揭示Apoptin对肿瘤细胞的放射增敏作用及分子机制。首先，我们发现Apoptin通过增加放射诱导的DNA损伤，增强肿瘤细胞的放射敏感性。我们进一步采用iTRAQ和LC-MS/MS联合的蛋白质组学方法，探索Apoptin放疗增敏的分子机制，由此发现Apoptin下游的三个重要DNA损伤修复蛋白，POLE、MNAT1和Ku80。此外，GFP-HR/NHEJ-报告基因系统表明Apoptin主要通过抑制DNA损伤修复的NHEJ通路，并且Ku80作为NHEJ的关键分子，在Apoptin放射增敏过程中发挥了至关重要的作用。最后，在人宫颈癌裸鼠移植瘤模型中，Apoptin与射线联合作用下，可通过抑制Ku80蛋白水平，从而延缓移植肿瘤生长，延长动物生存期。综上所述，本研究对评价Apoptin作为肿瘤放疗增敏剂具有重要的意义，为开发Apoptin用于恶性肿瘤的放疗增敏提供实验依据，为开拓新的临床放射增敏剂提供了重要的理论基础。