E1A通过E6/E6AP增强宫颈癌放疗敏感性的分子机理研究

Radiation resistance of cervical cancer has become an important clinical problem in current treatment which to be resolved. It is one of the urgent problxems to explore a good adjunctive treatment which has better effect of increasing radiosensitivity. Our preliminary tests suggest that gene therapy which mediated by E1A can enhance the cancer's radiosensitivity effectively and the improvement of the radiosensitivity has relevance to apoptosis of cervical cancer cells result from the upregulation of p53 expression.However, the molecular mechanism for E1A increasing the radiosensitivity should be investigated. 99% of the patients with cervical cancer are positive for HPV infection. The E6 protein which encoded by high-risk HPV form a compound with E6 associated protein(E6AP) induce p53 degradation, which is the main reason why p53 is expressed lowly in HPV positive cells. In our study, we try to demonstrate that E1A affects the degradation of p53 through interfering the process of E6 and E6AP forming complexes in vitro and in vivo so that E1A can increase the radiosensitivity. Then, we want to illustrate that E1A and E6AP can be combined with each other by immunoprecipitate and the laser scanning confocal microscope. Furthermore, we will identify the combined domain of E1A and E6AP in cancer cells assisted by the plasmids which express different gene functional domain of E1A and E6AP. In the end, we plan to adopt the in vitro ubiquitination degradation analysis to demonstrate the procedure in which the E1A repress the p53 degradation through the E6AP pathway. Our study will demonstrate the molecular mechaniam of E1A increasing the radiosensitivity and offer an experimental fundament for new radio-biotherapy treatments of cevical carcinoma.

宫颈癌放疗抵抗是亟待解决的重要临床问题，探索有效的放疗增敏手段具有重要的临床意义。我们的前期工作表明：利用E1A进行宫颈癌的基因治疗可有效增强细胞的放疗敏感性，观察到的增敏效应与p53表达升高导致的宫颈癌细胞凋亡有关，但E1A放疗增敏的分子机制仍有待深入探讨。99%的宫颈癌患者为HPV阳性，高危型HPV编码的E6蛋白与E6相关蛋白(E6AP)形成复合物降解p53是HPV阳性细胞内p53低水平表达的主要原因。本项目拟通过体内外实验阐明E1A通过干扰E6-E6AP复合物的形成，影响p53降解途径，实现其放疗增敏作用；并通过免疫共沉淀和激光共聚焦显微技术，证明E1A与E6AP的结合；利用表达不同E6AP和E1A基因区域的质粒在细胞中确定E1A与E6AP的相互结合区域；采取体外泛素化降解分析技术，阐明E1A通过E6AP抑制P53降解过程的分子机制，从而为宫颈癌的新的放疗/生物治疗手段提供实验基础。

宫颈癌放疗抵抗是亟待解决的重要临床问题，探索有效的放疗增敏手段具有重要的临床意义。99%的宫颈癌患者为HPV阳性，高危型HPV编码的E6蛋白与E6相关蛋白(E6AP)形成复合物降解p53是HPV阳性细胞内p53低水平表达的主要原因。我们的研究工作表明：利用E1A进行宫颈癌的基因治疗可有效增强HPV阳性宫颈癌细胞系的放疗敏感性，观察到的增敏效应与p53表达升高导致的宫颈癌细胞凋亡有关，E1A通过干扰E6-E6AP复合物的形成，与E6AP相互结合使得p53泛素化降解减少，实现其放疗增敏作用，具体的结合结构域可能在CR1这个保守区域。本研究为宫颈癌新的放疗/生物治疗手段提供实验基础。