已知HPV16╱18E6致癌蛋白始p53基因去活化在子宮頸癌的腫瘤化過程扮演了重要的角色。為了了解HPV16╱18E6與p53表現在肺腫瘤化過程中所扮演的角色，本計畫共收集122個肺癌患者的腫瘤組織以免疫組織化學染色分析兩者間的相關性，結果發現HPV16╱18E6與p53蛋白表現呈負相關，且在連續切片中亦可得到驗證。此外並以即時偵測反轉錄聚合連鎖反應偵測p53下游基因p21WAF1╱CIP1及mdm-2mRNA的表現量，結果發現p21WAF1╱CIP1及mdm-2mRNA的表現量在可測得HPV16╱18E6表現者低於HPV16╱18E6不表現者。為了進一步證明HPV16╱18E6可造成p53蛋白得去活化，本實驗室由肺癌患者胸水中成功的建立具有HPV16感染及不感染的肺腺癌細胞株，在肺癌細胞株的研究中發現HPVHPV16╱18E6表現的肺癌細胞株其p53蛋白的表現量低於不表現者且其下游基因p21WAF1╱CIP1及mdm-2mRNA的表現量也相對較低。免疫沉澱法的分析結果亦發現HPV16╱18E6確實與p53蛋白結合而造成其去活化，而當把細胞中的HPV16╱18E6以siRNA方式抑制後p53蛋白、p21WAF1╱CIP1及mdm-2mRNA的表現量均有回復的現象，根據組織及細胞的研究結果證實HPV確實存在於肺腫瘤組織中，並可透過始p53蛋白去活化而參與肺腫瘤化。此外為了證明HPV16╱18E6使p53蛋白去活化對MGMT基因甲基化的影響，本研究亦利用siRNA的方式及轉染突變形式的p53基因到肺癌細胞株中，並分析當p53蛋白去活化時對MGMT基因轉錄起始區甲基化的影響，結果亦發現當p53蛋白去活化確實會造成MGMT基因轉錄起始區甲基化。因此本研究認為HPV16╱18E6除透過使p53蛋白去活化而使細胞腫瘤化外亦可造成抑癌基因的甲基化而使細胞癌化。 Inactivrucial role in cervical tumorigenesis. To investigate the involvement of HPV 16╱18 in lung tumorigenesis, the association between HPV 16 or 18 E6 and p53 protein expression in 122 lung tumors was evaluated by immunohistochemistry and data showing that HPV 16╱18 E6 expression correlated inversely with p53 expression, which was further confirmed by tissue in situ immunostaining. Real-time RT-PCR analysis indicated that E6-positive tumors had lower p21WAF1╱CIP1 and mdm-2 mRNA levels than E6-negative tumors. To elucidate the role of E6 on p53 inactivation, we successfully established lung adenocarcinoma cell lines with or without HPV 16 infection from patients’ pleural effusions. Western blotting showed that E6 protein was indeed expressed in HPV16-infected cells and a lower level of p53 protein was observed in E6-postive cells compared to E6-negative cells. Moreover, the levels of p21WAF1╱CIP1 and mdm-2 mRNA in E6-positive cells were lower than in E6-negative cells. The interaction of E6 with p53 protein was revealed by immunoprecipitation assay showing that p53 could be inactivated by E6 protein. Conversely, p53 proteins, p21WAF1╱CIP1 and mdm-2 mRNA expressions were restored in E6 knockdown cells by RNA interference compared with vector control cells. These results reveal that HPV 16╱18 E6 may be partially involved in p53 inactivation to downregulate p21WAF1╱CIP1 and mdm-2 transcription. In conclusion, HPV 16╱18 E6 is indeed expressed in HPV DNA–positive lung tumors and is involved in p53 inactivation to contribute to HPV-mediated lung tumorigenesis. To elucidate whether p53 participates in promoter methylation, we engineered three cell lines: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished promoter methylation, whereas the MGMT promoter methylation status was unchanged by expression of L194R. Therefore, we concluded that HPV 16╱18 E6 could inactivate p53 protein to induced MGMT gene promoter hypermethylation to promote lung tumorgenesis.
