An atempt of gene therapy for oral squamous cell carcinoma

我们研究了ElAF和Bax的激活，因为ElAF被证明可以激活细胞周期抑制分子p21waf1/cip1的启动子活性。将bax报告质粒、ElAF、野生型（wt）或突变型（mt） p53表达质粒转染到p53缺乏细胞系中，采用CAT （Chloramphenicol acetyl transferase）检测bax的启动子活性。CAT活性随ElAF呈剂量依赖性增加，并且在wt p53和ElAF中观察到启动子活性的协同增加。Western blotting证实了Bax蛋白在转染ElAF细胞中的表达。生长抑制实验显示，ElAF转染的p53缺乏细胞中菌落形成减少。这些结果表明，ElAF与p53通过激活bax诱导细胞凋亡。E4orf6是一种新发现的腺病毒癌蛋白。E4orf6显著增强转化大鼠BRK细胞和人293细胞在裸鼠体内形成肿瘤的能力。E4orf6蛋白已被证明与p53和p73基因产物相互作用，并阻断由这些反基因介导的细胞凋亡的诱导。我们研究了E4orf6是否可以影响p53家族以外的诱导凋亡的分子，因为p53独立的凋亡途径也被提出。我们证明了腺病毒E4orf6蛋白抑制bbnip3的凋亡活性，bbnip3是Bcl-2家族的一组凋亡启动子。E4orf6的表达抑制bnip3介导的细胞凋亡及其线粒体易位。E4orl6与BNIP3相互作用。此外，E4orf6的核输出信号对于E4orf6的这些功能也很重要。我们的数据表明，Bcl-2家族的促凋亡蛋白是转化细胞中E4orf6蛋白的靶标。

ElAF and Bax activation was investigated since ElAF was shown to activate promoter activity of p21waf1/cip1, a cell cycle inhibitory molecule.CAT (Chloramphenicol acetyl transferase) assay was carried out to determine the promoter activity of bax by transfectipn with bax reporter plasmid, ElAF, wild type (wt) or mutant (mt) p53 expression plasmids into a p53 diffident cell line. CAT activity increased with ElAF in a dose-dependent manner and synergistical increase of the promoter activity was observed with wt p53 and ElAF. Protein expression of Bax was confirmed in ElAF transfected cells by Western blotting. Growth suppression assay was performed and decreased colony formation was observed in ElAF transfected p53 diffident cells. These results suggest that ElAF associates with p53 to induce apoptosis through activating bax.E4orf6 is a newly identified viral oncoprotein of the adenovirus. E4orf6 markedly enhances the ability of transformed rat BRK and human 293 cells to form tumors in nude mice. The E4orf6 protein has been shown to interact with p53 and p73 gene products and to block the induction of apoptosis which is mediated by these antioncogenes. We investigated whether or not E4orf6 can influence molecules that induce apoptosis other than the p53 family, because p53-independent apoptosis pathways have also been proposed. We demonstrate that the adenovirus E4orf6 protein suppresses the apoptotic activity of BNIP3, a member of a group of an apoptosis initiators of the Bcl-2 family. Expression of E4orf6 inhibits BNIP3-mediated apoptosis and its mitochondrial translocation. E4orl6 interacts with BNIP3. In addition, the nuclear export signal of E4orf6 is important for these functions of E4orf6. Our data suggest that the pro-apoptotic protein of the Bcl-2 family is a target of the E4orf6 protein in transformed cells.