An atempt of gene therapy for oral squamous cell carcinoma

由于E1 AF能激活细胞周期抑制分子p21 waf 1/cip 1的启动子活性，因此我们研究了E1 AF和Bax的激活作用，通过将bax报告质粒、E1 AF、野生型（wt）和突变型（mt）p53表达质粒转染p53不表达细胞系，用CAT（氯霉素乙酰转移酶）法测定了bax的启动子活性。CAT活性随着ElAF以剂量依赖性方式增加，并且观察到启动子活性随着wt p53和ElAF的协同增加。Western blotting检测到Bax蛋白表达。进行生长抑制测定，并且在E1 AF转染的p53缺陷细胞中观察到集落形成减少。这些结果表明，E4 orf 6是一种新发现的腺病毒癌蛋白。E4 orf 6显著增强转化的大鼠BRK和人293细胞在裸鼠中形成肿瘤的能力。E4 orf 6蛋白已被证明与p53和p73基因产物相互作用，并阻断由这些抗癌基因介导的细胞凋亡的诱导。我们研究了E4 orf 6是否可以影响p53家族以外的诱导细胞凋亡的分子，因为p53独立的细胞凋亡途径也被提出。我们证明腺病毒E4 orf 6蛋白抑制BNIP 3的凋亡活性，BNIP 3是Bcl-2家族的一组凋亡启动子的成员。E4 orf 6的表达抑制BNIP 3介导的细胞凋亡及其线粒体易位。E4或16与BNIP 3相互作用。此外，E4 orf 6的核输出信号对于E4 orf 6的这些功能是重要的。我们的数据表明，Bcl-2家族的促凋亡蛋白是转化细胞中E4 orf 6蛋白的靶点。

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.