【作者】 胡宝利；

【导师】 齐义鹏；

【作者基本信息】 武汉大学 ， 微生物学， 2004， 博士

【摘要】 TRAIL作为TNF家族成员,最显著的特征是只选择性的对肿瘤细胞具有凋亡诱导作用,而对正常的组织和细胞不具有明显的毒性效应,体内实验也表明,TRAIL能够明显抑制肿瘤细胞生长,甚至使肿瘤消退,却对宿主细胞没有损害。本室邱松波博士构建了携带人TRAIL基因的重组腺病毒ZD55-hTRAIL,该重组病毒中E1B55KD基因突变,仅能够在p53阴性的细胞中复制。体内、体外实验结果表明,ZD55-hTRAIL单独或联合化疗药物5-FU杀伤肿瘤的效果优于经典的溶瘤病毒ONYX015。为了进一步提高携带TRAIL基因的重组腺病毒治疗肿瘤的效果及其分子机理,本论文研究了腺病毒早期基因(E1A、E1B19KD、E3)与TRAIL诱导调亡之间的相互关系。这些研究将为设计更加合理有效的肿瘤治疗载体提供理论依据,同时也初步揭示了腺病毒感染与宿主细胞之间的分子机制。 首先,我们运用一种新的细菌内同源重组的方法构建了3株非复制型重组腺病毒,Ad-CMV、Ad-E1A和Ad-TRAIL。在这3株重组腺病毒基因组中,E1、E3区完全缺失,插入了带有GFP报告基因的表达盒。外源基因TRAIL和E1A分别插入在重组病毒基因组CMV启动子下的多克隆位点中。经过E.coli BJ5183内同源重组和293细胞中的病毒包装,得到重组病毒,以应用下面的研究中。 虽然TRAIL能有效的诱导肿瘤细胞凋亡而不诱导正常细胞凋亡,但有些肿瘤细胞系或同一肿瘤细胞系中的部分细胞对TRAIL诱导的细胞凋亡具有抗性。以往的研究显示,腺病毒早期基因E1A能够调控许多病毒和宿主细胞基因的转录表达。因此,我们探讨了E1A是否能够通过某种机制增强肿瘤细胞或正常细胞对更多还原

【Abstract】 TRAIL (TNF α-Related-Apoptosis-Inducing-Ligand)/Apo-2 ligand was first described as a TNF-related ligand that selectively induce apoptosis of cancer cells, but not to normal cells. In addition, TRAIL has been shown to be capable of inhibiting tumor development and regression, but has minimal or no toxicity against normal tissues, as examined both in vivo. Our previous data suggested that a novel recombinant adenovirus (ZD55-hTRAIL) with E1B55 kD mutant, which can replicate in the tumor cells with the non-functional p53, alone or in combination with 5-FU has potentially clinical implication compared to the typical oncolytic ONYX-015. However, little is known about the mechanism of its actions. Here we investigated the relationship between adenovirus early genes (E1A、 E1B19KD、E3) and TRAIL-induced apoptosis. Our results may underlie the selection of the optimal adenovirus mutant as effective delivering vector for cancer therapy; and also discover that the molecular mechanism of adenovirus infection and host cell response.Firstly, three replication-deficient adenoviruses, Ad-CMV, Ad-EIA and Ad-TRAIL which all express green fluorescent protein (GFP> as marker, were generated using the AdEasy adenoviral vector system. The recombinant adenoviruses, Ad-El A and Ad-TRAIL, encode Ad5 E1A and human TRAIL gene expressed from the CMV promoter, respectively. The recombinant adenovirus, Ad-CMV, non-encoding any interest gene, was used as virus controls. All of them were confirmed by sequence and western blotting analyses, and were prepared to use for following experiments.Although TRAIL is able to induce apoptosis in the tumor cells but not in normal cells, many tumor cell lines, different cell types or partial cells of same type, were found to be resistant to TRAIL-induced apoptosis. Previous reports demonstrated that adenovirus E1A can regulate many viral genes and host cells response. Therefore, we asked whether E1A enhances tumor cells orsensitizes normal cells to TRAIL-induced apoptosis. Here data suggested that TRAIL obviously mediated apoptosis in HeLa cells after co-transfection with pIRES-EGFP-TRAIL and pcDNA3-El A. Though HepG-2 cancer cells were resistant to Ad-TRAIL infection in the different MOI (0, 1,10, 100), this resistance could be overcome by the co-infection with Ad-TRAIL and Ad-El A. Furthermore, TRAIL dramatically induced apoptosis of normal primary human lung fibroblast cells (P-HLF) that expressed ElA following either infection with Ad-EIA or transfection with pcDNA3-ElA. These results indicated that the possibility that the combination of ElA with TRAIL could be used in the treatment of human malignancy. However, the normal cells and tissues need to be protected from El A/TRAIL mediated apoptosis using the tumor special promoters.To determine whether there is any correlation between the sensitivity of TRAIL by ElA and basal level of TRAIL and its receptors expression in the HLF cells tested, we carried out a semiquantitative RT-PCR, Western blot and at the level of cell surface expression by flow cytometry analysis for each of them. Semiquantitative RT-PCR analysis was used to study the relative abundance of distinct TRAIL receptors mRNA in ElA treated or untreated HLF cells. The expression of ElA slightly decreased the abundance of OPG and DcRl mRNA in Ad-EIA infected and pcDNA3-ElA transfected HLF cells. DcR2 mRNA was not detected basally in treated or untreated cells. However, endogenous TRAIL mRNA and protein were not found in P-HLF cells in the presence or absence of ElA. Importantly, treatment with ElA resulted in a significant increase in DR5 mRNA and protein expression, but that of no significant change in DR4. Previous reports suggested that the abundance of membrane-binding TRAIL is indispensable for the cells killing effect of the TRAIL gene. Our results revealed that exogenous TRAIL mRNAs were dramatically expressed in ElA-transfected and ElA-infected HLF cells by semi-quantitative RT-PCR analysis. The results in this study demonstrated that the co-infection with Ad-TRAIL and Ad-EIA leaded to TRAIL protein obvious production by Western blot analysis and flow cytometry analysis. Taken together, our data may imply a novel basis that adenovirus ElA sensitizes normal cell to TRAIL-induced apoptosis.Previous studies demonstrated that the molecular mechanism of TRAIL-mediated apoptosis involved death receptor mediated signal pathway or combining with mitochondria-dependent signal pathway in some tumor cells derived from different tissues. Here we studied the signalpathway of ElA/TRAIL-madiated apoptosis in P-HLF cells by a series of methods involving using the dominant negative mutant, caspase inhibitors and western blot analyses. Herein data indicated that FADD is necessary for apoptosis in the co-infected P-HLF cells with Ad-TRAIL and Ad-El A, based on this apoptotic effect to be inhibited by FADD-DN containing the death domain but not the death effector domain. We found that the apoptosis of P-HLF cells co-infected with Ad-TRAIL and Ad-EIA was clearly inhibited by the specific inhibitor of caspase 8 (Z-EETD-FMK) but not by caspase 9 inhibitor (Z-LEHD-FMK), which involved procaspase 3 cleavage and activation. These results demonstrate that ElA/TRAIL-madiated apoptosis in P-HLF cells is typical death receptor-dependent pathway involvement of sufficient TRAIL, DR5 upregulation, recruitment of FADD, capase 8 and caspase 3 activation, as well as degradation of cellular proteins and DNA. Our data may provide evidences for understanding the function of adenovirus El A and TRAIL, the relationship of virus infection and host cell response.Since the induction of apoptosis early after infection would severely limit virus production and reduce or even eliminate the spread of progeny virus in the host, most viruses have evolved strategies to evade or delay early apoptosis in an attempt to allow production of high yields of progeny virus. Therefore, viruses are also known to encode products that directly block the apoptotic signal cascade. To test the hypothesis that whether enhancement of TRAIL-induced apoptosis by El A expression in the cell lines is blocked by these adenoviral proteins, we constructed the recombinant adenovirus, r3/Ad, which was a mutant of E1B55K and contained E1B19K and complete E3 region. The results shown that r3/Ad-infected HLF cells infected with Ad-El A and Ad-TRAIL at the same time, reduce the TRAIL-dependent apoptosis compared with the uninfected cells with r3/Ad. Further studies demonstrated that r3/Ad infection efficiently inhibited the Ad-TRAIL dependent apoptosis of HLF cells in the presence of El A by down-regulation of DR5 and TRAIL expression. Taken together, we speculate that the ability of adenovirus early genes (E1A, ElB19KDs E3) may regulate viral life cycle and host cell ’s death or survival; TRAIL may play an important role in limiting virus infections.更多还原