| The application of gene therapy has brought new hopes to the cure of cancer and other severe diseases in the past decades. The core strategy of cancer gene therapy is the apoptosis induction of the carcinoma cells through deliverying the useful DNA fragments into the carcinoma cells by means of the gene recombination. So, how to choose the specific, efficient and minimal side-effect aim gene had become a most important step in tumor gene therapy.TNF (tumor necrosis factor) is a multifunctional antitumor activity cytokine produced predominantly by macrophages. TNF elicits apoptosis through direct interactions with carcinoma cells, it is known to exert pleiotropic activities that are mediated through two high-affinity receptor: the TNF-R55(TNFR1), which mainly transduces cytotoxic signals, and the TNF-R75(TNFR2), which is related to proinflammatory activity. TNF induces a typical apoptotic cell death program in various cell lines after oligomerization of the TNFR1 or the Fas receptor by the respective ligands. Both death receptors contain a homologous C-terminal cytoplasmic death domain (DD) involved in apoptosis.After bind by interacting with the TNFR1.A key step in the pathway to apoptosis is activation of procaspases. Activation of these cysteinyl aspartate-specific proteases is initiated by formation of a death-inducing signaling complex (DISC)ing of TNF to TNFR1, the clustered DDs recruit the TNFR1-associated DD-containing protein TRADD.TRADD in turn recruits Fas-associated death domain protein (FADD) gene by DD-DD interaction. Besides its C-terminal DD, FADD contains a death effector domain (DED) implicated in the recruitment of procaspase-8. Oligomerization of procaspase-8 leads to proximity-induced autocatalytic 2003-05activation followed by direct or indirect downstream activation of executionary caspases, which cleave substrates involved in apoptotic morphology. So TNFR1 and FADD gene play an important role in the apoptotic signal transduction pathway mediated by TNF.Tongue carcinoma is one of the most common malignant neoplasma in head and neck region, the high-grade tongue carcinoma is characterized by biological aggression, high incidence of metastasis and poor prognosis. Treatment of tongue carcinoma is surgery, chemotherapy or radiotherapy nowadays, and sometimes it is not effect enough, especially in the cases with high-grade lesion. But as we know, most of tongue carcinomas are superfacial, and can be visible easily, so they are suited for gene therapy, hi our present research, we attempted to amplify human FADD and TNFR1 gene by RT-PCR , then the fusion gene TFL which could expressed TNFR1/DED fusion protein activity was constructed by recombinant PCR. The fusion gene TFL included the EC (extracellular domain),TM (transmembrane domain) and MPR (membrane-proximal region) of TNFR1 gene and DED (death effector domain) of FADD gene respectively. The FADD and TFL genes were then cloned into eukaryotic expression vectors pIRES2-EGFP and pcDNAS .hi order to investigate the pro-apoptotic activities of the FADD gene in human tongue carcinoma cell (Tca-8113), we transfected the green fluoresent protein expression plasmid pIRES2-EGFP-FADD transiently into Tca-8113 cells, the expression of FADD protein was verified by indirect immuno-fluorescence labeled assay and western blot . Using fluorescence microscope and inverted microscope, we observed that Tca-8113 cells transfected with FADD gene became round and shrinked 24h after transfection, which differ from the cells transfcted with pIRES2-EGFP void vector. The nuclei of transfected cells displayed the hallmarks of apoptosis by D API staining: chromatin condensation and clustering along the nuclei membrane. Those hallmarks were also confirmed by electronic microscopic observation: dying cells displayed bubbly membrane, condensation of chromatin,formation of apoptotic bodies, which are all typical features of apoptotic cells. Tca-8113 cell cycle were examined following the induction of FADD gene, and the...
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