Role Of GJIC Of C6 Lines On The Bystander Effect Mediated By HSV-TK/GCV Approach And Its Regulation

Purpose The therapy of tumor suicide gene has made up for a loss in the gene therapy induced by virus. The transfected cells with suicide gene can kill its neighboring nontransduced cells, in a phenomenon known as the bystander effect. The suicide gene system has almost the bystander effect, but its mechanism has yet been unclear. Gap Junctiunal intercellular communication(GJIC) may play an important part in bystander effect. This so-called byslander effect has recently been shown to be dependent on connexin-mediated intercellular communication in vivo or in vitro. So we can enhance the bystander effect by over expressing connexin or by transduceing connexin or by treating with agent of gap junction in tumor cells with low levels of connexin expression. Guided by this theory and practice above, we attempted to explore them as follows: â‘  further to demonstrate the functions of HSV-TK/GCV system and its effected factors. â‘¡ to investigate the gap Junctiunal intercellular communication of C₆ lines and its changes modulated by chemical agent. â‘¢ further to search for the distinguishing biological feature of C₆ lines after transfected or treated. â‘£ to explore the influence of C₆ lines and the bystander effect induced by HSV-TK/GCV. in order to gain a result for the gene therapy of primary brain tumors.Methods Gene engineering techniques, Scrape-loading dye transferassay, FCM, Methods of MTT, TUNEL and Fluorescent microscope were used to determine the GJIC, apoptosis, cell-cycle and proliferation of C6 lines (C6-pcDNA3-TK, C6-pcDNA AND C6). Wild-type C6 cells were transfected in vitro with HSV-TK gene were generated by transfection of C(, with LipofectAMINE?Regent-HSV-TK plasmid DNA. Results The results showed as follows:1. Aretrovirus eukaryocytic expression vector pcDNA3-TK was successfully constructed through gene engineering techniques. DNA sequencing result showed that the terminative code of PCDNA3-TK gene in pcDNAs-TK has been mutated from ATG to TGA.2. The susceptiveness to HSV-TK/GCV approach and the magnitude of bystander effect were higher in C6-TK+ than in C6-TK' and C(, (P<0.01).Meanwhile, apigenin more markedly enhanced the bystander effect (p<0.05).3. The GJIC of C6 cells was low. Apigenin, which was reported as GJIC up-regulator, could significantly improved the GJIC of C6 lines.4. The growth of C6 lines were inhibited evidently after treated with apigenin. The cells showed characters of apoptosis and differentiation observed by light microscopy and FCM. The rates of apoptosis increased after treated with GCV and more markedly increased after treated with apigenin/GCV in C6-TK+ group than others (p<0.01). We observed the cells in apigenin/GCV group was inhibited in G0/G1 phase, the cellular number in S phase remarkably decreased (p<0.01).Conclusion1. The fusion protein of PCDNA3-TK is stably, continuously and high efficiently expressed in transfected C(, lines.2. The GJIC of C6 lines is low. Apigenin, which was reported as GJIC up-regulator, significantly improved the GJIC of C6 lines. Meanwhile,apigenin can inhibit the growth, proliferation and differentiation of C(, lines and induce its apoptosis.3. The C6 lines transfected with TK gene are susceptive to GCV and have a higher magnitude of bystander effect. Apigenin can enhance the bystander effect more remarkably in transfected C6 lines than in anther group.4. The results show the GJIC play an important part in bystander effect of tumor suicide gene therapy.