Augmentation Of The Efficacy Of HSV-tk Gene Therapy In Glioma Treatment Via Upregulating Gap Junctional Intercellular Communication

Gliomas are the most common type of tumor in the central nervous system, and refractory to nearly all the modern therapeutic strategies. Being confined to the tumor deposit with rare metastasis, gliomas appear to be appealing candidates for gene therapy. Under the current circumstance of cancer gene therapy, herpes simplex virus thymidine kinase (HSV-tk) suicide gene therapy is regarded as a promising strategy because it is independent of the genetic background of carcinogenesis, which is not clear yet, and more important, the hallmark of HSV-tk gene therapy, bystander effect, compensates for the low transduction efficiency of current gene therapy vectors, which had turned out to be a main obstacle in current gene therapy. Controversial is the mechanism of bystander effect, there is compelling evidence that gap junctional intercellular communication (GJIC) does play a substantial role by mediating the transfer of cytotoxic GCV triphosphate between tumor cells. GJIC deficiency, however, is a common phenotype of malignancy. Therefore, it would be efficient and feasible to enhance bystander effect, and consequently, the antitumor efficacy of HSV-tk gene therapy via upregulating GJIC.From the gene therapeutic point of view, it is a classical protocol to overexpress exogenous gap junctional protein encoding gene, connexin, in glioma cells in order to enhance the deficient GJIC. While alternatively, GJIC between tumor cells could also be upregulated by pharmacological manipulation. In this study, both methods were used to augment the bystander effect and the therapeutic effect of HSV-tk gene therapy in glioma treatment.As a beginning, by culturing the cells in medium with GCV of gradient concentrations, the clone expressing highest level of HSV-tk was selected from heterogenous HSV-tk transfectants of C6 rat glioma cell, and was named C6tk. The integration of HSV-tk gene in the genomic DNA of C6tk cells and other GCV resistant clones were also examined with PCR. The results of HSV-tk PCR were positive in all the clones, which means the foreign gene might not be expressed properly even being integrated into the genomic DNA of a host cell. The bystander effect in glioma HSV-tk gene therapy was tested in vitro by mixing C6 glioma cells and C6tk cells at different ratios and plating into 96 well tissue culture plates at the densities of 1000, 2000, 5000 cell/well respectively, followed by 7...