The expression of connexin43, the primary gap-junction constituent of glial cells, was evaluated at the messenger RNA and protein levels in different grades of astrocytoma to investigate the relevance of gap junctions in herpes simplex virus-thymidine kinase (HSV-tk)-mediated gene therapy of brain tumors. Transduction of the retroviral-mediated HSV-tk gene into tumor cells with subsequent administration of ganciclovir has recently been used as an experimental therapeutic strategy for treatment of brain tumors. One aspect of this approach is the bystander effect, which augments the efficacy of this therapeutic approach. Glioblastoma cells with minimum levels of connexin43 protein were transfected with a connexin43 complementary DNA. These cells manifested a marked increase in the in vitro bystander effect, supporting the contention that the in vitro bystander effect is a consequence of metabolic cooperation between cells mediated by gap junctions. To assess relative levels of gap-junction protein expression in the relevant tumor type, we examined primary astrocytomas, primary astrocytoma cell cultures, and glioblastoma cell lines. Although most astrocytoma tumor samples expressed connexin43, they differed in the level of expression, with the greatest variation exhibited in high-grade astrocytomas. Primary glioblastoma cell cultures and established glioblastoma cell lines also displayed some variability in connexin43 levels. In aggregate, our results anticipate that glioblastomas will have a varied bystander effect during HSV-tk gene therapy depending on the level of connexin43 expression.
Delivery and expression of the herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir is currently being evaluated for the treatment of many types of cancer. After initial phosphorylation by HSVtk, cellular kinases generate the toxic triphosphate form of ganciclovir (GCV). To further define the role of GCV metabolism in cells expressing HSVtk, two human tumor cell lines, UMSCC29 and T98G, were transduced with HSVtk and screened for insertion of one or two copies of the viral transgene by Southern blot analysis. Both the relative capacities for incorporating labeled GCV and the levels of GCV metabolites were determined for each of the parental cell lines and their derivatives containing either one or two copies of the HSVtk gene. The efficiency of GCV killing and the magnitude of the bystander effect were compared for the single-and double-copy HSVtk cell lines. Consistently, cells that expressed two copies of HSVtk metabolized GCV more efficiently, were more sensitive to GCV, and demonstrated improved bystander killing relative to single-copy HSVtk cells. The implications of these results for future and current therapies employing HSVtk and GCV are discussed. Cancer Gene Therapy (2000) 7, 240 -246
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