ACTIONS OF HSVTK AND CONNEXIN43 GENE DELIVERY ON GAP JUNCTIONAL COMMUNICATION AND DRUG SENSITIZATION IN HEPATOCELLULAR-CARCINOMA

We have previously demonstrated that transfected hepatot cellular carc inoma cells (Hepa1-6) with one copy (pAG0) and two copies (pYED) of th e HSVtk gene, using liposomes, induced cell death of untransfected cel ls in the presence of ganciclovir (GCV). This phenomenon is called the 'bystander effect'. To determine whether an elevated level of connexi n43 increases the bystander effect, we have cotransfected Hepa1-6 cell s with a plasmid contain ing the HSVtk gene driven by the alpha-fetopr otein promoter (pFTK) or pAG0 or pYED and connexin-43. The results sho wed that, after GCV treatment, the percentage of growth inhibition was higher (25-30%) in cells cotransfected with HSVtk gene. The IC50 of G CV on cells transfected transfected with pFTK alone. To improve these results, stable connexin43 transduced Hepa1-6 cells were transfected w ith pFTK followed by GCV treatment. In this case, the cell growth was markedly inhibited as compared with parental cells. Furthermore, we ha ve studied the correlation between the expression of the HSVtk and the connexin43 proteins. Using flow cytometric analysis, scrape loading/d ye transfer and immunoblotting assay we found that the cells transfect ed separately by pAG0, pYED, pFTK and pLTR-Cx43 showed an increase of connexin43 protein. This study indicates that transfecting Hepa1-6 cel ls with both connexin43 and HSVtk genes up-regulates connexin43 expres sion which enhances the bystander effect and subsequently tumor cell d eath.