Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma

Abstract

We have previously demonstrated that transfected hepatotransfected with pFTK alone. To improve these results, cellular carcinoma cells (Hepa1-6) with one copy (pAG0) stable connexin43 transduced Hepa1-6 cells were transand two copies (pYED) of the HSVtk gene, using lipofected with pFTK followed by GCV treatment. In this case, somes, induced cell death of untransfected cells in the the cell growth was markedly inhibited as compared with presence of ganciclovir (GCV). This phenomenon is called parental cells. Furthermore, we have studied the correthe 'bystander effect'. To determine whether an elevated lation between the expression of the HSVtk and the conlevel of connexin43 increases the bystander effect, we nexin43 proteins. Using flow cytometric analysis, scrape have cotransfected Hepa1-6 cells with a plasmid containloading/dye transfer and immunoblotting assay we found ing the HSVtk gene driven by the ␣-fetoprotein promoter that the cells transfected separately by pAG0, pYED, pFTK (pFTK) or pAG0 or pYED and connexin43. The results and pLTR-Cx43 showed an increase of connexin43 proshowed that, after GCV treatment, the percentage of tein. This study indicates that transfecting Hepa1-6 cells growth inhibition was higher (25-30%) in cells cotranswith both connexin43 and HSVtk genes up-regulates confected with HSVtk and connexin43 than in cells transfected nexin43 expression which enhances the bystander effect only with HSVtk gene. The IC 50 of GCV on cells transfected and subsequently tumor cell death. with pFTK/Connexin43 was 17.85-fold lower than cells

Gene directed enzyme/prodrug therapy of cancer: Historical appraisal and future prospectives

“…Gap junctional communication, for instance, can be enhanced by regulating the production of connexins (Cx), membrane proteins considered to be the building blocks of gap junctions and the major factors responsible for the gap junctionmediated bystander phenomenon (Mesnil and Yamasaki, 2000). The transfer of Cx-encoding genes (Cx43, Cx32, Cx40) or chemically induced Cx-overexpression has been shown in tissue culture (in vitro) (Elshami et al, 1996;Ghoumari et al, 1998;Kunishige et al, 1998;Carystinos, et al, 1999;Andrade-Rozental et al, 2000) and in vivo (Dilber et al, 1997;Park et al, 1997;Du¯ot-Dancer et al, 1998;Touraine et al, 1998) to increase intercellular communication and the transfer of toxic agents. It is important to notice that in some human tumour cells not only expression but also correct surface localisation of Cx43 are necessary components of the bystander effect (McMasters et al, 1998), and that Cx-cotransfection appears not be applicable to all tumour systems (Cirenei et al, 1998).…”

The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy

“…Although much work supports the view that GJIC and Cx43 expression are closely correlated with bystander cell killing in the HSVtk /GCV treatment, 35,46,64 our data suggest that pathways other than intercellular communication are also involved. Indeed, the killing of the MDA -MB -435 bystander cells occurs in vitro specifically with the HSVtk / GCV system, despite the fact that these tumor cells lack GJIC.…”

Gene therapy of hepatocarcinoma: a long way from the concept to the therapeutical impact

“…Moreover, in some cancer cells, it has been proposed to overcome the absence of relevant gap junctions by overexpressing selected connexins along with the HSV-TK gene. [110][111][112][113] However, the bystander effect has also been shown to be efficient in normal cells and could emphasize the hepatic toxicity of the HSV-TK/ GCV treatment when normal cells are fortuitously transduced. In some immunocompetent animal models of HCC, a distant bystander effect relying on the activation of the immune system has been described, which synergizes with the direct elimination of tumor cells.…”