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This paper focuses on the influence of chemoresistance on the herpes simplex virus (HSV-tk)/ganciclovir (GCV)-induced bystander effect (BE), as studied in a human small cell lung cancer (SCLC) cell line (GLC4) and its sublines with in vitro acquired resistance to adriamycin (GLC4/ADR), mitoxantrone (GLC4/MITO) and cisplatin (GLC4/CDDP). Chemoresistance for adriamycin, mitoxantrone and cisplatin significantly changed GCV sensitivity. A significant BE was found in all GLC4 cell lines. Compared to the parental GLC4 cell line, the BE was significantly higher only for the GLC4/ADR cell line. No expression of the nucleoside transporters MRP4 and MRP5 was detected. In all cell lines expression of connexin 43 was found, but modulation of gap junctional intercellular communication (GJIC) by 18-alpha-glycyrrhetinic acid did not significantly change the BE in any of the GLC4 cell lines. In conclusion, chemoresistance can influence the HSV-tk/GCV-induced BE, which seems not to be related to differences in MRP4/MRP5 expression or to differences in GJIC.

The p53 protein is mutated in 50% of all human tumors and plays a key role in apoptosis, cell cycle, and the expression of several genes. We investigated if p53 mutations influence the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV)-induced bystander effect (BE). Additionally, we studied some of the underlying mechanisms. GCV sensitivity and BE were studied in a human ovarian carcinoma cell line transfected with an empty vector (A2780/cmv) or a vector containing a p53 hotspot mutation at codon 175 (A2780/m175) or 248 (A2780/m248). In addition, expression levels of two nucleoside analogue transporters, multidrug resistance-related protein 4 and 5 (MRP4/MRP5), were determined. Finally, differences in gap junctional intercellular communication (GJIC) were studied by determining connexin 43 (Cx43) expression and by modulating GJIC with 18-alpha-glycyrrhetinic acid. Our results showed that compared to A2780/cmv, GCV sensitivity was significantly decreased in A2780/m175 and A2780/m248. Additionally, a significant BE (relative increase in cell kill) was found in A2780/cmv and A2780/m248. In contrast, an increased survival was observed in A2780/m175. No MRP4 or MRP5 expression was detected. However, all A2780 cell lines expressed Cx43. Modulating the GJIC significantly decreased the BE in A2780/cmv and A2780/ m248. In conclusion, HSV-tk/GCV-induced BE is influenced by p53 mutations. Differences in GJIC could be one of the underlying mechanisms.

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect., (Copyright 2001 Wiley-Liss, Inc.)

Antitumor suicide gene therapy is one of the emerging strategies against cancer. It consists of the introduction into cancer cells of a gene capable of converting a nontoxic prodrug into a cytotoxic drug. Because this therapeutic gene cannot be easily introduced into the whole cell population of a tumor, the successful eradication of tumors depends on a phenomenon called the "bystander effect," by which the introduced gene can affect even cells in which it is not itself present. From a therapeutic point of view, it may be crucial to enhance this phenomenon through various means to achieve tumor eradication. One such suicide gene, the thymidine kinase gene from the herpes simplex virus, in combination with the prodrug ganciclovir, has been extensively and successfully used in some animal models exhibiting a strong bystander effect. Among the mechanisms involved in this phenomenon, gap junctional intercellular communication (GJIC) is directly involved in the transfer of the toxic metabolites of ganciclovir, which pass directly from herpes simplex virus thymidine kinase-expressing cells to surrounding cells that do not express it. Because GJIC appears to be a mediator of the bystander effect both in vitro and in vivo, here we review possible molecular strategies for enhancing the extent of tumor cell death by increasing the intratumoral GJIC capacity.

Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.

We evaluated the gain in long-term survival of BDIX rats bearing DHDProB colon cancer developed in the peritoneal cavity after in vivo therapy with the tk gene and GCV. The sensitivity and the bystander effect of DHDProB cells stably transduced with the tk gene evaluated in vitro were low, as one tk+ cell killed two tk- cells. This correlated with the low ability of a fluorescent dye to diffuse through gap junctions. In vivo, more than 75% of tk-transduced cells were required and at least 100 mg/kg/day of GCV had to be injected no later than day 5 after tumor implantation to obtain a curative effect. A partial protection of the cured animals against rechallenge with the parental cells was also observed. Based on these results, a protocol of in vivo gene therapy was designed in which the tk/GCV treatment was combined with IL-2 gene expression. When the tk- and IL-2 encoding plasmids were injected twice i.p. with DOTAP and the animals treated with GCV, three of five rats were cured. This antitumoral activity resulted from the combined toxic effects of DNA/DOTAP and tk/GCV plus a potential immune response mediated by IL-2.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.