A tumor suppressor gene, Cx26, also mediates the bystander effect in HeLa cells (original) (raw)
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A Tumor Suppressor Gene , Cx 26 , Also Mediates the Bystander Effect in HeLa Cells 1
2006
The connexin 26 (Cx26) gene suppresses the growth of HeLa cells in vitro and in vivo. We explored the possibility that the Cx26genenot only suppresses growth but can also mediate the bystander effect that is observed in some gene therapy. In gene therapy mediated by the herpes simplex virus thymidine kinase, the toxicity of ganciclovir affects not only the cells transduced with the gene but also affects neighboring tumor cells; it has been suggested that gap junctional intercellular communication (GJIC) may play a role in such a bystander effect. HeLa cells expressing the Cx26 gene(Cx26―@) or not expressingthe Cx26genewere transfected with the herpes simplex virus thymidine kinase (tki gene, producing Cx26-tlC, Cx26'-tk―, Cx26@-tk', and Cx26@-tk' cells. By making different kinds of cocultures of these cells, we observed a clear bystander killing effect, assessed by the neutral red toxicity test, in the coculture of Cx26@-tk/Cx26@-tk@ cells. The bystander effect was m...
Gene Therapy, 1998
Gene therapy via the herpes simplex virus thymidine kin-tumors were palpable, fewer animals developed tumors, ase (tk) gene and ganciclovir (GCV) treatment eliminates even after a longer period, if the injected cells were mixexperimental tumors. In this approach, cells expressing the tures of Cx43 +-tk + and Cx43 +-tk − while tumor growth was tk gene (tk +) and neighboring tumor cells which do not not prevented with mixtures of HeLa cells not expressing express the gene are killed. We have demonstrated this Cx43, ie Cx43 −-tk + /Cx43 −-tk −. When GCV was given after bystander effect is enhanced in vitro by gap junctional the appearance of tumors, the size of the tumors from intercellular communication (GJIC). In order to extend our Cx43 − cells was 30% reduced for 3 weeks if 50% of the in vitro results into in vivo situations, we injected into nude injected cells were tk +. However, for cells expressing Cx43, mice different ratios of tk + /tk − HeLa cells, either lacking or the tumor size was 66% reduced if 10% of the cells were transfected with connexin43 (Cx43), a gene coding for a tk +. Such a reduction demonstrates a long-term bystander gap junction protein. When GCV was administered before effect which is dependent on Cx43 expression.
Cancer research, 2000
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 ...
Human Gene Therapy, 1998
A bystander effect is described when nontransduced or genetically unmodified cells are killed during death of genetically modified tumor cells transduced with a suicide gene. The "bystander effect" greatly enhances the efficacy of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy approach for cancer. The mechanism of the bystander effect is controversial. In this study, we examined the role of intercellular gap junction communication (GJIC) for the bystander effect in human gastrointestinal tumor cells. Our results show that the extent of the bystander effect varied amongst the tumor cell lines; pancreatic cancer cells BXPC-3 exhibited excellent bystander effects in vitro and in vivo studies whereas other gastrointestinal tumor cell lines such as pancreatic cancer cells MIAPACA-2, and colon cancer cells HT-29 showed poor bystander effects. Bystander effects were only found in the presence of cell-to-cell contact. The extent of the bystander effect was independent of the level of HSV-TK activity in the transduced tumor cells and was correlated with GJIC as demonstrated by an in vitro dye-transfer assay. Expression of the mRNA levels of gap junction protein connexin 43 was 8-to 26-fold or greater and connexin 26 gene expression was 2-to 229fold greater in BXPC-3 cells compared to HT-29, MIAPACA-2, and PANC3 cells. Our results suggest that intercellular communication is essential for the bystander effect. The correlation between GJIC and the extent of the bystander effect suggest a role for GJIC in mediating the bysatnder effect. Analysis of tumors for GJIC or expression of gap junction proteins may identify the subset of patients suitable for gene therapy with the HSV-TK/GCV approach. OVERVIEW SUMMARY correlation between GJIC and the extent of the bystander effect suggest a role for GJIC in mediating the bystander The bystander effect greatly enhances the efficacy of the effect. herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy approach for cancer. In this study, we examined the role of intercellular gap junction commu
Gene Therapy, 1997
Bystander' killing of adjacent wild-type tumor cells was was found in both herpes thymidine kinase transduced and seen when tumors transduced with the herpes thymidine unmodified cells, while 'resistant' cell combinations showed kinase gene were treated with the antiviral agent ganciclo-little or no transfer of phosphorylated GCV between cells. vir (GCV). Some tumors were 'bystander-sensitive' while The capacity of intracellularly produced nucleotide toxin to others were 'bystander-resistant'. Mixtures of different spread from cell to cell within a tumor mass effectively 'sensitive' tumor lines showed cross-transfer of bystander amplifies the apparent efficiency of gene transfer in the killing, while in mixtures of 'resistant' with 'sensitive' tumor and makes feasible the use of this system for thertumors, the resistant phenotype was predominant. Using apy of localized cancer. 3 H-GCV with 'sensitive' mixtures, phosphorylated 3 H-GCV
Clinical cancer research : an official journal of the American Association for Cancer Research, 1999
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 bystand...
Proceedings of the National Academy of Sciences, 1995
Herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) viral-directed enzyme prodrug gene therapy causes potent, tumor-selective cytotoxicity in animal models in which HSV-tk gene transduction is limited to a minority of tumor cells. The passage of toxic molecules from HSV-tk+ cells to neighboring HSV-tk- cells during GCV therapy is one mechanism that may account for this "bystander" cytotoxicity. To investigate whether gap junction-mediated intercellular coupling could mediate this bystander effect, we used a flow cytometry assay to quantitate the extent of heterocellular coupling between HSV-tk+ murine fibroblasts and both rodent and human tumor cell lines. Bystander tumor cytotoxicity during GCV treatment in a coculture assay was highly correlated (P < 0.001) with the extent of gap junction-mediated coupling. These findings show that gap junction-mediated intercellular coupling contributes to the in vitro bystander effect during HSV-tk/GCV therapy and that ...
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 1996
is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in viva tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homocypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals; they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms. COMP BIOCHEM PHYSIOL 113C, 247-256, 1996.