Connexin 26 enhances the bystander effect in HSVtk/GCV gene therapy for human bladder cancer by adenovirus/PLL/DNA gene delivery (original) (raw)
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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, 1998
In tumors, gap junctional intercellular communication cantly more slowly than control U-87 cells and lose their (GJIC) is usually down-regulated and the expression of tumorigenicity when injected subcutaneously in nude mice. connexins, membrane proteins constituting gap junction When the Cx43 gene was transduced in vitro in U-87 cells channels, is often low or altered. GJIC, allowing the inter-by a retroviral producer cell line (N3.2.ii, titer cellular diffusion of ganciclovir (GCV) triphosphate, is also 1.5 × 10 6 c.f.u./ml) in vivo results were similar. However, one mediator of the 'bystander effect', the phenomenon by only when U-87 cells were co-injected with N3.2.ii cells in which herpes simplex virus thymidine kinase (HSVtk)-nude mice in a 1:5 ratio, a 50% reduction in tumor size transduced, neoplastic cells kill surrounding HSVtk-nega-was obtained during the first 3 weeks. Moreover the cotive cells when treated with GCV. We set up experiments injection of U-87 cells with N3.2.ii and SBA cells (a retrovito evaluate the effects of retrovirus-mediated in vivo gene ral producer cell line expressing the HSVtk gene), was not transfer of connexin 43 in malignancies with low GJIC able to potentiate the effects of GCV administration, sugcapacity. We found that U-87 human glioblastoma cells gesting that Cx43 gene transfer requires more efficient transfected in vitro by the human Cx43 cDNA grow signifi-vectors to increase the bystander effect in vivo.
Journal of Biological Chemistry, 2002
The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (HBL100); (b) low levels of Cx43 and sparse intercellular communication (MDA-MB-231); and (c) significant levels of Cx43 and moderate intercellular communication (Hs578T). Although retroviral delivery of Cx43 and Cx26 cDNAs to MDA-MB-231 cells did not achieve an expected substantial rescue of intercellular communication, overexpression of connexin genes did result in a dramatic suppression of tumor growth when connexin-expressing MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Subsequent immunolocalization studies on xenograph sections revealed only cytoplasmic stores of Cx43 and no detectable gap junctions. Moreover, DNA array and Western blot analysis demonstrated that overexpression of Cx43 or Cx26 in MDA-MB-231 cells down-regulated fibroblast growth factor receptor-3. Surprisingly, these results suggest that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. Moreover, our studies show that retroviruses are effective vehicles for delivering connexins to human breast tumor cells, facilitating potential gene therapy applications.
Advances in Gene Therapy for Bladder Cancer
Current Gene Therapy, 2003
The efficacy of various currently available therapeutic strategies for bladder cancer is not always sufficient, especially for the advanced disease, recurrent superficial cancer, and treatment-resistant carcinoma in situ. Advances in genetic and molecular biology have led to novel approaches for cancer treatment. Gene therapy is currently one of the most promising strategies against various malignancies, and several clinical trials have been approved worldwide. Various strategies for modulating the genetic state have been applied in bladder cancer treatment, and encouraging results have been demonstrated both in vitro and in vivo. Although the therapeutic genes work dramatically when the transgenes are effectively expressed in the targeted cells, however, a sufficient rate of transduction cannot always be achieved. The most significant obstacle for clinical application of cancer gene therapy might be the method for sufficient delivery and expression of the therapeutic genes. Bladder is an easily accessible organ because of its anatomy; however, a glycosaminoglycan (GAG) layer on the bladder mucosa may protect integration of exo-delivered genetic vectors. Various strategies are applied for improving the transduction efficacy of the therapeutic genes into the bladder cancer cells. These strategies include the modification of adenoviral fibers, cotransduction of the materials for enhancing the viral infectivity, and disruption of the GAG layer. Recent advances in the field of gene therapy for bladder cancer are briefly summarized in this review.
Molecular Therapy - Oncolytics
Glioblastoma multiforme (GBM) is the most invasive form of primary brain astrocytoma. Gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) is a new strategy for GBM treatment. As the connexin 43 (Cx43) levels are downregulated in GBM cells, it seems that the upregulation of Cx43 could improve the efficacy of the gene therapy. This study aims to evaluate the effect of clenbuterol hydrochloride (Cln) as a b2-adrenergic receptor agonist on HSV-TK/ GCV gene therapy efficacy in human GBM cells using olfactory ensheathing cells (OECs) as vectors. The lentivirus containing the thymidine kinase gene was transduced to OECs and the effective dose of GCV on cells was measured by MTT assay. We found that Cln upregulated Cx43 expression in human GBM cells and OECs and promoted the cytotoxic effect of GCV on the co-culture cells. Western blot results showed that Cln increased the cleaved caspase-3 expression and the Bax/ Bcl2 ratio in the co-culture of GBM cells and OEC-TK. Also, the flow cytometry results revealed that Cln increased apoptosis in the co-culture of GBM cells and OEC-TK cells. This study showed that Cln via upregulation of Cx43 expression could enhance the bystander effect of HSVTK-GCV gene therapy in human GBM cells.