Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although the treatment results for some forms of haematologic malignancies are excellent, especially for the childhood acute leukaemias, there is still a significant fraction of patients that will not benefit from the therapy available today. The identification of new techniques, such as gene therapy, may therefore be of great importance for future therapeutic applications. Suicide gene therapy is one of several gene therapeutic approaches to treat cancer. A suicide gene is a gene encoding a protein, frequently an enzyme, that in itself is nontoxic to the genetically modified cell. However, when a cell is exposed to a specific nontoxic prodrug, this is selectively converted by the gene product into toxic metabolites that kill the cell. The suicide gene most commonly employed, both in experimental and a clinical settings, is herpes simplex thymidine kinase (HSVtk). Some suicide gene products also induce a so-called 'bystander effect', i.e. a toxic effect on adjacent nongene modified tumour cells and sometimes also on more distant tumour cells. The bystander effect is most evident in tumour cells that have a high number of gap junctions, cellular channels build up by proteins called connexins. Many tumours, amongst them many haematological ones, have a low number of gap junctions. Therefore, it is important to develop gap junction independent drug delivery systems. Suicide gene technology may also be used for the ex vivo purging of tumour cells in bone marrow or peripheral blood stem cell autografts or for inactivation of effector cells, such as antitumour T donor lymphocytes in allogeneic transplantation to prevent severe graft versus host reactions. New constructs, e.g. combining suicide genes and immune response enhancing genes or suicide genes and connexin inducing genes may further improve the value of suicide gene therapy.
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PMID:Suicide gene therapy: possible applications in haematopoietic disorders. 1129 56

We previously showed that gap junction intercellular communication mediates the bystander effect in anticancer gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir. Because most cancer cell lines have lost their ability to communicate through gap junctions, we investigated whether we could induce such a communication by transferring a gene for a gap junction. We transfected a vector carrying the HSV-tk (tk) and gap junction (connexin (Cx) 32) genes (Cx32(+)tk(+)) into noncommunicating HeLa cells. We compared the cytotoxicity of ganciclovir with mixtures of these cells and HeLa cells that expressed (Cx32(+)) or did not express (Cx32(-)) the Cx32 gene. The bystander effect was strong when the two mixed cell types expressed Cx32 (i.e., Cx32(+)tk(+) cells and Cx32(+)tk(-) cells). Only 25% of cells survived in this communicating mixture, even when only 10% of the cells were Cx32(+)tk(+). There was also a moderate bystander effect when the Cx32(+)tk(+) cells were mixed with noncommunicating HeLa cells in a 50% ratio. These results demonstrated that the bystander effect is enhanced by Cx32 and suggested that expression of Cx in only one cell type in a mixture can cause a bystander effect. Mol. Carcinog. 30:176--180, 2001.
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PMID:Induction of a bystander effect in HeLa cells by using a bigenic vector carrying viral thymidine kinase and connexin32 genes. 1130 78

Tumoricidal "bystander effect" observed in the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy was studied between different rat glioma cell lines (9L and C6 cells) under both in vitro and in vivo conditions. For that purpose, mixed populations of wild-type cells (9Lwt and C6wt) and respective HSVtk gene-transduced cells (9Ltk and C6tk) were examined for their sensitivity to GCV. A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Because the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target (wild-type) cells but not in effector (HSVtk gene-bearing) cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells.
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PMID:Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells. 1149 61

In tumor models, the killing by ganciclovir of a fraction of tumor cells transfected with the thymidine kinase (TK) gene has been shown to induce complete regression of the tumor. The mechanism responsible for this bystander effect is thought to be the diffusion of toxic metabolites or apoptotic signals across gap junctions. Connexin 43 (Cx43) is the major component of astrocyte gap junctions. We investigated the susceptibility of two rat glioma cell lines (CNS1 and C6) to thymidine kinase/ganciclovir, before and after transfection with the Cx43 gene. We report a close correlation between the level of Cx43 expression, the extent of gap junctional communication and the amplitude of the bystander effect. Transfection of C6 cells (which display a weak bystander effect and low levels of connexin) with a Cx43 construct induced a strong bystander effect. Inhibition of gap junction activity by 18-alpha-glycyrrhetinic acid abolished the metabolic interaction between TK(+) and TK(-) cells. This metabolic interaction was also abolished if TK(+) and TK(-) cells were separated by a semipermeable membrane. Surprisingly, the transfection of only one of these two interacting cell types with the Cx43 gene was sufficient to induce a bystander effect, although this effect was weaker than that observed if both TK(+) and TK(-) cells expressed Cx43. Finally, Cx43 expression increased sensitivity to contact inhibition. Overall, our data provide evidence that the restoration of gap junctional communication may potentiate HSV/tk-based cancer treatment and suggest that this strategy may have wider application in cancer therapy.
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PMID:Connexin 43-mediated bystander effect in two rat glioma cell models. 1185 32

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase (TK) gene from herpes simplex virus (HSV) after treatment with the antiviral drug, ganciclovir (GCV). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV-TK-expressing transduced cells in the culture resulted in >90% tumor cell death/stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication (GJIC) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell-cell contact of parental and HSV-TK-transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK+/TK- homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK+/TK - cells showed that the level of the bystander killing obtained was characteristic of the TK-bystander cells. The in vitro findings were confirmed in vivo with GCV-treated homologous and chimeric tumors composed of TK+/TK- cells. Day 21 mean tumor volumes (MTVs) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high-GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin-43, in facilitating the bystander effect was demonstrated with the HT29 low-GJIC cell line. When the TK-nontransduced cell population expressed connexin-43, a better bystander kill was achieved compared to the parental counterpart.
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PMID:Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells. 1191 47

Connexins are proteins that form gap junctions between cells in various mammalian tissues. Because of their role in intercellular communication, connexins are important in the bystander cell death seen in Herpes simplex virus-thymidine kinase (HSV-TK) gene therapy for brain tumors. A selective review of connexin transduction/transfection studies with particular emphasis to central nervous system tumor cells is presented. In addition, specific references to studies with cell types that demonstrate low gap junction intercellular communication are presented. Data are included with the HT-29 colorectal tumor cell line to support the concept that enhancing gap junction protein expression in otherwise low gap junction communicating HT-29 cells increases bystander cell death and reduces tumor burden beyond what might be expected from HSV-TK and ganciclovir (GCV) treatment alone. Maximum in vitro bystander cell death was always produced when GCV treated co-cultures of TK-transduced and non-TK-transduced HT-29 cell lines were also transduced with connexin-43. When connexin was present in only one group of cells in the co-culture, there was more bystander cell death observed with connexin transduced into the non-TK-transduced cells, rather than the TK-transduced cells. The data presented reinforces conclusions made from earlier findings from cell line mixing experiments in which the non-TK-transduced cell population determined the level of bystander cell death (Burrows et al., 2002).
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PMID:Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects. 1264 1

Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We investigated the bystander effect of retrovirus-mediated gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell line (neuro-2a) in vitro and in vivo, and we examined whether the mechanism of bystander effect in neuroblastoma would also depend on connexin-dependent gap junction and/or immune response. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to ganciclovir (GCV) killing. Implanted mixtures of wildtype cells and HSV-TK transduced cells showed a potent bystander effect upon administration of GCV in A/J mice. HSV-TK/GCV system in murine neuroblastoma induced systemic immunity. Immunohistochemical staining showed many CD4+ and CD8+ cell infiltration but did not show anti-connexin 43+ cells. In conclusion, a strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma might be dependent on immune response and/or on other mechanism such as protein phosphorylation or transfer of apoptotic vesicle, rather than connexin-dependent gap junction.
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PMID:Bystander-mediated regression of murine neuroblastoma via retroviral transfer of the HSV-TK gene. 1496 51

The role of gap junctional intercellular communication (GJIC) in bystander killing with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) was evaluated in U251 cells expressing a dominant-negative connexin 43 cDNA (DN14), and in HeLa cells, reportedly devoid of connexin protein. These cell lines both exhibited 0% GJIC when assayed by Lucifer Yellow fluorescent dye microinjection. Bystander cytotoxicity was still apparent in 50:50 cocultures of DN14 and HSV-TK-expressing U251 cells, but not in 50:50 cocultures of HeLa cells. However, the sensitivity of HeLa HSV-TK-expressing cells to GCV decreased nearly 100-fold (IC90=109 microM) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC90=1.2 microM). A more sensitive flow cytometry technique to measure GJIC over 24 h revealed that the DN14 and HeLa cells exhibited detectable levels of communication (29 and 23%, respectively). Transfer of phosphorylated GCV to HeLa bystander cells occurred within 4 h after drug addition, and GCV triphosphate (GCVTP) accumulated to 213+/-84 pmol/10(6) cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (867+/-33 pmol/10(6) cells) compared to 100% cultures of HSV-TK-expressing cells (1773+/-188 pmol/10(6) cells). The half-life of GCVTP in the HSV-TK-expressing cells was approximately four times that measured in the bystander cells (12.3 and 3.1 h, respectively). These data suggest that the lack of bystander cytotoxicity in HeLa cocultures is due to low transfer of phosphorylated GCV and a rapid half-life of GCVTP in the bystander cells. Thus, GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate either bystander cell killing or sparing of HSV-TK-positive cells, depending upon the cell specific drug metabolism.
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PMID:GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity. 1578 60

The thymidine kinase/ganciclovir (TK/GCV) cancer gene therapy approach is based on inducing GCV metabolite cytotoxicity in tumor cells expressing the herpes simplex virus TK gene and exposed to GCV. A bystander effect, mediated by gap junctions, accounts for the transfer of toxic metabolites from TK-expressing cells to neighboring cells. It has been proposed that E-cadherin participates in the formation and function of such gap junctions. In this study we investigate the influence of E-cadherin on TK/GCV suicide therapy with a panel of cellular and in vivo models of pancreatic ductal adenocarcinoma. We observed a strong correlation of E-cadherin expression and the TK/GCV bystander effect, associated with the modulation of gap junction communication and connexin expression or localization. Importantly, the co-expression of TK and E-cadherin genes in the adenoviral vector AdTat8TKIE improved TK/GCV cytotoxicity and triggered a potent antitumoral effect, superior to standard AdTat8TK/GCV in MIAPaCa-2 xenografts. The increased expression of E-cadherin resulted in the reduction of the bcl-2 content. Interestingly, the knockdown of bcl-2 sensitized cells to TK/GCV. Thus, we propose that by restoring E-cadherin in pancreatic tumor cells we will improve TK/GCV therapy, both by enhancing the bystander effect and by facilitating the induction of apoptosis.
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PMID:E-cadherin contributes to the bystander effect of TK/GCV suicide therapy and enhances its antitumoral activity in pancreatic cancer models. 2072 May 74

Medulloblastomas (MBs) are the most common malignant brain tumor in children. The current therapeutic strategies are ineffective against the infiltrative and disseminative nature of MBs in about one-third of patients. Based on studies which have revealed the tumor-tropic characteristic of neural stem cells (NSCs), we used an immortalized neural stem cell line C17.2 as a cellular therapeutic delivery system to evaluate the antitumor effect of herpes simplex virus thymidine kinase gene (HSVtk) on MBs. We first stably transfected the HSVtk gene into C17.2 cells to produce C17.2tk cells, and then mixed C17.2tk with the human MB cell line Daoy at various ratios supplemented with ganciclovir (GCV). Both in vitro and in vivo experiments yielded promising results. Even at a C17.2tk: Daoy ratio as low as 1:16, more than 25% cells were killed in vitro. In vivo co-implantation study showed that when C17.2tk: Daoy ratio was 1:8, tumor growth inhibition was still evident and the mice had significantly prolonged survival. These results might partially be explained by the inherent tumor-tropic properties of NSCs and the bystander effect coupled with expression of connexin-43 (Cx43) between C17.2tk and Daoy cells. Our study clearly showed for the first time that immortalized neural stem cells used as vectors to deliver HSVtk gene therapy have a strong tumoricidal effect on MBs.
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PMID:Bystander effect in suicide gene therapy using immortalized neural stem cells transduced with herpes simplex virus thymidine kinase gene on medulloblastoma regression. 2107 65


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