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)

Replication-defective retroviral vectors were created that contained chimeric genes composed of either the albumin (ALB) or the alpha-fetoprotein (AFP) transcriptional regulatory sequences linked to the coding domain of the thymidine kinase gene from Varicella zoster virus (VZV TK). These viruses were used to infect the human hepatoblastoma cell line, HepG2. Subsequent to infection, the infected cells were single-cell cloned. The level of expression of VZV TK from the chimeric genes correlated with the level of endogenous expression of ALB or AFP in most clones, indicating that the transcription of the chimeric VZV TK gene is controlled in a similar manner to the endogenous ALB or AFP genes, and that sites of viral integration are less important to overall gene expression. Most importantly, as the expression of the endogenous ALB gene was modified, so was expression of VZV TK from the ALB/VZV TK chimeric gene. This demonstrates that retroviruses can deliver a chimeric gene containing tissue-specific transcriptional regulatory sequences that can respond to endogenous cell regulatory signals resulting in regulated gene expression.
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PMID:Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virus-directed enzyme prodrug therapy (VDEPT) and other gene therapy applications. 886 54

We have further characterized the most distal of the three alpha-fetoprotein (AFP) enhancers required for expression of the AFP gene in fetal hepatocytes and yolk sac endodermal cells. Almost total rat AFP enhancer 3 (E3) activity is driven by a 160-bp fragment at -6 kb containing three target regions for nuclear proteins that cooperate to stimulate transcription from the AFP and the thymidine kinase promoters in HepG2 hepatoma cells. Region 1, recently shown to be crucial for correct function of the enhancer in liver of transgenic mice, is recognized by two sets of transcription factors that bind to partly overlapping sites, 1a and 1b, in a noncooperative and nonexclusive manner. Site 1a contains a motif, AGGTCA, which is recognized by chicken ovalbumin upstream promoter transcription factors (COUP-TFs), but not by hepatocyte nuclear factor 4. Hepatocyte nuclear factor 3 (HNF3) and CCAAT/enhancer binding protein (C/EBP), which bind to regions 2 and 3, respectively, are likely responsible for the liver-specific E3 action. They play a key role by acting in synergy. The participation of nuclear receptors such as COUP-TFs, with C/EBP and HNF3, in the tight control of the distal AFP enhancer is a new, and perhaps key, step toward understanding the regulation and function of this enhancer, which may remain active throughout development.
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PMID:Chicken ovalbumin upstream promoter-transcription factor, hepatocyte nuclear factor 3, and CCAAT/enhancer binding protein control the far-upstream enhancer of the rat alpha-fetoprotein gene. 898 20

Many efforts have been devoted to the development of gene therapy for primary liver tumors. This has been hampered by the absence of efficient gene transfer vectors for delivering genes into hepatoma cells in vivo. Also it remains to determine which type of gene has to be used to achieve complete tumor regression. Recent studies have documented improvements obtained using recombinant adenoviral vectors carrying the herpes simplex virus thymidine kinase gene as well as the ability to specifically target gene expression into the tumor cells by using the alpha-fetoprotein gene regulatory sequence. Therefore it seems reasonable to expect the development of clinical protocols in the near future.
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PMID:[Gene therapy of primary cancers of the liver: hopes and realities]. 923 70

About 70% of hepatocellular carcinomas are known to express alpha-fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the alpha-fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.
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PMID:Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice. 939 Nov 23

We have previously reported that a retrovirus vector (LNAF0.3TK) carrying a herpes simplex virus thymidine kinase gene regulated only by the 0.3-kb human alpha-fetoprotein (AFP) promoter provides ganciclovir (GCV)-mediated cytotoxicity in high AFP-producing human hepatoma cells but not in low AFP-producing cells. In the present study, a retrovirus vector (LNAF0.3(E+)TK), in which herpes simplex virus thymidine kinase gene expression is under the control of a human AFP enhancer directly linked to its promoter, was constructed and compared with LNAF0.3(E+)TK. In the intermediate and low AFP-producing human hepatoma cells PLC/PRF/5 and huH1/cl.2, respectively, as well as in the high AFP-producing human hepatoma cells (HepG2), LNAF0.3(E+)TK sensitized these cells to GCV in vitro but did not affect cell growth in nonhepatoma cells (HeLa). In an animal model using athymic mice harboring PLC/PRF/5 cells, GCV treatment resulted in more pronounced growth inhibition in the LNAF0.3(E+)TK virus-infected cells than in the LNAF0.3(E+)TK virus-infected cells. These results indicate that the human AFP enhancer that is directly linked to its promoter involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.
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PMID:Retrovirus-mediated gene therapy for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by human alpha-fetoprotein enhancer directly linked to its promoter. 982 49

Gene therapy using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of the 0.3-kb human alpha-fetoprotein (AFP) gene promoter (LNAF0.3TK virus) in combination with ganciclovir (GCV) treatment was performed in athymic mice harboring AFP-producing HuH-7 human hepatoma cells. GCV treatment resulted in pronounced growth inhibition of the virus-infected HuH-7 xenograft in mice, but did not affect growth of the parental xenograft. These results indicate that the AFP gene promoter sequence allows enough therapeutic gene expression to induce the GCV-mediated cytotoxicity in vivo in AFP-producing human hepatoma cells.
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PMID:Retrovirus-mediated gene therapy for human hepatocellular carcinoma transplanted in athymic mice. 985 81

We previously reported that the retroviral vector (LNAFW0.3TK) expressing the herpes simplex thymidine kinase (HSVtk) gene under the control of the 0.3 kb human alpha-fetoprotein (AFP) promoter provided the ganciclovir (GCV)-mediated cytotoxicity in the high AFP-producing (HuH-7) but not in the low AFP-producing (huH-1/cl.2) human hepatoma cells. In the present study, we constructed the retroviral vector (LNAFM0.3TK) in which the HSVtk gene expression is regulated by the variant-type of the 0.3 kb human AFP promoter with a G-to-A substitution at nucleotide -119, a point mutation responsible for hereditary persistence of human AFP and the vector was applied to three human hepatoma cell lines HuH-7, huH-1/cl.2 and intermediate AFP-producing cells (PLC/PRF/5). By the reporter gene transfection assay, the activity of the variant-type of the promoter was much higher than that of the wild-type of the promoter in both HuH-7 and huH-1/cl.2 cells. Consistent with this, LNAFM0.3TK infection could sensitize huH-1/cl.2 cells, as well as HuH-7 and PLC/PRF/5 cells to GCV, but did not affect cell growth of nonhepatoma cells (HeLa). In addition, the bystander effect was achieved more efficiently by LNAFM0.3TK infection than LNAFW0.3TK infection in HuH-7 cells. These results suggest that the variant-type of the human AFP promoter ensures the therapeutic gene expression in gene therapy particularly for the low AFP-producing hepatoma cells.
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PMID:Utilization of variant-type of human alpha-fetoprotein promoter in gene therapy targeting for hepatocellular carcinoma. 1047 6

Tissue-specific transcriptional regulatory sequences have been inserted in retrovirus vectors for therapeutic gene expression in cancer gene therapy. However, the transcriptional activity of these sequences is generally low, and the proviral DNA integration appears to increase the possibility of genomic DNA alteration in nontarget cells. Therefore, retrovirus-mediated targeted gene transduction into human hepatoma cells was evaluated using transient expression of an ecotropic receptor gene, mouse cationic amino acid transporter-1 (MCAT-1). Two recombinant adenoviruses, AxCAMCAT and AxAFMCAT, carrying the MCAT-1 gene under the control of the CAG and human alpha-fetoprotein (AFP) promoter, respectively, were generated. The preinfection with AxCAMCAT allowed highly efficient ecotropic retroviral infection of human cells. In addition, after AxAFMCAT infection, retroviral infection occurred only in AFP-producing hepatoma cells, resulting in selective cytotoxicity induced by the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system. Transient expression of the MCAT-1 gene under the control of the AFP promoter permits ecotropic retrovirus-mediated gene transduction specifically in AFP-producing human hepatoma cells, resulting in selective induction of the suicide killing effect while the therapeutic gene is driven from ubiquitously expressed promoters.
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PMID:Hepatoma-specific gene therapy through retrovirus-mediated and targeted gene transfer using an adenovirus carrying the ecotropic receptor gene. 1055 7

Transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene followed by the administration of ganciclovir (GCV) into hepatocellular carcinoma (HCC)-derived cell lines either in vitro or transplanted into nude mice has been shown to provide a potential strategy for HSV-tk-based gene therapy of HCC. We report herein an analysis of the antitumoral efficacy of two recombinant adenoviruses (Ads), Ad.CMVtk and Ad.AFPtk, in a relevant model of multifocal hepatic lesions induced in rats by a potent alkylating chemical carcinogen, diethylnitrosamine. Two routes of administration of the Ad were studied: intratumoral and intrahepatic artery injections. Both recombinant Ads, Ad.CMVtk and Ad.AFPtk, express the HSV-tk gene under the control of the early enhancer/promoter cytomegalovirus and alpha-fetoprotein regulatory gene sequences, respectively. The antitumor response was assessed by magnetic resonance imaging and by autopsy and histological analysis following postmortem. Tumor growth cessation was demonstrated by magnetic resonance imaging in large tumor nodules of size 5-8 mm treated by intratumoral administration of 2x10(9) pfu Ad.CMVtk plus i.p. treatment with GCV. We also show an antitumor efficacy in small tumor nodules of size <3 mm treated with 2x10(9) pfu Ad.CMVtk plus GCV by the intrahepatic artery route, albeit associated with an adverse toxicity. In vivo targeting of the HSV-tk gene to diethylnitrosamine-induced HCC cells with the recombinant Ad.AFPtk suppresses the hepatic toxicity in the nontumoral liver. The lower antitumor response would argue for the use of multiple injections of such adenoviral constructs. These observations may lead to potential approaches for designing gene therapy destined for early treatment of dysplastic nodules or advanced HCC in cirrhosis.
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PMID:Evaluation of HSV-tk gene therapy in a rat model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes. 1070 15

Gene therapy for hepatocellular carcinoma (HCC) has shown some promise, but its evaluation requires relevant experimental models. With this aim, we present an evaluation of the interest of using the woodchuck model of HCC to assess in vivo gene transfer efficiency. We tested the transduction efficacy of the adenoviral vectors directing lacZ gene product expression under the control of the cytomegalovirus and alpha-fetoprotein (AFP) regulatory sequences. We have also investigated whether an adenoviral cytomegalovirus-thymidine kinase (Tk) vector might induce an antitumoral effect in this model. Our results demonstrate that with direct intratumoral and intrahepatic arterial injections, transduction of a significant proportion of tumor cells occurred even in large HCC nodules. Furthermore, due to intra-arterial anastomoses, direct intratumoral injection led to transduction of some noninjected HCC nodules. Moreover, direct intratumoral injection of a herpes simplex virus-1 Tk-encoding vector induced, on ganciclovir administration, a significant antitumoral effect in the two animals evaluated. However, in one animal, massive hepatic failure occurred due to Tk expression in nontumor cells. These results emphasize the need to target the expression of the Tk gene to tumor cells using a hepatoma-specific promoter such as AFP promoter. However, we showed that, in vivo, lacZ expression as driven by the AFP promoter was extremely low, thus emphasizing some potential pitfalls when using this approach. Altogether, our data stress the need to test gene therapy-based strategies in such in vivo animal models of HCC and evaluate gene transduction, expression, and biological activity, as well as its potential toxicity.
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PMID:Transduction efficacy, antitumoral effect, and toxicity of adenovirus-mediated herpes simplex virus thymidine kinase/ ganciclovir therapy of hepatocellular carcinoma: the woodchuck animal model. 1083 Jul 12


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