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)

An approach involving retroviral-mediated gene therapy for the treatment of neoplastic disease is described. This therapeutic approach is called "virus-directed enzyme/prodrug therapy" (VDEPT). The VDEPT approach exploits the transcriptional differences between normal and neoplastic cells to achieve selective killing of neoplastic cells. We now describe development of the VDEPT approach for the treatment of hepatocellular carcinoma. Replication-defective, amphotrophic retroviruses were constructed containing a chimeric varicella-zoster virus thymidine kinase (VZV TK) gene that is transcriptionally regulated by either the hepatoma-associated alpha-fetoprotein or liver-associated albumin transcriptional regulatory sequences. Subsequent to retroviral infection, expression of VZV TK was limited to either alpha-fetoprotein- or albumin-positive cells, respectively. VZV TK metabolically activated the nontoxic prodrug 6-methoxypurine arabinonucleoside (araM), ultimately leading to the formation of the cytotoxic anabolite adenine arabinonucleoside triphosphate (araATP). Cells that selectively expressed VZV TK became selectively sensitive to araM due to the VZV TK-dependent anabolism of araM to araATP. Hence, these retroviral-delivered chimeric genes generated tissue-specific expression of VZV TK, tissue-specific anabolism of araM to araATP, and tissue-specific cytotoxicity due to araM exposure. By utilizing such retroviral vectors, araM was anabolized to araATP in hepatoma cells, producing a selective cytotoxic effect.
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PMID:Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy. 165 55

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). The administration of 3'-MeDAB in combination with 1-(2-tetrahydrofuryl)-5-fluorouracil and uracil (UFT) delayed the appearance of oval cells and the formation of hyperplastic nodules, which were observed in the liver from 3 and 5 weeks, respectively, after the onset of 3'-MeDAB feeding, and also delayed the transient increase of serum alpha-fetoprotein level, which transiently peaked at 5 weeks, and completely suppressed the transient increase of tissue thymidylate synthetase activity, but not thymidine kinase, which were induced by 3'-MeDAB at 5 weeks, and finally reduced markedly the incidence of hepatocarcinomas. These results indicate that the suppression of de novo synthesis in pyrimidine metabolism prevents hepatocarcinogenesis.
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PMID:Inhibition by 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil of hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene in rats. 171 76

The upstream transcription control region of the rat alpha-fetoprotein (AFP) gene was analyzed using transient expression of CAT genes in HepG2 cells which express the gene; H4C3 cells which repress the AFP gene but express the albumin gene; and four nonexpressing cell lines. Deletion analysis based on the DNA sequence resolved three upstream enhancers corresponding to the mouse AFP enhancers, but showed additional weak effects from flanking sequences. Quantitative experiments demonstrated that the three enhancers were additive when acting through a single promoter and did not confirm the presence of a distal upstream repressor. All three enhancers stimulated the AFP, albumin, or thymidine kinase (tk) promoter in HepG2, but only the tk and albumin promoters in H4C3. Deletion of a proximal repressor region near the AFP promoter allowed expression in H4C3 cells with the AFP promoter. Thus, the liver-specific developmental repressor is near the AFP promoter, and H4C3 cells provide an in vitro system for analysis of this repressor in transfection assays. The repressor region also blocked expression of the SV40 enhancer through the AFP promoter in hepatic and nonhepatic cell lines, but when this enhancer was combined with an AFP promoter from which the repressor region was deleted, the combination showed expression in all six cell lines studied. AFP expression results from a combination of enhancer, promoter, and repressor activities, and the repressor is functional with a heterologous enhancer in a variety of cells.
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PMID:Enhancer, repressor, and promoter specificities combine to regulate the rat alpha-fetoprotein gene. 171 40

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). In the present study, we investigated serum levels of alpha-fetoprotein (AFP) and thymidine kinase (TK), DNA-synthesizing enzyme in the salvage pathway, and tissue TK and its isozyme activities in the liver of rats treated with 3'-MeDAB. Serum TK activities rose abruptly right after the onset of 3'-MeDAB treatment, peaking after one week and then gradually decreasing. At 3 weeks, though serum TK was decreasing, serum AFP and tissue TK began to increase, and oval cells appeared in the liver. At 5 weeks, though serum TK reached a nadir, serum AFP and tissue TK formed transient peaks, and oval cells occupied a major part of the hepatic lobules with hyperplastic nodules. Thereafter, serum TK continued to increase, and serum AFP and tissue TK, after transiently decreasing, re-increased; at 20 weeks, each value was at high level, and mixed type hepatocarcinoma was observed. The liver TK isozymes were separated into 3 types by DEAE-cellulose column chromatography. A 3'-MeDAB induced a remarkable increase in activity of cytosolic and fetal type isozyme in non-tumorous regions of livers at 5 weeks and tumorous regions at 20 weeks. These results indicate that biochemical changes in 3'-MeDAB-treated rat liver may provide a valuable insight into two step process in hepatocarcinogenesis.
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PMID:[Thymidine kinase activities in sera and liver tissues during hepatocarcinogenesis in rats treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)]. 207 89

Three enhancer elements spanning a distance of 7 kilobases have been found at the 5' end of the alpha-fetoprotein (AFP) gene. These elements were identified by transient expression assay after the introduction of a modified mouse AFP gene with variable amounts of 5' flanking sequence into a human hepatoma cell line, Hep G2. These regulatory elements function in a position-independent and orientation-independent manner that is typical of enhancers. All three elements will stimulate transcription from the promoter of the herpes simplex virus thymidine kinase gene. In Hep G2 cells, transcriptional activation from the heterologous promoter was approximately 25- to 50-fold higher than the basal levels obtained in the absence of AFP enhancer elements. In HeLa cells, the increase in thymidine kinase gene transcription varied from 6- to 14-fold, indicating that the enhancer elements exhibit some cell type specificity. Deletion analysis of the region proximal to the AFP transcription initiation site identified an essential region between 85 and 52 bases upstream of the site of initiation of transcription whose removal resulted in almost complete extinction of transcriptional activity. This region, which has been shown to be dispensable for transcription in HeLa cells, defines a second tissue-specific regulatory region in the gene.
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PMID:Multiple regulatory elements in the intergenic region between the alpha-fetoprotein and albumin genes. 243 Dec 69

Cytoplasmic hybrid (cybrid) cell lines were formed by fusing whole cells of rat yolk sac tumor cell line (EST-II) with cytoplasts of mouse fibroblastic cell line (B-82cap), a variant of mouse L cell line that is deficient in thymidine kinase (TK-) and resistant to chloramphenicol (capr). The cybrid cell line with the nucleus of EST-II and cytoplasma of B-82cap was successfully obtained using double selection with HAT and chloramphenicol-containing medium. The cybrid's ability to synthesize proteins such as albumin, alpha-fetoprotein, gamma-EST, and gamma-GTP was found to be approximately one-fourth that of the nuclear donor, EST-II, at a early time of growth in culture, but this was followed by a gradual increase during the period of observation. The nude mice undergoing subcutaneous transplantation of 1 X 10(6) cells of EST-II and cybrid were killed by the tumor growth, with the survival time being 56 +/- 11 and 105 +/- 25 days, respectively. The histologic findings of cybrid tumor closely resembled those of the nuclear donor, EST-II. These facts suggest that factors from both the nucleus and cytoplasma will be able to affect the gene expression of the cybrid cell line.
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PMID:Protein synthesis and tumorigenicity of the cytoplasmic hybrid between rat yolk sac tumor and mouse fibroblastic cell line. 658 79

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and transcriptionally silent in adult tissues, but can be abnormally reactivated in hepatocellular carcinoma (HCC). We linked 7.6 kb of 5'-flanking DNA from the mouse AFP gene to the herpes simplex virus (HSV) thymidine kinase gene (tk), and a line of transgenic mice was produced that expressed TK in a pattern similar to endogenous AFP. When these AFP/tk transgenic mice were crossed to another transgenic line that develops multifocal HCC due to expression of a SV40 large T-antigen transgene under regulation of the albumin promoter/enhancer complex, a significant delay of tumor progression could be achieved by administration of ganciclovir (GCV), a cytotoxic compound that is a substrate for phosphorylation by viral, but not mammalian, TK. Control animals carrying only the tk gene were unaffected by GCV treatment. These results illustrate the feasibility of prophylactic gene therapy for ablation of cancer, utilizing a strategy in which the tk gene is regulated by a promoter expected to be active only in tumor cells.
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PMID:Delayed morbidity and mortality of albumin/SV40 T-antigen transgenic mice after insertion of an alpha-fetoprotein/herpes virus thymidine kinase transgene and treatment with ganciclovir. 751 48

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but is reactivated in hepatocellular carcinoma. It has been shown that the positive and negative transcriptionally regulatory elements of the human AFP gene, which play an important role in its developmental regulation, exist over the quite extended region (4 kb). We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of the 0.3-kb human AFP gene promoter and inserted it into a retroviral vector. When AFP-producing hepatoma cells were infected with this recombinant retrovirus (LNAF0.3TK virus), the cells expressed HSV-tk gene and exhibited increased sensitivity to ganciclovir parallel with the ability of AFP production. On the other hand, the retroviral infection had little effect on ganciclovir-mediated cytotoxicity in AFP-nonproducing hepatoma or non-hepatoma cells. Moreover, the addition of dexamethasone increased the cytotoxicity of aciclovir to the virus-infected, AFP-producing cells through a glucocorticoid-responsive element in the AFP promoter, although aciclovir, by itself, had little cytotoxicity. These results demonstrate that the AFP promoter sequence alone can provide enough tumor-specific activity for therapeutic gene expression and induce selective growth inhibition by ganciclovir in the virus-infected, AFP-producing human hepatoma cells. In addition, it is possible that expression of the therapeutic gene is modulated by administration of dexamethasone or other agents that alter AFP promoter activity after gene transduction.
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PMID:Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter. 754 12

We have investigated whether adenovirally mediated gene transfer of the herpes simplex thymidine kinase gene to human hepatocellular carcinoma (HCC) cell lines can sensitize these cells to the prodrug ganciclovir and thereby provide a therapeutic option for this intractable cancer. Two replication-deficient adenoviruses encoding for the herpes simplex virus type-1 (HSV) thymidine kinase (TK) gene were generated in which expression of TK is under the control of either the human cytomegalovirus immediate early promoter (CMV) or the human alpha-fetoprotein (AFP) promoter/enhancer. We demonstrate that the combination of adenovirally mediated TK gene transfer and ganciclovir treatment effectively inhibits proliferation and causes cell death of HCC cells in vitro and that in vivo TK gene transfer and ganciclovir treatment inhibits hepatocellular tumor growth in a mouse model of this cancer. Furthermore, we show that expression of the TK gene can be restricted to those HCCs that express the tumor marker AFP through the incorporation of the AFP enhancer/promoter within an adenoviral vector.
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PMID:Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene. 852 62

We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms.
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PMID:Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. 867 52


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