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)

Human immunodeficiency virus (HIV) resistance to the nonnucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant HIV generally emerges in patients only after more-prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, we have utilized replication-defective HIV encoding reporter genes. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of a cell line derived from one such cell has demonstrated decreased accumulation of AZT triphosphate, increased phosphorylation of thymidine to thymidine triphosphate, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line. Attempts to detect and characterize the mechanisms responsible for early viral infection after initiation of AZT therapy may result in the development of new strategies for prolonged suppression of viral infection prior to the emergence of drug-resistant virus.
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PMID:Sanctuary growth of human immunodeficiency virus in the presence of 3'-azido-3'-deoxythymidine. 785 95

Moloney murine leukemia virua (MoMuLV)-derived retroviral vectors were engineered to express human immunodeficiency virus type 1 (HIV-1) packaging (psi) signal and Rev response element (RRE) sequences in either sense or antisense orientation. The RRE sequences were expressed under the control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter fused to the HIV-1 trans-activation-responsive (TAR) element, while the psi signal sequences were expressed under control of the HSV tk promoter. Both RRE and psi signal sequences were expressed as part of the 3' untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants, harboring proviral vector DNA expressing one to two copies of HIV-1 RRE and psi signal in either antisense or sense orientation, were each tested for their susceptibility to HIV-1 infection. Compared to the results obtained with the control cells lacking any of the test DNA sequences, the rate of HIV-1 production remained unaltered in RRE1+ (sense RNA containing a single copy of RRE) RNA-containing cells, whereas it was delayed in cells expressing both RRE2+ (sense RNA containing two copies of RRE) and RRE1- (antisense RNA containing a single copy of RRE) RNA-expressing cells. In cells expressing HIV-1 psi signal, HIV-1 production remained unaltered in psi + RNA-expressing cells, whereas it was delayed by up to 30 days in psi - RNA-expressing cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of HIV-1 multiplication in a human CD4+ lymphocytic cell line expressing antisense and sense RNA molecules containing HIV-1 packaging signal and Rev response element(s). 791 62

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are two members of a family of carcinogenic heterocyclic amines (HAs) found in cooked meats that form DNA adducts after activation to N-acetoxy derivatives. The ability of IQ- and PhIP-DNA adducts to inhibit gene expression was investigated using a human growth hormone (hGH) reporter gene in a pUC12-based mammalian expression vector under the control of either the herpes simplex virus-1 thymidine kinase promoter or the human immunodeficiency virus-1 long terminal repeat. The plasmids were treated in vitro with 0, 5, 10, or 40 microM N-hydroxy-IQ or N-hydroxy-PhIP in the presence of a 10-fold molar excess of acetic anhydride to generate the N-acetoxy derivatives in situ. The adduct levels in the plasmids were quantitated by the 32P-postlabeling method. The adducted (and control) plasmids were each transfected into repair-deficient or -proficient Chinese hamster ovary cells, and expression of hGH was measured by immunoassay of growth hormone secreted into the cell medium. The results showed that IQ- and PhIP-DNA adducts inhibited gene expression in both plasmids and that the degree of inhibition of hGH production was proportional to the levels of IQ- and PhIP-DNA adducts. The degree of inhibition, however, was independent of the promoter, despite the differences in the strengths of the two promoters to drive hGH production. Repair capacity influenced the extent of inhibition of gene expression by HA adducts since, in general, fewer adducts were needed to inhibit reporter gene expression in repair-deficient cells than in repair-proficient cells. In both cell lines, DNA adducts of PhIP appeared to be more potent in inhibiting hGH expression than adducts of IQ. Whether alteration of gene expression by HA adducts plays a role in the carcinogenicity of these compounds deserves further study.
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PMID:Inhibition of plasmid reporter gene expression in CHO cells by DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. 818 27

Inhibitors of the ribonucleotide reductase of herpes simplex viruses (HSV) potentiate the activity of acyclovir in vitro and in animal studies. In addition, the combination of the ribonucleotide reductase inhibitor 348U87 and acyclovir has synergistic therapeutic effects against infections in mice due to thymidine kinase-deficient, thymidine kinase-altered, and DNA polymerase mutants of HSV. We performed a pilot study of topical combination therapy with 348U87 (3%) and acyclovir (5%) cream for acyclovir-resistant, anogenital HSV infections in ten human immunodeficiency virus (HIV)-infected patients. Our results, with lack of complete reepitheliazation of lesions in all patients and poor virologic response, suggest that this therapy is unlikely to be useful for this indication.
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PMID:Potential for combined therapy with 348U87, a ribonucleotide reductase inhibitor, and acyclovir as treatment for acyclovir-resistant herpes simplex virus infection. 824 82

The identification of human immunodeficiency virus (HIV) as the etiologic agent of AIDS has led to the proposal of novel intervention strategies to block HIV infection and viral replication or eliminate HIV-infected cells. We have produced recombinant retroviruses for a molecular ablation system, whereby a toxin gene can be delivered to hematopoietic cells for the specific elimination of HIV Tat-expressing cells. For this cell-specific ablation, we have coupled the conditional toxin herpes simplex virus type 1 thymidine kinase (tk) gene to the HIV-2 promoter and Tat responsive region (TAR) in order that transcriptional activity be under the absolute control of HIV and simian immunodeficiency virus Tat trans-activator proteins. Since the HIV-2 promoter has a considerable level of basal expression in the absence of Tat, we constructed a number of modifications in the HIV-2 promoter to minimize the risk of cytotoxicity to cells not containing HIV Tat. We demonstrate that certain promoter modifications reduce basal transcription while maintaining high trans-activated levels of expression when transfected or transduced by retroviral vectors into several different cell lines. In mouse and human cells infected with HIV-2 tk retroviruses, we show that Tat-induced expression from the HIV-2 promoter results in differential ablation and a massive reduction in Tat-positive cells after ganciclovir treatment. Thus, the retroviruses produced in these studies may be applicable to HIV ablative therapy.
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PMID:Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses. 827 95

9-(cis-3-Hydroxymethyl-2-methylenecyclobutyl)guanine (3b) and 9-(3-methylene-trans-2-hydroxymethylcyclobutyl)guanine (4b) were prepared from N2-isobutyryl-9-[trans-trans-2,3-bis(hydroxymethyl)cyclobutyl]guanine (2f) or 2,3-bis(hydroxymethyl)-1-cyclobutanol (7b). Carbocyclic oxetanocin analogues (A, 1d; G, 2d) and related compounds including 4b were assayed against a broad variety of viruses. It appeared that the activity of 2d against herpes simplex virus (HSV) and varicella-zoster virus (VZV) at least partially depends on phosphorylation by the virus-induced thymidine kinase (TK). Although 1d and 2d are inhibitory to the replication of human immunodeficiency virus (HIV), they are quite toxic to proliferating human T-lymphocytes.
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PMID:Synthesis and antiviral activity of carbocyclic oxetanocin analogues (C-OXT-A, C-OXT-G) and related compounds. II. 838 94

A palindromic element (site B) located between bases -328 and -347 (relative to the start site of transcription) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) was shown in a gel mobility shift assay to bind the human retinoic acid receptors (RARs). Greatly enhanced binding to this site was observed in the presence of both RAR and the retinoid X receptor. Retinoic acid responsiveness in F9 cells could be conferred on a thymidine kinase promoter by the presence of single or multiple copies of site B and responsiveness was abolished when this sequence was mutated to a form that could not bind RARs. However, the presence of this sequence did not render the HIV-1 LTR responsive to retinoic acid in F9 cells.
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PMID:A palindromic element in the human immunodeficiency virus long terminal repeat binds retinoic acid receptors and can confer retinoic acid responsiveness on a heterologous promoter. 838 9

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a protein with a predicted molecular weight of 51,540 presenting amino acid sequence homology with the immediate-early regulatory protein ICP27 of herpes simplex virus type 1. To investigate the regulatory properties of the ORF4 gene product, we performed a series of transient expression assays in Vero cells, using a plasmid expressing ORF4 as effector and several VZV genes and heterologous genes as targets. The VZV target plasmids contained promoter/regulatory regions from genes belonging to the three putative VZV kinetic classes fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target plasmids consisted of promoter/regulatory regions of human cytomegalovirus, Rous sarcoma virus, and human immunodeficiency virus type 1 fused to the reporter gene. These experiments demonstrated that the ORF4 gene product activated expression of ORF62 in a dose-dependent fashion but had no effect on the expression of the three other putative immediate-early genes (ORF4, ORF61, and ORF63). When various amounts of ORF4 were transfected in the presence of early gene promoters, dose-dependent transactivation was evidenced with the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters; interestingly, little activity was detected with the promoter of the DNA polymerase gene (ORF28). No activation of late gene expression, represented by the glycoprotein I and glycoprotein II genes, was seen even over a wide range of concentrations of input ORF4 plasmid. Expression of pCMVCAT, pRSVCAT, and pHIVCAT was also stimulated by the ORF4 gene product. CAT mRNA analysis showed that activation of VZV target promoters occurs at the transcriptional and/or posttranscriptional level.
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PMID:Characterization of the regulatory functions of varicella-zoster virus open reading frame 4 gene product. 838 35

We have previously shown that the Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a modular transcriptional activator that can be targeted upstream of either a synthetic promoter or the intact HIV promoter to activate transcription. This activation was shown to be largely dependent on the presence of consensus binding sites for the cellular transcription factor Sp1. Since the use of heterologous promoters may provide further insight into Tat-mediated transactivation, we have analyzed the transactivation of the thymidine kinase promoter of herpes simplex virus by Tat and by the acidic transcriptional transactivator VP16. The effects of mutations of defined upstream promoter elements show that Tat transactivation is dependent on Sp1 binding sites in a site-specific manner. In contrast, transactivation by the acidic transactivator VP16 is completely independent of any of the defined promoter elements upstream of the TATA box. These results suggest that Tat and the classically defined modular acidic transcriptional activators have different modes of transactivation. In addition, the substitution of the HIV-1 TATA box for the thymidine kinase TATA box substantially increases Tat transactivation, indicating that Tat transactivation may also ultimately involve TATA box-associated cellular transcription factors.
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PMID:Activation of a heterologous promoter by human immunodeficiency virus type 1 Tat requires Sp1 and is distinct from the mode of activation by acidic transcriptional activators. 841 86

The T-cell line Jurkat E6-1 was rendered resistant to zidovudine (AZT) in vitro by exposure to low but gradually increased concentrations of the drug. Biochemical pharmacology studies of [3H]AZT in the AZT-resistant T-cell lines showed a significant reduction of AZT phosphorylation to the mono-, di-, and triphosphate anabolites. Peripheral blood mononuclear cells (PBMCs) from pediatric patients with human immunodeficiency virus type 1 (HIV-1) infection showed a similar pattern of decreased AZT anabolism. Enzymatic studies with purified thymidine kinase (TK) preparations from these cell lines showed a gradual decline in Vmax related to their level of resistance to AZT. The Jurkat/AZT-20 and Jurkat/AZT-100 cells were studied in greater detail with reverse transcriptase/polymerase chain reaction (RT/PCR) cloned probes to determine possible molecular mechanisms of resistance to AZT. TK mRNA was significantly decreased (approximately 5- to 10-fold) in the AZT-resistant T-cell lines. Southern blot analyses indicated that there were no major rearrangements or deletions of the TK gene, but the 5' end of the gene in the AZT-resistant cells is highly methylated when compared to wild-type cells. No apparent differences were seen in thymidylate kinase (dTMPk) mRNA levels in the same T-cell lines. Thus the decreased expression of TK mRNA and resultant TK enzymatic activity is responsible for the observed reduction in the AZT anabolism in the resistant T-cell lines. Decreased T-cell TK activity could allow wild-type, AZT-sensitive HIV-1 to replicate in the presence of subinhibitory AZT triphosphate (AZT-TP) cellular concentrations enabling a genetic variant with drug resistance to emerge and outgrow the AZT-sensitive, wild-type virus.
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PMID:Development of zidovudine (AZT) resistance in Jurkat T cells is associated with decreased expression of the thymidine kinase (TK) gene and hypermethylation of the 5' end of human TK gene. 854 39


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