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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The composition and response of the retinoid signaling pathway in a human cell line (CC-1), representative of a low grade cervical carcinoma, were evaluated. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated expression of cytoplasmic retinol binding protein, CRBPI, cytoplasmic retinoic acid binding protein, CRABPII, and nuclear retinoic acid receptors, RAR alpha, RARgamma, RXR alpha, and RXRbeta, but not CRABPI or RARbeta. This pattern is similar to that of the ectocervix. Activation of endogenous nuclear receptors was evaluated in a reporter subline of CC-1, called CC-B, containing a reporter gene controlled by a retinoic acid responsive element (RARE) and thymidine kinase promoter. Retinoid treatment of CC-B resulted in dose-dependent increases in reporter gene expression. Retinoids inhibited growth at concentrations greater than 100 nM. 9-cis retinoic acid (1 nM) significantly stimulated growth. Immunohistochemical analysis of CC-B organotypic cultures demonstrated a high level of epidermal growth factor receptor (EGF-R) expression that was decreased by retinoids. The degree of RARE transactivation induced by retinoids significantly correlated with the degree of inhibition of growth (R = -0.96) and EGF-R expression (R = -0.92). The dose-dependent and retinoid-specific responses of CC-1 at the molecular and biological levels demonstrate the utility of this reporter cell line for evaluation of retinoid activities.
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PMID:Biological assay for activity and molecular mechanism of retinoids in cervical tumor cells. 923 31

Previously, we described a nonviral cytoplasmic gene therapy vector system based on the T7 autogene concept. This system has been shown to achieve rapid and high levels of gene expression in a variety of animal cells and tissues. To test the utility of the system in vivo tumor ablation, a T7 cancer gene therapy plasmid vector, pT7T7/T7TK, was constructed. This nonviral vector contains a T7 autogene, T7T7, and a human herpes simplex virus thymidine kinase (HSV-TK) gene driven by a second T7 promoter (T7TK). When co-transfected with T7 RNA polymerase (T7 RNAP) into cultured human osteosarcoma 143B cells, abut 10-20% of the cells were found to express HSV-TK, and more than 90% of the cells were killed in the presence of 1 microM ganciclovir (GCV) within 4 days after DNA transfection. The increase in killing above the transfection frequency is due to a "bystander" effect among transfected and untransfected 143B cells. Direct injections of pT7T7/T7TK into 143B tumors grown in nude mice resulted in TK gene expression in tumor cells located near the injection sites as revealed by the immunohistochemical staining. Repeated tumor injections of the pT7T7/T7TK vector and intraperitoneal (i.p.) injections of GCV resulted in inhibition of tumor growth and in tumor shrinkage in 6 out of 10 treated nude mice. Three of those six tumors fully regressed shortly after the end of the GCV injections. All of the full tumor regressions were found to be permanent and no apparent tumor relapses were observed for the rest of the lives of the treated nude mice after the initial tumor ablations. These results, combined with the nonviral and rapid cytoplasmic gene expression features, suggest that the T7 vector may be a good candidate for cancer gene therapy and other medical and biological applications.
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PMID:Cancer gene therapy by direct tumor injections of a nonviral T7 vector encoding a thymidine kinase gene. 955 20

We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy.
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PMID:L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity? 989 5

Human salivary gland adenocarcinoma cells (HSG) express nuclear receptors, all-trans-retinoic acid (at-RA) receptors (RARs), and retinoid X/9-cis-retinoic acid (9-c-RA) receptors (RXRs). In order to investigate whether the endogenous RARs or RXRs of HSG cells can induce transcription activation, the thymidine kinase promoter (TK)-driven luciferase reporter gene containing the retinoic acid response element (RARE), of RARbeta, betaRARE2-TK-Luc, was transfected into HSG cells and ligand-dependent transcription activation was examined. Luciferase activity of cell lysate increased by the treatment with either at-RA or 9-c-RA. Co-transfection of RARalpha and (or) RXRalpha-expression plasmids with the reporter gene enhanced the luciferase activity, suggesting that endogenous RARs and RXRs work as ligand-dependent transfactors in HSG cells. Reverse transcriptase - polymerase chain reaction analysis revealed that HSG cells express chicken ovalbumin upstream promoter - transcription factor I (COUP-TFI). Co-transfection of COUP-TFI-expression plasmid suppressed the at-RA-induced transcription activation of the reporter gene. Similar results were shown using a chromatin-integrated reporter gene system, using a stably transfected beta-RARE2-TK-beta-galactosidase (beta-Gal) reporter gene. The at-RA-dependent increase in the beta-Gal expression was completely inhibited by COUP-TFI. The transfection of antisense oligonucleotide of COUP-TFI squelched the RA-dependent growth inhibition induced by RAR-RXR heterodimers. Conclusively, RARs and RXRs of HSG cells are functional and play roles as transactivators in at-RA-sensitive processes such as the proliferation or differentiation of cells. COUP-TFI very likely regulates these processes by repressing the functions of these transactivators.
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PMID:Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG. 1066 29

There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.
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PMID:Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal. 1071 33

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.
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PMID:Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection. 1079 Jan 10

The novel antitumor compound NC-190 strongly inhibited the growth of FM3A cells with an IC50 of 0.019 microg/ml (0.042 microM) when cultured with NC-190 for 48 h. NC-190 potently suppressed DNA synthesis, with 90% inhibition observed at 0.1 microg/ml of NC-190. RNA and protein syntheses were also suppressed under the same conditions, but to a lesser extent. We then measured the cellular enzymatic activities of DNA polymerase alpha, RNA polymerase, thymidine kinase, thymidylate synthase and Leu-tRNA synthetase of FM3A cells cultured with or without NC-190. Of these 5 enzymes, the activity of thymidine kinase was most strongly suppressed by NC-190, by 77%. Although NC-190 did not directly inhibit the activitiy of thymidine kinase in a cell-free system, expression of mRNA of thymidine kinase was suppressed by 75% in NC-190-treated cells. These results indicate that NC-190 can suppress the expression of the gene for thymidine kinase and the inhibition of thymidine kinase contributes to the inhibition of cell growth by NC-190 together with the inhibition of topoisomerase II.
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PMID:The topoisomerase II-inhibitor NC-190 reduces the level of thymidine kinase mRNA in murine tumor cells. 1463 16

Novel N-1-sulfonylpyrimidine derivatives have a strong antiproliferative activity and an ability to induce apoptosis in treated tumor cells. The purpose of this study was to elucidate the effects of two N-1-sulfonylpyrimidine nucleobases on catalytic activity of tumor cells' enzymes involved in DNA and RNA synthesis, and in de novo and salvage pyrimidine and purine syntheses. Investigations were performed in vitro on colon carcinoma cells (Caco2). The biosynthetic activity of the tumor cells' enzymes was determined using sensitive radio-assays. Enzyme activity in treated cells was calculated relative to untreated control cells. Both of the investigated compounds, 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) inhibited activities of specific enzymes involved in nucleic acid synthesis. BMsU strongly inhibited activities of DNA polymerase alpha (53%), thymidine kinase (68%), thymidilate synthase (43%), and ribonucleotide reductase (46%). De novo biosynthesis of pyrimidine and purine was reduced by 20%. TsC was able to inhibit RNA polymerase (37%), orotate phosphoribosyltransferase (39%), uridine kinase (44%), ribonucleotid reductase (47%), and de novo purine synthesis (61%). Antitumor activity of 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) is closely associated with their inhibitory activity on enzymes that play an important role in the metabolism of tumor cells.
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PMID:Metabolic effects of novel N-1-sulfonylpyrimidine derivatives on human colon carcinoma cells. 1591 14

The aim of the present study was to estimate the expression of mRNA, specific for thymidine kinase 1 (TK1), deoxycytidine kinase (dCK), and thymidine phosphorylase (dThdPase), i.e. enzymes involved in pyrimidine and purine metabolism in human papillary thyroid carcinoma (PTC) tissue. Additionally, the expression of dCK was estimated, in medullary thyroid carcinoma (MTC). For control, the RNA expression levels for all the enzymes were measured in macroscopically unchanged thyroid tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) and densitometry were employed for mRNA expression measurements, with the beta-actin gene as a control housekeeping gene. The levels of mRNA expression for TK1, dCK and dThdPase in human PTC, as well as mRNA expression for dCK in MTC, were significantly higher than mRNA expressions for those enzymes found in macroscopically unchanged thyroid tissue. It is concluded that an increased expression of mRNA, specific for TK1, dCK and dThdPase, may be involved in carcinogenic processes in the human thyroid.
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PMID:Increased expression of mRNA specific for thymidine kinase, deoxycytidine kinase or thymidine phosphorylase in human papillary thyroid carcinoma. 1597 30

Duck enteritis virus (DEV) is classified to the family Herpesviridae, but has not been grouped into any genus so far. Four overlapped fragments were amplified from the DEV genome with polymerase chain reaction (PCR). The assembled length of the four fragments was 6,202 bp, which contained the genes encoding unique long (UL) 24, thymidine kinase (TK) and glycoprotein H (gH) proteins. The UL24 overlapped with TK by 64 nucleotides (nt), in a head-to-head transcription orientation, and the TK and gH had the same transcription orientation. The comparison of amino acid sequences of these 3 deduced DEV proteins with other 12 alphaherpesviruses displayed 5 highly conserved sites in the UL24, as well as another 5 consensus regions in the TK and 4 consensus regions in the gH. The RNA polymerase II transcriptional control elements were identified in all the UL24, TK and gH of DEV. These elements included core promoters, TATA motifs and polyadenylation sites. Phylogenetic analysis for the genetic classification of DEV in the Alphaherpesvirinae subfamily with other 12 alphaherpesviruses was computed. The result showed that DEV was more closely related to avian herpesviruses, except infectious laryngotracheitis virus (ILTV), than to other alphaherpesviruses. Conclusively, according to the phylogenesis-based analysis and the homology comparison of functional domains of UL24, TK and gH, DEV should be classified to a separate genus of the Alphaherpesvirinae subfamily in the family Herpesviridae.
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PMID:Characterization of the genes encoding UL24, TK and gH proteins from duck enteritis virus (DEV): a proof for the classification of DEV. 1697 38


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