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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/T)GCA, was defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oliginucleotides defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrated that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repression was abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.
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PMID:Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor. 875

Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1. Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background. Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation.
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PMID:Role of zinc-finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human synaptobrevin II gene. 877 32

Increases in transforming growth factor beta1 (TGF-beta1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-beta1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-beta1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-beta1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-beta1 promoter regions each containing an Egr-1 consensus site were shown to be important for IE2-induced transactivation in a cell type that displayed greatly reduced nonspecific activity. Furthermore, transfer of an Egr-l site from between positions -125 and -98, but not point mutant versions of this site, to a heterologous promoter also conveyed IE2 responsiveness. Addition of an IE2 expression vector or use of the U373 A45 astrocytoma cell line expressing IE2 also produced synergistic stimulation of GAL4-Egr-l-mediated activation of a target promoter containing GAL4 binding sites. The 80-kDa IE2 protein present in A45 cells proved to selectively bind to glutathione S-transferase (GST)-Egr-1 beads. The results of in vitro protein binding assays also revealed that an intact in vitro-translated IE2 protein bound directly to the GST-Egr-1 fusion protein through the zinc finger domain of the Egr-1 protein and that this binding activity was abolished by deletion of parts of the zinc finger DNA-binding domain. Similarly, the Egr-1 protein was found to associate preferentially with a small region within the C-terminal half of the IE2 protein adjacent to the DNA-binding and dimerization domains that are important for both transactivation and downregulation. We conclude from these observations that IE2 may regulate transcription of the TGF-beta1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA-binding protein.
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PMID:The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor beta1 gene through an Egr-1 binding site. 879 51

Rat glutathione transferase P (GST-P) is expressed at low levels in the normal liver but becomes highly expressed in hyperplastic nodules and in hepatocellular carcinomas during chemical hepatocarcinogenesis. To understand the regulation mechanisms of this gene, we have characterized the 5'-flanking region and have found that GST-P gene is regulated by at least two elements: one is a strong enhancer and the other is a silencer. GST-P enhancer I (GPEI), located at -2.5 Kb, consists of two TPA-responsive element (TRE)-like sequences that are palindromically oriented with 3 bp in between. It is well known that TRE is activated by two nuclear oncogenes, c-Jun and c-Fos. Although GPEI is trans-activated by these oncogenes, it is also active in F9 embryonal carcinoma cells that lack c-Jun protein, suggesting that it can function with some trans-activator other than AP-1 (c-Jun/c-Fos heterodimer). Indeed, another protein is identified from the F9 nuclear extract. We have also identified a silencer element at 300 bp upstream from the cap site. There are several cis-elements in this region and at least three trans-acting factors bind to these elements. We purified SF-A (silencer factor A) which binds to several regions in this silencer, and determined the partial amino acid sequence. Interestingly, SF-A seemed to be a related protein to NF1 (nuclear factor 1) which is an activator for the transcription and DNA replication. Another factor SF-B (silencer factor B) has been cloned and found to be the same as LIP (liver inhibitory protein) which is a competitor for LAP (liver activator protein), both are from the same gene designated as C/EBP beta. By transfection analysis using GAL4 DNA binding domain we found LIP is not only a competitor but a direct repressor. In the normal liver, another C/EBP family member, C/EBP alpha also acts as a negative regulator, and this expression decreases during hepatocarcinogenesis, resulting in the loss of silencer function. We carried out the carcinogenesis experiments using transgenic rats harboring a chloramphenicol acetyltransferase (CAT) reporter gene with -2900 to + 59 of the GST-P gene. Liver foci and nodules produced by chemical carcinogens were found to express high levels CAT activity by both CAT assay and immunohistochemical study, while normal liver cells did not express any CAT activity. These results demonstrate that the GST-P gene is trans-activated locus-independently during rat hepatocarcinogenesis. Moreover, the similar results were obtained using transgenic rats carrying GPEI-CAT, indicating that GPEI is an important cis-element for activation of GST-P gene during hepatocarcinogenesis.
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PMID:[Regulation mechanism of specific expression of tumor marker gene during carcinogenesis]. 883 Dec 56

Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Two dietary components capable of mediating chemopreventive activity in animal models by modulation of drug-metabolizing enzymes are sulforaphane, an aliphatic isothiocyanate, and brassinin, an indole-based dithiocarbamate, both found in cruciferous vegetables. We currently report the synthesis and activity of a novel cancer chemopreventive agent, (+/-)-4-methylsulfinyl-1-(S-methyldithiocarbamyl)-butane (trivial name, sulforamate), an aliphatic analogue of brassinin with structural similarities to sulforaphane. This compound was shown to be a monofunctional inducer of NAD(P)H:quinone oxidoreductase [quinone reductase (QR)], a Phase II enzyme, in murine Hepa 1c1c7 cell culture and two mutants thereof. Induction potential was comparable to that observed with sulforaphane (concentration required to double the specific activity of QR, approximately 0.2 microM), but cytotoxicity was reduced by about 3-fold (IC50 approximately 30 microm). In addition, sulforaphane, as well as the analogue, increased glutathione levels about 2-fold in cultured Hepa 1c1c7 cells. Induction of QR was regulated at the transcriptional level. Using Northern blotting techniques, time- and dose-dependent induction of QR mRNA levels were demonstrated in Hepa 1c1c7 cell culture. To further investigate the mechanism of induction, HepG2 human hepatoma cells were transiently transfected with QR-chloramphenicol acetyltransferase plasmid constructs containing various portions of the 5'-region of the QR gene. Sulforaphane and the analogue significantly induced (P < 0.0001) CAT activity at a concentration of 12.5 microM by interaction with the antioxidant responsive element (5-14-fold induction) without interacting with the xenobiotic responsive element. Moreover, both compounds significantly induced mouse mammary QR and glutathione S-transferase activity (feeding of 3 mg/mouse intragastric for 4 days), whereas the elevation of hepatic enzyme activities was less pronounced. Both sulforaphane and the analogue were identified as potent inhibitors of preneoplastic lesion formation in carcinogen-treated mouse mammary glands in organ culture (84 and 78% inhibition at 1 microm, respectively). On the basis of these results, the sulforaphane analogue can be regarded as a readily available promising new cancer chemopreventive agent.
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PMID:Cancer chemopreventive potential of sulforamate, a novel analogue of sulforaphane that induces phase 2 drug-metabolizing enzymes. 900 May 67

Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant derived from the propolis of honeybee hives. CAPE was shown to inhibit the formation of intracellular hydrogen peroxide and oxidized bases in DNA of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated HeLa cells and was also found to induce a redox change that correlated with differential growth effects in transformed cells but not the nontumorigenic parental ones. Mediated via the electrophile or human antioxidant response element (hARE), induction of the expression of NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase Ya subunit genes by certain phenolic antioxidants has been correlated with the chemopreventive properties of these agents. Here, we determined by Northern analysis that CAPE treatment of hepatoma cells stimulates NQO1 gene expression in cultured human hepatoma cells (HepG2), and we characterized the effects of CAPE treatment on the expression of a reporter gene either containing or lacking the hARE or carrying a mutant version of this element in rodent hepatoma (Hepa-1) transfectants. A dose-dependent transactivation of human hARE-mediated chloramphenicol acetyltransferase (cat) gene expression was observed upon treatments of the Hepa-1 transfectants with TPA, a known inducer, as well as with CAPE. The combined treatments resulted in an apparent additive stimulation of the reporter expression. To learn whether this activation of cat gene expression was effected by protein kinase C in CAPE-treated cells, a comparison was made of cat gene activity after addition of calphostin, a protein kinase C inhibitor. Calphostin reduced the cat gene induction by TPA but not by CAPE, suggesting that stimulation of gene expression in this system by these agents proceeds via distinct mechanisms. Band-shift experiments to examine binding of transactivator proteins from nuclear extracts of treated and untreated cells to a hARE DNA probe showed that TPA exposure increased the binding level. In contrast, binding of factors to this probe was inhibited after either in vivo treatment of cells with CAPE or in vitro addition of this compound to the nuclear extract. In view of the clear stimulation by CAPE of gene expression mediated by hARE, possible explanations of this result are discussed.
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PMID:Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene. 901 71

Green tea polyphenols, major constituents of green tea, are potent chemopreventive agents in a number of experimental models of cancer in animals. The mechanisms of cancer protection by these agents are not clear, but may involve modulation of the enzyme systems responsible for the detoxification of chemical carcinogens. The present studies show that a green tea polyphenol extract (GTP) induces chloramphenicol acetyltransferase (CAT) activity in human heptoma HepG2 cells transfected with a plasmid construct which contains an antioxidant-responsive element (ARE) and a minimal glutathione S-transferase Ya promoter linked to the CAT reporter gene. This indicates that GTP stimulates the transcription of Phase II detoxifying enzymes through the ARE. To explore the upstream signaling pathways leading to gene expression, we studied the involvement of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1). Potent activation of ERK2 was seen following treatment of HepG2 cells with different concentrations of GTP. Similar to ERK2, JNK1 was also strongly activated by treatment with GTP, although to a lesser extent and in a different dose-dependent fashion. Kinetic studies revealed that GTP activation of JNK1 was delayed and sustained, whereas ERK2 activation was rapid and transient. Furthermore, GTP treatment also increased mRNA levels of the immediate-early genes c-jun and c-fos, as determined by reverse transcriptase-coupled polymerase chain reaction. Taken together, these studies provide insights into the action of GTP and suggest that the stimulation MAPKs may be the potential signaling pathways utilized by GTP to activate ARE-dependent genes.
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PMID:Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression. 905 42

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.
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PMID:Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells. 906 51

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.
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PMID:Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression. 919 27

A panel of HepG2-derived cell lines (CAT-Tox [L] assay, Xenometrix), harboring stress genes consisting of a sequence for chloramphenicol acetyltransferase (CAT) under the transcriptional regulation from mammalian promoters and response elements, was exposed for 18-24 hr to aqueous suspensions of urban dusts (SRM-1648, SRM-1649, EHC-93) or PM2.5 particles (particulate matter < 2.5 micron). Expression of CAT protein was measured by enzyme-linked immunosorbent assay. Induction of the CAT genes was verified with benzo[a]pyrene (CYP1A1, cytochrome P450 1A1 promoter; GSTYa, glutathione transferase subunit Ya promoter; XRE, xenobiotic response element), cadmium sulfate, and copper sulfate (HMTIIa, metallothionein IIa promoter; HSP70, heat shock protein 70 promoter). The urban dust suspensions were active on CYP1A1, GSTYa, and XRE cell lines. SRM-1648 and SRM-1649 were twice as potent as EHC-93 per unit mass in inducing the xenobiotic-dependent responses, which correlated with contents in polycyclic aromatic hydrocarbons. These three reference particles, as well as six PM2.5 preparations collected on hi-vol filters in the Great Lakes basin, were also found to induce HMTIIa and HSP70, the magnitude of the responses correlating closely with the amount of soluble copper in the particulate preparations. The results indicate that bioavailable chemical species in the unfractionated particles can directly and quantitatively induce xenobiotic, metal, and stress-dependent responses in a target cell model, resulting in patterns of gene induction consistent with the chemical compositions of the environmental materials. We propose that cell culture models could be helpful for toxicodynamic inferences in adjunct to environmental monitoring and exposure assessments.
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PMID:Regulation of promoter-CAT stress genes in HepG2 cells by suspensions of particles from ambient air. 932 24


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