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)

We have isolated the 5'flanking regions of two human Alpha class glutathione S-transferase genes, GSTA1 and GSTA2. The two genes share 95% sequence identity between nucleotide positions -1,300 and +500 from the transcriptional start site. Various DNA fragments from the 5' flanking region of the GSTA1 gene were fused to the chloramphenicol acetyltransferase reporter gene and transfected into HepG2 cells. The results indicated that negative regulatory and enhancer elements are located in the sequence upstream of the GSTA1 gene. Sequence analysis and functional assays have not found any evidence for xenobiotic- or antioxidant-responsive elements previously described in rodent Alpha class genes. Thus the transcriptional regulation of the human Alpha class glutathione S-transferase genes may be dramatically different from the regulation of Alpha class glutathione S-transferase genes in rodents.
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PMID:Structure and function of the 5' flanking sequences of the human alpha class glutathione S-transferase genes. 818 23

Phenobarbital is an inducer of xenobiotic-metabolizing enzymes, such as cytochrome P-450, glutathione S-transferases (GSTs) and NAD(P)H:quinone reductase, as well as being a promoter of hepatocarcinogenesis. The molecular mechanisms regulating these biological activities are, however, unknown. In this paper we show that induction by phenobarbital of GST Ya and quinone reductase gene expression is mediated by regulatory elements, EpRE and ARE respectively, which are composed of two adjacent AP-1-like binding sites. EpRE was recently found to be activated by a Fos/Jun heterodimeric complex (AP-1). Here we show that phenobarbital induces an increase in AP-1 binding activity in nuclear extracts of cultured hepatoma cells. Furthermore, we observe that the induction of chloramphenicol acetyltransferase (CAT) activity from an EpRE Ya-cat gene construct and of AP-1 binding activity by phenobarbital is inhibited by the thiol compounds N-acetyl-L-cysteine and glutathione. These results suggest that the phenobarbital induction of AP-1 activity, leading to the AP-1-mediated transcriptional activation of the GST Ya and quinone reductase genes, may involve production of reactive oxygen species and an increase in intracellular oxidant levels, which is prevented by thiol compounds. In view of the involvement of AP-1 in the control of cell proliferation and transformation, the induction by phenobarbital of AP-1 binding activity observed here provides a possible molecular mechanism for the tumour-promoting activity of this drug.
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PMID:Phenobarbital induction of AP-1 binding activity mediates activation of glutathione S-transferase and quinone reductase gene expression. 845 90

17 beta-Estradiol (E2) induces cathepsin D mRNA levels and intracellular levels of immunoreactive protein in MCF-7 human breast cancer cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression in this cell line; however, in cells cotreated with TCDD and E2, TCDD inhibited E2-induced cathepsin D mRNA levels, the rate of gene transcription, and levels of immunoreactive protein. The inhibitory responses were observed within 30 to 120 min after the cells were treated with TCDD. TCDD also inhibited E2-induced secreted alkaline phosphatase activity in aryl hydrocarbon (Ah)-responsive MCF-7 and wild-type mouse Hepa 1c1c7 cells cotransfected with the human estrogen receptor (hER) and the pBC12/S1/pac plasmid, which contains the 5' promoter region (-296/+57) of the cathepsin D gene and an alkaline phosphatase reporter gene. The E2-responsive ER/Sp1 sequence (-199 to -165) in the cathepsin D 5' region contains an imperfect GTGCGTG (-175/-181) xenobiotic responsive element (XRE); the role of this sequence in Ah responsiveness was investigated in gel electrophoretic mobility shift assays and with plasmid constructs containing a wild-type ER/Sp1 oligonucleotide or a mutant ER/Sp1-"XRE" oligonucleotide containing two C-->A mutations in the XRE sequence (antisense strand). In plasmid constructs which contained a chloramphenicol acetyltransferase reporter gene and the wild-type ER/Sp1 promoter sequence, E2-induced chloramphenicol acetyltransferase activity and mRNA levels were inhibited by TCDD whereas no inhibition was observed with the mutant ER/Sp1-"XRE" plasmids. Electrophoretic mobility shift assays showed that the nuclear or transformed cytosolic Ah receptor complex blocked formation of the ER-Sp1 complex with the wild-type but not the ER/Sp1 mutant oligonucleotide. Moreover, incubation of the wild-type bromodeoxyuridine-substituted ER/Sp1 oligonucleotide with the nuclear Ah receptor complex gave a specifically bound cross-linked 200-kDa band. These data demonstrate that Ah receptor-mediated inhibition of E2-induced cathepsin D gene expression is due to disruption of the ER-Sp1 complex by targeted interaction with an overlapping XRE.
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PMID:Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. 852 36

We have isolated genomic DNA clones containing rat UDP-glucuronosyltransferase family 1 (UGT1) sequences and have shown drug-responsive and tissue-specific alternative expression of multiple first exons (Emi, Y., Ikushiro, S., and Iyanagi, T. (1995) J. Biochem. (Tokyo) 117, 392-399). The UGT1 locus encodes at least nine UGT1 isoforms. UGT1A1 is a major 3-methylcholanthrene (MC)-inducible form in rat liver. In this report, we have identified a cis-acting element necessary for transcriptional activation of UGT1A1 in hepatocytes. A promoter region was fused to a chloramphenicol acetyltransferase gene, and the resultant construct was transiently transfected into hepatocytes. A DNA fragment carrying 1,100 nucleotides derived from the 5'-flanking region of the UGT1A1 gene was enough for MC induction. Unidirectional deletion of this region revealed that there existed one xenobiotic responsive element (XRE), TGCGTG, between -134 and -129. When a single base substitution was introduced into the XRE, MC-induced expression of the UGT1A1 gene was completely abolished. In addition, an XRE-deleted construct failed to respond to MC. Gel mobility shift assays showed MC-inducible binding of the nuclear aromatic hydrocarbon receptor-ligand complex to this motif. Gel shift-coupled DNase I protection analyses revealed that the GCGTG-core sequence was a target site of the liganded aromatic hydrocarbon receptor. These results suggest that the XRE participates in induction of the rat UGT1A1 gene by MC.
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PMID:Xenobiotic responsive element-mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex. 863 18

As an approach to understanding the molecular mechanism(s) of thymic gene expression mediated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we investigated the effect of TCDD on expression of prostaglandin G/H synthase-2 (PGHS-2) in rat thymocytes by reverse transcription-polymerase chain reaction. Incubation of thymocytes with increasing doses of TCDD resulted in inhibition of PGHS-2 gene expression in a concentration-dependent manner, with an IC50 of 10 nM. In contrast, TCDD had no appreciable effect on expression of glyceraldehyde phosphate dehydrogenase. Because the xenobiotic-responsive element is conserved in the PGHS-2 promoter from several animal species, it seems likely that inhibition of PGHS-2 expression by TCDD may occur at the level of transcription. To test this hypothesis in cultured thymocytes, we characterized the Ah receptor in the thymoma cell line WEHI 7.1. Reverse transcription-polymerase chain reaction experiments indicated that TCDD inhibited PGHS-2 expression in this cell line. Sucrose density gradient centrifugation experiments indicated that WEHI 7.1 cytosol exhibited 9 to 10S ligand-binding activity characteristic of the Ah receptor. The viability of WEHI 7.1 cells incubated with TCDD was comparable to that of control cells, whereas dexamethasone induced toxicity in a concentration-dependent manner. Transient transfection experiments using PGHS-2 promoter fragments ligated into a chloramphenicol acetyltransferase reporter plasmid suggested that TCDD inhibits PGHS-2 transcription, and deletion of the xenobiotic-responsive element failed to exhibit this repression. These results demonstrate that TCDD is a potent inhibitor of PGHS-2 gene expression, and they represent the first mechanistic evidence for TCDD-dependent inhibition of transcription in thymocytes.
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PMID:2,3,7,8-tetrachlorodibenzo-p-dioxin modulates expression of the prostaglandin G/H synthase-2 gene in rat thymocytes. 896 84

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

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

Transcriptional regulation of human UGT1A6, a UDP glucuronosyltransferase isoform conjugating a wide variety of planar phenols, has been studied using transfection experiments with plasmids containing its 3-kb 5' upstream region and chloramphenicol acetyltransferase as reporter gene. Previously, two modes of expression of the isoform have been described: in colon carcinoma Caco-2 cells UGT1A6 was found to be TCDD-inducible, whereas in lung carcinoma A549 cells it was constitutively expressed. Therefore functional analysis of UGT1A6 regulation was carried out using these two cell lines. In the upstream region of human UGT1A6 one xenobiotic-responsive element (XRE) was found between-1498 and -1502 bp. In Caco-2 cells the reporter gene activity of the entire plasmid and of deletion mutants containing the XRE were TCDD-inducible, in contrast to experiments with a deletion mutant which did not contain the XRE. TCDD induction was marginal in transfection studies with A549 cells. Gel mobility shift analysis indicated that the aryl hydrocarbon receptor and its partner Arnt bind to the XRE. Furthermore, primer extension studies suggest cell-specific use of multiple TATA boxes. Hence, regulation of human UGT1A6 appears to be cell-specific including both constitutive and aryl hydrocarbon receptor-controlled expression.
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PMID:Aryl hydrocarbon receptor-inducible or constitutive expression of human UDP glucuronosyltransferase UGT1A6. 946 22

The crystal structure of the xenobiotic acetyltransferase from Pseudomonas aeruginosa PA103 (PaXAT) has been determined, as well as that of its complex with the substrate chloramphenicol and the cofactor analogue desulfo-coenzyme A. PaXAT is a member of the large hexapeptide acyltransferase family of enzymes that display tandem repeated copies of a six-residue hexapeptide repeat sequence motif encoding a left-handed parallel beta helix (L betaH) structural domain. The xenobiotic acetyltransferase class of hexapeptide acyltransferases is composed of microbial enzymes that utilize acetyl-CoA to acylate a variety of hydroxyl-bearing acceptors. The active site of trimeric PaXAT is a short tunnel into which chloramphenicol and the cofactor analogue desulfo-CoA project from opposite ends. This tunnel is formed by the flat parallel beta sheets of two separate L betaH domains and an extended 39-residue loop. His 79 of the extended loop forms hydrogen bonds from its imidazole NE2 atom to the 3-hydroxyl group of chloramphenicol and from its ND1 group to the peptide oxygen of Thr 86. The interactions of this histidine residue are similar to those found in the structurally unrelated type III chloramphenicol acetyltransferase and suggest that His 79 of PaXAT may be similarly positioned and tautomerically stabilized to serve as a general base catalyst.
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PMID:Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa. 957 52

We have previously demonstrated that H2O2 downregulates CYP1A1 and CYP1A2 transcription in isolated rat hepatocytes (C. W. Barker, et al., 1994, J. Biol. Chem. 269, 3985-3990). In the present study, induction of chloramphenicol acetyltransferase (CAT) expression driven by 3.1 kb of rat CYP1A1 upstream regulatory sequences was suppressed by 56% in Hepa-1 cells treated with H2O2. Similarly, H2O2 inhibited CAT expression from vectors containing two copies of either xenobiotic-response element (XRE) 1 or XRE2. H2O2 did not inhibit basal CAT expression in cells that were not treated with the inducer beta-napthoflavone. Electrophoretic mobility shift assays demonstrated that the suppression of XRE-dependent transcription by H2O2 was not due to changes in nuclear aryl hydrocarbon (Ah) receptor DNA binding activity. Several types of experiments indicated that modulation of XRE enhancer strength by various means could modify H2O2-dependent suppression of CAT expression. Conditions that increased the transactivation potential of the Ah receptor (increase in XRE copy number or shortening of the distance between XREs and the minimal CYP1A1 promoter) attenuated the action of H2O2, while conditions that reduced XRE-mediated transactivation potential (decrease in XRE copy number, increase of the distance between the XRE and the promoter, or reduction of the number of bound Ah receptors by lowering the concentration of inducer) potentiated the inhibitory action of H2O2.
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PMID:Suppression of CYP1A1 transcription by H2O2 is mediated by xenobiotic-response element. 970 4


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