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)

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

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.
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PMID:cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression. 901 60

The antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in several cell lines using transient transfection assays and constructs containing 5'-regulatory sequences from the estrogen (E2)-responsive vitellogenin (Vit) A2 gene linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. TCDD significantly inhibited CAT activity induced by E2 in MCF-7 human breast cancer cells transiently transfected with 5'-deletion plasmids containing the homologous promoter [(-821/+14)- and (-482/+14)-CAT] or the heterologous thymidine kinase (tk) promoter [(-821/-87)tk-, (-482/-87)tk-, (-397/-87)tk-, and (-331/-87)tk-CAT]. In parallel experiments using wild-type mouse Hepa 1c1c7 and human HeLa cells cotransfected with a human estrogen receptor expression plasmid, TCDD also inhibited E2-induced CAT activity. The role of the nuclear Ah receptor complex was confirmed by results of the following studies using MCF-7 or mouse Hepa 1c1c7 cells transiently transfected with E2-responsive Vit A2 gene 5'-promoter constructs: (i) for a series of Ah receptor ligands, there was a correlation between their antiestrogenic activity in MCF-7 cells and their rank order binding affinity for the Ah receptor; (ii) alpha-naphthoflavone, an Ah receptor antagonist, inhibited the antiestrogenic activity of TCDD in MCF-7 cells; and (iii) TCDD inhibited E2-induced CAT activity in Ah-responsive wild-type but not in Ah-nonresponsive class 2 mutant Hepa 1c1c7 cells. The antiestrogenic activity of TCDD was also observed in cells which transiently overexpressed the human estrogen receptor (ER), suggesting that the mechanism does not involve downregulation of the ER by TCDD.
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PMID:Inhibition of estrogen-induced activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-7 human breast cancer and other cell lines transfected with vitellogenin A2 gene promoter constructs. 901 89

Several studies have reported a correlation between expression of the estrogen receptor (ER) and aryl hydrocarbon (Ah) responsiveness in human breast cancer cell lines. MDA-MB-231 cells are ER-negative and Ah-nonresponsive; however, initial studies showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA levels (5.8-fold) and chloramphenicol acetyltransferase activity (2.6-fold) in high passage (Hp, >50 passages) cells transiently transfected with an Ah-responsive plasmid. In contrast, no induction responses were observed in low passage (Lp, <20 passages) cells. The Ah responsiveness of Hp compared to Lp MDA-MB-231 cells was associated with a >2-fold increased expression of the Ah receptor in Hp cells. Further analysis revealed that the apparent molecular weight of the Ah receptor mRNA transcript and immunoreactive protein were comparable in Lp MDA-MB-231 and Ah-responsive human HepG2 cells. In contrast, RT-PCR analysis of the Ah receptor nuclear translocator (Arnt) protein showed that HepG2 cells expressed the expected 2.6-kb transcript, whereas a 1.3-kb transcript was the major product in MDA-MB-231 cells. Western blot analysis confirmed that HepG2 cells primarily expressed a 97-kDa wild-type form of Arnt, whereas a dominant 36-kDa variant was expressed in MDA-MB-231 cells. Complete sequence analysis of the variant form of Arnt revealed a major deletion of the C-terminal region of the protein (aa 330 to 789). Like HepG2 cells, the wild-type 2.6-kb transcript was detected in ER-positive (Ah-responsive) MCF-7 cells, whereas the low-molecular-weight variant Arnt was dominant in ER-negative MDA-MB-231, MDA-MB-435, and Adriamycin-resistant MCF-7 cells. These results suggest that expression of this protein may be useful as a prognostic factor in breast cancer.
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PMID:Aryl hydrocarbon (Ah) nonresponsiveness in estrogen receptor-negative MDA-MB-231 cells is associated with expression of a variant arnt protein. 932 85

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

Cultured mouse hepatoma Hepa lclc7 cells were treated with either estradiol or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or in combination to assess the role of estradiol in the process of Cypla-1 induction. Estradiol at a concentration as high as 1 microM slightly increased the activity of Cypla-1-specific 7-ethoxyresorufin O-deethylase (EROD); in contrast, TCDD-induced EROD activity and Cypla-1 mRNA levels were markedly reduced in the concomitant treatment of TCDD and estradiol in a dose-dependent manner. Treatment with tamoxifen, an anti-estrogen which acts through the estrogen receptor, did not affect the suppressive effects of estradiol on TCDD-induced EROD activity. Electrophoretic mobility shift assay using nuclear extract of cells revealed that estradiol reduced transformation of the Ah receptor to the form capable of specifically binding to an oligonucleotide containing dioxin-response element (DRE) sequence. Consistent with this, estradiol decreased TCDD-induced increased chloramphenicol acetyltransferase (CAT) activity from a DRE-containing CAT reporter plasmid after transient transfection into the cells. The levels of the cytosolic [3H]TCDD-Ah receptor complex were reduced by estradiol in competitive Ah receptor binding assay using [3H]TCDD. This study demonstrated that estradiol acts as an antagonist to TCDD and can regulate Cyp1a-1 expression in an Ah receptor-dependent manner but not through estradiol receptor in Hepa 1c1c7 cells.
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PMID:Suppressive effects of estradiol on 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated transcriptional activation of murine Cyp1a-1 in mouse hepatoma Hepa 1c1c7 cells. 1007 67

Diflubenzuron (DFB) belongs to a group of compounds called benzoyphenyl ureas acting as chitin synthesis inhibitors, which also inhibit growth of B16 murine melanomas. The present study was designed to investigate the effect of this insecticide, on CYP1A1 expression and induction in human hepatoma cells HepG2. Treatment of HepG2 cells over 72 h with noncytotoxic concentrations of DFB resulted in a strong dose-dependent decrease in constitutive ethoxyresorufin-O-deethylase activity. Moreover, DFB significantly decreased CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) after 24 h exposure, as demonstrated by ethoxyresorufin-O-deethylase (EROD) activity and Northern blot analysis. Additional studies were performed both on parental HepG2 cells and HepG2-241c.1, which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene, cloned under the control of the human CYP1A1 promoter (-1140 to +59). Ribonuclease protection assays (RPA) analysis clearly demonstrated an inhibition of CYP1A1 transcription in both cell lines. Surprisingly, in corresponding experiments using 3-methylcholanthrene (3-MC) as a CYP1A1 inducer, DFB was less effective. Finally, in competitive binding studies using a 9S-enriched fraction of HepG2 cytosol, DFB was capable of displacing [(3)H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from its Ah receptor binding site. Taken together, these results support the involvement of a transcriptional mechanism in the inhibition of CYP1A1 expression in HepG2 cells by DFB, possibly via an Ah receptor antagonism.
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PMID:Diflubenzuron, a benzoyl-urea insecticide, is a potent inhibitor of TCDD-induced CYP1A1 expression in HepG2 cells. 1079 37


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