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)

Differences in expression of the CYP1A1 gene have previously been observed in human breast carcinoma cell lines exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an expression vector containing the functional 5'-regulatory region of human CYP1A1 (up to -1140) fused to the reporter gene CAT (for chloramphenicol acetyltransferase), the breast carcinoma cell lines, MCF-7, T47-D and ZR-75-1, classified as highly responsive to TCDD, were highly responsive to TCDD in the chloramphenicol acetyltransferase assay as well. Gel mobility shift assays have shown that these cell lines express a nuclear protein that binds the aryl hydrocarbon (Ah) receptor responsive element. The low or non-responsive cell lines, AL-1, BT-20 and CAMA-1, were low or non-responsive to TCDD in the chloramphenicol acetyltransferase assay, suggesting that the low-responsive phenotype is caused by altered trans-acting factors. However, the mechanism appears to differ among the cell lines. Whereas no induction was observed in AL-1, a fivefold induction in activity was observed in BT-20 and CAMA-1. The TCDD concentration giving half-maximum induction differed greatly between CAMA-1 and BT-20. The gel mobility shift assay showed the presence of a protein that bound specifically to the Ah responsive element in the non-responsive cell line AL-1, as well as the low-responsive cell lines, BT-20 and CAMA-1. The high basal activity but low induction observed in CAMA-1 may be due to an Ah receptor constitutively bound to the Ah responsive element.
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PMID:Differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1A1 expression in human breast carcinoma cell lines involve altered trans-acting factors. 202 91

We have investigated the transcriptional regulation of 3-methylcholanthrene (3MC)-inducible P-450c gene which is involved in the metabolic activation of polycyclic aromatic carcinogens. Reverse genetic study using the fusion gene composed of the 5' upstream sequence of P-450c gene and the structure gene for bacterial chloramphenicol acetyltransferase (CAT) and a cultured cell line of Hepa-1 cells localized two kinds of cis-acting regulatory DNA elements. One is designated XRE or xenobiotic responsive element which is responsible for the inducibility of the gene and is distributed 5 times in the region from -3.0 to -0.5 kb. The other is BTE or basal transcription element whose deletion reduces a low level of the constitutive CAT expression to a background level, and which is localized immediately upstream of the TATA sequence. Both kinds of regulatory elements are necessary for a high level of inducible expression. Gel mobility shift assay strongly suggests that the binding protein to the XRE is an Ah receptor with a specific affinity for 3MC or 2,3,7,8,tetrachlorodibenzo-p-dioxin (TCDD). Without inducer treatment, cryptic form of the binding protein occurs only in the cytoplasm of the Hepa-1 cells. Upon treatment with the inducer, the cryptic form of the binding protein exhibits binding activity to XRE and, at the same time, translocates to the nuclei. The BTE-binding protein is an ubiquitous nuclear factor and its cDNA cloning reveals the DNA-binding feature with zinc finger motifs.
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PMID:Transcriptional regulation of 3-methylcholanthrene-inducible P-450 gene responsible for metabolic activation of aromatic carcinogenes. 213 75

Glutathione S-transferase (GST) Ya subunit gene expression is induced in mammalian tissues by two types of chemical agents: (i) planar aromatic compounds (e.g., 3-methylcholanthrene, beta-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p- dioxin) and (ii) electrophiles (e.g., trans-4-phenyl-3-buten-2-one and dimethyl fumarate) or compounds easily oxidized to electrophiles (e.g., tert-butylhydroquinone). To study the mechanism of this induction, we have introduced deletions in the 5' flanking region of a mouse GST Ya subunit gene, fused it to the coding sequence for chloramphenicol acetyltransferase (CAT) activity, and transfected the Ya-CAT genes for expression into hepatoma cells. We show that a single cis-regulatory element, between nucleotides -754 and -713 from the start of transcription, is responsible for the induction by both planar aromatic and electrophilic compounds. Using murine hepatoma cell mutants defective in either the Ah-encoded aryl hydrocarbon receptor (BPrc1 mutant) or in cytochrome P1-450 gene (c1 mutant), we show that induction by planar aromatic but not by electrophilic inducers requires a functional Ah receptor and cytochrome P1-450 activity. From this it is concluded that Ya gene activation by planar aromatic compounds involves metabolism of these inducers by the phase I xenobiotic-metabolizing cytochrome P1-450 system into electrophilic compounds, which is consistent with a recently proposed model [Prochaska, H. J. & Talalay, P. (1988) Cancer Res. 48, 4776-4782]. Therefore, the regulatory sequence of the Ya gene should be considered an electrophile-responsive element (EpRE) activated exclusively by inducers containing an electrophilic center. An EpRE-containing 41-bp oligonucleotide ligated at the -187 site of the Ya gene promoter confers upon it an increase in basal activity and xenobiotic inducibility. The basal activity augments with the number of EpRE copies. DNase I protection patterns show the protection of the EpRE domain by a nuclear factor(s) that becomes more abundant upon exposure of Hepa 1c1c7 cells to tert-butylhydroquinone.
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PMID:Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. 216 52

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
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PMID:Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene. 217 7

The foreign chemical tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with the aromatic hydrocarbon receptor and, in turn, activate transcription of the mouse P1450 and P3450 genes. Various lengths of DNA upstream from the human P1450 gene were inserted into the promoterless pSVO-cat prokaryotic expression vector and compared with mouse P1450 upstream sequences similarly treated. The constructs were cotransfected with pSV2-neo into human, mouse, and monkey liver- and nonliver-derived cell lines. After selection in G418, the transformed colonies were treated with control medium, TCDD, or, in some cases, cycloheximide. Pooled transformants were then assayed for chloramphenicol acetyltransferase activity. The data are consistent with the presence of several functional regulatory regions within the upstream DNA: a promoter region, a region that is negatively autoregulated, and a region further upstream that activates transcription and is dependent upon a functional aromatic hydrocarbon receptor. Compared with 1604 base pairs of human P1450 upstream sequences, 1646 base pairs of mouse P1450 upstream sequences exhibit an increased sensitivity to TCDD; this effect was found to require both trans-acting protein factors and cis-acting DNA elements. Our results demonstrate the successful interaction of mouse trans-acting factors with human P1450 upstream sequences and human trans-acting factors with mouse P1450 upstream sequences.
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PMID:Comparison of human mouse P1450 upstream regulatory sequences in liver- and nonliver-derived cell lines. 345 96

In liver of adult responsive C57BL/6J (B6) mice the aromatic hydrocarbon receptor (AHR) has high affinity for specific halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as nonhalogenated aromatic hydrocarbons (PAHs), such as benz[a]anthracene (BA) or 3-methylcholanthrene (MC). In livers of adult nonresponsive DBA/2J (D2) mice TCDD binds to a low-affinity variant form of AHR. Both TCDD and MC induce aryl hydrocarbon hydroxylase (AHH) in adult B6 mice, whereas adult D2 mouse liver is nonresponsive to MC. In fetal cell cultures derived from D2 mice AHH is induced by PAHs such as MC or BA, and these PAHs bind to cytosolic AHR (P.A. Harper, C.L. Golas, and A.B. Okey. Mol. Pharmacol. 40: 818-826, 1991). We compared AHR from fetal cell cultures with AHR from adult livers to determine whether there was some structural differences in receptors expressed in fetal cell culture that might permit cells from "nonresponsive" mice to respond to PAHs. The apparent molecular mass of AHR from cells cultured from 18-day fetuses is identical with that from adult liver within each strain of inbred mice tested (M(r) approximately 95 kDa in B6 and approximately 105 kDa in D2 mice). The AHR in D2 fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence (DRE-CAT) when the cells were treated with TCDD or MC. The potency of CAT expression in D2 fetal cells was similar to that in B6 fetal cells. Our data suggest that the responsiveness of fetal cells from "nonresponsive" mice is likely mediated by AHR in these cells but is not due to expression of a different allelic form of AHR ligand-binding subunit in fetal cells versus adult liver.
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PMID:Aromatic hydrocarbon receptor in cultured fetal cells from C57BL/6J and DBA/2J mice: similarity in molecular mass to receptors in adult livers. 760 Apr 48

Arnt (Ah receptor nuclear translocator) is a member of a transcription factor family having characteristic motifs designated bHLH (basic helix-loop-helix) and PAS and was originally found as a factor forming a complex with Ah receptor (AhR) to bind the specific xenobiotic responsive element (XRE) sequence for induction of drug-metabolizing P4501A1. We have examined interaction of Arnt with other PAS proteins--Drosophila Per, Sim, and AhR--by the coimmunoprecipitation method. Arnt formed a homodimer with itself as well as heterodimers with the others by means of the PAS and HLH domains in a cooperative way. The Arnt homodimer binds the sequence of adenovirus major late promoter (MLP) with the E box core sequence CACGTG, suggesting that the CAC half of the XRE, CACGCN(A/T), recognized by the AhR-Arnt heterodimer is a target for Arnt. Cotransfection experiments using CV-1 cells with an Arnt expression plasmid and a MLP chloramphenicol acetyltransferase (CAT) reporter plasmid revealed that Arnt markedly activated CAT expression, indicative of a newly discovered regulatory role of Arnt.
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PMID:Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation. 789 3

The Ah receptor (AHR) is a ligand-activated transcription factor that is structurally related to its dimerization partner, the Ah receptor nuclear translocator (ARNT), and two Drosophila proteins, SIM and PER. All four proteins contain a region of homology now referred to as a PAS homology domain. In addition, the AHR, ARNT, and SIM harbor a basic region helix-loop-helix motif in their N termini, whereas PER does not. Previous mapping studies of the AHR have demonstrated that the PAS domain contains sequences required for ligand recognition, dimerization, and interaction with the 90-kDa heat shock protein. They also have confirmed that the basic region helix-loop-helix domain plays a role in both dimerization and sequence-specific DNA binding. To identify domains involved in transactivation of target genes, we generated chimeras of AHR/ARNT deletion mutants with the DNA binding region of the yeast Gal4 protein, transiently expressed these in COS-1 cells, and monitored their capacity to activate the chloramphenicol acetyltransferase reporter gene under the control of a minimal promoter driven by enhancer elements recognized by Gal4. Extensive analysis of these fusions revealed that the AHR and ARNT harbor potent transactivation domains within their C termini. Importantly, the amino-terminal halves of both the AHR and ARNT were found to be devoid of transactivation activity.
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PMID:Potent transactivation domains of the Ah receptor and the Ah receptor nuclear translocator map to their carboxyl termini. 798 19

The gene for cytochrome P4501A2 is constitutively expressed in the liver of vertebrates and shows induced expression when an organism is exposed to polycyclic aromatic hydrocarbons and halogenated hydrocarbons. To identify DNA elements regulating transcription of the human CYP1A2 gene, transient transfection experiments were conducted in the human hepatoma cell line HepG2. Dissection of the 5'-flanking portion of the CYP1A2 gene identified two regions that contributed to the overall induction by 3-methylcholanthrene. One region located at -2532/-2423 contains an xenobiotic-responsive element-like sequence, termed X1, that binds a nuclear 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible protein in HepG2 and wild type mouse Hepa-1 cells, but not in the Ah receptor nuclear translocation defective mouse C- mutant c4 cells. In addition, deletion of this region of the CYP1A2 gene reduces the 3-methylcholanthrene (3-MC)-initiated induction of chloramphenicol acetyltransferase activity in both promoter- and enhancer-specific constructs. The second responsive region is located at -2259/-1987. This region of the gene contains a second xenobiotic-responsive element-like element, but this element does not associate with the nuclear Ah receptor. However, there does exist several potential AP1 binding sites and a conserved TATA box. A DNA fragment from -2259/-1970 that contains these elements was shown to function as an efficient eukaryotic promoter, in addition to supporting 3-MC-induced promoter activity. These results suggest that Ah receptor-specific and promoter-specific elements regulate the expression of the human CYP1A2 gene.
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PMID:The human CYP1A2 gene and induction by 3-methylcholanthrene. A region of DNA that supports AH-receptor binding and promoter-specific induction. 812 57

alpha-Naphthoflavone (alpha NF) is a weak aryl hydrocarbon (Ah) receptor agonist and inhibits the induction of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. It has been suggested that the Ah receptor antagonist activity is due to the formation of alpha NF-cytosolic Ah receptor complexes that fail to undergo transformation. This hypothesis is consistent with data obtained in this and other studies using alpha NF concentrations from 10 to 1000 nM. However, 10 microM alpha NF exhibited Ah receptor agonist activity in several assays. Incubation of rat hepatic cytosol with 10 microM alpha NF caused transformation of the Ah receptor, as determined in a gel retardation assay using a 32P-labeled oligonucleotide containing a single dioxin-responsive element (DRE). Incubation of rat hepatoma (H-4-II E) cells with 10 microM alpha NF not only resulted in the induction of CYP1A1 mRNA levels but also increased chloramphenicol acetyltransferase activity from a DRE-containing chloramphenicol acetyltransferase reporter plasmid. Moreover, the DRE-transformed cytosolic Ah receptor complex liganded with either alpha NF or 2,3,7,8-tetrachlorodibenzo-p-dioxin did not undergo significant dissociation at 4 degrees. These data confirm that alpha NF is an Ah receptor agonist and, based on the results of previous studies, exhibits partial antagonist activity via competition for receptor binding sites.
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PMID:alpha-Naphthoflavone-induced CYP1A1 gene expression and cytosolic aryl hydrocarbon receptor transformation. 838 8


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