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)

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. The CYP composition of enterocytes could thus affect the potential toxicity or therapeutic efficacy of xenobiotics by modifying systemic uptake. We have characterized human enterocyte CYP composition to enable assessment of its functional roles. An isolation method for enterocytes from human small intestine was developed using EDTA buffer-mediated elution. Villous enterocytes were isolated in high yield, separated from crypt cells. Reverse transcriptase-polymerase chain reaction of total RNA from enterocytes revealed that CYP1A1, 1B1, 2C, 2D6, 2E1, 3A4, and 3A5 mRNA were expressed, but only CYP2C and 3A4 were detectable by Western immunoblotting in enterocyte microsomes from 10 human small intestines, whereas CYP1A1 was weakly detectable in two of eight intestines tested. Microsomal protein content decreased markedly along the small intestine from the duodenum to the ileum, whereas total CYP content and CYP3A4 erythromycin N-demethylase activity increased slightly in progressing from the duodenum to the jejunum and then decreased markedly toward the ileum. Levels of CYP3A4 and 2C protein did not decrease in concert as a function of length along the intestine distally. Maximal CYP content for the 10 intestines varied from 0.06 to 0.18 nmol/mg microsomal protein and maximal CYP3A4 erythromycin N-demethylase activity varied from 0.30 to 0.76 nmol/min/mg microsomal protein. In conclusion, CYP3A4 is the major form of CYP expressed in human small intestine enterocytes, CYP3A5 expression was not detected, CYP2C and, in some intestines, CYP1A1 were expressed. The highest metabolic activity occurred in the proximal intestine.
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PMID:Characterization of human small intestinal cytochromes P-450. 1038 24

The expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1 and 3A4) was studied in human bone marrow, bone-marrow-derived macrophages and blood monocyte-derived macrophages. Reverse transcriptase polymerase chain reaction (RT-PCR) detected expression of all five CYPs in each of these cell populations. All five CYPs were also expressed in the haemopoietic cell lines HL-60 and HEL and in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines. The data suggest that bone marrow macrophages and probably other bone marrow cell types are capable of metabolizing xenobiotics. This metabolic potential may play a role in the bone marrow damage induced by some drugs and chemicals.
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PMID:Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow-derived macrophages and human haemopoietic cell lines. 1065 38

Natural resistance-associated macrophage protein (Nramp) genes in rainbow trout, Oncorhynchus mykiss, were identified and characterized. The greatest mRNA level encoding these genes was in the developing ovary of rainbow trout. We evaluated the response of these genes to a certain aromatic hydrocarbon receptor (AHR) agonist. Adult rainbow trout were treated with beta-naphthoflavone (BNF) (50 and 100 mg/kg) for 48 h. Using reverse-transcriptase polymerase chain reaction with ovary and head kidney RNA and specific alpha and beta Nramp primers, a 400 bp Nramp-alpha- and a 400 bp Nramp-beta-specific cDNA were obtained. There were no changes in the alpha and beta Nramp mRNA levels in the ovary following BNF administration. CYP1A1 mRNA was increased in the ovary and kidney, suggesting the presence of AHR in rainbow trout ovary, while the AHR agonist produced no effect on Nramp mRNAs.
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PMID:Lack of effect of beta-naphthoflavone on induction of Nramp genes in adult rainbow trout Oncorhynchus mykiss. 1146 Jun 81

Cytochrome p4501A induction and subsequent enzyme expression is used as a biomarker for exposure to aryl hydrocarbon receptor active contaminants in fish and other species. In the present study, CYP1A cDNA (1912 bp, GenBank accession number AF364076) was cloned, sequenced and characterized from the liver of a beta-naphthoflavone (betaNF)-treated teleost, Atlantic salmon (Salmo salar), by reverse-transcriptase polymerase chain reaction (RT-PCR). The salmon CYP1A sequence contained a 5'-flanking region of 99 bp, an open reading frame of 1566 bp that encodes a 521 amino acid protein, a stop codon, and a 3'-untranslated region of 346 bp, and a single polyadenylated signal. The theoretical molecular mass and isoelectric point was 58.6 kDa and 6.17, respectively. Comparison of the deduced amino acid sequence of salmon cDNA with reported CYP1A genes showed identities of 73-88% with fish CYP1A, 51-54% with mammalian CYP1A1, 47-51% with mammalian CYP1A2 and 54% with frog p450. Phylogenetic analysis showed that salmon CYP1A clustered in the tree with rainbow trout CYP1A1 and eel CYP1A sequences. CYP1A mRNA induction in beta-naphthoflavone-treated salmon showed differential organ expression with a distinct single transcript pattern. A new specific molecular tool has been developed for the monitoring of environmental pollution using CYP1A mRNA from salmon as a biomarker.
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PMID:Complementary DNA cloning, sequence analysis and differential organ expression of beta-naphthoflavone-inducible cytochrome P4501A in Atlantic salmon (Salmo salar). 1245 89

Oltipraz, a promising cancer chemopreventive agent, has been recognized as a monofunctional inducer selectively activating phase II carcinogen-detoxifying enzymes via the antioxidant responsive element (ARE). However, we report here that oltipraz also induces rat glutathione S-transferase A5 (GSTA5), a potent phase II detoxifying enzyme, by means of the xenobiotic responsive element (XRE). Although an ARE sequence exists in the 5' upstream of the rGSTA5 gene, this cis-acting regulatory element loses its responsiveness to oltipraz treatment because of extensive mutations in its distal-half site. Our data indicate that a XRE sequence, located downstream of the transcription initiation site of the gene, is another oltipraz-responsive element. Electrophoretic mobility shift assay showed that oltipraz steadily induces XRE-aryl hydrocarbon receptor (AhR) binding, which can be blocked specifically by excess XRE oligonucleotides or by AhR antibody. By cloning different XREs into the pGL3-promoter vector, we found that oltipraz can activate XRE enhancers from several phase II drug metabolism enzymes, including rGSTA5, rGSTA2, NAD(P)H:quinone reductase, and it also activates XRE from the phase I metabolism enzyme CYP1A1. Oltipraz's effect on XRE is AhR-dependent and is independent of the presence of active CYP1A1. Reverse transcriptase-polymerase chain reaction experiments revealed that oltipraz induces gene expression of both phase I and II drug-metabolizing enzymes in rat hepatoma cells. Thus, we conclude that, like ARE, the XRE pathway constitutes an important part of the molecular mechanism contributing to oltipraz-induced expression of the phase II metabolism enzymes. Oltipraz is a bifunctional inducer, modulating both phase I and II drug-metabolizing enzymes to enhance carcinogen detoxification.
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PMID:Oltipraz is a bifunctional inducer activating both phase I and phase II drug-metabolizing enzymes via the xenobiotic responsive element. 1286 39

In contrast to the beneficial effects of tert-butylhydroquinone (tBHQ) as a food antioxidant, a number of studies have shown that chronic exposure to tBHQ may induce carcinogenicity. Therefore, we examined the ability of tBHQ to induce the cytochrome P450 1a1 (Cyp1a1), an enzyme known to play an important role in the chemical activation of xenobiotics to carcinogenic derivatives. A significant concentration-dependent increase in Cyp1a1 mRNA, protein, and activity occurred after treatment of murine hepatoma Hepa 1c1c7 cells with tBHQ. The increase in mRNA was apparent 3 h after treatment. The RNA polymerase inhibitor, actinomycin D, completely blocked the Cyp1a1 induction by tBHQ, indicating a requirement of de novo RNA synthesis through transcriptional activation. The protein synthesis inhibitor cycloheximide superinduced the tBHQ-mediated induction of Cyp1a1 mRNA and completely prevented the increase in Cyp1a1 activity, indicating that the induction of enzyme activity by tBHQ is dependent on de novo protein synthesis. In addition, the aryl hydrocarbon receptor (AHR) antagonist, resveratrol, inhibited the increase in Cyp1a1 activity by tBHQ. Gel electrophoretic mobility shift assays showed that tBHQ causes activation or transformation of the AHR in nuclear extracts, indicating that AHR-dependent mechanisms contributed to the Cyp1a1 induction. Similar to murine Hepa 1c1c7 cells, tBHQ caused a concentration-dependent increase in CYP1A1 at the mRNA and activity levels in human HepG2 cells. This is the first demonstration that the phenolic antioxidant, tBHQ, can directly induce Cyp1a1 gene expression in an AHR-dependent manner and may represent a novel mechanism by which tBHQ promotes carcinogenicity.
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PMID:tert-Butylhydroquinone is a novel aryl hydrocarbon receptor ligand. 1560 32

We show here that arsenite (As(3+)) elicits multiple effects on gene control, such as the interruption of cell cycle control by initiating G(2)/M arrest as well as inhibiting the aryl hydrocarbon (Ah) receptor-mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible expression of CYP1A1. This raises the question as to whether As(3+) is selectively inhibiting TCDD induction of CYP1A1 independent of cell cycle control. As(3+) stimulated a concentration-dependent increase in G(2)/M phase arrest that was detected at 12.5 microM As(3). However, cotreatment of HepG2 cells with TCDD and concentrations of As(3+) as low as 0.5 microM stimulated a pronounced decrease in the induction of CYP1A1-dependent ethoxyresorufin-O-deethylase activity and protein, indicating that the inhibition of CYP1A1 induction by As(3+) was considerably more sensitive than As(3+)-initiated cell cycle arrest. Low concentrations of As(3+) also initiate a dose-dependent reduction in TCDD-induced mouse Cyp1a1 as well as human CYP1A1 in primary hepatocytes cultured from transgenic CYP1A1N(+/-) mice. Because primary hepatocytes in culture are quiescent, these results indicate that the actions of As(3+) on TCDD-initiated induction of CYP1A1 are independent of cell cycle control. As(3+) does not impact on Ah receptor function as evaluated by nuclear transport and binding to xenobiotic responsive element sequences, but it does reduce TCDD-induced CYP1A1 mRNA, a property that is concordant with RNA polymerase II association to the gene and the reduction in transcriptional heteronuclear RNA. We conclude from these studies that interruption of CYP1A1-induced transcription by As(3+) is not dependent upon cell cycle arrest.
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PMID:Arsenite inhibition of CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin is independent of cell cycle arrest. 1563 80

Using chromatin immunoprecipitation assays, we studied the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated recruitment of the aryl hydrocarbon receptor (AhR) and several co-regulators to the CYP1A1 promoter. AhR displayed a time-dependent recruitment, reaching a peak at 75 min and maintaining promoter occupancy for the remainder of the time course. Recruitment of AhR was followed by TIF2/SRC2, which preceded CBP, histone H3 acetylation, and RNA polymerase II (RNAPII). Simultaneous recruitment to the enhancer and the TATA box region suggests the formation of a large multiprotein complex bridging the two promoter regions. Interestingly, estrogen receptor alpha (ERalpha) displayed a TCDD- and time-dependent recruitment to the CYP1A1 promoter, which was increased by co-treatment with estradiol. Transfection in HuH7 human liver cells confirmed previously reported ERalpha enhancement of AhR activity. In contrast, TCDD did not induce the recruitment of ERalpha to the estrogen-responsive pS2 promoter, and after 120 min of co-treatment with estradiol, ERalpha is still present on the CYP1A1 promoter but no longer at pS2. RNA interference studies with T47D cells support a role for ERalpha in TCDD-dependent CYP1A1 expression. Our data suggest that ERalpha acts as a coregulator of AhR-mediated transcriptional activation and that the recruitment of ERalpha by AhR represents a novel mechanism AhR-ERalpha cross talk.
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PMID:Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters. 1596 90

Cytochrome P450 (CYP) 1A1 and CYP1B1 are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) in the human breast cancer cell line, MCF-7. Since CYP1A1 was inducible to a much greater degree than CYP1B1, we hypothesized that there may be differences in coactivator recruitment to the promoter and/or enhancer regions of these genes. Dioxin treatment leads to recruitment of the aryl hydrocarbon receptor to the enhancer regions but not to the proximal promoter regions of both the CYP1A1 and CYP1B1 genes. On the other hand, dioxin treatment facilitated recruitment of RNA polymerase II to the promoters but not the enhancer regions. Dioxin treatment also elicited recruitment of the transcriptional coactivators, steroid receptor coactivator 1 (SRC-1) and steroid receptor coactivator 2 (SRC-2) and p300, which possess intrinsic histone acetyltranferase activities, to both genes, whereas Brahma (BRM)/Switch 2-related gene 1 (BRG-1), a subunit of nucleosomal remodeling factors, was recruited more robustly to CYP1A1 relative to CYP1B1. Small inhibitory RNA-mediated knockdown of p300 and SRC-2 adversely affected dioxin induction of both genes, whereas knockdown of BRM/BRG-1 reduced CYP1A1 induction but had little, if any, effect on CYP1B1 induction. These results suggest that nucleosomal remodeling is less significant for dioxin-mediated induction of CYP1B1 than that of CYP1A1 and may be related to the more modest inducibility of the former. Interestingly, simultaneous knockdown of SRC-2 and BRM/BRG-1 had no greater effect on CYP1A1 induction than knockdown of each coactivator individually, while simultaneous knockdown of p300 and BRM/BRG-1 had a much greater effect than knockdown of each individual gene, suggesting that the recruitment of SRC-2 to CYP1A1 depends upon BRM/BRG-1, while the recruitments of p300 and BRM/BRG-1 are independent of each other. These observations provide novel insights into the functional roles of the endogenous coactivators in dioxin induction of the human CYP1A1 and CYP1B1 genes in their natural chromosomal configurations.
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PMID:Roles of coactivator proteins in dioxin induction of CYP1A1 and CYP1B1 in human breast cancer cells. 1884 20

Recent reports have proposed that some naturally occurring phytochemicals can function as anticancer agents mainly through inducing phase II drug detoxification enzymes. Of these phytochemicals, isothiocyanates sulforaphane (SUL), present in broccoli, is by far the most extensively studied. In spite of its positive effect on phase II drug metabolizing enzymes, its effect on the phase I bioactivating enzyme cytochrome P450 1a1 (Cyp1a1) is still a matter of debate. As a first step to investigate this effect, Hepa 1c1c7 and HepG2 cells were treated with various concentration of SUL. Our results showed that SUL-induced CYP1A1 mRNA in a dose- and time-dependent manner. Furthermore, this induction was further reflected on the protein and catalytic activity levels. Investigating the effect of SUL at the transcriptional level revealed that SUL increases the Cyp1a1 mRNA as early as 1h. The RNA polymerase inhibitor actinomycin D (Act-D) completely abolished the SUL-induced Cyp1a1 mRNA. Furthermore, SUL successfully activated AhR transformation and its subsequent binding to the XRE. At the post-transcriptional level, SUL did not affect the levels of existing Cyp1a1 mRNA transcripts. This is the first demonstration that the broccoli-derived SUL can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SUL induces this enzyme.
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PMID:Sulforaphane induces CYP1A1 mRNA, protein, and catalytic activity levels via an AhR-dependent pathway in murine hepatoma Hepa 1c1c7 and human HepG2 cells. 1901 13


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