Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin versus 3-methylcholanthrene: comparative studies of Ah receptor binding, transformation, and induction of CYP1A1. 816 16

The aromatic hydrocarbon (Ah) receptor is a cytosolic protein that binds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and nonhalogenated ligands such as 3-methylcholanthrene (MC) and benzo[a]pyrene. The best characterized biological response mediated by the Ah receptor is induction of cytochrome P4501A1 (CYP1A1). Photoaffinity labeling of the Ah receptor has been reported only with halogenated ligands such as TCDD and some of its iodinated derivatives. In this study, photolabeling of the Ah receptor was achieved with the nonhalogenated aromatic hydrocarbon [3H]MC. Sources of Ah receptor were the mouse hepatoma cell line Hepa-1c1c9 and the human colon adenocarcinoma line LS180. Cytosolic fractions either were used in a crude form or were enriched by glycerol density gradient centrifugation. These then were incubated with [3H]MC, irradiated with UV light (> 300 nm), precipitated with acetone, and analyzed by SDS-polyacrylamide gel electrophoresis. The yield of photoadduct formation was lower with [3H]MC (approximately 1%) compared with [3H]TCDD (3.5%) in Hepa-1c1c9 cells. The same was true in LS180 cells, i.e. the yield was 0.2% for [3H]MC versus 5.48 +/- 0.26% for [3H]TCDD. The relative molecular mass of the [3H]MC-labeled receptor estimated by SDS-polyacrylamide gel electrophoresis was 94,600 +/- 2,400 (mean +/- S.E.) for Hepa-1c1c9 cells and 113,600 +/- 3,200 for LS180 cells; these are the same molecular masses as determined by photolabeling with [3H]TCDD. In velocity sedimentation assays of mouse cytosol, [3H]MC binds specifically to two cytosolic proteins: the 4 S carcinogen-binding protein and the Ah receptor (9 S). However, no photolabeling of the 4 S protein was detected in our experiments. [3H]MC photolabeling of the human Ah receptor from LS180 cells was detected only in experiments using enriched cytosolic preparations. In addition to the 95-kDa ligand-binding subunit, a specifically radiolabeled protein of 164,900 +/- 5,800 kDa was also detected in Hepa-1c1c9 cytosol photolabeled with [3H]MC, suggesting cross-linking, by MC, of another subunit of the multimeric Ah receptor complex to the ligand-binding subunit. Immunochemical analysis showed that the ligand-binding subunit of the Ah receptor is one component of the 165-kDa complex. The other protein in the complex could not be identified with antibodies to the heat shock proteins hsp90 or hsp70 or with antibodies to the p59 protein or Ah receptor nuclear translocator protein. The identity and function of the protein that becomes cross-linked to the ligand-binding subunit require further investigation.
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PMID:Photoaffinity labeling of the Ah receptor with 3-[3H]methylcholanthrene and formation of a 165-kDa complex between the ligand-binding subunit and a novel cytosolic protein. 816 17

A new synthetic route was utilized to prepare 6-substituted 3,4-benzocoumarins where the substituents were iodo, fluoro, trifluoromethyl, bromo, chloro, isopropyl, ethyl, t-butyl, methyl, hydrogen, amino, phenyl, or nitro; 3,4-naphthocoumarin was also synthesized. The relative affinities of these congeners for the aryl hydrocarbon (Ah) receptor were determined using rat hepatic cytosol and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD) as the radioligand. In addition, the Ah receptor agonist activity of the 6-substituted 3,4-benzocoumarins was determined from their concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast with many other structural classes of halogenated aromatics, there was not a correlation between the structure-binding versus structure induction relationships for the 6-substituted 3,4-benzocoumarins. These results suggested that some of these congeners may exhibit partial Ah receptor antagonist activities and this was investigated by determining the inhibitory effects of 6-substituted 3,4-benzocoumarins on TCDD-induced EROD activity in rat hepatoma H4II E cells in culture. Only four compounds (6-isopropyl, 6-phenyl, 6-fluoro, and 6-t-butyl) inhibited the TCDD-induced response (21.7 to 64.4% inhibition) and the mechanism of action of the most active inhibitor, 6-t-butyl-3,4-benzocoumarin, was further investigated. In contrast, with other partial Ah receptor antagonists such as alpha-naphthoflavone, cotreatment of rat hepatoma H4II E cells with 1 nM TCDD plus 1 and 10 microM 6-t-butyl-3,4-benzocoumarin did not result in decreased levels of the Ah receptor complex (liganded with TCDD). In addition, there was not significant inhibition of TCDD-induced CYP1A1 mRNA levels or protein as determined by Northern and Western blot analyses. The results suggest that 6-t-butyl-3,4-benzocoumarin or one of its metabolites is a post-translational inhibitor of CYP1A1-dependent enzyme (EROD) activity in this cell line and thus represents a novel Ah receptor-independent inhibition of CYP1A1.
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PMID:6-substituted 3,4-benzocoumarins: a new structural class of inducers and inhibitors of CYP1A1-dependent activity. 821 8

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

We have used a ligation-mediated polymerase chain reaction technique to analyze protein-DNA interactions at a dioxin-responsive enhancer upstream of the CYP1A1 gene in intact mouse hepatoma cells. In its inactive state, the enhancer binds few, if any, proteins within the major DNA groove in vivo. Thus, the inactive enhancer is relatively inaccessible to DNA-binding proteins. Exposure of cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to the binding of the liganded Ah receptor at six sites within the major DNA groove of the enhancer. The receptor-enhancer interactions occur rapidly and do not require ongoing transcription, consistent with their role in regulating CYP1A1 gene expression. The liganded receptor, which is a heteromer composed of at least two basic helix-loop-helix proteins, is probably the only DNA-binding transcription factor necessary to activate the enhancer in vivo. The small size and irregular distribution of receptor binding sites suggest that chromatin structure imposes substantial steric constraints upon the function of the receptor-enhancer system in intact cells.
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PMID:Mechanism of dioxin action: receptor-enhancer interactions in intact cells. 838 88

The DNA upstream of the dioxin-inducible CYP1A1 gene contains six distinct sites to which the liganded Ah receptor binds in intact mouse hepatoma cells. Here, we have analyzed these six bona fide receptor-binding sites in order to study the relationships between DNA sequence, receptor binding, and dioxin responsiveness. Gel retardation studies reveal that the sites vary by about 7-fold in their relative affinities for the liganded receptor. Within this range, there is no obvious association between the strength of receptor binding and the degree of dioxin responsiveness, as measured in transfection experiments. In fact, one site binds the receptor well but fails to respond to dioxin. This observation implies that the receptor-DNA binding event per se is not sufficient to confer dioxin responsiveness upon a linked gene. Comparison of the DNA sequences of the six receptor-binding sites permits the derivation of a "functional consensus" recognition sequence, which is more extended in length than the "core"-binding sequence previously described. In corroboration of these results, protein-DNA cross-linking studies indicate that the liganded receptor contacts base pairs beyond the core sequence. Our observations also indicate that the liganded receptor can tolerate limited sequence heterogeneity at its DNA-binding site and still elicit a response to dioxin. This finding might reflect corresponding heterogeneity in the amino acid sequence of the liganded receptor's DNA-binding domain.
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PMID:Protein-DNA interactions at a dioxin-responsive enhancer. Analysis of six bona fide DNA-binding sites for the liganded Ah receptor. 838 16

In the presence of halogenated and polycyclic aromatic hydrocarbons, the CYP1A1 gene is regulated through induction after ligand binding to the cytosolic Ah receptor (AhR). Ligand-dependent AhR activation leads to nuclear translocation and binding of the receptor to dioxin-responsive element (DRE) sequences, an event that initiates transcriptional activation of the CYP1A1 gene. We recently established a human hepatoma cell line stably integrated with the human CYP1A1 promoter and 5'-flanking enhancer sequences fused to the firefly luciferase gene. This cell line, 101L, was used to determine whether the induction of CYP1A1 by omeprazole, a gastric proton pump inhibitor, is AhR mediated. Treatment of 101L cells with either 50 microM omeprazole or 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin for 12-72 hr resulted in maximal activity at 24 hr for both inducers. A dose-response curve for omeprazole induction at 24 hr was determined and the EC50 for omeprazole induction of the human CYP1A1 gene was estimated to be 100 microM. The induction of the CYP1A1 gene by omeprazole corresponds to increases in CYP1A1 mRNA. To examine whether omeprazole-initiated transcriptional activation of the CYP1A1 gene correlates with nuclear accumulation of the AhR, binding of nuclear proteins to the DRE was examined. When gel mobility shift assays were performed using nuclear extracts isolated from 101L cells treated with omeprazole or 2,3,7,8-tetrachlorodibenzo-p-dioxin, specific binding of the AhR to the DRE was observed. These studies demonstrate that omeprazole initiates AhR activation and that induction of the human CYP1A1 gene by omeprazole is AhR dependent.
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PMID:Nuclear uptake of the Ah (dioxin) receptor in response to omeprazole: transcriptional activation of the human CYP1A1 gene. 838 5

Immunoprecipitation experiments performed on cytosolic extracts of the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1) confirm that the 9-S, unliganded, cytosolic aryl hydrocarbon (Ah) receptor complex contains the 90-kDa heat shock protein and the Ah receptor protein but reveal that it does not contain the Ah receptor nuclear translocator (ARNT) protein. These experiments confirm that the 6-S liganded form of the receptor identified in nuclear extracts of cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contains the Ah receptor protein and ARNT but not the 90-kDa heat shock protein. The 6-S liganded Ah receptor complex activates transcription of the CYP1A1 gene via its binding to upstream xenobiotic-responsive elements (XREs). Treatment of cytosolic extracts of Hepa-1 cells with TCDD in vitro transforms the Ah receptor complex to the XRE-binding state. No such transformation occurs in a C- mutant deficient in ARNT activity. When in vitro synthesized ARNT was added concomitantly with TCDD to C- cytosolic extracts, it associated with the Ah receptor and restored Ah receptor-dependent XRE-binding activity to the extracts. Covalent cross-linking experiments in nuclear extracts of Hepa-1 and human LS180 cells treated with TCDD in vivo demonstrate that both ARNT and the Ah receptor bind directly to the XRE core sequence.
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PMID:Role of the aryl hydrocarbon receptor nuclear translocator protein in aryl hydrocarbon (dioxin) receptor action. 839 13

Previous studies from this laboratory have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased complement component C3 levels in female B6C3F1 mouse serum following in vivo acute or subchronic exposure (White et al., 1986). Since TCDD is a hepatotoxic compound and more than 90% of serum C3 is produced by the liver, studies were undertaken using mouse Hepa 1c1c7 (Hepa 1) hepatoma cell line to determine if TCDD acts directly on hepatocytes to inhibit C3 production. The C3-producing capacity of Hepa 1 cells was first examined. When confluent Hepa 1 cell monolayers were cultured in 24-well plates with serum-free medium, a detectable amount of C3 (14.1 +/- 0.8 ng/ml) was secreted as early as 1 h after culture and reached a plateau at 12 h (68.3 +/- 4.9 ng/ml). Furthermore, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that the molecular weight of C3 in culture supernatant corresponded to that present in mouse serum. Human recombinant IL-1 beta (hrIL-1 beta), a known inducer of complement C3, at doses as low as 1 unit/ml increased the C3 production to 158% of control after 24 h of incubation. The effect of hrIL-1 beta was dose dependent, and the maximum tested dose of 10 units/ml increased C3 production to 256% of control. When cells were directly exposed to TCDD at concentrations from 10(-10) to 10(-6) M, there was no inhibitory effect on production of C3. TCDD also failed to block the stimulatory effect of 10 units/ml hrIL-1 beta added to the culture 1 h later. To verify that cultured Hepa 1 cells were able to respond to TCDD, 7-ethoxyresorufin O-deethylase (EROD) activity was measured under the same conditions. TCDD dose-dependently increased EROD activity of Hepa 1 cells at 24 h following exposure. The activity reached 56.7 +/- 3.0 pmol/min/mg protein with 10(-9) M TCDD, compared with 10.7 +/- 1.7 pmol/min/mg protein of vehicle-exposed cells. Our results indicate that the direct interaction of TCDD with Hepa 1 cells does not affect their C3-producing capacity, although EROD activity, a characteristic response mediated by the cellular TCDD/Ah receptor, was induced. The lack of effect of TCDD in vitro suggests that the decrease of serum C3 levels observed in vivo may result from an indirect effect of TCDD on hepatocytes.
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PMID:Mouse Hepa 1c1c7 hepatoma cells produce complement component C3; 2,3,7,8-tetrachlorodibenzo-p-dioxin fails to modulate this capacity. 849 28

We have studied the response of genes in the dioxin-inducible [Ah] battery to three compounds that protect mouse hepatoma cells (Hepa-1c7c7 wild-type, wt) against menadione toxicity. Pretreatment of wt cells with 25 microM 5,10-dihydroindenol[1,2-b]indole (DHII), 25 microM tert-butylhydroquinone (tBHO) or 10 microM menadione itself, generated substantial protection against toxicity produced by subsequent menadione exposure. The gene response was examined in wt cells, and three mutant lines: CYP1A1 metabolism-deficient (c37 or P1-); nuclear translocation-impaired (c4 or nt-); and AHR-deficient (c2 or r-, containing < 10% of normal functional receptor levels). DHII treatment of wt cells for 12 hr markedly elevated the enzyme activities and mRNA levels of genes in the [Ah] battery: aryl hydrocarbon hydroxylase (Cyp1a1), NAD(P)H:menadione oxidoreductase (Nmol), cytosolic aldehyde dehydrogenase class 3 (Ahd4), and UDP-glucuronosyltransferase form 1*06 (Ugt1*06). Treatment of the c4 and c2 cells with DHII failed to induce mRNA levels of the genes, indicating that induction of the [Ah] gene battery by DHII is aromatic hydrocarbon receptor (AHR)-mediated. On the other hand, neither tBHO nor menadione caused increases in CYPlAl mRNA, but tBHQ significantly enhanced the NMO1, AHD4, and UGT1*06 mRNA levels in all three mutant cell lines. In conclusion, we expect one or more putative electrophile response elements (EpRE), previously found in the regulatory regions of the murine Nmol, Ahd4, and ugt1*06 genes, to be functional in responding to phenolic antioxidants.
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PMID:Response of [Ah] battery genes to compounds that protect against menadione toxicity. 861 69


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