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)

Mammalian organisms possess a variety of enzymes that catalyze the biotransformation of numerous chemicals with diverse structure. The gene superfamily comprising the cytochrome P-450 monooxygenases (P-450) are key participants in these reactions, and certain P-450 genes are highly inducible upon xenobiotic exposure. Many of the standard techniques used in the study of these systems rely on the disruption of tissues and cells, together with the preparation of subcellular particles. We have adopted a sensitive new technique, scanning laser cytometry, to monitor P-450-mediated O-dealkylation activities directly in cultured cells. Metabolism in single cells was quantified by fluorescence detection of resorufin, the P-450-mediated O-dealkylation product of alkoxyresorufin ether substrate probes. Functional activities associated with P-4501A1 and NADPH DT-diaphorase were compared among a human hepatoma (Hep G2) cell line and cells derived from mouse (Hepa 1clc7 wt) and rat (H4-II-E) hepatomas. Pretreating cells with the polyaromatic hydrocarbon inducer beta-naphthoflavone resulted in 50- to 100-fold increases in single cell rates of O-dealkylation of ethoxyresorufin (EROD activity). The use of scanning laser cytometry enabled in situ analysis of both constitutive and inducible biotransformation activities without disruption of cells or intracellular processes that determine the toxicologic fate of exogenous chemicals in vivo.
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PMID:Direct determination of functional activity of cytochrome P-4501A1 and NADPH DT-diaphorase in hepatoma cell lines using noninvasive scanning laser cytometry. 769 59

In this report we characterized the transcriptional regulation of the rat mdr1b gene by xenobiotics. The expression of this gene was increased in primary rat hepatocytes and in the H4-II-E hepatoma cell line by exposure to carcinogens such as aflatoxin B1, N-acetoxy-2-acetylaminofluorene, and methyl methanesulfonate. Nuclear run-on experiments indicated that the higher steady-state levels of mdr1b mRNA were due to an increase in transcription. The 5'-flanking region of the mdr1b gene was isolated, sequenced, and functionally characterized in transient and stable transfection assays. A single transcription start site was identified for this gene; no alternate start sites were used after induction with aflatoxin B1. Deletion analysis of this promoter demonstrated that the sequence between nt -214 and -178 was critical for basal promoter activity. This region did not contain any consensus-binding sites for previously identified transcription factors. A negative regulatory region was also identified between nt -940 and -250. No specific carcinogen-responsive element was identified; the xenobiotic response required a large part of the promoter. These data suggest that the carcinogen induction of mdr1b expression is mediated through sequences that overlap or that are identical to the basal promoter element.
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PMID:Characterization of the basal and carcinogen regulatory elements of the rat mdr1b promoter. 776 10

The hydrolytic metabolism of cocaine into benzoylecgonine, ecgonine methyl ester, and ecgonine was studied in the human hepatoma cell line Hep-G2 and in the nontumorigenic fetal hepatic cell line WRL-68. Also, the toxicological response of these cells to cocaine was compared to previously published results obtained with perfused liver cells and in vivo systems. Our experiments indicated that Hep-G2 appear to have similar metabolic and toxicological patterns to in vivo and perfused cell systems. The WRL-68 tissue culture system was found to be less similar. These results suggest Hep-G2 cells can be utilized to study cocaine metabolism and toxicology, and possibly in studies involving other xenobiotic compounds.
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PMID:Response of the human hepatic tissue cultures Hep-G2 and WRL-68 to cocaine. 776 18

A genomic clone encoding the hamster CYP1A1 gene was isolated from a hamster EMBL-3 genomic library and characterized. The CYP1A1 gene contained seven exons including the noncoding first exon as determined for CYP1A1 of other species. DNA sequence analysis up to -2307 bp of the CYP1A1 gene revealed the occurrence of five consensus xenobiotic responsive elements (XREs) and one basal transcription element (BTE) in addition to the canonical TATA box. For functional analysis, transfection experiments were performed in human hepatoma HepG2 cells with reporter gene constructs consisting of fragments with various lengths of the 5'-flanking region of the CYP1A1 gene and bacterial chloramphenicol acetyltransferase (CAT) gene. External deletion of the upstream region from the reporter gene resulted in a stepwise decrease of the CAT activity, suggesting that XREs were responsible for inducible expression of CYP1A1 gene by 3-methylcholanthrene (MC). A negative regulatory element (NRE) was also identified in the 5'-flanking region at -833 to -642. Removal of the NRE from the CYP1A1-CAT fusion gene resulted in about 3-fold increase of MC-inducible CAT activity. Using gel retardation assays with HepG2 nuclear extract, we demonstrated the presence of a specific protein which bound to the NRE fragment. Further competition analysis and methylation interference assays revealed that the nuclear protein bound to a 22-base fragment (from -688 to -709) of the NRE region, whose sequences were conserved among hamster, human, and rat CYP1A1 genes.
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PMID:Characterization of hamster CYP1A1 gene: inducible expression and negative regulation. 788 54

Cytochrome P4501A1 and its associated aryl hydrocarbon hydroxylase activity are highly inducible in the mouse hepatoma cell line, Hepa-1, by substrates of the enzyme and related compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mutants of this cell line, deficient in P4501A1 inducibility, were isolated. Some of the mutants show a dominant phenotype. Such mutants may have resulted from a genetic alteration leading to the inappropriate activation of a repressor gene that normally functions to restrict high level inducibility to the liver and certain other organs or to certain developmental stages. One dominant mutant was shown to express a protein that prevents binding of the liganded aryl hydrocarbon (Ah) receptor (which mediates induction of P4501A1) to its recognition sequence in DNA (the xenobiotic responsive element, or XRE). The majority of mutants are recessive, and were assigned to four different complementation groups (which probably correspond to four different genes). Gene A corresponds to the structural gene (Cyp1a-1) for P4501A1. Mutations in genes B, C and D all affect functioning of the Ah receptor. A cDNA for gene C was cloned. The encoded protein (ARNT) is required for ligand-dependent translocation of the Ah receptor to the nucleus and its binding to the XRE. ARNT and the Ah receptor form a heterodimeric complex which binds the XRE in a fashion such that both subunits bind the XRE directly. Both ARNT and the Ah receptor contain basic helix-loop-helix motifs. Such motifs have been identified in several transcription factors that bind DNA as heterodimers or homodimers. The roles of the proteins corresponding to the B and D genes are presently under investigation.
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PMID:A genetic analysis of processes regulating cytochrome P4501A1 expression. 794 73

We have recently demonstrated that release of normal human epithelial cells from cell-substratum and/or cell-cell adhesion generates cellular signals that induce the expression of CYP1A1 in the absence of xenobiotic polycyclic aromatic hydrocarbons (Sadek, C. M., and Allen-Hoffmann, B. L. (1994) J. Biol. Chem. 169, 16067-16074). To directly test the involvement of the Ah receptor signal transduction pathway in CYP1A1 induction following suspension of epithelial cells, we analyzed wild-type Hepa 1c1c7 cells, a subclone of the Hepa-1c1 mouse hepatoma line, and two mutant Hepa 1c1c7 lines, Class I and Class II. Suspension of wild-type Hepa 1c1c7 cells for 4 h led to an induction of steady state levels of CYP1A1 mRNA, similar to that obtained following treatment of adherent cells with 10(-9) M 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mutants of the Hepa 1c1c7 cells defective in different aspects of the Ah receptor signal transduction pathway exhibited negligible (Class I) or no (Class II) suspension-mediated induction of CYP1A1 mRNA. Gel mobility shift analysis of nuclear extracts from suspended or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated wild-type cells showed that both treatments produced identical shifts in the mobility of an XRE-containing probe. Antibody supershift experiments confirmed that the Ah receptor was a component of the DNA-protein complex from suspended wild-type Hepa 1c1c7 cells. These data directly demonstrate that suspension of wild-type Hepa 1c1c7 cells leads to nuclear localization and activation of the Ah receptor to a DNA-binding form.
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PMID:Suspension-mediated induction of Hepa 1c1c7 Cyp1a-1 expression is dependent on the Ah receptor signal transduction pathway. 798 17

1. The applicability of the human hepatoma cell line, HepG2, as a cell culture model for studying xenobiotic liver toxicity has been investigated using the well-characterized hepatotoxic chemical, bromobenzene. 2. Bromobenzene caused a concentration- (0-10 mM) and time-dependent (0-180 min) decrease in HepG2 cell viability. The degree of toxicity was dependent upon the culture medium composition and the state of cell growth. Toxicity in Modified Earle's and Williams' E Media was maximal at 7 days growth compared with 3 and 10 days, and was greater in Williams' than in Earle's medium. Toxicity in Dulbecco's medium was apparent only at 10 days growth and was less than the maximum toxicity in the other media. 3. Bromobenzene was detoxified by epoxide hydrase. The question of metabolic activation by P450 remained unresolved, but any involvement of P450 was by forms not inhibited by ketoconazole. 4. The mechanism of bromobenzene toxicity did not appear to involve lipid peroxidation, depletion of reduced glutathione, calcium-mediated proteolysis or metabolic activation by prostaglandin synthetase, but may have involved direct solvent-induced cell damage. 5. This study demonstrates the potential usefulness of HepG2 cells in toxicity testing and highlights the importance of standardizing culture conditions.
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PMID:Bromobenzene detoxification in the human liver-derived HepG2 cell line. 800 89

The dioxin receptor is a ligand-dependent transcription factor that binds to target DNA sequences (xenobiotic responsive elements, XREs) following ligand-dependent dimerization with its partner factor, Arnt (aryl hydrocarbon receptor nuclear translocator). Both factors contain an N-terminal basic region helix-loop-helix motif mediating dimerization and subsequent DNA binding. In this study we investigate the possible role of Arnt in agonistic and antagonistic effects of the dioxin receptor ligand alpha-naphthoflavone (ANF). Using specific antisera for the ligand binding dioxin receptor and Arnt, respectively, we show that exposure of the dioxin receptor to ANF in vitro induced recruitment of Arnt, thus stimulating binding of the heteromeric complex to XRE. In transient transfection assays, ANF at high concentrations stimulated expression of an XRE-driven reporter gene. This agonistic effect of ANF is, therefore, most likely attributable to ANF stimulation of dioxin receptor-Arnt heterodimerization and subsequent binding of the complex to XRE. Using a minimal XRE-driven reporter gene construct, we could further confirm earlier studies showing that ANF antagonizes the effect of a dioxin receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Next we employed chimeric receptor constructs containing amino acids 1-500 of the human glucocorticoid receptor fused to dioxin receptor fragments lacking the very N-terminal basic region helix-loop-helix dimerization and DNA binding motif. These chimeric receptor constructs show dioxin responsiveness upon transient transfection into mutant Arnt-deficient hepatoma cells and are, thus, functionally uncoupled from Arnt. Importantly, dioxin-dependent activation of the chimeric receptors was inhibited in the presence of ANF, demonstrating that dimerization of dioxin receptor with Arnt was not necessary for manifestation of the antagonistic effect of ANF. Rather, dioxin receptor sequences, which confer dioxin regulation upon a heterologous DNA binding and transactivating domain, also mediated the antagonistic effects of ANF.
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PMID:Agonistic and antagonistic effects of alpha-naphthoflavone on dioxin receptor function. Role of the basic region helix-loop-helix dioxin receptor partner factor Arnt. 803 60

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.
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PMID:Down-regulation of nuclear aryl hydrocarbon receptor DNA-binding and transactivation functions: requirement for a labile or inducible factor. 806 2

The intracellular basic helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). In analogy to nuclear receptors that are members of the steroid hormone receptor superfamily the dioxin receptor is a ligand-inducible transcriptional regulator that directly binds to response elements within regulated genes. The most commonly studied dioxin receptor ligands are dioxin itself and structurally related environmental contaminants. A physiological ligand has not yet been identified. Interestingly, however, indolo[3,2-b]carbazole, a compound formed from precursors in the diet, has been shown to bind the murine dioxin receptor with high affinity in vitro. In the present study we show that this compound and its methylated derivative 5,11-dimethylindolo[3,2-b]carbazole very potently activated transcription from a dioxin or xenobiotic response element (XRE)-driven reporter gene in both murine and human hepatoma cells. This effect was not observed in mutant, dioxin-resistant hepatoma cells which are either deficient in expression of dioxin receptor or the bHLH receptor partner factor Arnt. In vitro indolocarbazoles induced XRE binding activity by the human dioxin receptor-Arnt complex in a dose-dependent manner. Thus, both dioxin- and indolocarbazole-activated forms of dioxin receptor regulate target gene expression by the same mechanism involving recruitment of the bHLH factor Arnt and recognition of the XRE element. Finally, the indolo[3,2-b]carbazole-activated human dioxin receptor appeared to generate more stable complexes with the XRE target sequence relative to those produced by the dioxin-activated receptor form, indicating interesting mechanistic differences between different classes of dioxin receptor ligands in their abilities to modulate human dioxin receptor function.
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PMID:Regulation of human dioxin receptor function by indolocarbazoles, receptor ligands of dietary origin. 810 94


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