UMLS:C0019204 - FACTA Search

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Query: UMLS:C0019204 (hepatocellular carcinoma)
71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Ah receptor regulates induction of cytochrome P450IA1 and mediates certain toxicities of polyhalogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been characterized previously in continuous cell lines, notably the mouse hepatoma line Hepa 1, the human squamous cell carcinoma line A431, and the human liver cell line Hep G2. The present work extends our knowledge of the Ah receptor in continuous human liver cell lines. Ah receptor can be detected in Mz-Hep-1, a hepatitis B virus-negative cell line derived from a Thorotrast-induced hepatocellular carcinoma. The mean concentration of Ah receptor in Mz-Hep-1 cells was 341 +/- 22 fmol/mg cytosol protein (mean +/- SEM, nine separate determinations). This is equivalent to approximately 30,000 sites per cell. The concentration of Ah receptor in Mz-Hep-1 cells is similar to that in Hepa 1 cells and approximately three times higher than that in Hep G2 cells. The Mz-Hep-1 Ah receptor sedimented in continuous sucrose gradients at approximately 9 S. Specificity of binding by [3H]TCDD was demonstrated by competitive binding of non-radiolabeled 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene (MC), and dibenz[a,h]anthracene in 50-fold molar excess. Phenobarbital, which is not a substrate for P450IA1, did not compete with [3H]TCDD for binding to Mz-Hep-1 Ah receptor. Dexamethasone and estradiol also did not compete with [3H]TCDD for binding, suggesting non-identity of Ah receptor with glucocorticoid or estrogen receptor. In separate experiments, glucocorticoid receptor was identified in Mz-Hep-1 cells. By Scatchard plot analysis, the apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Mz-Hep-1 Ah receptor was estimated to be 4.4 nM, compared to 0.8 nM in Hepa 1 cells. By Woolf plot analysis the Kd was 5.4 nM, compared to 1.2 nM in Hepa 1 cells. The [3H]TCDD.Ah receptor complex extracted from nuclei of Mz-Hep-1 cells incubated with [3H]TCDD in culture at 37 degrees sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was detectable in Mz-Hep-1 cells after pretreatment with inducing chemicals. Mz-Hep-1 cells have the highest concentrations of Ah receptor in any continuous human liver cell line thus far investigated. The Mz-Hep-1 Ah receptor is similar physicochemically to that described in murine systems. AHH activity is inducible in Mz-Hep-1 cells.
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PMID:Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1). Detection by binding with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase. 165 Feb 14

The photoaffinity labeling of the nuclear aryl hydrocarbon (Ah) receptor from mouse Hepa 1c1c7, rat hepatoma H-4-II E, and human liver Hep G2 cells was investigated using two high affinity ligands, namely 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) and 7-[125I]iodo-2,3,-dibenzo-p-dioxin ([125I]DBDD). Irradiation of nuclear [3H]TCDD-Ah receptor complexes from the three cell lines for 5 min gave 47, 38, and 62% yields of trichloroacetic acid-precipitable photoadducts from the Hepa 1c1c7, H-4-II E, and Hep G2 cell lines, respectively; denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation followed by autoradiography gave one major Ah receptor photoadduct for each cell line with apparent molecular masses at 97, 100, and 110 kDa, respectively. [125I]DBDD could also be used as a photoaffinity label for the nuclear Ah receptor from the three cell lines; although the maximum net yield of photoaffinity labeled nuclear Ah receptor from the rodent nuclear Ah receptor preparations was relatively low (0.5-2.5%), a greater than 15% yield of photoadduct was obtained from the human Hep G2 cells. Both [3H]TCDD and [125I]DBDD were utilized to photoaffinity label the nuclear Ah receptor in Hepa 1c1c7 cells in suspension and the net yield of photoadducts with these ligands was 94.6 and 3.0%, respectively. The cytosolic Ah receptor from the three cell lines was photolabeled with [125I]DBDD and the net yield of photoadducts varied from 3.3 to 14.7%. The functional activity of the photoaffinity-labeled nuclear TCDD-Ah receptor complexes from the cell lines was also determined by comparing relative binding affinities of the photolyzed and unphotolyzed complexes with a synthetic dioxin-responsive element (DRE) using a gel retardation assay. The photolyzed and unphotolyzed complexes from the three cell lines all bound with the DRE in the gel shift assay; however, the gel mobilities of the rodent and human nuclear receptor-DRE complexes were different. Quantitative analysis of the DRE binding showed that there were no significant differences between the photolyzed and unphotolyzed nuclear receptor complexes from the rodent cells, whereas there was a significant 27% decrease in the DRE binding of the photolyzed versus the unphotolyzed nuclear receptor complex from the human Hep G2 cells. These studies demonstrate the utility of [3H]TCDD and [125I]DBDD as photoaffinity labels for the Ah receptor and illustrate the structural and photochemical differences between the rodent and the human nuclear Ah receptor complexes.
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PMID:In situ and in vitro photoaffinity labeling of the nuclear aryl hydrocarbon receptor from transformed rodent and human cell lines. 165 3

The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.
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PMID:Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180. 165 65

Mouse hepatoma Hepa 1c1c7 cells and nonresponsive mutants have been extensively used as models for investigating the molecular mechanism of induction of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Incubation of cytosolic [3H]TCDD-aryl hydrocarbon (Ah) receptor from wild-type Hepa 1c1c7 cells for 16 h at 4 degrees C in 0.4 M KCl resulted in the formation of transformed liganded receptor which exhibited increased binding affinity on DNA-Sepharose columns. The elution properties of the peak with the highest DNA binding affinity were similar to the elution profiles of the nuclear receptor complex isolated from wild-type cells. TAOc1BPrcl (class I) nonresponsive mutant cells were characterized by relatively low levels of the cytosolic and nuclear Ah receptor complex. The BPrcl (class II) variant cell line contained levels of cytosolic receptor which were comparable to those observed in the wild-type cells; however, significantly reduced levels of nuclear receptor complex were observed in the class II variant cell line. Incubation of the nuclear or transformed liganded cytosolic Ah receptor from wild-type cells with a consensus 32P-labeled dioxin responsive element (DRE) in a gel shift assay gave a retarded band associated with the receptor-DRE complex. Incubation of the cytosolic receptor complex from the class I and II mutant cells for 16 h at 4 degrees C in 0.4 M KCl or for 2 h at 20 degrees C did not yield complexes with increasing binding affinities on DNA-Sepharose columns. Moreover, incubation of these complexes with 32P-labeled DRE did not give a retarded band in a gel shift assay. However, coincubation of the liganded class II mutant cytosol with cytosol from class I cells resulted in transformation of the liganded receptor and this was confirmed in both the DNA-Sepharose and gel retardation assays. These results suggest that the failure of class II mutant cells to respond to TCDD is due to a defect in the factors responsible for transformation of the cytosolic receptor complex.
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PMID:DNA binding properties of the Ah receptor in wild-type and variant mouse hepatoma cells. 165 77

alpha-Naphthoflavone (ANF) has previously been shown to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the Ah receptor under conditions in vitro. However, ANF also prevents TCDD-elicited cytochrome P450lA1 induction, immunosuppression, and down-regulation of the estrogen receptor in vivo and within intact isolated cells. These data suggest that ANF is a TCDD antagonist. This study investigated the ability of ANF to transform the Ah receptor contained in rat hepatic cytosol or mouse hepatoma cells to a form that recognizes the dioxin-responsive enhancer element (DRE) upstream of the cytochrome P450lA1 gene. Gel retardation analysis indicated that TCDD- or beta-naphthoflavone (BNF)-bound receptor was able to bind to the DRE, whereas essentially no receptor-DRE complexes were observed using cytosol incubated with ANF concentrations as high as 1000 nM. Furthermore, an excess of ANF, when added to cytosol just before TCDD, blocked, in a concentration-dependent manner, the ability of TCDD to transform the receptor to a form that bound to the DRE. These studies indicated that ANF binds to the receptor and confers on it a conformation that cannot recognize the DNA recognition sequence contained in the DRE. Although an excess of the agonist 2,3,7,8-tetrachlorodibenzofuran (TCDF) readily reversed the inhibitory actions of ANF, ANF was unable to reverse the effects of TCDD, TCDF, or BNF on the receptor. These studies suggested that TCDD binding, unlike that of ANF, results in a receptor conformation that has higher affinity for the ligand. Treatment of mouse hepatoma Hepa 1c1c7 cells with TCDD or BNF resulted in receptor contained in nuclear extracts that bound to the DRE. Only a very minor ligand-dependent protein-DNA complex was detected when cells were treated with ANF. These data indicated that ANF acts as an antagonist of TCDD by directly binding to the Ah receptor and eliciting a protein conformation that has very low affinity for DNA.
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PMID:Alpha-naphthoflavone acts as an antagonist of 2,3,7, 8-tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor. 165 99

The aromatic hydrocarbon (Ah) receptor behaves as a ligand-dependent transcription factor in the induction of cytochrome P450IA1. In cells exposed to the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the Ah receptor undergoes a transformation from a form with low affinity for nucleic acids (cytosolic receptor) into a form that preferentially associates with the cell nucleus (nuclear receptor). We followed the fate of the Ah receptor in mouse hepatoma cells during short-term exposure to [3H]TCDD by analyzing both cytosolic and nuclear fractions for specific binding. Nuclear Ah receptor levels increased over the first 2 h of treatment and then decreased to about 50% of maximal concentrations by 5 h after start of treatment. The decrease in nuclear receptor was not accompanied by a reappearance of detectable Ah receptor in the cytosolic fraction; further incubation with [3H]TCDD in cytosols from lysed cells did not label any additional receptor sites in cytosolic extract. By the 6th h of incubation, the total receptor population in the cell was only about 15-20% of that detected at the start of the incubation. The levels of specific binding detected were unaffected by up to 20 h of incubation with the vehicle DMSO, confirming that the presence of TCDD is required for the observed downregulation to occur. These results indicate that there is a substantial ligand-dependent loss in total Ah receptor during short-term exposure of cells to TCDD in culture.
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PMID:Downregulation of the Ah receptor in mouse hepatoma cells treated in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin. 166 93

The polymorphism of mammalian aromatic hydrocarbon (Ah) responsiveness appears to be correlated with genetic differences in risk of bronchogenic carcinoma caused by cigarette smoking. The human polymorphism has been uncovered, largely as the result of corresponding genetic differences characterized first in the mouse. The murine Ah locus has been defined as the gene encoding the aromatic hydrocarbon-responsive (Ah) receptor, responsible for the inducibility of a battery of at least six genes, two of which encode P450 enzymes. The high-affinity receptor and, hence, more highly induced levels of P450, can result in greater concentrations of polycyclic aromatic reactive intermediates that form DNA adducts and, ultimately, mutation fixation (tumour initiation). The Ah receptor is also likely to participate in growth and differentiation signal transduction pathways (tumour promotion). Positive and negative control regions flanking the murine Cyp 1a-1 and human CYP1A1 (cytochrome P(1)450) genes have been identified. A DNA motif approximately 1 kb upstream of the transcription start site appears to affect the translatability of the CYP1A1 mRNA and activity of the enzyme. Expression of the CYP1A1 or CYP1A2 enzyme in mouse hepatoma Hepa-1 cells lacking endogenous CYP1A1 activity represses constitutive transcription of not only the endogenous Cyp1a-1 gene but other genes in the dioxin-inducible [Ah] battery. Human polymorphisms involving a Msp I site 450 bp downstream from the last CYP1A1 exon have been described in Japan, the Eastern Mediterranean, Norway and the USA. The '1.9 allele' is associated with an increased incidence of Kreyberg Type I bronchogenic carcinomas in Japan and has recently been correlated with a valine-to-isoleucine substitution at position 462 in the haeme-binding region. This allele is about 3 times more frequent in Japan than in Caucasians of Norway and the USA, in which no correlation has been found between this allele and lung cancer. More work is needed to clarify these findings. Isolation and sequencing of the human Ah receptor cDNA, and the subsequent screening of populations for polymorphisms, hold great promise for predicting interindividual risk of cancer caused by smoking and other environmental pollutants.
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PMID:Human AH locus polymorphism and cancer: inducibility of CYP1A1 and other genes by combustion products and dioxin. 184 73

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.
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PMID:Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: characterization of 6-[125I]methyl-8-iodo-1,3-dichlorodibenzofuran-Ah receptor complexes. 184 13

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein had been implicated in regulating the expression of rat cytochrome P450IA1 which is most closely associated with aryl hydrocarbon hydroxylase (AHH). We have now investigated the presence of both the 4 S PAH-binding protein and the 8 S Ah receptor in rat hepatoma H4-II-E cells as well as the induction of P450IA1 upon their exposure to PAH's such as benzo[a]pyrene (BP) and 3-methylcholanthrene (3MC), and halogenated dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDBF). Sucrose density gradient analyses and hydroxylapatite assays indicate that, in addition to the 8 S protein, the 4 S PAH binding protein is present in these cells. This protein interacts in a saturable and high affinity manner with BP and 3MC, but not with TCDD or TCDBF. Using a P450IA1 probe, the induction of gene expression was observed by Northern blot analysis of total cellular RNA after exposure of the H4-II-E cells to BP, 3MC, or TCDBF. Since the 4 S protein was observed to interact only with BP and 3MC, these results suggest that this protein may also play a role in the PAH-induced expression of cytochrome P450IA1 gene expression in H4-II-E.
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PMID:Presence of the 4 S polycyclic hydrocarbon-binding protein in H4-II-E cells. 184 70

The Ah receptor is a soluble protein complex that mediates carcinogenesis by a wide range of environmental pollutants, including polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds. The best understood activity of the receptor concerns its role in the induction of cytochrome P450IA1. We undertook a somatic cell genetic analysis of P450IA1 induction using the mouse hepatoma cell line, Hepa-1. Clones of Hepa-1 were isolated that are defective in induction of P450IA1. Evidence was obtained that the clones are mutational in origin. Cell fusion experiments demonstrated that a few of the mutants are dominant, while the majority are recessive. The dominant mutants were shown to synthesize a repressor of P450IA1 transcription. The recessive mutants were assigned to 4 complementation groups (probably corresponding to 4 different genes). Complementation group A corresponds to the P450IA1 structural gene. Mutations in the B, C and D genes all affect functioning of the Ah receptor. A 'reverse selection procedure', whereby cells that express P450IA1 inducibility can be selected from a majority population of cells lacking inducibility, was developed. The reverse selection procedure was used to isolate transfectants of representative recessive mutants in which the mutational defects are complemented by exogenously applied genomic DNA. A human DNA-derived transfectant of a C- mutant was used to clone the human C gene. The C gene is not the ligand-binding subunit of the Ah receptor but is a protein that is required for translocation of Ah receptor-ligand complexes from cytoplasm to nucleus. In analogous experiments the dominant gene from one of the dominant mutants was transfected into wild-type Hepa-1 cells. Success in transfecting the dominant gene should provide the means for cloning it.
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PMID:Genetic and molecular analysis of the Ah receptor and of Cyp1a1 gene expression. 185 44

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