Biomedical subjects

O Hankinson

Publications and source records attributed to O Hankinson.

At least 19 recordsLinked to original sources

PubMed2001

Identification of genes differentially induced by hypoxia in pancreatic cancer cells.

A hypoxic microenvironment is characteristic of many solid tumors, including pancreatic cancer, the fifth leading cause of cancer death in the United States. Hypoxia causes the stabilization of the HIF-1 (hypoxia-inducible factor-1) transcription factor and the induction of many genes that promote angiogenesis, tumor growth, and metastasis. We performed representational difference analysis (RDA) using mRNA extracted from hypoxic and normoxic Capan-2, a human pancreatic cancer cell line. cDNAs corresponding to hypoxia-inducible genes were cloned and sequenced. We identified GPI/NLK/AMF (glucose phosphate isomerase/neuroleukin/autocrine motility factor) as a hypoxic inducible gene. In addition, hexokinase II and DEC1/Stra13, genes known to be hypoxia inducible in other systems, were found to be hypoxia inducible in our pancreatic cancer system. We thus identified three genes that are induced by hypoxia in a human pancreatic cancer, including GPI/NLK/AMF, which was not previously known to be hypoxia inducible in any other system. These genes may provide new targets for diagnosis and treatment of pancreatic cancer.

Basic Helix-Loop-Helix Proteins↗

PubMed2001

Effect of diesel exhaust particles and their components on the allergen-specific IgE and IgG1 response in mice.

Increased antigen-specific IgE expression is a hallmark of the allergic response in mice. IgG1 may also be involved. Co-injection of mice with diesel exhaust particles (DEP) and ovalbumin three times over a 2 week period lead to a rapid and marked elevation of ovalbumin-specific IgE, IgG1 and also IgG2a, compared with ovalbumin alone. When DEP were injected 1 day before or after ovalbumin on each occasion, their adjuvant effect was considerably muted, suggesting that the adjuvant effect of DEP is short-lived, or that a physical interaction between ovalbumin and DEP is required. DEP were extracted with methylene chloride. Both the resulting core carbon particles and the organic extract enhanced ovalbumin specific IgE and IgG1 levels. Thus the adjuvant effect of DEP in this model is due both to the physical and the chemical attributes of the particles. The tricyclic hydrocarbons phenanthene (the most prevalent polycyclic aromatic hydrocarbon in DEP) and anthracene were both capable of enhancing antigen-specific IgE and IgG1 production. The phenolic antioxidant, butylated hydroxyanisole, which can affect gene expression via the antioxidant responsive element (ARE), had a lesser effect. Two agonists for the aryl hydrocarbon receptor, 3-methychloranthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin, either were without effect or suppressed the response, suggesting that DEP adjuvancy may not be mediated by this receptor.

Adjuvants, Immunologic↗

PubMed2001

CYP1A1 levels in lung tissue of tobacco smokers and polymorphisms of CYP1A1 and aromatic hydrocarbon receptor.

Induction of a polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 isoform CYP1A1 is regulated by aromatic hydrocarbon receptor (AHR). High inducibility of CYP1A1, possibly due to genetic polymorphisms, has been considered to be a risk factor for lung cancer in tobacco smokers. The relationship between low or high pulmonary expression of CYP1A1 and polymorphic genotypes of CYP1A1 and AHR was investigated in 73 active smokers. CYP1A1 expression was determined in surgical lung samples by measuring ethoxyresorufin O-deethylase (EROD) activity and by immunostaining for CYP1A1 protein. The most common allelic variants of CYP1A1 and AHR in Finns, i.e. the MspI variant (CYP1A1*2A), I462V variant (CYP1A1*2B), and -459C to T variant of CYP1A1 and the R554K variant (AHR*2) of AHR were studied using polymerase chain reaction based methods. EROD activity correlated positively with the daily cigarette consumption (r = 0.45). There was additional variation in EROD activity independent of the amount of smoking e.g. among those who smoked one pack per day until the day of operation, EROD activity ranged from 4-142 (median 48) pmol/min/mg. The frequencies of the MspI, 462V, and -459T variant alleles of CYP1A1 and 554K variant allele of AHR were 0.158, 0.055, 0.055 and 0.075, respectively. No differences were observed in the frequencies of polymorphic genotypes between the smokers with low and those with high expression, when the relationship was studied using a regression analysis adjusted for cigarette consumption. Our results thus indicate that the interindividual variation of CYP1A1 levels in smokers' lung tissue is not attributable to genetic polymorphisms of CYP1A1 or AHR tested in this study.

Cytochrome P-450 CYP1A1↗

PubMed2001

Loss of cyp1a1 messenger rna expression due to nonsense-mediated decay.

Clones of the mouse hepatoma cell line Hepa1c1c7 (Hepa-1) with lesions in the Cyp1a1 gene were isolated previously. A subset of these clones fails to express CYP1A1 mRNA even when treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which induces this mRNA in wild-type Hepa-1 cells. The current investigation sought an explanation for this phenotype in one of these clones, c33. Loss of mRNA expression in c33 was shown to be caused by mutational changes in the Cyp1a1 gene rather than by its epigenetic silencing. No mutations were identified in the 5' flanking region of the Cyp1a1 gene, containing the promoter and dioxin-responsive enhancer sequences. A single nucleotide insertion occurred at nucleotide 418 in the coding region of one Cyp1a1 allele, and a single nucleotide insertion occurred at nucleotide 465 in the other allele in c33. These sequence alterations were confirmed in the genomic DNA of the clone. Both insertions generate a premature termination codon at codon 172. This termination codon occurs in a position within the intron/exon structure of the Cyp1a1 gene such that the encoded mRNA should be subject to "nonsense-mediated decay" (NMD). Inhibition of protein synthesis is known to reverse NMD. The protein synthesis inhibitors cycloheximide and puromycin fully restored CYP1A1 mRNA expression to c33 cells, supporting the notion that NMD degrades CYP1A1 mRNA in this strain. The mutations identified in the coding region of c33 provide an explanation, therefore, for its loss of both CYP1A1 enzymatic activity and inducible CYP1A1 mRNA expression.

Animals↗

PubMed2001

Induction and regulation of the carcinogen-metabolizing enzyme CYP1A1 by marijuana smoke and delta (9)-tetrahydrocannabinol.

Induction of the carcinogen-metabolizing enzyme cytochrome P4501A1 (CYP1A1) is a key step in the development of tobacco-related cancers. To determine if marijuana smoke activates CYP1A1, a murine hepatoma cell line expressing an inducible CYP1A1 gene (Hepa-1) was exposed in vitro to tar extracts prepared from either tobacco, marijuana, or placebo marijuana cigarettes. Marijuana tar induced higher levels of CYP1A1 messenger RNA (mRNA) than did tobacco tar, yet resulted in much lower CYP1A1 enzyme activity. These differences between marijuana and tobacco were primarily due to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychoactive component of marijuana. Here we show that Delta(9)-THC acts through the aryl hydrocarbon receptor complex to activate transcription of CYP1A1. A 2-microg/ml concentration of Delta(9)-THC produced an average 2.5-fold induction of CYP1A1 mRNA, whereas a 10- microg/ml concentration of Delta(9)-THC produced a 4.3-fold induction. No induction was observed in Hepa-1 mutants lacking functional aryl-hydrocarbon receptor or aryl-hydrocarbon receptor nuclear translocator genes. At the same time, Delta(9)-THC competitively inhibited the CYP1A1 enzyme, reducing its ability to metabolize other substrates. Spiking tobacco tar with Delta(9)-THC resulted in a dose-dependent decrease in the ability to generate CYP1A1 enzyme activity as measured by the ethoxyresorufin-o-deethylase (EROD) assay. This inhibitory effect was confirmed by Michaelis-Menton kinetic analyses using recombinant human CYP1A1 enzyme expressed in insect microsomes. This complex regulation of CYP1A1 by marijuana smoke and the Delta(9)-THC that it contains has implications for the role of marijuana as a cancer risk factor.

Animals↗

PubMed2000

An uncommon phenotype of poor inducibility of CYP1A1 in human lung is not ascribable to polymorphisms in the AHR, ARNT, or CYP1A1 genes.

Cigarette smoking can induce CYP1A1 in the lung. Induction requires the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins. Lung samples from seven of 75 Finnish patients who smoked until the time of surgery exhibited absent or low levels of CYP1A1 protein, mRNA and enzymatic activity, suggesting that these individuals might be genetically non or poorly inducible for CYP1A1. All seven lung samples expressed normal levels of AHR mRNA and ARNT mRNA, indicating that they did not carry inactivating polymorphisms in the 5' upstream regulatory regions of these genes. Sequencing of cDNAs encompassing the complete coding regions of AHR and ARNT identified a previously known codon 554 polymorphism in AHR, which was present in the homozygous state in one individual. This polymorphism, which leads to an amino acid substitution, has previously been reported either to have no effect or to enhance CYP1A1 induction. Previously unreported silent single nucleotide polymorphisms were identified in codon 44 of AHR and codon 189 of ARNT. 1500 bp of genomic sequence from the 5' upstream regulatory sequence of the CYP1A1 gene was also sequenced in the non-inducible individuals. A nucleotide substitution polymorphism at position -459 was detected in the heterozygous state in two individuals. This polymorphic site does not reside in any known regulatory sequence. The complete CYP1A1 coding sequence and intron/exon boundaries were then sequenced. None of the non or poorly inducible individuals exhibited any polymorphisms, either homozygous or heterozygous compared to representative inducible individuals or the previously published CYP1A1 sequence. Thus, no polymorphisms in the AHR, ARNT or CYP1A1 genes were identified that could be responsible for the non/low inducibility phenotype observed.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed1997

A mutation in the aryl hydrocarbon receptor (AHR) in a cultured mammalian cell line identifies a novel region of AHR that affects DNA binding.

Introduction of a retroviral expression vector for the aryl hydrocarbon receptor (AHR) restores CYP1A1 inducibility to a mutant derivative of the Hepa-1 cell line that is defective in induction of CYP1A1 by ligands for the receptor. An AHR protein with normal ligand binding activity is expressed in the mutant but ligand treatment of mutant cell extract fails to induce binding of the AHR. ARNT (aryl hydrocarbon receptor nuclear translocator) dimer to the xenobiotic responsive element (XRE). AHR cDNAs derived from the mutant encode a protein that is unimpaired in ligand-dependent dimerization with ARNT, but the AHR.ARNT dimer so formed is severely impaired in XRE binding activity. The mutant cDNAs contain a C to G mutation at base 648, causing a cysteine to tryptophan alteration at amino acid 216, located between the PER-ARNT-SIM homology region (PAS) A and PAS B repeats. Introduction of the same mutation in the wild-type AHR sequence by site-directed mutagenesis similarity impaired XRE binding activity. Substitution with the conservative amino acid, serine, had no effect on XRE binding. The tryptophan mutation, but not the wild-type allele, was detectable in genomic DNA of the mutant. The implication that an amino acid within the PAS region may be involved in DNA binding indicates that the DNA binding behavior of AHR may be more anomalous than previously suspected.

Alleles↗

PubMed1997

ARNT-deficient mice and placental differentiation.

We used homologous recombination in embryonic stem cells to generate mice heterozygous for an aryl hydrocarbon nuclear translocator (ARNT) null mutation. These mice were intercrossed, but no live homozygous Arnt-/- knockout mice were produced among 64 newborns. Homozygotes die in utero between 9.5 and 10.5 days of gestation. Abnormalities included neural tube closure defects, forebrain hypoplasia, delayed rotation of the embryo, placental hemorrhaging, and visceral arch abnormalities. However, the primary cause of lethality appears to be failure of the embryonic component of the placenta to vascularize and form the labyrinthine spongiotrophoblast. This may be related to ARNT's known role in hypoxic induction of angiogenesis. We found no defects in yolk sac circulation.

Animals↗

PubMed1997

Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth.

Recent studies of tissue culture cells have defined a widespread system of oxygen-regulated gene expression based on the activation of a heterodimeric transcription factor termed hypoxia-inducible factor-1 (HIF-1). To determine whether the HIF-1 transcriptional response is activated within solid tumors and to define the consequences, we have studied tumor xenografts of a set of hepatoma (Hepa-1) cells that are wild type (wt), deficient (c4), and revertant (Rc4) for an obligatory component of the HIF-1 heterodimer, HIF-1beta. Because HIF-1beta is also essential for the xenobiotic response (in which it is termed the aryl hydrocarbon receptor nuclear translocator), we also studied c31 cells, which have a different defect in the xenobiotic response and form the HIF-1 complex normally. Two genes that show different degrees of HIF-1-dependent hypoxia-inducible expression in cell culture were selected for analysis-the glucose transporter, GLUT3, and vascular endothelial growth factor (VEGF). In situ hybridization showed intense focal induction of gene expression in tumors derived from wt, Rc4, and c31 cells, which was reduced (VEGF) or not seen (GLUT3) in those derived from c4 cells. In association with these changes, tumors of c4 cells had reduced vascularity and grew more slowly. These findings show that HIF-1 activation occurs in hypoxic regions of tumors and demonstrate a major influence on gene expression, tumor angiogenesis, and growth.

Animals↗

PubMed1997

Two murine homologs of the Drosophila single-minded protein that interact with the mouse aryl hydrocarbon receptor nuclear translocator protein.

Drosophila single-minded, which acts as a positive master gene regulator in central nervous system midline formation in Drosophila, its two mouse homologs SIM1 and SIM2, and the mammalian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins are members of the basic-helix-loop-helix.PAS family of transcription factors. In the yeast two-hybrid system, we demonstrate strong constitutive interaction of ARNT with SIM1 and SIM2 and fully ligand-dependent interaction of ARNT with AHR. Both the helix-loop-helix and the PAS regions of SIM1 and of ARNT are required for efficient heterodimerization. SIM1 and SIM2 do not form homodimers, and they do not interact with AHR. We also failed to detect homodimerization of ARNT. The interaction of ARNT with SIM1 was confirmed with in vitro synthesized proteins. Like AHR, in vitro synthesized SIM1 associates with the 90-kDa heat shock protein. SIM1 inhibits binding of the AHR.ARNT dimer to the xenobiotic response element in vitro. Introduction of SIM1 into hepatoma cells inhibits transcriptional transactivation by the endogenous AHR.ARNT dimer. The mouse SIM1. ARNT dimer binds only weakly to a proposed DNA target for the Drosophila SIM.ARNT dimer. In adult mice mRNA for SIM1 was expressed in lung, skeletal muscle, and kidney, whereas the mRNA for SIM2 was found in the latter two. ARNT is also expressed in these organs. Thus mouse SIM1 and SIM2 are novel heterodimerization partners for ARNT in vitro, and they may function both as positive and negative transcriptional regulators in vivo, during embryogenesis and in the adult organism.

Animals↗

PubMed1996

Induction of cytochrome P4501A1 by photooxidized tryptophan in Hepa lclc7 cells.

Mouse hepatoma Hepa-lclc7 (Hepa-1) cells were cultivated in the presence of UV-irradiated amino acids. The results demonstrated that all of the amino acids tested, UV-oxidized tryptophan caused the highest induction of 7-ethoxyresorufin O-deethylase (EROD) activity compared with the controls (P < 0.01). The induction of EROD activity by oxidized tryptophan was dose dependent, and maximal induction was obtained at 12 hr after administration. Studies with various Hepa-1 mutants, which are defective in either the aryl hydrocarbon (Ah) receptor or Ah receptor nuclear translocator protein, indicated that the induction of EROD activity by oxidized tryptophan occurs through the Ah receptor. Gel mobility shift assays using nuclear extracts of Hepa-1 cells revealed that oxidized products of tryptophan can induce both Ah receptor transformation and binding of the liganded Ah receptor complex to its specific DNA recognition site. CYP1A1 mRNA, quantified by reverse transcription-polymerase chain reaction, and CYP1A1 protein were induced markedly in the oxidized tryptophan group compared with the controls. Injection of isolated oxidized tryptophan products into adult male rats caused significant induction of EROD activity in the pulmonary and hepatic microsomes compared with the controls (P < 0.01). These results demonstrated that oxidized tryptophan induces Ah receptor activation and binding of the liganded Ah receptor complex to its specific DNA recognition site, thereby initiating transcription and translation of the CYP1A1 gene with concomitant increase of EROD activity in Hepa-1 cells. Induction of EROD activity in the liver and lungs after injection of isolated oxidized tryptophan products into rats suggests that a similar mechanism may be operative in vivo.

Animals↗

PubMed1996

The role of the aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells.

Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix Per-AHR-ARNT-Sim (PAS) proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, had been recognized previously as the aryl hydrocarbon receptor nuclear translocator (ARNT), an essential component of the xenobiotic response. In the present work, ARNT-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of ARNT/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and transcriptional activity of HIF-1 was absent in the mutant cells, indicating an essential role for ARNT/HIF-1beta. Analysis of deleted ARNT/HIF-1beta genes indicated that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, were necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrated the critical importance of ARNT/HIF-1beta in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persisted in these mutant cells, clearly distinguishing ARNT/HIF-1beta-dependent and ARNT/HIF-1beta-independent mechanisms of gene activation by both these stimuli.

Animals↗

PubMed1996

Functional characterization of DNA-binding domains of the subunits of the heterodimeric aryl hydrocarbon receptor complex imputing novel and canonical basic helix-loop-helix protein-DNA interactions.

The aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) belong to a novel subclass of basic helix-loop-helix transcription factors. The AHR.ARNT heterodimer binds to the xenobiotic responsive element (XRE). Substitution of each of four amino acids in the basic region of ARNT with alanine severely diminishes or abolishes XRE binding, intimating that these amino acids contact DNA bases. Three of these amino acids are conserved among basic helix-loop-helix proteins, and the corresponding amino acids of Max and USF are known to contact DNA bases. Alanine scanning mutagenesis of the basic domain of AHR and substitution with conservative amino acids at particular positions in this domain and in a more amino-proximal AHR segment previously shown to be required for XRE binding (Fukunaga, B. N., and Hankinson, O. (1996) J. Biol. Chem. 271, 3743-3749) demonstrate that the most carboxyl-proximal amino acid position of the basic domain and a position within the amino-proximal segment are intolerant to amino acid substitution with regard to XRE binding, suggesting that these two amino acids make base contacts. Amino acid positions in these AHR regions and in the ARNT basic region less adversely affected by substitution are also identified. The amino acids at these positions may contact the phosphodiester backbone. The apparent bipartite nature of the DNA binding region of AHR and the identity of those of its amino acids that apparently make DNA contacts impute a novel protein-DNA binding behavior for AHR.

Amino Acid Sequence↗

PubMed1996

Identification of a novel domain in the aryl hydrocarbon receptor required for DNA binding.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the AHR nuclear translocator protein (ARNT). Both proteins possess basic helix-loop-helix motifs. ARNT binds to the side of the xenobiotic responsive element (XRE) that resembles an E-box (the sequence recognized by the majority of other basic helix-loop-helix proteins), whereas AHR binds to the side of the XRE that does not conform to the E-box sequence. The basic region of ARNT closely resembles those of other E-box-binding proteins, whereas the "nominal basic region" of AHR (amino acids 27 39), although required for XRE binding, deviates from this consensus. By extensive mutational analysis it is shown here that an additional block of amino acids of AHR (from tyrosine 9 to lysine 20) that contains a highly basic segment is required for XRE binding and transcriptional activation. Deletion of the first nine amino acids negates XRE binding. Substitution of either tyrosine 9 or arginine 14 with alanine eliminates XRE binding, whereas alanine substitutions at certain other sites within the block reduce but do not eliminate binding. The reported absence of the first nine amino acids in the purified protein may therefore be artifactual. These results suggest that the amino acids of AHR involved in binding to the XRE constitute a novel DNA-binding domain, comprising amino acids located within and amino-terminal to the nominal basic region.

Alanine↗

PubMed1996

Basis for the loss of aryl hydrocarbon receptor gene expression in clones of a mouse hepatoma cell line.

Rare benzo[a]pyrene-resistant clones were previously isolated from the mouse hepatoma cell line, Hepa-1 (Hepa1c1c7), and shown to be deficient in induction of CYP1A1 mRNA by ligands for the aryl hydrocarbon receptor (AHR). Clones belonging to complementation group B were shown to have reduced levels of ligand binding to AHR. It is shown here that all 15 independently derived B clones analyzed had much reduced levels of AHR mRNA, but in each case, the mRNA was normal in size. Infection of B clones with a retroviral expression vector for AHR restores CYP1A1 inducibility (although viral AHR expression is progressively silenced and CYP1A1 expression progressively diminishes as the cells are maintained in culture). Treatment of the B clones with the histone deacetylase inhibitors sodium butyrate or trichostatin A restores AHR expression and also restores CYP1A1 inducibility to nearly 100% of the cells in the treated cultures. Fusion of a representative B clone with a rat hepatoma cell line restores expression to the mouse AHR gene encoded by the B clone's genome. These results demonstrate that the loss of CYP1A1 inducibility in B clones is probably totally ascribable to their reduced levels of AHR and that the clones are most probably not mutated in the AHR gene but are deficient in its expression. The evidence suggests that the reduction in expression of mRNA encoded by the endogenous AHR gene in the B clones is not due to an epigenetic alteration in chromatin structure but that the clones are probably defective either in a transcription factor for the AHR gene or in a protein required for generating an open chromatin configuration over the gene.

Animals↗

PubMed1995

Identification of functional domains of the aryl hydrocarbon receptor.

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding was localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt (Reisz-Porszasz, S., Probst, M.R., Fukunaga, B. N., and Hankinson, O. (1994) Mol. Cell. Biol. 14, 6075-6086) compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than the corresponding region of Arnt.

Animals↗

PubMed1995

The aryl hydrocarbon receptor complex.

The heteromeric unliganded aryl hydrocarbon receptor complex (AHRC) contains the aryl hydrocarbon receptor monomer (AHR). Binding of polycyclic or halogenated aromatic hydrocarbon (PAH and HAH) ligand causes release of AHR, which then associates with the AHR nuclear translocator protein (ARNT) to generate the heterodimeric "transformed" AHRC. AHR and ARNT belong to a novel subclass of basic helix-loop-helix-containing transcription factors. The transformed AHRC binds xenobiotic responsive elements in responsive genes and turns on their transcription. Certain of these genes encode enzymes involved in the metabolic activation of PAHs to mutagenic derivatives. HAHs are not genotoxic: Their pathogenicity depends on the AHRC but not on their metabolism. Current research includes investigations directed towards delineating the pathways of HAH pathogenesis, ascertaining whether AHR can mediate signal transduction independently of DNA binding, understanding the mechanism of transcriptional activation, and investigating the potential roles of AHR and ARNT in development.

Animals↗

PubMed1995

Orientation of the heterodimeric aryl hydrocarbon (dioxin) receptor complex on its asymmetric DNA recognition sequence.

The 2,3,7,8-tetrachlorodibenzo-p-dioxin-transformed aryl hydrocarbon receptor (AHR) complex binds to xenobiotic-responsive element (XRE) sequences in the 5' flanking region of the CYP1A1 gene, resulting in initiation of transcription. Both components of the transformed AHR complex [the ligand-binding AHR monomer and the AHR nuclear translocator (ARNT)] directly contact the XRE. These proteins belong to a novel subclass of basic helix-loop-helix transcription factors. The binding sites of AHR and ARNT on the asymmetric XRE were determined using nuclear extracts of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated Hepa-1c1c7 cells and a panel of double-stranded oligonucleotides containing XRE1 of the CYP1A1 gene (5'-TTGCGTGAGAA-3'), in which all combinations of three, two, or one of the thymines indicated were substituted by the photoreactive thymine analog 5-bromodeoxyuracil. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AHR or ARNT demonstrated that ARNT directly contacts the 3'-most thymine position, that AHR directly contacts the second thymine position, and that neither protein contacts the 5'-most thymine position. The thymine position contacted by ARNT lies within a three-nucleotide sequence (5'-GTG-3') identical to a half-site of an E-box element (5'-CACGTG-3') that is recognized by a number of other basic helix-loop-helix transcription factors. AHR binds to a portion of the XRE that does not resemble an E-box. Additional experiments demonstrated that neither protein loops over to contact residues located beyond the other's binding site.

Animals↗