Browsing by keyword "Receptors, Aryl Hydrocarbon"
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Glycine N-methyltransferase is an example of functional diversity. Role as a polycyclic aromatic hydrocarbon-binding receptorThe cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 269, 5750-5756). The role of GNMT as a 4 S PAH-binding protein in mediating the induction of cytochrome P-4501A1 has been investigated further. GNMT cDNA, which was cloned into a pMAMneo vector containing the Rous sarcoma virus promoter and the neomycin resistance gene, was stably transfected into D422 Chinese hamster ovary (CHO) cells. Several positive clones were selected by reverse transcription-polymerase chain reaction and assayed for the expression of recombinant protein. Western blot analysis indicated the expression of significant levels of the 4 S protein in the stably transfected CHO cells (CHO-GNMT). Cytosolic preparations from the CHO-GNMT showed high benzo[a]pyrene (B[a]P) binding but no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity when compared with clones transfected with the pMAMneo vector alone (CHO-neo) or the parental CHO cells. Challanging the CHO-GNMT cells with 4 microM B[a]P resulted in elevated levels of CYP1A1 mRNA. Equally effective in inducing CYP1A1 mRNA were benzo[e]pyrene and 3-methylcholanthrene. On the other hand, TCDD did not induce CYP1A1 gene expression in these cells. B[a]P-treated CHO-GNMT, expressing the 4 S protein, also showed CYP1A1 protein by Western blotting and exhibited ethoxyresorufin-O-deethylase activity; neither the CHO-neo or parental CHO cells were positive for any of these measures. No Ah receptor message or protein was detectable in the parental CHO, CHO-neo, or CHO-GNMT cells. Furthermore, no XRE binding activity was observed in TCDD-treated cytosolic preparations or nuclear extracts from CHO-GNMT cells that were treated with TCDD. These studies unequivocally establish that GNMT is a PAH-binding protein that can mediate the induction of CYP1A1 by PAHs such as B[a]P through an Ah receptor-independent pathway.
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Transcriptional regulation of the human CYP1B1 gene. Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expressionThe cytochrome P450 1B1 gene (CYP1B1) is expressed constitutively and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the human breast adenocarcinoma cell line MCF-7 but not in the human hepatoma cell line HepG2. Genomic DNA isolated from both cell lines was digested with the methylation-sensitive restriction enzyme isoschizomers MspI and HpaII, and subjected to Southern analysis with a probe for the CYP1B1 promoter/enhancer region. Although differences were observed in methylation patterns for the CYP1B1 gene from MCF-7 and HepG2 cells, treatment with the demethylating agent 5-azacytidine (10 microM for 6 days) did not activate CYP1B1 mRNA expression in HepG2 cells. Furthermore, treatment with the histone deacetylase inhibitor trichostatin A (100 nM for 24 h) did not activate CYP1B1 mRNA expression in HepG2 cells. Comparative analysis of the constitutive expression of luciferase/1B1 reporter constructs containing a series of deletions in the 5' enhancer region indicated that in MCF-7 cells the region from -987 to -732 (relative to the transcription start site) was necessary for maximal levels of activity. Mutation of the aryl hydrocarbon receptor response elements (dioxin response elements) in this region showed that the dioxin response elements located at -833 is essential for constitutive gene expression in MCF-7 cells. In HepG2 cells, reporter gene activity was at least equal or greater than the activity observed in MCF-7 cells, which is in marked contrast to the expression of the native CYP1B1 gene. Taken together these findings indicate that the observed cell-specific differences in CYP1B1 constitutive expression are not mediated by DNA promoter/enhancer methylation, but are likely due to either 1) inaccessibility of the 5'-enhancer region in HepG2 cells to transcriptional activators due to a higher order chromatin structure that does not involve histone acetylation, or 2) the action of a repressor protein at cis-elements located outside of the -2296 to +25 region examined with the CYP1B1 reporter constructs. Furthermore, at least one of the dioxin response elements in the enhancer region is required for constitutive expression of CYP1B1.