Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene.
It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.
Department of Pharmacology, University of Toronto, Ontario, Canada.
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Li
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Harper
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Tang
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Okey
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D015152:Blotting, Northern; D002384:Catalysis; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D004790:Enzyme Induction; D006801:Humans; D016015:Logistic Models; D008748:Methylcholanthrene / Q000494:pharmacology*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics; D014407:Tumor Cells, Cultured
C497396:ARNT protein, human; D001551:Benz(a)Anthracenes; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D008748:Methylcholanthrene; C030935:benz(a)anthracene; D019388:Cytochrome P-450 CYP1A2