Suspension-mediated induction of Hepa 1c1c7 Cyp1a-1 expression is dependent on the Ah receptor signal transduction pathway

• Published in: The Journal of biological chemistry 1994-12, Vol.269 (50), p.31505-31509
• Main Authors: Sadek, C.M., Allen-Hoffmann, B.L.
• Format: Article
• Language: English
• Subjects: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Cell Adhesion; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System - metabolism; DNA-Binding Proteins; Enzyme Induction; Gene Expression Regulation - drug effects; In Vitro Techniques; Liver Neoplasms, Experimental - enzymology; Liver Neoplasms, Experimental - pathology; Mice; Oxidoreductases - metabolism; Polychlorinated Dibenzodioxins - pharmacology; Receptors, Aryl Hydrocarbon - physiology; RNA, Messenger - genetics; Signal Transduction; Transcription Factors - metabolism; Tumor Cells, Cultured - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)31723-X

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.