Transient Expression of CYP1A1 in Rat Epithelial Cells Cultured in Suspension Scott A. Monk, Michael S. Denison, and Robert H. Rice 1 Department of Environmental Toxicology, One Shields Avenue, University of California, Davis, California 95616-8588 Received April 18, 2001, and in revised form June 12, 2001; published online August 7, 2001 Suspension of human epidermal cells in methylcel- lulose-containing medium induces CYP1A1 by a mech- anism requiring functional Ah receptor (AhR). In present work CYP1A1 mRNA was induced in a variety of cultured rat epithelial cells by suspension, but the induction was transient, with CYP1A1 mRNA reaching maximal levels by 5 h and disappearing by 12 h. Though the methylcellulose itself contained no detect- able ligand, (a) suspension activated the AhR, as judged by mobility shift assays, (b) the AhR competi- tive inhibitor -naphthoflavone inhibited suspension- mediated induction, and (c) induction was dependent upon dioxin responsive transcriptional elements in the CYP1A1 promoter. The rapid disappearance of CYP1A1 mRNA after 5 h of suspension was unaffected by the addition of TCDD but was prevented by the inclusion of the protein synthesis inhibitor cyclohexi- mide. Thus the downregulation appears to be medi- ated by a novel short-lived protein induced or acti- vated by suspension. © 2001 Academic Press Key Words: CYP1A1; keratinocytes; methylcellulose; Ah receptor. Cytochrome P450 monooxygenases (P450s) consti- tute a superfamily of heme-proteins that catalyze the monooxygenation of both endogenous and exogenous compounds. P450s generally serve a protective func- tion against exogenous compounds, yielding products that are more water-soluble, better substrates for Phase II conjugative reactions, and more easily ex- creted from the body. However, in some cases P450 metabolism results in products that are much more reactive and toxic than the parent compound. Cyto- chrome P4501A1 (CYP1A1) 2 is one of the most in- tensely studied P450s, in part due to its ability to metabolize a variety of polycyclic aromatic hydrocar- bons (PAHs) to products that are highly reactive and form adducts with cellular macromolecules, including DNA. Expression of CYP1A1 is normally extremely low but is highly inducible by treatment with PAHs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and re- lated compounds. CYP1A1 gene expression is under complex transcriptional control, with several defined trans-acting factors regulating constitutive expression, tissue- and differentiation-specific control, and PAH/ TCDD induction (1–3). The best characterized trans- acting factor involved in CYP1A1 regulation is the aryl hydrocarbon receptor (AhR). The AhR is a highly evolutionarily conserved, ligand- dependent transcription factor. In its inactive form, the AhR is localized in the cytoplasm and is bound by two molecules of hsp90 (4) as well as a recently identified 37-kDa protein referred to as AIP, XAP2, or ARA9 (5–7). Exogenous ligands for the AhR diffuse across the plasma membrane and bind to the AhR, resulting in its translocation into the nucleus. Once in the nucleus, the AhR dissociates from hsp90 and XAP2, and the ligand- bound AhR dimerizes with another basic helix–loop– helix protein, the aryl hydrocarbon receptor nuclear- translocation (ARNT) protein (8 –11). The AhR:ARNT heterodimer forms an active transcription factor that binds with high affinity to its specific DNA recognition site, the dioxin responsive element (DRE) (12). DNA 1 To whom correspondence should be addressed. Fax: (530) 752- 3394. E-mail: rhrice@ucdavis.edu. 2 Abbreviations used: AhR, aromatic hydrocarbon receptor; ARNT, AhR nuclear translocator; CHX, cycloheximide; CYP1A1, cytochrome P4501A1; DMSO, dimethyl sulfoxide; DRE, dioxin responsive ele- ment; DTT, dithiothreitol; GAPDH, glyceraldehyde phosphate dehy- drogenase; HAH, halogenated aromatic hydrocarbon; HEDG, 25 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol; MMTV, mouse mammary tumor virus; NF, -naphthoflavone; PAH, polycyclic aromatic hydrocarbon; TCDD, 2,3,7,8-tetrachloro- dibenzo-p-dioxin. 154 0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 393, No. 1, September 1, pp. 154 –162, 2001 doi:10.1006/abbi.2001.2475, available online at http://www.idealibrary.com on