title: Tightly regulated and inducible expression of rabbit CYP2E1 using a tetracycline-controlled expression system.
abstract: A tetracycline (Tc)-controlled gene expression system that quantitatively controls gene expression in eukaryotic cells () was used to express cytochrome P-450 2E1 (CYP2E1) in HeLa cells in culture. The rabbit CYP2E1 cDNA was subcloned into the Tc-controlled expression vector (pUHD10-3) and transfected into a HeLa cell line constitutively expressing the Tc-controlled transactivator, a positive regulator of expression in the absence of Tc. The expression of CYP2E1 was tightly regulated. There was a time-dependent induction of CYP2E1 after removal of Tc. In the absence of Tc, the enzyme was induced more than 100-fold and expressed about 18 pmol of CYP2E1/mg microsomal protein. At maximal levels of expression the enzyme catalyzed the formation of 158 pmol 6-hydroxychlorzoxazone/min/mg total cellular protein. In addition, the level of the enzyme could be modulated by the concentration of Tc in the media. In the absence of Tc, exposure of cells to N-nitrosodimethylamine caused a significant dose-dependent decrease in cell viability. In contrast, menadione, a redox cycling toxicant, was less toxic to the cells after induction of CYP2E1 when compared with noninduced cells. Pulse-chase studies conducted 72 h after removal of Tc indicated a rapid turnover of CYP2E1 with a half-life of 3.9 h. Addition of the ligand, 4-methylpyrazole, and the suicide substrate, 1-aminobenzotrizole, decreased the degradation of CYP2E1. This cell line offers a useful system to examine the role of CYP2E1 in the cytotoxicity of xenobiotics and to investigate post-translational regulation of the enzyme.
authors: Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
authors: J Y
authors: JY
authors: Huan
authors: D R
authors: DR
authors: Koop
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004128:Dimethylnitrosamine / Q000494:pharmacology; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006367:HeLa Cells; D006490:Hemostatics / Q000633:toxicity; D006801:Humans; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D014812:Vitamin K / Q000633:toxicity
chemical_list: D000477:Alkylating Agents; D006490:Hemostatics; D017382:Reactive Oxygen Species; D014812:Vitamin K; D019392:Cytochrome P-450 CYP2E1; D013752:Tetracycline; D004128:Dimethylnitrosamine