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Research ArticleArticle

Tightly Regulated and Inducible Expression of Rabbit Cyp2e1 Using A Tetracycline-Controlled Expression System

Jian-Ya Huan and Dennis R. Koop
Drug Metabolism and Disposition April 1999, 27 (4) 549-554;
Jian-Ya Huan
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Dennis R. Koop
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Abstract

A tetracycline (Tc)-controlled gene expression system that quantitatively controls gene expression in eukaryotic cells (Gossen and Bujard, 1992) 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 toN-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.

Footnotes

  • Send reprint requests to: Dr. Dennis R. Koop, Department of Physiology and Pharmacology, L334, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201. E-mail: koopd{at}ohsu.edu

  • Supported by National Institute on Alcohol Abuse and Alcoholism Grants AA08608 and AA05462.

  • Abbreviations used are::
    CYP2E1
    cytochrome P-450 2E1
    Tc
    tetracycline
    6-OH-CZ
    6-hydroxychlorzoxazone
    MTT
    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
    NDMA
    N-nitrosodimethylamine
    4MP
    4-methylpyrazole
    ABT
    1-aminobenzotriazole
    DCF
    2′,7′-dichlorofluorescein
    H2DCFDA
    2′,7′-dichlorodihydrofluorescein diacetate
    tTA
    Tc-controlled transactivator
    tetR
    tetracycline repressor protein
    DMEM
    Dulbecco’s modified Eagle’s medium
    ROS
    reactive oxygen species
    • Received November 19, 1997.
    • Accepted December 23, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 27 (4)
Drug Metabolism and Disposition
Vol. 27, Issue 4
1 Apr 1999
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Research ArticleArticle

Tightly Regulated and Inducible Expression of Rabbit Cyp2e1 Using A Tetracycline-Controlled Expression System

Jian-Ya Huan and Dennis R. Koop
Drug Metabolism and Disposition April 1, 1999, 27 (4) 549-554;

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Research ArticleArticle

Tightly Regulated and Inducible Expression of Rabbit Cyp2e1 Using A Tetracycline-Controlled Expression System

Jian-Ya Huan and Dennis R. Koop
Drug Metabolism and Disposition April 1, 1999, 27 (4) 549-554;
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