Tightly regulated and inducible expression of rabbit CYP2E1 using a tetracycline-controlled expression system

Jian Ya Huan, Dennis R. Koop

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


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 levels 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.

Original languageEnglish (US)
Pages (from-to)549-554
Number of pages6
JournalDrug Metabolism and Disposition
Issue number4
StatePublished - 1999

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science


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