TY - JOUR
T1 - Tightly regulated and inducible expression of rabbit CYP2E1 using a tetracycline-controlled expression system
AU - Huan, Jian Ya
AU - Koop, Dennis R.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1999
Y1 - 1999
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0032893473&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032893473&partnerID=8YFLogxK
M3 - Article
C2 - 10101151
AN - SCOPUS:0032893473
SN - 0090-9556
VL - 27
SP - 549
EP - 554
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
IS - 4
ER -