MAMMALIAN PC-12 CELL GENETICALLY-ENGINEERED FOR HUMAN CYTOCHROME-P450 2E1 EXPRESSION

被引:35
作者
MAPOLES, J
BERTHOU, F
ALEXANDER, A
SIMON, F
MENEZ, JF
机构
[1] FAC MED BREST, EQUIPE ACCUEIL DRED, BIOCHIM LAB, BP-815, F-29285 BREST, FRANCE
[2] UNIV COLORADO, VAMC, DENVER, CO 80202 USA
[3] UNIV COLORADO, HEPATOBILIARY RES CTR, DENVER, CO 80202 USA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 214卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1993.tb17975.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol - min-1 . mg microsomal protein- 1 vs 68 +/- 48 nmol . min-1 . mg-1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol . min-1 - mg microsomal protein-1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.
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页码:735 / 745
页数:11
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