Cloning and stable expression of the human mitochondrial cytochrome P45011B1 cDNA in V79 Chinese hamster cells and their application for testing of potential inhibitors.
V79 Chinese hamster cells are being genetically engineered to express human mitochondrial cytochromes P450 as an analytical tool for studying adrenal steroid synthesis. Here, a V79 derived cell line is presented expressing the enzymatically active human cytochrome P45011B1 (CYP11B1) in a stable and constitutive manner. Full length CYP11B1 cDNA was obtained from surgically removed normal adrenal gland by polymerase chain reaction. The cDNA was recombined with a SV40 early promoter containing plasmid for stable integration and expression in V79 cells upon gene transfer. The presence of the human CYP11B1 cDNA in the genome of the transfected cells was confirmed by Southern analysis. CYP11B1 cDNA directed expression was detected by Northern analysis. CYP11B1 dependent hydroxylation of deoxycorticosterone and 11-deoxycortisol was measured by HPLC analysis. Interestingly, the nonsteroidogenic lung fibroblast derived V79 Chinese hamster cell line was able to support human CYP11B1 mediated steroid hydroxylation without simultaneous heterologous expression of the human electron transfer system, adrenodoxin, and adrenodoxin reductase. CYP11B1 inhibitory potency of metyrapone, spironolactone, and the azole derivatives ketoconazole, clotrimazole, miconazole, and fluconazole was measured using the newly established V79MZh11B1 cell line. Thus, beside steroid metabolism studies in general, this cell line may also serve as an in vitro tool for monitoring the interference of drugs with 11 beta-hydroxylase activity.
Denner
K
K
Fachbereich Chemie, Freie Universität Berlin, Germany.
Vogel
R
R
Schmalix
W
W
Doehmer
J
J
Bernhardt
R
R
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003022:Clotrimazole / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D018076:DNA, Complementary; D015725:Fluconazole / Q000494:pharmacology; D015870:Gene Expression; D006854:Hydrocortisone / Q000378:metabolism; D006900:Hydroxylation; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008797:Metyrapone / Q000494:pharmacology; D008825:Miconazole / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015183:Restriction Mapping; D013148:Spironolactone / Q000494:pharmacology; D013252:Steroid 11-beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013148:Spironolactone; D008825:Miconazole; D015725:Fluconazole; D013252:Steroid 11-beta-Hydroxylase; D003022:Clotrimazole; D007654:Ketoconazole; D006854:Hydrocortisone; D008797:Metyrapone