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 studyin g adrenal steroid synthesis. Here, a V79 derived cell line is presente d expressing the enzymatically active human cytochrome P45011B1 (CYP11 B1) in a stable and constitutive manner, Full length CYP11B1 cDNA was obtained from surgically removed normal adrenal gland by polymerase ch ain reaction, The cDNA was recombined with a SV40 early promoter conta ining 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, CPP11B1 cDN A directed expression was detected by Northern analysis, CYP11B1 depen dent hydroxylation of deoxycorticosterone and 11-deoxycortisol was mea sured by HPLC analysis, Interestingly, the nonsteroidogenic lung fibro blast derived V79 Chinese hamster cell line was able to support human CYP11B1 mediated steroid hydroxylation without simultaneous heterologo us expression of the human electron transfer system, adrenodoxin, and adrenodoxin reductase, CYP11B1 inhibitory potency of metyrapone, spiro nolactone, and the azole derivatives ketoconazole, clotrimazole, micon azole, and fluconazole was measured using the newly established V79MZh 11B1 cell line, Thus, beside steroid metabolism studies in general, th is cell line may also serve as an in vitro tool for monitoring the int erference of drugs with 11 beta-hydroxylase activity.