HIGH-LEVELS OF RECOMBINANT CYP3A4 EXPRESSION IN CHINESE-HAMSTER OVARYCELLS ARE MODULATED BY COEXPRESSED HUMAN P450 REDUCTASE AND HEMIN SUPPLEMENTATION

Expression of recombinant cytochrome P450s (P450s) in mammalian cells has been used as a powerful tool to study these enzymes, However, the activity of CYP3A4 expressed in several stable mammalian cell lines wa s much lower than native enzyme in human liver, The low level of recom binant CYP3A4 may have been due to the low copy number of the cDNA, In addition, the low activity is caused by the low level of P450 reducta se in these cells, To achieve high levels of CYP3A4 expression, we emp loyed gene amplification of the CYP3A4 cDNA in Chinese hamster ovary ( CHO) cells followed by transfection of the P450 reductase cDNA. Using this strategy, we have obtained a cell line, designated D3A4, with hig h levels of recombinant CYP3A4, The content of spectrally active P450 was 14 pmol/mg total cellular protein, Hemin treatment increased the P 450 content 2-fold, Upon coexpression of P450 reductase in DHR/3A4 cel ls, enzyme activity of CYP3A4 was stimulated 15-fold, despite a 40% de crease in spectrally active P450, Interestingly, the latter effect was not due to a decrease in CYP3A4 mRNA, Treatment of these cells with h emin, however, counteracted the P450 reductase-mediated decrease of sp ectrally active P450, These data demonstrate that P450 reductase has a strong influence on the levels of recombinant P450 holoenzyme, possib ly by modulating the level of heme in CHO cells, Concomitantly our res ults show that the gene amplification strategy provides a powerful app roach to obtain a high level of functional recombinant P450. (C) 1997 Academic Press.