Estrogen Synthetase Stimulation by Dibutyryl Cyclic Adenosine 3',5'-Monophosphate in Human Choriocarcinoma Cell Culture: The Relative Biosynthetic Rate of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced Form)-Cytochrome c Reductase Component*

Publisher Website
Google Scholar
Abstract
Human trophoblast cells, derived from choriocarcinoma (JAr line), in continuous culture with 1 mM (Bu)2cAMP plus 1 mM theophylline (dbT) in the culture medium secrete increased amounts of estrogen compared with cells grown without dbT, and after 72 h with dbT, they demonstrate a 6- to 8-fold increase in the specific activity of estrogen synthetase (aromatase; a cytochrome P-450 monooxygenase enzyme system) and an increased cytochrome P-450 concentration. Furthermore the dbT stimulation of aromatase requires protein and RNA synthesis, suggesting that an increase in the biosynthetic rate of at least one of the aromatase component proteins is involved in the mechanism for dbT stimulation of aromatase. This hypothesis was tested for the NADPH-cytochrome c reductase component of aromatase after 25 h growth with dbT, while aromatase activity is increasing linearly, by pulse labeling control and dbT-grown cells for 4 h with a 3H- or 14C-labeled amino acid mixture and by measuring the relative rate of amino acid incorporation into this aromatase component. A 15–20% stimulation in the biosynthetic rate of NADPH-cytochrome c reductase in dbT-grown cells relative to the biosynthetic rate in control cells was detected using a double-antibody immunoprecipitation procedure with rabbit antiserum made against purified human placental microsomal NADPH-cytochrome c reductase as the primary antibody, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This 15–20% stimulation of the biosynthetic rate of reductase 24 h after addition of dbT cannot account for the approximately 2.5-fold higher rate of aromatase activity increase in dbT-grown cells relative to control cells, unless the NADPH-cytochrome c reductase component is compartmentalized so that less than 10% of the total cellular reductase functions in aromatization. Since we know of no evidence for this kind of partitioning, we conclude that some other protein, either the aromatase cytochrome P-450 or an “activator” protein, is responsible for the aromatase stimulation caused by dbT.

This publication has 10 references indexed in Scilit: