Time-dependent inhibition of aromatase in trophoblastic tumor cells in tissue culture.

Human choriocarcinoma trophoblast cells (JAr line) were utilized as whole cell preparations in tissue culture to evaluate the effects of mechanism-based inactivation or "suicide inhibition" of estrogen biosynthesis. A C-19 acetylenic analog (MDL 18,962) of the substrate, androstenedione, was evaluated in competitive and time-dependent assays. Product formation was determined by accumulation of 3H2O resulting from the stereo-specific elimination of 1 beta-tritium from the androgen substrate. Trophoblast cells exhibited initial linear kinetics for at least 3 h following addition of [1-3H]androstenedione. The Km for androstenedione was 35.1 nM with Vmax of 3.7 pmol/h/10(6) trophoblast cells. Kinetic analysis of time-dependent inhibition of aromatase in trophoblast cells revealed an apparent Ki of 0.6 nM for MDL 18,962 and at t 1/2 of inactivation of 26 min at infinite inhibitor concentration. These studies suggest that a suicide aromatase inhibitor can cause irreversible inhibition of estrogen biosynthesis in intact trophoblast cells. In the presence of 1 nM or 10 nM MDL 18,962, trophoblast cells exhibited initial linear kinetics for estrogen biosynthesis during the first hour following co-incubation with inhibitor and 33 nM substrate. During the subsequent 30 min the rate of estrogen biosynthesis precipitously declined from 104 +/- 24 fmol/min/10(6) cells in control cells to 24 +/- 13 and 8 +/- 4 fmol/min/10(6) trophoblasts treated with 1 or 10 nM MDL 18,962, respectively. This significant decrease in aromatase activity (P less than or equal to 0.01) implied irreversible inactivation, which was supported by prolonged inhibition of aromatase activity in trophoblast cells incubated for 6-48 h following removal of medium containing 3 nM or 30 nM MDL 18,962.