Medium was replaced and cells were dosed with PZ (0.03-30 |xM), DHT (0.1 nM), DHT (0.1 nM) + PZ (0.03-30 |xM), DHT (0.1 nM) + hydroxyflutamide (1 |xM) as an antiandrogen control, or vehicle control (ethanol) and incubated overnight. Cells were washed using Dulbecco PBS (14080-055; Gibco BRL) and lysed using 25 |xl of E1531 lysis buffer (Promega, Madison, WI). Luciferase readings were measured using a Dyn-Ex MLX microtiter plate luminometer.
As a measure of cytotoxicity we determined the capacity of PZ to inhibit the ability of MDA cells to convert a tetrazolium salt (MTT) to blue formazan crystals as described in. MDA cells were plated and PZ was administered with and without 0.1 nM DHT as described above for the transcriptional activation assay. Blue crystal formation was quantified using a Bio-Tek UV900 HDi microplate reader.
Binding Assay Using Homogenized Rat Prostate
The ability of PZ to compete with 1 nM [3H] R1881 for binding to the rat AR was evaluated as described in using ventral prostate tissue obtained from 90-day-old rats that had been castrated 24 h before tissue removal.