We used prochloraz (45631; Riedel-deHaen; lot 9060X, CAS 06774709-5, [C15H16Cl3N3O2] 99.4% pure by high-performance liquid chromatography, formula weight 376.67 g/mol), corn oil (C 8267; Sigma Chemical Co., St. Louis, MO; lot 81K2204, CAS 8001-30-7, density = 0.9 g/ ml), and dihydrotestosterone (DHT; Sigma; >99% purity). Hydroxyflu-tamide was provided by R.O. Neri (Schering Corp., Bloomfield, NJ).
AR-Dependent Gene Transcriptional Activation Assay
PZ, but not its metabolites, was reported to inhibit AR-mediated gene transcription. We evaluated the effects of PZ on AR-mediated gene transcription using MDA-kb2 cells stably transfected with a reporter gene construct (pMMTV.neo.luc). Cells were maintained without CO2 at 37°C in L-15 media (Gibco BRL) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 |xg/ml streptomycin, and 0.25 |xg/ml amphotericin B. AR-dependent transcriptional activation assays were conducted as described in. Briefly, cells were plated at 5 X 104 cells per well in 96-well Costar luminometer plates and allowed to attach.