Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation.

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in receptor deficient monkey kidney cells CV-1 in culture examine the ability of dihydrotestosterone (DHT) and of the antiandrogens hydroxylflutamide (HO-FLU), cyproterone acetate (Cypro.A) and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). CV-1 cells cotransfected with wild type hAR (hAR1-910) and MMTV-CAT, were treated with varying concentrations of DHT. DHT stimulated transcription activation of MMTV-CAT gene in a dose-dependent fashion. Cypro.A though only partially, also stimulated the transcription activation of MMTV-CAT. In the absence of steroids, HO-FLU induced the MMTV-CAT transcription in transfectants only 4% above the basal level. RU 23908-10 revealed the least agonistic activity at concentrations between 10 nM and 1 microM. Despite this, 100- to 1000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A and RU 23908-10 competed with [3H]DHT for AR binding with hAR expressed in CV-1 cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in extracts prepared from hAR1-910 transfected CV-1 cells. Incubation of labeled synthetic palindromic androgen responsive element (ARE) with the hAR containing CV-1 cell extracts followed by u.v. cross-linking demonstrated the specificity of AR-DNA interaction. Analysis by gel mobility shift assays showed that the interaction of AR-antiandrogen complexes with labeled ARE was specific.