Effects of dihydrotestosterone and hydroxyflutamide on androgen receptors in cultured human breast cancer cells (EVSA-T).

The purpose of our study was to evaluate the effects of 5 alpha-dihydrotestosterone (DHT) and hydroxyflutamide (HF), alone or in combination, on androgen receptor (AR) dynamics and on cellular growth in cultured breast cancer cells (EVSA-T). The incubation of cells with DHT increased the concentration of nuclear AR after 24 and 48 h. HF was also able to promote the nuclear accumulation of AR after 24 and 48 h of treatment. When HF-treated cells are incubated with DHT, the nuclear AR concentration is lower than that found in cells treated with DHT alone. We conclude that HF acts by increasing nuclear accumulation of receptor-antiandrogen complexes. Moreover, DHT stimulates cell growth while HF has an inhibitory effect. Thymidine incorporation in cells also increased after DHT treatment and decreased after HF incubation. The HF-induced inhibition of cell growth persisted both after renewal of the medium and after the addition of DHT to cultures. It may be hypothesized that either DHT is converted to inactive metabolites or that HF exerts a persistent inhibitory effect. In the latter case, the antiandrogen action of HF could be exerted by retention of high levels of antiandrogen in cells or by such a depressed protein synthesis that the renewal of growth is slower than the 48 h period studied.     

Androgen receptor acetylation sites differentially regulate gene control

Androgen receptor (AR) function is modulated by post-translational modifications such as acetylation, ubiquitylation, sumoylation, and phosphorylation. Concerning acetylation, three lysines residues located in a consensus KxKK motif of the AR hinge domain have been identified. For a better evaluation of the role of this modification, the activity of AR modified at different acetylation sites was determined by comparing the effects on natural and synthetic promoters. We found that mutation of AR acetylation sites affected both potency and efficacy of androgen-dependent response. Remarkably, elimination of all three acetylation sites was still compatible with strong AR activity on the PSA and MMTV promoters, but not on the Pem promoter. This differential effect was seen at various wild-type (wt) to mutant AR receptor ratios and at changing hormone concentrations. Subcellular localization studies showed that both mutated and wt AR efficiently translocated into the cell nucleus. Plasmid immunoprecipitation revealed comparable binding of both receptor forms to the Pem promoter. The differential effects observed for the Pem promoter were partially due to an androgen response element (ARE) named ARE-1 which was only poorly stimulated by the AR acetylation site mutant. Finally, AR mutants impaired in their N/C interaction elicited intact stimulation of the Pem promoter, suggesting that AR acetylation was not influenced by this inter-domain communication. The promoter-selective effects seen for the AR acetylation site mutants strongly suggest this post-translational modification to be important in the fine-tuning of the effects of androgens on different target genes.