Figure 9. Effects of cyclic-AMP on AR transcriptional activity.

Dose-dependence of androgen-dependent and androgen-independent AR transcriptional activity in response to cAMP was measured in (A) LNCaP-C4-2, and (B) DU145 prostate cancer cells. Cells were transfected in 12 well plates using Effectene (Qiagen) with 10 ng/well pSG5 (p5) empty vector or 10 ng/well pCMV-AR with 0.25 µg/well PSA-Enh-Luc in 5% charcoal-stripped serum medium. Cells were treated for 24 h in 5% charcoal-stripped serum containing media in the absence and presence of 1 nM DHT and 0, 0.5, 2 and 8 mM db-cAMP as indicated. Luciferase activity is representative of three independent experiments. (C) Inhibition of cAMP-stimulated endogenous AR transcriptional activity in CWR-R1 cells using MAGE-11 siRNA. CWR-R1 cells (1.6 × 10⁵/well in 12 well plates) were plated in serum containing growth medium and transferred the next day into antibiotic-free medium containing 5% charcoal-stripped serum. Cells were transfected using Lipofectamine-2000 Reagent (Invitrogen) with 0.1 µg MMTVΔ-421–-364-Luc/well (an MMTV-Luc reporter with deletion of a negative response element) in the presence of 2 nM MAGE-11 siRNA-3, which does not inhibit MAGE-11 expression, and 2 nM MAGE-11 siRNA-2, which reduces MAGE-11 expression (20). Cells were placed the next day in serum-free, phenol red-free medium, and 24 h later the medium was replaced with serum-free medium with and without 0.1 nM DHT and 0.5 mM dibutyryl-cAMP (db-cAMP). Cells were cultured for 24 h and luciferase activity was measured. The results are representative of four independent experiments.