Effects of androgens on Cdc25C and cell cycle protein levels in PCa cells.

<p>(A) LNCaP C-33 and C-81 cells were seeded at a density of 8×10³ and 6×10³ cells/cm², respectively, in duplicates for 3 days in regular medium. Cells were steroid-starved for 48 hr in SR medium and then treated with 10 nM DHT for 24 hr and 48 hr. Total cell lysate proteins were harvested and analyzed for Cdc25C, Cdc25B, cPAcP, Cyclin B1 and Cyclin D1 proteins. β-actin was detected for serving as a loading control. (B) AS LNCaP C-33 and (C) VCaP cells were plated at a density of 8×10³ and 2×10⁴ cells/cm², respectively, for 3 days, and then steroid starved for 48 hr in SR medium. Cells were treated with 10 nM DHT with or without 10 µM Casodex for 48 hr. (B) For C-33 cells, total cell lysate proteins were analyzed for AR, Cdc25A, Cdc25B, Cdc25C, cPAcP, PSA, Cyclin D1 and Cyclin B1 protein levels. (C) For VCaP cells, total cell lysate proteins were analyzed for AR, Cdc25C, Cyclin D1, Cyclin B1 and PSA protein levels. β-actin was analyzed in each experiment and used as a loading control. (D) Effect of DHT <i>vs</i>. EGF on Cdc25C protein level in C-33 cells. Steroid-starved C-33 cells were treated with 10 nM DHT or 10 ng/ml EGF for 24 hr, and control cells were treated with solvent alone. All cells were harvested for analyzing Cdc25C protein level. As controls, cPAcP, ErbB-2 and its tyrosine phosphorylation at Y877, AR and its phosphorylation at S81, and Cyclin B1 were also analyzed. Tubulin was detected for serving as a loading control.