Differential effect of androgen on the regulation of ATM/ATR downstream targets in LNCaP cells.

<p>(A) Androgen down regulates p53 protein expression in LNCaP cells. LNCaP cells were treated with R1881 for 24 hours and harvested for Western blotting analysis on p53 protein expression (Left panel). p53 mRNA levels in androgen-treated LNCaP cells were examined by RT-PCR and GAPDH expression level was used as a loading control (Right panel). (B) Androgen promotes p53 protein degradation in LNCaP cells. Degradation profile of p53 protein in LNCaP cells with or without R1881 (1 nM) treatment was examined by blocking protein synthesis with CHX (50 µg/ml). p53 protein level was measured at the indicated time points by Western blotting. Signal intensity of the Western blotting result was measured by gel documentation system and the reading was normalized as percentage to that of the initial p53 level (level at time = 0). (C) Log₁₀ of the percentage was plotted against time and the half-life of the p53 protein was calculated as the time corresponding to the log₁₀ of 50%. (D) Androgen fails to down regulate p53 in the presence of proteasome inhibitor. LNCaP cells were treated with 1 nM R1881 for 24 hrs. At 16 hrs of R1881 treatment, 2 µM of the proteasome inhibitor (MG132) was added. At the end of the treatment, cells were lysed for western blotting analysis using p53 antibody.