FIGURE 3.

Suppression of androgen receptor trans-activation by 17-HASs. A, PC3 clones that stably express the WT AR protein were transiently transfected with the reporter vector pARE4-Luciferase and pCMV-Renilla. 24 h later, cells were stimulated with R1881 (1 nm) and treated with serial dilutions of TOK-001, Casodex, or abiraterone alcohol (n = 6 ± S.E.). The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU). Solid lines represent the best-fit sigmoidal dose response (variable slope). IC_50[TOK-001] = 1.3 μm; IC_{50[abiraterone alcohol]} = 4.7 μm; IC_50[Casodex] = 0.89 μm. B, PC3 clones that stably express the wild type (black), W741C (white), or W741L (gray) AR proteins were transiently transfected with the reporter vector pARE-4X-Luciferase and then stimulated with R1881 (1 nm). Cells were co-treated with the indicated concentrations of TOK-001. The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU). C, PC3 clones that stably express the WT, W741C, or W741L AR proteins were transiently transfected with the reporter vector pARE-4-Luciferase. The cells were untreated (mock or control), treated with 1 nm R1881, or treated with 10 μm Casodex, 10 μm TOK-001, or 10 μm abiraterone in the absence of R1881. The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU). D, LNCaP cells were either pretreated with DMSO (TOK-) or pretreated with 20 μm TOK-001 (TOK+) for 1 h. Cells were then stimulated with either 1 nm R1881 (R1881+) or DMSO (R1881-) for an additional 2 h before fractionation of the cytoplasmic (C) and nuclear (N) proteins by differential lysis and centrifugation. A representative Western blot is depicted showing the levels of AR in the C and N fractions. The levels of PARP and α-tubulin indicate the quality of the fractionation procedure.