Figure 6.

Characterization Y-AR: a new breast cancer cell line stably expressing AR. (a) AR levels and function of Y-AR. Inset: whole cell extracts of Y-AR and MCF-7 cells were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose and probed with the anti-AR antibody PG-21 (Upstate Biotechnology). The 110 kDa AR band is shown. Main figure: Y-AR and control T47D-Y cells were transiently transfected PRE₂/luciferase and treated with DHT, MPA, and R5020 at concentrations from 0.01 to 1000 nmol/l for 20 hours. Luciferase activity is shown as a percentage of 1 mmol/l DHT (which was set at 100%). Data shown are the mean of triplicate determinations ± standard error. (b) AR immunocytochemistry in Y-AR cells. Y-AR breast cancer cells were treated with ethanol or 1 μmol/l DHT for 30 min, or 2 and 20 hours. ARs were immunologically visualized using green fluorescent FITC (row A), and the cell nucleus was labeled with blue DAPI (row B). Cells were visualized using confocal microscopy and photographed at 100×, as described in the Materials and method section. (c) PCR of the PSA transcript. RNA was harvested from Y-AR cells treated 6 and 12 hrs with ethanol, or 1 μmol/l R5020, MPA, or DHT. RT-PCR was performed using PSA-specific primers. GAPDH is shown as a control. Representative results are shown. AR, androgen receptor; DHT, dihydrotestosterone; EtOH, ethanol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MPA, medroxyprogesterone acetate; PR, progesterone receptor; PSA, prostate-specific antigen.