Figure 1

Expression of androgen receptor (AR) in human breast epithelial cells. (A) MCF-10A cells were stably transfected with an AR cDNA, and lysates from single-cell clones were probed for AR expression by western blotting. As a negative control, MCF-10A cells were also transfected with empty vector (10A plus vector) and isolated as single-cell clones. MDA-MB-453 and LNCaP cell lysates served as positive controls for AR expression. All lysates were also probed with GAPDH antibody as a loading control. (B) Androgen Receptor In Breast Epithelium (ARIBE) cells and control cells were transfected with luciferase reporter plasmids containing consensus DNA binding sites and mutated controls for AR as described in Methods. At the time of transfection, cells were treated with 1 nmol/l R1881 or vehicle (ethanol), and analyzed 48 hours later. Firefly luciferase measurements were normalized to Renilla luciferase measurements in all samples. The relative luciferase units (RLU) ratio was calculated as the luciferase expression comparing wild-type ARE/mutant ARE in R1881 versus vehicle-treated control cells (R1881 wtARE/mutARE: ETOH wtARE/mutARE). Error bars represent the standard deviation of three independent experiments. Luciferase expression in each ARIBE clone compared with control cell lines was significant by one-way analysis of variance (ANOVA) (P < 0.05).(C) cDNA was made from RNA of cells treated with 1 nmo/l R1881 or vehicle for 48 hours. Quantitative real-time PCR using SYBR Green was performed on triplicate samples of each cell line using intron-spanning primers for each of three androgen-response genes. All cycle threshold numbers were normalized to a control gene, TATA binding protein (TBP). Ratio is expression in cells treated with drug versus vehicle. Error bars represent the standard deviation of four independent experiments. In both ARIBE lines, induction of all genes after drug treatment was significant by one-way ANOVA compared with control cell lines (P < 0.001).