In vitro studies have demonstrated that androgen signaling may counteract the proliferative effect of estrogens in AR positive breast cancer cells, while over expression of the AR mark edly decreases ER alpha transcriptional activity in ER positive breast cancer the site cells. In these latter models, basal as well as estradiol induced proliferation are inhibited by the non aromatizable androgen 5 a dihydrotestosterone. through an in crease in AR protein cell content, along with a block in G1 to S transition of the cell cycle. AR induced apoptosis has also been reported in these cell lines. Recently, we have shown a direct down regulation of the cyclin D1 gene expression by AR activation via interaction with the orphan nuclear receptor DAX1, as an additional mechanism involved in the androgen dependent Inhibitors,Modulators,Libraries inhibition of ER positive breast cancer cell growth.
Given the ability of Inhibitors,Modulators,Libraries AR to function as an anti pro liferative effecter by antagonizing ER signaling, the aim of this study was to investigate whether ER beta may also be a target of androgen actions. Here, we demonstrate that mibolerone, a synthetic non metabolizable androgen, up regulates ER beta expres sion and gene promoter activity, through a direct binding of AR to a newly identified androgen response element located in the human ER beta gene promoter. Methods Chemicals and reagents Dulbeccos Modified Eagles Medium Nutrient Mixture F 12 Ham, DMEM, 100 bp DNA ladder, l glutamine, penicillin, streptomycin, bovine serum albumin and phosphate buffered saline were purchased from Invitrogen, and Sephadex G50 spin columns and poly from Roche.
Inhibitors,Modulators,Libraries GoTaq DNA polymerase, T4 polynucleotide kinase, dual luciferase kit, FuGENE 6 and CMV renilla luciferase plasmid were provided by Promega. Inhibitors,Modulators,Libraries The RETROscript kit and DNase I were purchased from Ambion. Aprotinin, leupeptin, phenyl methylsulfonyl fluoride, sodium orthovanadate, formal dehyde, NP 40, 3 2,5 diphenyl tetrazolium bromide, dimethyl sulfoxide, protein ase K, tRNA, IGF 1, DHT, bicatulamide were from Sigma. Antibodies against cyclin D1, p21, GAPDH, and polymerase II were provided by Santa Cruz Biotechnology. Antibody anti ER beta, 1 1000 dilution was purchased from Millipore. The ECL System, 3H thymidine and ATP were purchased from PerkinElmer, salmon sperm DNA protein A agarose was from UBI, and triazol, SYBR Green Universal PCR Master Mix was from Biosystems. Cell cultures The three human breast cancer cell lines MCF 7, ZR 75 and MDA MD 231 were acquired in 2010 from American Type Culture Collection Inhibitors,Modulators,Libraries where they were authenticated, selleck products stored according to the suppliers instructions, and used within a month after fro zen aliquots were resuscitated.