Molecular and Cellular Endocrinology 160 (2000) 39 – 49 A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects Be ´atrice Te ´rouanne a , Bouchra Tahiri a , Virginie Georget a , Charles Belon b , Nicolas Poujol a , Christophe Avances a,c , Francesco Orio Jr b , Patrick Balaguer a , Charles Sultan a,b, * a INSERM Unite ´ 439, Pathologie Mole ´culaire des Re ´cepteurs Nucle ´aires, 70 rue de Naacelles, 34090 Montpellier, France b Laboratoire d’Hormonologie du De ´ eloppement et de la Reproduction, Ho ˆpital Lapeyronie, Montpellier, France c Serice d’Urologie -Andrologie (Pr. P. Costa), Hopital G. Doumergue, 5 rue Hoche 30000 Nı ˆmes, France Received 18 August 1999; accepted 9 December 1999 Abstract We developed a new stable prostatic cell line expressing the human androgen receptor (AR) and the AR-responsive reporter gene to generate a powerful tool for investigating androgen action and for rapid screening of agonists and antagonists. The AR-deficient PC-3 cells were stably transfected with pSG 5 -puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, one highly inducible clone was isolated and named PALM, for PC-3-Androgen receptor-Luciferase-MMTV. The expression of hAR was confirmed by western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after incubation of cells with increasing concentrations of synthetic R1881 or natural androgens (DHT and testosterone). The three agonists had the same maximal activity at 0.1 M and the fold induction was equal to 20. The agonist and antagonist activities of the steroidal antiandrogens (cyproterone acetate and RU2956) and the non-steroidal antiandrogens (nilutamide, bicalutamide, inocoterone and hydroxyflutamide) measured with the PALM cells were in good correlation with the results obtained with transiently transfected cells. The selectivity in steroid transactivation was demonstrated with estradiol, progesterone, cortisol, dexamethasone and aldosterone. Spironolactone and RU486 showed partial agonist and antagonist activities, whereas R5020 presented only a partial antagonist activity. We here demonstrate that this stable transfectant provides an accurate tool for studying wild-type human AR activation and its regulation by androgens and antiandrogens in a human prostatic epithelial cell, which is routinely available and remains androgen-responsive in vitro. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human androgen receptor; Antiandrogen; PC-3 cells; Prostate cancer; Stable transfectant www.elsevier.com/locate/mce 1. Introduction The presence of androgens is essential for normal prostate differentiation, growth, and function, as well as for the proliferation of steroid-sensitive prostate cancer. The androgen action is mediated in target cells via the androgen receptor (AR), which is a ligand-de- pendent transcription factor. In the presence of ligand, AR is activated, resulting in stimulation or repression of genes that are under steroid hormone control. An- drogen antagonists can disrupt this process. Pharmaco- logical treatment of prostate cancer is mostly based upon inhibition of androgen-regulated gene expression by blocking AR function with antiandrogens. This treatment is apparently effective for a short period although antiandrogen resistance occurs in most cases within a few years. A detailed characterization of the antiandrogen substances is of interest for a better un- derstanding of their potential therapeutic use. Numer- ous studies have investigated the hormone action mediated by AR in many cell lines, such as the human prostate carcinoma LNCaP cell line (Horoszewicz et al., 1980). This cell line expresses high levels of AR but it possesses a mutation in the ligand-binding domain * Corresponding author. Tel.: +33-467-338696; fax: +33-467- 338327. E-mail address: chsultan@u439.montp.inserm.fr (C. Sultan) 0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII:S0303-7207(99)00251-8