title: Development of a novel cell based androgen screening model.
abstract: The androgen receptor (AR) mediates the majority of androgen effects on target cells. The DNA cis-regulatory elements that respond to AR share sequence similarity with cis-regulatory elements for glucocorticoid, mineralocorticoid and progesterone receptors (GR, MR and PR, respectively). As a result, many of the current AR screening models are complicated by inaccurate activation of reporters by one of these receptor pathways. Identification of more selective androgen testing systems would be beneficial for clinical, pharmacological and toxicologic screening of AR activators. The present study describes the development of a selective androgen-responsive reporter cell line that expresses AR but does not express GR, MR and PR. CV1 cells were stably transduced to express human AR and an androgen-responsive gaussia luciferase gene. Clonal populations of AR expressing cells were isolated. Quantitative RT-PCR (qPCR) and western analysis confirmed stable integration of AR in the most responsive clonal line which was named 'CV1-ARluc'. Stimulation of CV1AR-luc with androgenic ligands (testosterone and 5α-dihydrotestosterone) for 18h caused an increase in luciferase activity in a dose-dependent manner. Other steroid hormones including aldosterone, cortisol, and progesterone did not stimulate luciferase response. The CV1-ARluc also increased luciferase activity when treated with human serum extracts. In conclusion, the CV1-ARluc cells provide a novel model system for screening of new AR agonists and antagonists and can determine the androgenic activity of human serum samples.
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal Medicine, University of Michigan, Medical School, Ann Arbor, MI, USA; Department of Pharmacy Health and Nutritional Sciences, University of Calabria, Rende (CS), Italy.
authors.forename: Carmela
authors.initials: C
authors.lastname: Campana
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal Medicine, University of Michigan, Medical School, Ann Arbor, MI, USA.
authors.forename: Juilee
authors.initials: J
authors.lastname: Rege
authors.affiliation: Division of Metabolism, Diabetes, and Endocrinology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Adina F
authors.initials: AF
authors.lastname: Turcu
authors.affiliation: Department of Pharmacy Health and Nutritional Sciences, University of Calabria, Rende (CS), Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Pezzi
authors.affiliation: Endocrine Section, Department of Medicine, G.V. (Sonny) Montgomery VA Medical Center and University of Mississippi Medical Center, Jackson, USA.
authors.forename: Celso E
authors.initials: CE
authors.lastname: Gomez-Sanchez
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Robins
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal Medicine, University of Michigan, Medical School, Ann Arbor, MI, USA. Electronic address: wer@umich.edu.
authors.forename: William E
authors.initials: WE
authors.lastname: Rainey
mesh_terms: D000328:Adult; D000728:Androgens / Q000097:blood* / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D000882:Haplorhini; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D055815:Young Adult
chemical_list: C410798:AR protein, human; D000728:Androgens; D011944:Receptors, Androgen; D013256:Steroids; D008156:Luciferases
keywords: AR selectivity; Androgen activity; Human serum; Luciferase; Stable in vitro bioassay