Development of a novel cell based androgen screening model

• We have developed a new cell line, named CV1-ARluc, which stably express the human AR and an androgen-driven gaussia luciferase reporter.
• The androgen specificity of the assay was tested using high concentrations of different steroids such as cortisol, progesterone and aldosterone, which did not active reporter expression.
• The transactivation assay developed with the help of the cell line is highly sensitive as it detects androgen activity at 0.1 nM of DHT and 0.3 nM of T.
• This assay provides a valid and practical method to analyze male and female serum samples for androgenic activity.

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 18 h 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.