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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
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A multifunctional androgen receptor screening assay using the high-throughput Hypercyt flow cytometry system.

Megan K Dennis1, Harmony J C Bowles, Debra A MacKenzie

  • 1Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|March 15, 2008
PubMed
Summary

A new Multifunctional Androgen Receptor Screening (MARS) assay uses flow cytometry to efficiently identify chemicals that modulate androgen receptor activity. This high-throughput method accurately detects androgenic and antiandrogenic compounds, aiding in the discovery of new modulators.

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Area of Science:

  • Endocrinology
  • Molecular Biology
  • Pharmacology

Background:

  • The androgen receptor (AR) is crucial for male development and function.
  • Dysregulation of AR activity is linked to various diseases, particularly in androgen-sensitive tissues.
  • Existing reporter systems for screening AR modulators can be limited in throughput and flexibility.

Purpose of the Study:

  • To develop and validate a novel, high-throughput assay for screening modulators of androgen receptor transcriptional activity.
  • To establish a flexible and sensitive method for identifying novel androgenic and antiandrogenic compounds.

Main Methods:

  • Development of the Multifunctional Androgen Receptor Screening (MARS) assay using androgen-independent PC3 cells.
  • Transient cotransfection with wild-type human AR and an androgen-sensitive promoter driving destabilized enhanced GFP (dsEGFP) expression.
  • High-throughput screening utilizing flow cytometry and the HyperCyt system, with stimulation by known agonists and antagonists.

Main Results:

  • The MARS assay successfully identified known AR agonists, inducing dsEGFP expression in a dose-dependent manner.
  • AR antagonists were accurately detected by their ability to block dsEGFP induction by a submaximal agonist concentration, also in a dose-dependent fashion.
  • The assay demonstrated high sensitivity, flexibility, and compatibility with high-throughput screening.

Conclusions:

  • The MARS assay is a valuable and novel tool for identifying modulators of AR transcriptional activity.
  • The assay's high-throughput format and sensitivity make it suitable for drug discovery and research.
  • Flow cytometry combined with the MARS assay offers a robust platform for assessing AR activity.