Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and mutation-specific effects.
BACKGROUND
Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors.


RESULTS
We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5-24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear "speckling" were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions.


CONCLUSIONS
HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations.
Szafran
Adam T
AT
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.
Szwarc
Maria
M
Marcelli
Marco
M
Mancini
Michael A
MA
D000726:Androgen Antagonists / Q000494:pharmacology; D059002:Androgen Receptor Antagonists; D000728:Androgens / Q000494:pharmacology*; D000813:Anilides / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008024:Ligands; D009154:Mutation / Q000502:physiology; D009570:Nitriles / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D011944:Receptors, Androgen / Q000235:genetics* / Q000502:physiology*; D045165:Testosterone Congeners / Q000494:pharmacology; D014105:Tosyl Compounds / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D000726:Androgen Antagonists; D059002:Androgen Receptor Antagonists; D000728:Androgens; D000813:Anilides; D001578:Benzopyrans; D002087:Butyrates; D008024:Ligands; D009570:Nitriles; D010080:Oxazoles; D011944:Receptors, Androgen; D045165:Testosterone Congeners; D014105:Tosyl Compounds; C053541:bicalutamide; C101278:decursin; C025643:vinclozolin