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Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and
Adam T Szafran1, Maria Szwarc, Marco Marcelli
1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.
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.
Methodology/Principal Findings:
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/Significance:
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.
Insights
A new high-throughput imaging assay quantifies androgen receptor (AR) function and its response to various compounds. This method aids in developing therapies for AR-related diseases like prostate cancer and personalizing treatments for androgen insensitivity syndrome (AIS).
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Androgen receptor (AR) function is crucial in diseases like prostate cancer and androgen insensitivity syndrome (AIS).
- Understanding AR modulation by external factors is key for developing effective therapies and analyzing environmental endocrine disruptors.
Purpose of the Study:
- To develop a high-throughput (HT) image-based assay for quantifying AR subcellular localization, subnuclear distribution, and transcriptional activity.
- To enable cell cycle-dependent analysis of AR function in unsynchronized cell populations.
Main Methods:
- Developed an HT image-based assay measuring AR distribution and reporter gene activity cell-by-cell.
- Integrated DNA content analysis for cell cycle determination.
- Selective analysis of cells expressing physiological AR levels to ensure assay accuracy.
Main Results:
- Achieved high assay quality with EC50 coefficients of variation of 5-24% and Z' values up to 0.91.
- Demonstrated that AR nuclear translocation and speckling correlate with transcriptional output.
- Showed differential effects of ligands on wild-type versus mutant AR, indicating distinct mechanisms of action.
- Successfully applied HT imaging to patient-derived AIS mutations for personalized medicine, identifying ligands to restore AR function.
Conclusions:
- HT imaging-based multiplex screening offers a rapid, systems-level approach for analyzing compounds and RNAi.
- This method can differentiate effects on wild-type AR versus clinically relevant AR mutations.
- Facilitates personalized medicine strategies for AR-related disorders.
