In vitro Androgen Receptor Modulation Assay

Androgen Receptor modulation and its measurement in vitro

The androgen receptor (AR) is a nuclear receptor activated by binding either testosterone or dihydrotestosterone in the cytoplasm and translocating to the nucleus¹.
Environmental contaminants such as industrial and agricultural chemicals have been shown to alter androgen receptor (AR) function through agonistic or antagonistic modulation.
Androgen disruptors can lead to reduced sperm count, infertility, prostate cancer and can interfere with normal male development.
AR agonists and some antagonists induce nuclear translocation. This can be detected using GFP-tagged proteins which are monitored in the cytoplasm and nucleus of the cells. The assay can also detect nuclear foci (punctuate distribution in the nucleus) which is characteristic of AR agonists.
The in vitro Androgen Receptor (AR) modulation assay can be applied as a therapeutic screening assay (e.g., in the development of prostate cancer therapies) or to assess potential endocrine disruption effects.

The AR is a ligand-dependent transcription factor that controls the expression of specific genes. The binding of the AR to its native ligands 5α-dihydrotestosterone (DHT) and testosterone initiates male sexual development and differentiation.

²Tan MHE et al., (2015) Androgen receptor: structure, role in prostate cancer and drug discovery. Acta Pharmacologica Sinica 36: 3-23

Protocol

In vitro Androgen Receptor Modulation Assay

Test Method	AR Redistribution Assay (ThermoScientific)
Cell Type	Recombinant U2OS cells stably expressing human androgen receptor
Test Article Concentrations	10 µM in quadruplicate (alternative concentrations or dose curves available on request)
Test Article Exposure	Overnight
Reference Controls	Dihydrotestosterone (agonist) Mifepristone (antagonist)
Typical Vehicle Control	0.5% DMSO
Analysis Method	ThermoScientific ArrayScan^® VTi High Content Imaging
Data Delivery	% Effect compared to control (single concentration) EC₅₀ (dose response)

Data

Data from the Androgen Receptor Modulation Assay

Figure 1
High content screening images illustrating the effect of the agonist dihydrotestosterone and the antagonist mifepristone on the translocation of AR-GFP into the nucleus.

Both the agonist dihydrotestosterone and the antagonist mifepristone induce nuclear translocation of AR. However, dihydrotestosterone can be identified as an agonist by the punctuated distribution pattern in the nuclei (also known as nuclear foci).

Figure 2
Spotfire visualization of reproducibility of the vehicle control (0.5% DMSO) and dihydrotestosterone (20nM) in agonist mode.

The data in Figure 2 show the reproducibility of the positive control agonist (dihydrotestosterone) and negative vehicle control across eight different plates (different colors represent different plates) and across the same plate (same colors represent sixteen positive control wells and sixteen negative control wells within the same plate).

Mean_CircRingAvgIntenDiff is the difference between the average intensity of the nucleus (circle) and the cytoplasm (ring) of the cell.

Figure 3
Spotfire visualization of the vehicle control (0.5% DMSO) and 17-AAG (25µM) in antagonist mode (in the presence of dihydrotestosterone).

The data in Figure 3 show the reproducibility of the positive control antagonist 17-AAG (17-allylamino-17-demethoxygeldanamycin) and negative vehicle control across eight different plates (different colors represent different plates) and across the same plate (same colors represent sixteen positive control wells and sixteen negative control wells within the same plate).

Mean_CircRingAvgIntenDiff is the difference between the average intensity of the nucleus (circle) and the cytoplasm (ring) of the cell.

References

¹ Hua Y, et al, (2014) High-content positional biosensor screening assay for compounds to prevent or disrupt androgen receptor and transcriptional intermediary factor 2 protein-protein interactions. ASSAY Drug Dev Technol 12(7); 395-418
² Tan MHE et al., (2015) Androgen receptor: structure, role in prostate cancer and drug discovery. Acta Pharmacologica Sinica 36; 3-23