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Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and mutation-specific effects.

Szafran AT, Szwarc M, Marcelli M, Mancini MA - PLoS ONE (2008)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.

ABSTRACT

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.

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Related in: MedlinePlus

Dose dependent effects measured in panel of steroid compounds.The differential effects of various steroidal compounds on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR. Cells transfected with pARR-2PB-dsRED2skl reporter vector and maintained in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound for 18 hr in 5%SD-FBS. Results normalized to negative (no treatment) and positive (R1881) controls. Tested compounds include known AR agonist R1881 (A), mibolerone (B), and DHT(C) as well as other steroidal compounds estradiol (D), progesterone (E) and corticosterone (F). EC50 values calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error (G). Data represents average of 8 experiments.
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pone-0003605-g004: Dose dependent effects measured in panel of steroid compounds.The differential effects of various steroidal compounds on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR. Cells transfected with pARR-2PB-dsRED2skl reporter vector and maintained in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound for 18 hr in 5%SD-FBS. Results normalized to negative (no treatment) and positive (R1881) controls. Tested compounds include known AR agonist R1881 (A), mibolerone (B), and DHT(C) as well as other steroidal compounds estradiol (D), progesterone (E) and corticosterone (F). EC50 values calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error (G). Data represents average of 8 experiments.

Mentions: To demonstrate the ability to use the assay as a screening tool, known AR agonists that have similar high affinity for AR [28] were tested over a wide range of concentrations, including R1881, mibolerone, and DHT. Whereas DHT can rapidly be metabolized [29], the synthetic androgens R1881 and mibolerone are relatively stable [30]. All three compounds induced GFP-AR nuclear translocation, nuclear hyperspeckling and dsRED2skl transcriptional reporter gene activity in a dose-dependent manner (Fig. 4 and Supplementary Table S6). Using R1881, the calculated EC50 concentration for nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity were 0.96±0.03 nM, 30.7±4.5 nM, and 28.2±4.2 nM, respectively. The AR agonists DHT and mibolerone demonstrated similar effects as compared to R1881, differing only in that DHT was approximately 10-fold less efficient in inducing nuclear translocation. It is interesting to note that the EC50 for hyperspeckling and transcriptional reporter gene activity were both ∼30-fold higher than that of nuclear translocation, indicating that AR translocation and hyperspeckling/transcriptional reporter gene activity are distinct biological steps and that highly quantitative data can be culled from this multiplex imaging-based approach.


Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and mutation-specific effects.

Szafran AT, Szwarc M, Marcelli M, Mancini MA - PLoS ONE (2008)

Dose dependent effects measured in panel of steroid compounds.The differential effects of various steroidal compounds on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR. Cells transfected with pARR-2PB-dsRED2skl reporter vector and maintained in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound for 18 hr in 5%SD-FBS. Results normalized to negative (no treatment) and positive (R1881) controls. Tested compounds include known AR agonist R1881 (A), mibolerone (B), and DHT(C) as well as other steroidal compounds estradiol (D), progesterone (E) and corticosterone (F). EC50 values calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error (G). Data represents average of 8 experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g004: Dose dependent effects measured in panel of steroid compounds.The differential effects of various steroidal compounds on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR. Cells transfected with pARR-2PB-dsRED2skl reporter vector and maintained in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound for 18 hr in 5%SD-FBS. Results normalized to negative (no treatment) and positive (R1881) controls. Tested compounds include known AR agonist R1881 (A), mibolerone (B), and DHT(C) as well as other steroidal compounds estradiol (D), progesterone (E) and corticosterone (F). EC50 values calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error (G). Data represents average of 8 experiments.
Mentions: To demonstrate the ability to use the assay as a screening tool, known AR agonists that have similar high affinity for AR [28] were tested over a wide range of concentrations, including R1881, mibolerone, and DHT. Whereas DHT can rapidly be metabolized [29], the synthetic androgens R1881 and mibolerone are relatively stable [30]. All three compounds induced GFP-AR nuclear translocation, nuclear hyperspeckling and dsRED2skl transcriptional reporter gene activity in a dose-dependent manner (Fig. 4 and Supplementary Table S6). Using R1881, the calculated EC50 concentration for nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity were 0.96±0.03 nM, 30.7±4.5 nM, and 28.2±4.2 nM, respectively. The AR agonists DHT and mibolerone demonstrated similar effects as compared to R1881, differing only in that DHT was approximately 10-fold less efficient in inducing nuclear translocation. It is interesting to note that the EC50 for hyperspeckling and transcriptional reporter gene activity were both ∼30-fold higher than that of nuclear translocation, indicating that AR translocation and hyperspeckling/transcriptional reporter gene activity are distinct biological steps and that highly quantitative data can be culled from this multiplex imaging-based approach.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus