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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

Differential responses of the F764L AR mutation to a panel of agonist.The effects of the F764L mutation on AR nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional reporter gene activity (C) in HeLa cells with treated with R1881 (red), mibolerone (black), or DHT (green). Cells stably expressing F764L form of AR were 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. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error. Data represents average of 4 experiments.
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pone-0003605-g008: Differential responses of the F764L AR mutation to a panel of agonist.The effects of the F764L mutation on AR nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional reporter gene activity (C) in HeLa cells with treated with R1881 (red), mibolerone (black), or DHT (green). Cells stably expressing F764L form of AR were 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. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error. Data represents average of 4 experiments.

Mentions: Parallel to studying mutations associated with prostate cancer, we were also interested in characterizing the inactivating mutations associated with androgen insensitivity syndrome. In particular, we sought in compounds that are able to induce a normal response from the mutant receptor. The F764L mutation was isolated from a patient with complete AIS and was previously characterized as having an abnormally high ligand dissociation rate [21]. After generating a HeLa GFP-AR F764L stable cell line, we examined the responses of the mutation when cells were treated with DHT, R1881, and mibolerone at concentrations ranging between 200 nM to 0.02 nM. Consistent with the disease phenotype, DHT failed to induce a strong hyperspeckling or transcriptional reporter gene response even at the highest concentrations tested (Fig. 8 A–C). DHT was able to induce nuclear translocation of the mutant receptor, but only at a high concentration (EC50 = 66.1±7.4 nM). In contrast, when the cells were treated with either R1881 or mibolerone, a marked response was observed in all three parameters examined (Fig. 8 A–C). For R1881, the maximal responses ranged between 40–60% of that observed with the WT AR, but occurred at higher concentrations (EC50 range = 10.6–159.4 nM). Strikingly, higher concentrations of mibolerone were able to induce maximal responses between 85–105% of that observed with the WT receptor (EC50 range = 2.6–71.6 nM). These results not only demonstrate why the disease phenotype is present (e.g., no response to endogenous DHT), but also establishes a rapid and specific ability to identify therapeutically-relevant compounds that may rescue receptor function.


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)

Differential responses of the F764L AR mutation to a panel of agonist.The effects of the F764L mutation on AR nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional reporter gene activity (C) in HeLa cells with treated with R1881 (red), mibolerone (black), or DHT (green). Cells stably expressing F764L form of AR were 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. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error. Data represents average of 4 experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g008: Differential responses of the F764L AR mutation to a panel of agonist.The effects of the F764L mutation on AR nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional reporter gene activity (C) in HeLa cells with treated with R1881 (red), mibolerone (black), or DHT (green). Cells stably expressing F764L form of AR were 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. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitting tool and presented±std. error. Data represents average of 4 experiments.
Mentions: Parallel to studying mutations associated with prostate cancer, we were also interested in characterizing the inactivating mutations associated with androgen insensitivity syndrome. In particular, we sought in compounds that are able to induce a normal response from the mutant receptor. The F764L mutation was isolated from a patient with complete AIS and was previously characterized as having an abnormally high ligand dissociation rate [21]. After generating a HeLa GFP-AR F764L stable cell line, we examined the responses of the mutation when cells were treated with DHT, R1881, and mibolerone at concentrations ranging between 200 nM to 0.02 nM. Consistent with the disease phenotype, DHT failed to induce a strong hyperspeckling or transcriptional reporter gene response even at the highest concentrations tested (Fig. 8 A–C). DHT was able to induce nuclear translocation of the mutant receptor, but only at a high concentration (EC50 = 66.1±7.4 nM). In contrast, when the cells were treated with either R1881 or mibolerone, a marked response was observed in all three parameters examined (Fig. 8 A–C). For R1881, the maximal responses ranged between 40–60% of that observed with the WT AR, but occurred at higher concentrations (EC50 range = 10.6–159.4 nM). Strikingly, higher concentrations of mibolerone were able to induce maximal responses between 85–105% of that observed with the WT receptor (EC50 range = 2.6–71.6 nM). These results not only demonstrate why the disease phenotype is present (e.g., no response to endogenous DHT), but also establishes a rapid and specific ability to identify therapeutically-relevant compounds that may rescue receptor function.

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