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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

Changes in AR expression level can alter magnitude of responses, but not the concentration at which they occur.To generate a population of cells with a wide range of expression levels, HeLa cells were transiently transfected with GFP-AR and the pARR-2PB-dsRED2skl construct and treated for 18 hr with a R1881 titration. Cells were fixed, DAPI stained, and imaged using the IC100 HTM. Because of image artifacts generated due to the range of expression observed with transient transfections, images were analyzed using Pipeline Pilot software package. After cells were analyzed, the population was subdivided into low, medium low, medium high, and high based on total GFP-AR expression. A. Dose response curves for nuclear translocation failed to demonstrate any affects on the response by AR expression level. B. Dose response curves for nuclear hyperspeckling demonstrated that medium-high and high AR expression was associated with increased amount of hyperspeckling; however, the calculated EC50 values were not significantly different between the populations. C. Analysis of cells treated with 100 nM R1881 demonstrates that transcriptional reporter gene activity in cells with medium-high to high levels of AR expression was significantly (p<0.001) reduced despite increased hyperspeckling.
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pone-0003605-g005: Changes in AR expression level can alter magnitude of responses, but not the concentration at which they occur.To generate a population of cells with a wide range of expression levels, HeLa cells were transiently transfected with GFP-AR and the pARR-2PB-dsRED2skl construct and treated for 18 hr with a R1881 titration. Cells were fixed, DAPI stained, and imaged using the IC100 HTM. Because of image artifacts generated due to the range of expression observed with transient transfections, images were analyzed using Pipeline Pilot software package. After cells were analyzed, the population was subdivided into low, medium low, medium high, and high based on total GFP-AR expression. A. Dose response curves for nuclear translocation failed to demonstrate any affects on the response by AR expression level. B. Dose response curves for nuclear hyperspeckling demonstrated that medium-high and high AR expression was associated with increased amount of hyperspeckling; however, the calculated EC50 values were not significantly different between the populations. C. Analysis of cells treated with 100 nM R1881 demonstrates that transcriptional reporter gene activity in cells with medium-high to high levels of AR expression was significantly (p<0.001) reduced despite increased hyperspeckling.

Mentions: The ability to perform cell-by-cell analysis allowed us to test the hypothesis that expression level alters the observed responses. At higher levels of expression, an increased magnitude of hyperspeckling was observed with no effect on nuclear translocation (Fig. 5A and 5B). At these higher levels of AR expression (elevated by 2- to 4-fold), transcriptional reporter gene activity was repressed significantly despite the elevated hyperspeckling (Fig. 5C), and completely abolished as expression levels increased. These measurements were taken from cells transiently transfected with GFP-AR in order to poll a range of AR expression beyond that observed in our HeLa stable cell lines. These results suggest that the reason why we are able to achieve high assay quality is largely due to the ability to examine a narrow range of AR expression that is essentially free from potential over-expression artifacts.


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)

Changes in AR expression level can alter magnitude of responses, but not the concentration at which they occur.To generate a population of cells with a wide range of expression levels, HeLa cells were transiently transfected with GFP-AR and the pARR-2PB-dsRED2skl construct and treated for 18 hr with a R1881 titration. Cells were fixed, DAPI stained, and imaged using the IC100 HTM. Because of image artifacts generated due to the range of expression observed with transient transfections, images were analyzed using Pipeline Pilot software package. After cells were analyzed, the population was subdivided into low, medium low, medium high, and high based on total GFP-AR expression. A. Dose response curves for nuclear translocation failed to demonstrate any affects on the response by AR expression level. B. Dose response curves for nuclear hyperspeckling demonstrated that medium-high and high AR expression was associated with increased amount of hyperspeckling; however, the calculated EC50 values were not significantly different between the populations. C. Analysis of cells treated with 100 nM R1881 demonstrates that transcriptional reporter gene activity in cells with medium-high to high levels of AR expression was significantly (p<0.001) reduced despite increased hyperspeckling.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g005: Changes in AR expression level can alter magnitude of responses, but not the concentration at which they occur.To generate a population of cells with a wide range of expression levels, HeLa cells were transiently transfected with GFP-AR and the pARR-2PB-dsRED2skl construct and treated for 18 hr with a R1881 titration. Cells were fixed, DAPI stained, and imaged using the IC100 HTM. Because of image artifacts generated due to the range of expression observed with transient transfections, images were analyzed using Pipeline Pilot software package. After cells were analyzed, the population was subdivided into low, medium low, medium high, and high based on total GFP-AR expression. A. Dose response curves for nuclear translocation failed to demonstrate any affects on the response by AR expression level. B. Dose response curves for nuclear hyperspeckling demonstrated that medium-high and high AR expression was associated with increased amount of hyperspeckling; however, the calculated EC50 values were not significantly different between the populations. C. Analysis of cells treated with 100 nM R1881 demonstrates that transcriptional reporter gene activity in cells with medium-high to high levels of AR expression was significantly (p<0.001) reduced despite increased hyperspeckling.
Mentions: The ability to perform cell-by-cell analysis allowed us to test the hypothesis that expression level alters the observed responses. At higher levels of expression, an increased magnitude of hyperspeckling was observed with no effect on nuclear translocation (Fig. 5A and 5B). At these higher levels of AR expression (elevated by 2- to 4-fold), transcriptional reporter gene activity was repressed significantly despite the elevated hyperspeckling (Fig. 5C), and completely abolished as expression levels increased. These measurements were taken from cells transiently transfected with GFP-AR in order to poll a range of AR expression beyond that observed in our HeLa stable cell lines. These results suggest that the reason why we are able to achieve high assay quality is largely due to the ability to examine a narrow range of AR expression that is essentially free from potential over-expression artifacts.

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