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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

HTM analysis allows selection of cells expressing physiologically relevant levels of GFP-AR.A and B. Representative images of LNCaP cells (A) and HeLa cells stably transfected with GFP-AR (B) were immunolabeled and imaged to determine the GFP-AR signal that corresponds to endogenous LNCaP AR expression levels. Exposure levels for the GFP (left) and anti-AR labeling (right) is identical for both cell types. C. Image analysis was performed on the LNCaP antibody images to quantify the total anti-AR labeling per cell, derived from the standard 10×10 field of cells, containing >500 cells. The dashed lines indicate the 10% and 90% percentile of the population. D. Similar image analysis was performed on the HeLa GFP-AR antibody images to quantify the total anti-AR labeling per cell. The dashed lines indicate the 10%–90% expression range found in the LNCaP cell line. These cut-offs were empirically determined to remove the outliers, either barely-detectable GFP, or grossly over-expressing cells. As can be seen in the histogram from the ∼500 cell quantitation, removing the very heterogenous top 10%, or the more homogeneous bottom 10%, only eliminates the extremes, which can have an untoward influence on the bulk of the population. E. HeLa GFP-AR cells with AR expression within this range were selected and total GFP intensity per cell was determined and plotted. GFP expression corresponding to the 10% and 90% percentile in this population were determined (dashed lines) and used as lower and upper limits of GFP-AR expression in all subsequent experiments. This analysis enables the selection of HeLa GFP-AR cells with AR expression levels similar to that endogenously expressed in LNCaP cell line.
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pone-0003605-g002: HTM analysis allows selection of cells expressing physiologically relevant levels of GFP-AR.A and B. Representative images of LNCaP cells (A) and HeLa cells stably transfected with GFP-AR (B) were immunolabeled and imaged to determine the GFP-AR signal that corresponds to endogenous LNCaP AR expression levels. Exposure levels for the GFP (left) and anti-AR labeling (right) is identical for both cell types. C. Image analysis was performed on the LNCaP antibody images to quantify the total anti-AR labeling per cell, derived from the standard 10×10 field of cells, containing >500 cells. The dashed lines indicate the 10% and 90% percentile of the population. D. Similar image analysis was performed on the HeLa GFP-AR antibody images to quantify the total anti-AR labeling per cell. The dashed lines indicate the 10%–90% expression range found in the LNCaP cell line. These cut-offs were empirically determined to remove the outliers, either barely-detectable GFP, or grossly over-expressing cells. As can be seen in the histogram from the ∼500 cell quantitation, removing the very heterogenous top 10%, or the more homogeneous bottom 10%, only eliminates the extremes, which can have an untoward influence on the bulk of the population. E. HeLa GFP-AR cells with AR expression within this range were selected and total GFP intensity per cell was determined and plotted. GFP expression corresponding to the 10% and 90% percentile in this population were determined (dashed lines) and used as lower and upper limits of GFP-AR expression in all subsequent experiments. This analysis enables the selection of HeLa GFP-AR cells with AR expression levels similar to that endogenously expressed in LNCaP cell line.

Mentions: To further characterize the cell lines, we analyzed them at the single cell level using fluorescence microscopy, and in each line, >90% of the cells were GFP positive. In the absence of ligand, GFP-AR was diffusely distributed cell-wide (Fig. 1B); GFP-AR T877A and GFP-AR F764L were predominantly located in the cytoplasm (Fig. 1C and 1D) in >95% of cells. Upon addition of the synthetic androgen R1881 (10 nM or 100 nM) for two hours, 95–97% had a majority of the signal in the nucleus (Fig. 1B–D). Despite single cell cloning, expression between single HeLa cells was heterogeneous and varied up to 12-fold. Therefore, we used immunofluorescence to determine the relative AR expression level in both stably transfected HeLa and LNCaP cells (Fig. 2A–D) to define a sub-population of HeLa expressing AR at levels similar to LNCaP (Fig. 2E). In subsequent analyses, only this refined, homogenous subpopulation of HeLa cells were analyzed to limit potential over-expression artifacts [3], [16].


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)

HTM analysis allows selection of cells expressing physiologically relevant levels of GFP-AR.A and B. Representative images of LNCaP cells (A) and HeLa cells stably transfected with GFP-AR (B) were immunolabeled and imaged to determine the GFP-AR signal that corresponds to endogenous LNCaP AR expression levels. Exposure levels for the GFP (left) and anti-AR labeling (right) is identical for both cell types. C. Image analysis was performed on the LNCaP antibody images to quantify the total anti-AR labeling per cell, derived from the standard 10×10 field of cells, containing >500 cells. The dashed lines indicate the 10% and 90% percentile of the population. D. Similar image analysis was performed on the HeLa GFP-AR antibody images to quantify the total anti-AR labeling per cell. The dashed lines indicate the 10%–90% expression range found in the LNCaP cell line. These cut-offs were empirically determined to remove the outliers, either barely-detectable GFP, or grossly over-expressing cells. As can be seen in the histogram from the ∼500 cell quantitation, removing the very heterogenous top 10%, or the more homogeneous bottom 10%, only eliminates the extremes, which can have an untoward influence on the bulk of the population. E. HeLa GFP-AR cells with AR expression within this range were selected and total GFP intensity per cell was determined and plotted. GFP expression corresponding to the 10% and 90% percentile in this population were determined (dashed lines) and used as lower and upper limits of GFP-AR expression in all subsequent experiments. This analysis enables the selection of HeLa GFP-AR cells with AR expression levels similar to that endogenously expressed in LNCaP cell line.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g002: HTM analysis allows selection of cells expressing physiologically relevant levels of GFP-AR.A and B. Representative images of LNCaP cells (A) and HeLa cells stably transfected with GFP-AR (B) were immunolabeled and imaged to determine the GFP-AR signal that corresponds to endogenous LNCaP AR expression levels. Exposure levels for the GFP (left) and anti-AR labeling (right) is identical for both cell types. C. Image analysis was performed on the LNCaP antibody images to quantify the total anti-AR labeling per cell, derived from the standard 10×10 field of cells, containing >500 cells. The dashed lines indicate the 10% and 90% percentile of the population. D. Similar image analysis was performed on the HeLa GFP-AR antibody images to quantify the total anti-AR labeling per cell. The dashed lines indicate the 10%–90% expression range found in the LNCaP cell line. These cut-offs were empirically determined to remove the outliers, either barely-detectable GFP, or grossly over-expressing cells. As can be seen in the histogram from the ∼500 cell quantitation, removing the very heterogenous top 10%, or the more homogeneous bottom 10%, only eliminates the extremes, which can have an untoward influence on the bulk of the population. E. HeLa GFP-AR cells with AR expression within this range were selected and total GFP intensity per cell was determined and plotted. GFP expression corresponding to the 10% and 90% percentile in this population were determined (dashed lines) and used as lower and upper limits of GFP-AR expression in all subsequent experiments. This analysis enables the selection of HeLa GFP-AR cells with AR expression levels similar to that endogenously expressed in LNCaP cell line.
Mentions: To further characterize the cell lines, we analyzed them at the single cell level using fluorescence microscopy, and in each line, >90% of the cells were GFP positive. In the absence of ligand, GFP-AR was diffusely distributed cell-wide (Fig. 1B); GFP-AR T877A and GFP-AR F764L were predominantly located in the cytoplasm (Fig. 1C and 1D) in >95% of cells. Upon addition of the synthetic androgen R1881 (10 nM or 100 nM) for two hours, 95–97% had a majority of the signal in the nucleus (Fig. 1B–D). Despite single cell cloning, expression between single HeLa cells was heterogeneous and varied up to 12-fold. Therefore, we used immunofluorescence to determine the relative AR expression level in both stably transfected HeLa and LNCaP cells (Fig. 2A–D) to define a sub-population of HeLa expressing AR at levels similar to LNCaP (Fig. 2E). In subsequent analyses, only this refined, homogenous subpopulation of HeLa cells were analyzed to limit potential over-expression artifacts [3], [16].

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