Limits...
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.

Show MeSH

Related in: MedlinePlus

Multiple×assay automated image analysis.A. The probasin proximal promoter element containing two AR binding sites duplicated and fused to a peroxisomes-targeted dsRED2skl protein reporter. B. Raw gray-scale image of HeLa cell nuclei stained with DAPI. White box indicates view used in rest of figure. C. Binary nuclear mask generated by non-linear least squares image filter and image field segmentation based on nuclear centroids and veronoi tessellation. This tessellation in combination with a user defined radius rule defines cytoplasmic compartment of each cell. D. Virtual pseudo color well image generated after GFP-AR data extraction. E. AR transcriptional reporter gene activity at single cell level determined by dsRED2skl image data extraction. All screen captures directly from Cytoshop with various features toggled on/off.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g003: Multiple×assay automated image analysis.A. The probasin proximal promoter element containing two AR binding sites duplicated and fused to a peroxisomes-targeted dsRED2skl protein reporter. B. Raw gray-scale image of HeLa cell nuclei stained with DAPI. White box indicates view used in rest of figure. C. Binary nuclear mask generated by non-linear least squares image filter and image field segmentation based on nuclear centroids and veronoi tessellation. This tessellation in combination with a user defined radius rule defines cytoplasmic compartment of each cell. D. Virtual pseudo color well image generated after GFP-AR data extraction. E. AR transcriptional reporter gene activity at single cell level determined by dsRED2skl image data extraction. All screen captures directly from Cytoshop with various features toggled on/off.

Mentions: To allow visualization of AR regulated transcriptional reporter gene activity, the HeLa cell lines were transiently transfected with the pARR2PB-dsRED2skl reporter construct, based on the AR-responsive composite probasin promoter (Fig. 3A), and then incubated for 18 hours with a 10-point titration (10−5 to 10−14 M) of the compounds of interest. The dsRED2skl gene encodes a red fluorescent protein that is targeted to the peroxisomes, which improves detection due to concentrating dsRED2skl in the small cytoplasmic organelles. When examining potential anti-androgenic activity, test compounds were titred against 10 nM R1881, a minimal dose found to be sufficient to generate a response in all key measurements. Cells were imaged using an automated microscope with a 40×/0.90 NA objective. For each field, three images were captured: DAPI (nuclei, blue), GFP (AR, green), and dsRED2skl (reporter protein, red). Cytoshop (Beckman Coulter) or Pipeline Pilot (Accelrys) image analysis software was used to identify individual cells in each image. For each cell, the DAPI channel (Fig. 3B) was used to identify the nucleus, and the remaining field was computationally segmented to determine the cytoplasmic compartment for each cell (Fig. 3B–C). These masks were then applied to the green and red images to determine cellular distribution of GFP-AR and transcriptional reporter gene activity (Fig. 3D–E). Cell populations were then sorted to remove nuclei clusters (bi- or multinucleate), abnormal nuclear shape and/or DNA density (apoptotic, mitotic), and to select for low expression levels as defined above. To analyze the GFP-AR subcellular trafficking and transcription results, three key features were determined for each cell: 1) degree of nuclear translocation (fraction of GFP signal localized in nucleus, FLIN):2) amount of nuclear hyperspeckling (nuclear variation of GFP signal intensity, NVAR):(where X is each nuclear GFP pixel intensity, γ is the average GFP pixel intensity, and N is the number of pixels in the nucleus) and, 3) transcriptional reporter gene activity (total amount of correlated channel 2/dsRED2skl signal, CORR2):All measurements are then normalized to those observed in untreated and treated (100 nM R1881) GFP-AR. The ability to measure the hyperspeckled patterning is important because it is thought to represent the formation of transient protein complexes by the receptor as it scans the DNA for androgen response elements [22], [23]. The ability of the IC-100 to rapidly focus using a high NA 40× objective was particularly important for these measurements.


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)

Multiple×assay automated image analysis.A. The probasin proximal promoter element containing two AR binding sites duplicated and fused to a peroxisomes-targeted dsRED2skl protein reporter. B. Raw gray-scale image of HeLa cell nuclei stained with DAPI. White box indicates view used in rest of figure. C. Binary nuclear mask generated by non-linear least squares image filter and image field segmentation based on nuclear centroids and veronoi tessellation. This tessellation in combination with a user defined radius rule defines cytoplasmic compartment of each cell. D. Virtual pseudo color well image generated after GFP-AR data extraction. E. AR transcriptional reporter gene activity at single cell level determined by dsRED2skl image data extraction. All screen captures directly from Cytoshop with various features toggled on/off.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003605-g003: Multiple×assay automated image analysis.A. The probasin proximal promoter element containing two AR binding sites duplicated and fused to a peroxisomes-targeted dsRED2skl protein reporter. B. Raw gray-scale image of HeLa cell nuclei stained with DAPI. White box indicates view used in rest of figure. C. Binary nuclear mask generated by non-linear least squares image filter and image field segmentation based on nuclear centroids and veronoi tessellation. This tessellation in combination with a user defined radius rule defines cytoplasmic compartment of each cell. D. Virtual pseudo color well image generated after GFP-AR data extraction. E. AR transcriptional reporter gene activity at single cell level determined by dsRED2skl image data extraction. All screen captures directly from Cytoshop with various features toggled on/off.
Mentions: To allow visualization of AR regulated transcriptional reporter gene activity, the HeLa cell lines were transiently transfected with the pARR2PB-dsRED2skl reporter construct, based on the AR-responsive composite probasin promoter (Fig. 3A), and then incubated for 18 hours with a 10-point titration (10−5 to 10−14 M) of the compounds of interest. The dsRED2skl gene encodes a red fluorescent protein that is targeted to the peroxisomes, which improves detection due to concentrating dsRED2skl in the small cytoplasmic organelles. When examining potential anti-androgenic activity, test compounds were titred against 10 nM R1881, a minimal dose found to be sufficient to generate a response in all key measurements. Cells were imaged using an automated microscope with a 40×/0.90 NA objective. For each field, three images were captured: DAPI (nuclei, blue), GFP (AR, green), and dsRED2skl (reporter protein, red). Cytoshop (Beckman Coulter) or Pipeline Pilot (Accelrys) image analysis software was used to identify individual cells in each image. For each cell, the DAPI channel (Fig. 3B) was used to identify the nucleus, and the remaining field was computationally segmented to determine the cytoplasmic compartment for each cell (Fig. 3B–C). These masks were then applied to the green and red images to determine cellular distribution of GFP-AR and transcriptional reporter gene activity (Fig. 3D–E). Cell populations were then sorted to remove nuclei clusters (bi- or multinucleate), abnormal nuclear shape and/or DNA density (apoptotic, mitotic), and to select for low expression levels as defined above. To analyze the GFP-AR subcellular trafficking and transcription results, three key features were determined for each cell: 1) degree of nuclear translocation (fraction of GFP signal localized in nucleus, FLIN):2) amount of nuclear hyperspeckling (nuclear variation of GFP signal intensity, NVAR):(where X is each nuclear GFP pixel intensity, γ is the average GFP pixel intensity, and N is the number of pixels in the nucleus) and, 3) transcriptional reporter gene activity (total amount of correlated channel 2/dsRED2skl signal, CORR2):All measurements are then normalized to those observed in untreated and treated (100 nM R1881) GFP-AR. The ability to measure the hyperspeckled patterning is important because it is thought to represent the formation of transient protein complexes by the receptor as it scans the DNA for androgen response elements [22], [23]. The ability of the IC-100 to rapidly focus using a high NA 40× objective was particularly important for these measurements.

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