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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

HeLa GFP-AR cell lines expressing wild type and mutant AR able to translocate into nucleus in response to agonist.A. Western blot analysis comparing HeLa (1), LNCaP (2), HeLa GFP-AR (3), HeLa GFP-AR T877A (4), and HeLa GFP-AR F764L (5) androgen receptor expression. Equal total protein levels were loaded for all cell abstracts and confirmed with β-actin control. B, C, and D. Deconvolution images of HeLa cell lines expressing stably integrated GFP-AR, GFP- AR T877A (LNCaP, ligand binding domain mutation), and GFP- AR T877A (AIS associated mutation, ligand binding domain mutation), shown without (top) and with 10 nM or 100 nM R1881 (bottom). The LBD mutation in GFP-AR T877A or GFP-F764L does not affect the ability to translocate into the nucleus.
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pone-0003605-g001: HeLa GFP-AR cell lines expressing wild type and mutant AR able to translocate into nucleus in response to agonist.A. Western blot analysis comparing HeLa (1), LNCaP (2), HeLa GFP-AR (3), HeLa GFP-AR T877A (4), and HeLa GFP-AR F764L (5) androgen receptor expression. Equal total protein levels were loaded for all cell abstracts and confirmed with β-actin control. B, C, and D. Deconvolution images of HeLa cell lines expressing stably integrated GFP-AR, GFP- AR T877A (LNCaP, ligand binding domain mutation), and GFP- AR T877A (AIS associated mutation, ligand binding domain mutation), shown without (top) and with 10 nM or 100 nM R1881 (bottom). The LBD mutation in GFP-AR T877A or GFP-F764L does not affect the ability to translocate into the nucleus.

Mentions: To examine how wild-type and mutant ARs (expressed at physiologically relevant levels) respond to various experimental manipulations, HeLa cell lines were generated that stably express either wild type (GFP-AR), mutant GFP-ART877A (LNCaP mutation; [13]), or GFP-ARF764L (AIS mutation; [21]) under control of the CMV promoter. The T877A and F764L mutations were selected due to known altered ligand responses [13]. Generated cell lines were characterized at the population level by western blot analysis which indicated that HeLa GFP-AR, HeLa GFP-AR T877A, and HeLa GFP-AR F764L expressed AR of the expected size and at levels approximately 1.1-, 2.1, and 0.8-fold of that found in LNCaP cell pools (Fig. 1A). Furthermore, microarray-based RNA expression analysis demonstrated that GFP-AR regulates (activation or repression) known endogenous AR-responsive genes in response to ligand, indicating the cellular machinery of HeLa readily supports AR transcription function (Supplementary Table S1).


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)

HeLa GFP-AR cell lines expressing wild type and mutant AR able to translocate into nucleus in response to agonist.A. Western blot analysis comparing HeLa (1), LNCaP (2), HeLa GFP-AR (3), HeLa GFP-AR T877A (4), and HeLa GFP-AR F764L (5) androgen receptor expression. Equal total protein levels were loaded for all cell abstracts and confirmed with β-actin control. B, C, and D. Deconvolution images of HeLa cell lines expressing stably integrated GFP-AR, GFP- AR T877A (LNCaP, ligand binding domain mutation), and GFP- AR T877A (AIS associated mutation, ligand binding domain mutation), shown without (top) and with 10 nM or 100 nM R1881 (bottom). The LBD mutation in GFP-AR T877A or GFP-F764L does not affect the ability to translocate into the nucleus.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g001: HeLa GFP-AR cell lines expressing wild type and mutant AR able to translocate into nucleus in response to agonist.A. Western blot analysis comparing HeLa (1), LNCaP (2), HeLa GFP-AR (3), HeLa GFP-AR T877A (4), and HeLa GFP-AR F764L (5) androgen receptor expression. Equal total protein levels were loaded for all cell abstracts and confirmed with β-actin control. B, C, and D. Deconvolution images of HeLa cell lines expressing stably integrated GFP-AR, GFP- AR T877A (LNCaP, ligand binding domain mutation), and GFP- AR T877A (AIS associated mutation, ligand binding domain mutation), shown without (top) and with 10 nM or 100 nM R1881 (bottom). The LBD mutation in GFP-AR T877A or GFP-F764L does not affect the ability to translocate into the nucleus.
Mentions: To examine how wild-type and mutant ARs (expressed at physiologically relevant levels) respond to various experimental manipulations, HeLa cell lines were generated that stably express either wild type (GFP-AR), mutant GFP-ART877A (LNCaP mutation; [13]), or GFP-ARF764L (AIS mutation; [21]) under control of the CMV promoter. The T877A and F764L mutations were selected due to known altered ligand responses [13]. Generated cell lines were characterized at the population level by western blot analysis which indicated that HeLa GFP-AR, HeLa GFP-AR T877A, and HeLa GFP-AR F764L expressed AR of the expected size and at levels approximately 1.1-, 2.1, and 0.8-fold of that found in LNCaP cell pools (Fig. 1A). Furthermore, microarray-based RNA expression analysis demonstrated that GFP-AR regulates (activation or repression) known endogenous AR-responsive genes in response to ligand, indicating the cellular machinery of HeLa readily supports AR transcription function (Supplementary Table S1).

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