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

Analysis of the relationship between observed responses, and cell cycle in asynchronous HeLa GFP-AR cells treated with R1881 (green square), o-hydroxyflutamide (red triangle), or thymidine (yellow diamond).A. Biparametric dot-plot analysis of total DNA content (total DAPI signal per nucleus) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Dotted lines represent thresholds used to divide cells with either low or high DNA content (vertical line) or cells positively stained for EdU (horizontal line). The percent of cells in each quadrant with each treatment are represented in the associated table. B. Concentration-response curves of the percent of cells in S phase (large markers) and G1/G2 ratio (small markers) after an 18 hr treatment. C and D. Concentration-response curves examining nuclear translocation response (C) and nuclear hyperspeckling (D) in G1, S phase, and G2 cells treated with R1881 and o-hydroxyflutamide. Results normalized to positive and negative controls (all cells).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2572143&req=5

pone-0003605-g007: Analysis of the relationship between observed responses, and cell cycle in asynchronous HeLa GFP-AR cells treated with R1881 (green square), o-hydroxyflutamide (red triangle), or thymidine (yellow diamond).A. Biparametric dot-plot analysis of total DNA content (total DAPI signal per nucleus) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Dotted lines represent thresholds used to divide cells with either low or high DNA content (vertical line) or cells positively stained for EdU (horizontal line). The percent of cells in each quadrant with each treatment are represented in the associated table. B. Concentration-response curves of the percent of cells in S phase (large markers) and G1/G2 ratio (small markers) after an 18 hr treatment. C and D. Concentration-response curves examining nuclear translocation response (C) and nuclear hyperspeckling (D) in G1, S phase, and G2 cells treated with R1881 and o-hydroxyflutamide. Results normalized to positive and negative controls (all cells).

Mentions: As expected, thymidine demonstrated a dose-dependent ability to block HeLa GFP-AR cells in G1 (EC50 = 2.6 mM±0.2 mM) and significantly reduced the occurrence of cells in S-phase (42.6%→0.5%, EC50 = 1.30±0.01 mM, p<0.01: Fig. 7A–B). Treatment of cells with R1881 also results in a dose dependent growth arrest of cells characterized by a G1/S block (EC50 = 0.10±0.03 nM) and concomitantly a significant reduction in S-phase cells (42.6%→0.7%, EC50 = 0.07±0.02 nM, p<0.01) (Fig. 7A–B). This response is similar to cell cycle effects of AR when is re-introduced to the PC3 prostate cancer cell line [38]; OHF did not have any appreciable effect on the cell cycle.


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)

Analysis of the relationship between observed responses, and cell cycle in asynchronous HeLa GFP-AR cells treated with R1881 (green square), o-hydroxyflutamide (red triangle), or thymidine (yellow diamond).A. Biparametric dot-plot analysis of total DNA content (total DAPI signal per nucleus) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Dotted lines represent thresholds used to divide cells with either low or high DNA content (vertical line) or cells positively stained for EdU (horizontal line). The percent of cells in each quadrant with each treatment are represented in the associated table. B. Concentration-response curves of the percent of cells in S phase (large markers) and G1/G2 ratio (small markers) after an 18 hr treatment. C and D. Concentration-response curves examining nuclear translocation response (C) and nuclear hyperspeckling (D) in G1, S phase, and G2 cells treated with R1881 and o-hydroxyflutamide. Results normalized to positive and negative controls (all cells).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003605-g007: Analysis of the relationship between observed responses, and cell cycle in asynchronous HeLa GFP-AR cells treated with R1881 (green square), o-hydroxyflutamide (red triangle), or thymidine (yellow diamond).A. Biparametric dot-plot analysis of total DNA content (total DAPI signal per nucleus) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Dotted lines represent thresholds used to divide cells with either low or high DNA content (vertical line) or cells positively stained for EdU (horizontal line). The percent of cells in each quadrant with each treatment are represented in the associated table. B. Concentration-response curves of the percent of cells in S phase (large markers) and G1/G2 ratio (small markers) after an 18 hr treatment. C and D. Concentration-response curves examining nuclear translocation response (C) and nuclear hyperspeckling (D) in G1, S phase, and G2 cells treated with R1881 and o-hydroxyflutamide. Results normalized to positive and negative controls (all cells).
Mentions: As expected, thymidine demonstrated a dose-dependent ability to block HeLa GFP-AR cells in G1 (EC50 = 2.6 mM±0.2 mM) and significantly reduced the occurrence of cells in S-phase (42.6%→0.5%, EC50 = 1.30±0.01 mM, p<0.01: Fig. 7A–B). Treatment of cells with R1881 also results in a dose dependent growth arrest of cells characterized by a G1/S block (EC50 = 0.10±0.03 nM) and concomitantly a significant reduction in S-phase cells (42.6%→0.7%, EC50 = 0.07±0.02 nM, p<0.01) (Fig. 7A–B). This response is similar to cell cycle effects of AR when is re-introduced to the PC3 prostate cancer cell line [38]; OHF did not have any appreciable effect on the cell cycle.

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