A High-Content Glucocorticoid Receptor Translocation Assay for Compound Mechanism-of-Action Evaluation

Agler, Michele; Prack, Margaret M.; Zhu, Yingjie; Kolb, Janet; Nowak, Kimberly; Ryseck, Rolf; Shen, Ding Ren; Cvijic, Mary Ellen; Somerville, John; Nadler, S.; Chen, Taosheng

doi:10.1177/1087057107309353

Cited by 22 publications

(17 citation statements)

References 57 publications

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“…The instrument uses an algorithm to calculate the fluorescence intensity difference between nucleus and cytosol, and a higher nuclear-cytoplasmic intensity difference denotes increased protein localization in the nucleus. A number of studies have used ArrayScan to monitor nuclear localization of various proteins (31)(32)(33). To measure ER␣ and ER␤ translocation, NSCLC cells were treated with E2 for 1 h, and cells were processed as described in Materials and Methods.…”

Section: Er␤ Does Not Translocate To the Nucleusmentioning

confidence: 99%

Estrogen Receptor β Functions through Nongenomic Mechanisms in Lung Cancer Cells

Zhang

Liu²,

Farkas³

et al. 2009

Molecular Endocrinology

103

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Recent studies have shown that estrogens promote the growth of lung cancer cells and may potentially be responsible for increased susceptibility to lung cancer in women. These observations raise the possibility of using antiestrogens in treating and preventing lung cancer. However, it is not clear how estrogen receptors (ERs) modulate the growth of non-small cell lung cancer (NSCLC) cells. Our Western blotting and real-time PCR analysis showed that NSCLC cells expressed ERbeta, but not ERalpha. In addition, ERbeta-specific ligands, but not ERalpha-specific ligands, promoted the growth of lung cancer cells. Furthermore, knockdown of ERbeta by short hairpin RNA constructs resulted in loss of estrogen-dependent growth of lung cancer cells. Interestingly, endogenous ERbeta failed to transcriptionally activate estrogen response element (ERE)-luciferase constructs in NSCLC cells, suggesting a lack of genomic function. Upon further investigation, ERbeta was found to be in the cytoplasm in all lung cancer cells and failed to translocate to the nucleus in the presence of estrogen, as observed by biochemical, ArrayScan, and confocal microscopy experiments. Nonetheless, estrogen caused rapid activation of cAMP, Akt, and MAPK signaling pathways in lung cancer cells. Immunohistochemical analysis of lung tumor biopsies showed strong ERbeta staining in the cytoplasm, whereas no staining was observed for ERalpha. In conclusion, our results suggest that that proliferative effects of estrogen in lung cancer cells is mediated primarily, if not exclusively, by the nongenomic action of ERbeta.

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Section: Er␤ Does Not Translocate To the Nucleusmentioning

confidence: 99%

Estrogen Receptor β Functions through Nongenomic Mechanisms in Lung Cancer Cells

Zhang

Liu²,

Farkas³

et al. 2009

Molecular Endocrinology

103

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“…More recently, a number of fluorescent indicators have been designed for ligand-mediated responses of SRs. Some of these quantify ligand-induced activity by nuclear translocation or nuclear mobility of fluorescently labeled receptors or receptor chimeras (39)(40)(41)(42). Others make use of ligand-induced conformational changes in the SR-LBDs (43).…”

Section: Introductionmentioning

confidence: 99%

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

et al. 2013

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Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands. ' 2013 International Society for Advancement of Cytometry Key terms androgen receptor; recurrent prostate cancer; ligand; N/C-interaction; quantitative live cell imaging; FRET

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“…[6][7][8] Microcarriers and arrays are used primarily for genetic analysis and clinical diagnostics, whereas high-content screening has recently been incorporated into early drug discovery for mechanism of action studies. 9,10 Conventional high-throughput cell-based reporter assays for lead identification or liability profiling are typically run in serial or parallel mode, with counterscreens to identify cytotoxicity and eliminate false-positives and recover false-negatives. Most reporter assays use stably transfected cell lines maintained in culture or involve transient transfections prior to running the assay.…”

Section: Cell Culture and Transient Transfectionmentioning

confidence: 99%

Multiplexing a High-Throughput Liability Assay to Leverage Efficiencies

Herbst

Anthony

Stewart

et al. 2009

ASSAY and Drug Development Technologies

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In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

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A High-Content Glucocorticoid Receptor Translocation Assay for Compound Mechanism-of-Action Evaluation

Cited by 22 publications

References 57 publications

Estrogen Receptor β Functions through Nongenomic Mechanisms in Lung Cancer Cells

Estrogen Receptor β Functions through Nongenomic Mechanisms in Lung Cancer Cells

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

Multiplexing a High-Throughput Liability Assay to Leverage Efficiencies

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