title: A high-content glucocorticoid receptor translocation assay for compound mechanism-of-action evaluation.
abstract: Ligand-induced cytoplasm to nucleus translocation is a critical event in the nuclear receptor (NR) signal transduction cascade. The development of green fluorescent proteins and their color variants fused with NRs, along with the recent developments in automated cellular imaging technologies, has provided unique tools to monitor and quantify the NR translocation events. These technology developments have important implications in the mechanistic evaluation of NR signaling and provide a powerful tool for drug discovery. The unique challenges for developing a robust NR translocation assay include cytotoxicity accompanied with chronic overexpression of NRs, basal translocation induced by serum present in culture medium, and interference from endogenous NRs, as well as subcellular dynamics. The authors have developed a robust assay system for the glucocorticoid receptor (GR) that was applied to a panel of nuclear receptor ligands. Using a high-content imaging system, ligand-induced, dose-dependent GR nuclear translocation was quantified and a correlation with other conventional assays established.
authors.affiliation: Bristol-Myers Squibb, Lead Discovery & Profiling, Wallingford, Connecticut 06492, USA. michele.agler@bms.com
authors.forename: Michele
authors.initials: M
authors.lastname: Agler
authors.forename: Margaret
authors.initials: M
authors.lastname: Prack
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Zhu
authors.forename: Janet
authors.initials: J
authors.lastname: Kolb
authors.forename: Kimberly
authors.initials: K
authors.lastname: Nowak
authors.forename: Rolf
authors.initials: R
authors.lastname: Ryseck
authors.forename: Ding
authors.initials: D
authors.lastname: Shen
authors.forename: Mary Ellen
authors.initials: ME
authors.lastname: Cvijic
authors.forename: John
authors.initials: J
authors.lastname: Somerville
authors.forename: Steve
authors.initials: S
authors.lastname: Nadler
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D016227:Benzoquinones / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D019556:COS Cells; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003907:Dexamethasone / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D020239:Receptor Cross-Talk / Q000187:drug effects; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D003907:Dexamethasone; C001277:geldanamycin