Development of a flow cytometric assay to study glucocorticoid receptor-mediated gene activation in living cells.
A flow cytometry-based reporter gene assay was developed and utilized to measure glucocorticoid receptor (GR)-mediated gene activation at the single cell level in living cells. A reporter gene was generated that contains two copies of the glucocorticoid response element and an E1b TATA box upstream of a destabilized enhanced green fluorescent protein. Glucocorticoid activation of the reporter gene in Cos-1 and HTC cell lines was measured in vivo by flow cytometry and was shown to be dose dependent, leading to an increase in total fluorescence of the cell population. Flow cytometric analysis indicated this increase in total fluorescence per sample resulted from an increase in the number of cells expressing the activated green fluorescent protein (GFP) reporter as well as an overall increase in the mean GFP fluorescence within cells. Activation of reporter gene activity was time dependent occurring as early as 1-2h after dexamethasone addition. Activation of the reporter gene was specific as it exhibited different sensitivities to a range of glucocorticoids and activation could be blocked with glucocorticoid receptor antagonists. Coexpression of the coactivator SRC-1a or P65 subunit of NF-kappa B with GR led to enhancement or repression, respectively. Taken together, these data suggest the reporter-based flow cytometry assay is an effective method for analyzing glucocorticoid receptor-mediated gene expression at the single cell level in living cells.
Necela
Brian M
BM
Department of Health and Human Services, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Building 101, MD F3-07, Research Triangle Park, NC 27709, USA.
Cidlowski
John A
JA
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006727:Hormone Antagonists / Q000494:pharmacology; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020218:Response Elements / Q000235:genetics; D013997:Time Factors; D015534:Trans-Activators / Q000494:pharmacology; D051996:Transcription Factor RelA; D015533:Transcriptional Activation; D014162:Transfection
D005938:Glucocorticoids; D006727:Hormone Antagonists; D008164:Luminescent Proteins; D016328:NF-kappa B; D011965:Receptors, Glucocorticoid; D015534:Trans-Activators; D051996:Transcription Factor RelA; D049452:Green Fluorescent Proteins; D003907:Dexamethasone