Flow Cytometric Analysis of Glucocorticoid Receptor Using Monoclonal Antibody and Fluoresceinated Ligand Probes

Conditions were established for single cell analysis of glucocorticoid receptor (GR) content by flow cytometry using several clones of a human leukemic cell line (CCRF-CEM). These included CEM-7A, 7R, Cl, and ICR 27 TkJ cells which were examined both by standard [3H|dexameth-asone radiometric binding and by two independent flow cytometry assays. The latter involved either mouse monoclonal antibody against GR (GR-MoAb) or fluoresceinated cortisol lipnd probes. For CEM-7A, 7R, and Cl cells, there was a correlation between GR-MoAb and radiometrically defined GR values. However, clone ICR-27 Tk3 with low [3H]dexameth-asone binding exhibited the highest GR-MoAb fluorescence. The flu-oresceinated cortisol assay correlated with dexamethasone binding values in all four clones. Thus, GR-MoAb identifies the total immunologically reactive GR present, while the fluoresceinated cortisol assay quantifies only the functionally intact GR in terms of its initial binding. Their combined use may reveal the cellular heterogeneity of GR expression and function also in human tumor samples, to which they have been successfully applied. When coupled with DNA counterstaining, GR expression can be related directly to frequently DNA-aneuploid tumor cells and cell cycle distribution.