An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells.
We have developed a rapid and convenient method for the quantitative assessment of nuclear translocation of glucocorticoid-receptor complexes in lymphoid cells. In this assay, cells are incubated with [3H]dexamethasone at 4 C or 21 C, treated for 15 min at 4 C with the sulfhydryl blocking reagent sodium tetrathionate or methyl methanethiolsulfonate, and then ruptured with the neutral detergent Triton X-100 to separate the nuclei from the cytoplasmic constituents. When cells are incubated with [3H]dexamethasone at 21 C, an average of 45-60% of the specifically bound cellular radiolabel remains with the nuclei. In contrast, less than 15% of the hormone remains with the nuclei when cells are labeled at 4 C. In rat thymocytes, quantitatively similar results were obtained using this new method and a conventional method of isolating nuclei (homogenization of cells in a hypotonic buffer). For both methods, preincubation of the labeled cells with the sulfhydryl blocking reagents was essential to prevent release of the nuclear hormone (or hormone-receptor complex) by a sulfhydryl-dependent labilizing activity apparently located in the cytoplasm. The new method has been successfully used to quantitate uptake of nuclear [3H]dexamethasone-receptor complexes in rat thymocytes, human lymphocytes, and human leukemia cells of lymphoid and myeloid origin. It should prove successful for separating nuclear from cytoplasmic glucocorticoid-receptor complexes in a wide variety of cells, many of which resist disruption by more conventional techniques.
D000315:Adrenalectomy; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008722:Methods; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism; D011987:Receptors, Steroid / Q000302:isolation & purification*; D013950:Thymus Gland / Q000378:metabolism
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone