An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells

Scott H. Kaufmann, Fauzia F. Quddus, Joel H. Shaper

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

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 hypo tonic 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.(Endocrinology 110: 708, 1982).

Original languageEnglish (US)
Pages (from-to)708-716
Number of pages9
JournalEndocrinology
Volume110
Issue number3
DOIs
StatePublished - Mar 1982

ASJC Scopus subject areas

  • Endocrinology

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