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

Scott H Kaufmann, F. F. Quddus, J. H. Shaper

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 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.

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

Fingerprint

Glucocorticoid Receptors
Lymphocytes
Sulfhydryl Reagents
Hormones
Thymocytes
Cell Nucleus
Dexamethasone
Tetrathionic Acid
Lymphoid Leukemia
Myeloid Leukemia
Octoxynol
Myeloid Cells
Cytoplasmic and Nuclear Receptors
Detergents
Buffers
Cytoplasm

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells. / Kaufmann, Scott H; Quddus, F. F.; Shaper, J. H.

In: Endocrinology, Vol. 110, No. 3, 1982, p. 708-716.

Research output: Contribution to journalArticle

@article{d60e16957ddd45ea9acb2a02ecf64c49,
title = "An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells",
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 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.",
author = "Kaufmann, {Scott H} and Quddus, {F. F.} and Shaper, {J. H.}",
year = "1982",
language = "English (US)",
volume = "110",
pages = "708--716",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "3",

}

TY - JOUR

T1 - An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells

AU - Kaufmann, Scott H

AU - Quddus, F. F.

AU - Shaper, J. H.

PY - 1982

Y1 - 1982

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0020048043&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020048043&partnerID=8YFLogxK

M3 - Article

C2 - 7056227

AN - SCOPUS:0020048043

VL - 110

SP - 708

EP - 716

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 3

ER -