IDENTIFICATION OF HUMAN-LEUKEMIC GLUCOCORTICOID RECEPTORS USING AFFINITY LABELING AND ANTI-HUMAN GLUCOCORTICOID RECEPTOR ANTIBODIES

  • 1 January 1984
    • journal article
    • research article
    • Vol. 44  (10) , 4540-4547
Abstract
Antisera raised against human lymphoid glucocorticoid receptors were used in combination with the glucocorticoid receptor affinity label [3H]dexamethasone 21-mesylate ([3H]DM) to identify the glucocorticoid receptors of the human B-lymphoblastoid cell line IM-9 and the human T-cell leukemic cell line CEM-C7. Antisera were obtained following immunization of New Zealand White rabbits with [3H]triamcinolone acetonide ([3H]TA)-glucocorticoid receptor complexes partially purified by 2-stage DNA-cellulose chromatography. The presence of anti-human glucocorticoid receptor antibodies was verified by: adsorption of [3H]TA-receptor-antibody complexes to Protein A; a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [3H]TA-receptor complexes incubated with immune serum; and the ability of immune serum to displace [3H]TA-receptor complexes on sucrose gradients. These antibodies also recognized rat liver and murine S49 cell glucocorticoid receptors. Sodium dodecyl sulfate[SDS]-polyacrylamide gel electrophoresis of [3H]DM-labeled IM-9 cytosol identified a major competable band with a MW of .apprx. 90,000, 3 minor competable components with MW of .apprx. 78,000, .apprx. 51,000 and .apprx. 38,500 and at least 21 other noncompetable components. Following immunoprecipitation of [3H]DM-labeled cytosol with immune serum, only the 90,000 and 78,000 MW components were seen. SDS-polyacrylamide gel electrophoresis of [3H]DM-labeled CEM-C7 cytosol revealed a larger number of [3H]DM-labeled components. After immunoprecipitation of [3H]DM-labeled CEM-C7 cytosol, a predominant competable component with a MW of 90,000 was easily identified. This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used. The combination of affinity labeling and anti-human glucocorticoid receptor antibodies evidently is capable of providing direct physical identification of human lymphoid glucocorticoid receptors [Implications with respect to the use of glucocorticoids as chemotherapeutic agents are presented.].

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