Identification of Human Leukemic Glucocorticoid Receptors Using Affinity Labeling and Anti-Human Glucocorticoid Receptor Antibodies

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-lymphoWastoid 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 [3Htriamcinolone acetonide ([3H]TA)-glucocorticoid receptor complexes partially purified by two-stage DNA-cellulose chromatography. The presence of anti-human glucocorticoid receptor antibodies was verified by: (a) adsorption of [3H]TA-receptor-antibody complexes to Protein A; (b) a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [3H]TA-receptor complexes incubated with immune serum; and (c) 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-polyacrylamide gel electrophoresis of [3H]DM-labeled IM-9 cytosol identified a major competable band with a molecular weight of -90, 000, three minor competable components with molecular weights of ∼78, 000, ∼51, 000, and ∼38, 500, and at least 21 other noncom-petable components. Following immunoprecipitation of [3H]DM-labeled cytosol with immune serum, only the Mr 90, 000 and 78, 000 components were seen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled CEM-C7 cytosol revealed a larger number of [3H]DM-labeled components. However, after immunoprecipitation of [inf>3H]DM-labeled CEM-C7 cytosol, a predominant competable component with a molecular weight of 90, 000 was easily identified. This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used. Thus, the combination of affinity labeling and anti-human glucocorticoid receptor antibodies is capable of providing direct physical identification of human lymphoid glucocorticoid receptors. © 1984, American Association for Cancer Research. All rights reserved.