Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.
Anti-peptide antibody to the human glucocorticoid receptor (GR) was produced and used to demonstrate that a subset of the GR population resides in the plasma membrane of human leukemic cells. Characterization of the antibody with intracellular GR (iGR) showed its ability to shift [3H]triamcinolone acetonide-labeled GR (4S protein) from two human leukemic cell lines to a higher density in sucrose gradients; Western and autoradiographic analysis of affinity-labeled ([3H]dexamethasone 21-mesylate) receptor revealed an immunoreactive and competitively labeled band of 94 kDa. CCRF-CEM cell membrane GR (mGR) resolved as a > 7S protein on density gradients and immunoselected cell surface protein labeled by whole cell biotinylation or affinity-labeling with [3H]dexamethasone 21-mesylate was approximately 145 kDa, demonstrating that mGR was larger in size than iGR, as has been shown previously for the mGR of mouse lymphoma cells. Analysis of mGR in lymphocytes of leukemic patients and the CCRF-CEM cell line indicated differences in levels of expression as shown by FACS and immunocytochemical analyses. We are currently using this system to study the correlation between the quantity of membrane-resident GRs and the glucocorticoid-induced lytic response, a relationship previously shown in the murine (S-49 cell) system.
Gametchu
B
B
Department of Pediatrics, Medical College of Wisconsin, Milwaukee 53226.
Watson
C S
CS
Wu
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S
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001345:Autoradiography; D001667:Binding, Competitive; D015153:Blotting, Western; D002051:Burkitt Lymphoma / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011233:Precipitin Tests; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000276:immunology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D005938:Glucocorticoids; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid