Use of [3H]cortivazol to characterize glucocorticoid receptors in a dexamethasone-resistant human leukemic cell line.
ICR 27 is a mutant derived from the glucocorticoid-sensitive human leukemic cell line CEM C7 that has been characterized as glucocorticoid receptor negative based on its ability to specifically bind [3H]dexamethasone ([3H]DEX). We used the pyrazolosteroid [3H]cortivazol ([3H]CVZ) to determine whether ICR 27 cells actually contain glucocorticoid receptors and, if so, whether these receptors can mediate physiological effects. Scatchard analysis of the binding of [3H]CVZ to cytosol from ICR 27 cells was consistent with a single class of receptors of uniform affinity (0.7 nM). Cytosolic [3H]CVZ complexes had a sedimentation coefficient of 4.6S on linear sucrose gradients and eluted from DEAE-cellulose columns at a potassium phosphate concentration of 250 mM. CVZ also competed with [3H]DEX mesylate for binding to a 96,000 mol wt protein. Incubation of ICR 27 cells with CVZ caused 50% growth inhibition and 50% maximal induction of glutamine synthetase activity at concentrations of 20 and 35 nM, respectively. Elution profiles of [3H]CVZ complexes from DEAE-cellulose columns showed that complexes formed upon thermal activation were relatively unstable, and little or no increase in binding of [3H]CVZ-receptor complexes to DNA-cellulose was observed. Thus, [3H]CVZ identifies functional glucocorticoid receptors in a cell line previously described as DEX resistant. Although the binding of [3H]CVZ to activated receptors in vitro appears unstable, high concentrations of CVZ may facilitate stabilization of activated complexes that can mediate both anabolic and catabolic effects.
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004351:Drug Resistance; D004790:Enzyme Induction / Q000187:drug effects; D004963:Estrenes / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis; D012739:Gonadal Steroid Hormones / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D015735:Mifepristone; D009363:Neoplasm Proteins / Q000096:biosynthesis; D011281:Pregnatrienes / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000032:analysis*; D013601:T-Lymphocytes / Q000032:analysis
D004963:Estrenes; D012739:Gonadal Steroid Hormones; D009363:Neoplasm Proteins; D011281:Pregnatrienes; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D003907:Dexamethasone; D005974:Glutamate-Ammonia Ligase; C005924:cortivazol