Biochemical evidence that glucocorticoid-sensitive cell lines derived from the human leukemic cell line CCRF-CEM express a normal and a mutant glucocorticoid receptor gene.
To characterize the immunoreactive glucocorticoid receptor (GR) protein present in "receptorless" (r-) mutants isolated from the glucocorticoid-sensitive (dexs) human leukemic cell line CEM-C7, binding of [3H]dexamethasone was determined in extracts prepared from the sensitive cell line 6TG1.1 and the r- mutant ICR27TK.3 after gentle freeze-thaw lysis and low-speed centrifugation. Under these conditions there was significant high-affinity binding activity in r- extracts assayed at 4 degrees C but not at 23 degrees C. Loss of binding at 23 degrees C was not a function of GR proteolysis or denaturation of the steroid-binding site and could be prevented by the addition of sodium molybdate. Dissociation of ligand from either activated or unactivated receptors in r- extracts was significantly more rapid than from receptors in extracts prepared from normal cells, suggesting that the defect in receptors in r- cells is the result of mutation in the ligand-binding site. While the rate of dissociation from unactivated receptors in r- extracts was linear, dissociation from receptors in extracts of 6TG1.1 cells was biphasic. Analysis of these dissociation curves, as well as dissociation from receptors in the B-cell line IM-9, indicated that the mutant gene present in r- cells is also present in the dexs parental cell line. This conclusion is consistent with our previous hypothesis (J.M. Harmon et al., Mol. Endocrinol., 3:734-743, 1989) that glucocorticoid-sensitive CCRF-CEM cells express both a normal (GR+) and a mutant (GR*) allele.
Palmer
L A
LA
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Harmon
J M
JM
D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D003907:Dexamethasone / Q000493:pharmacokinetics; D006801:Humans; D007938:Leukemia / Q000235:genetics* / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism; D009154:Mutation; D011110:Polymorphism, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013696:Temperature; D013997:Time Factors
D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D008982:Molybdenum; C024687:sodium molybdate(VI)