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Biochemical Evidence That Glucocorticoid-sensitive Cell Lines Derived from the Human Leukemic Cell Line CCRF-CEM Express a Normal and a Mutant Glucocorticoid Receptor Gene¹

Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

To characterize the immunoreactive glucocorticoid receptor (GR) protein present in "receptorless" (r^-) mutants isolated from the glucocorticoid-sensitive (dex^s) 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°C but not at 23°C. Loss of binding at 23°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 dex^s 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.

¹ This investigation was supported by USPHS Grant CA32226 awarded by the National Cancer Institute.

² Present address: Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892.

³ To whom requests for reprints should be addressed, at Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799.

Received 2/14/91. Accepted 7/19/91.

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