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Characterization of Glucocorticoid-specific Binding in the L₂C Leukemia¹

Departments of Physiology and Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104

Glucocorticoid-specific binding macromolecules were measured and characterized in L₂C cells, a B-lymphocyte guinea pig leukemia that is resistant to administered cortisol or adrenocorticotrophic hormone. L₂C cells were harvested by cardiac puncture followed by Ficoll:Hypaque separation techniques. The cells were lysed by sonication, and the cytosol was obtained after centrifugation at 106,000 x g for 60 min at 0°. Cytosol glucocorticoid receptor measurements were obtained by hydroxylapatite assay or column chromatography using Sephadex G-25. Maximal specifically bound [³H]triamcinolone acetonide in the cytosol fraction was 300 fmol/10⁸ cells. A Scatchard plot of specific L₂C cytosol binding gave a K_d of 18 nM and an estimate of 2000 binding sites/cell. The specificity of binding in L₂C cytosol was triamcinolone acetonide > dexamethasone > cortisol > progesterone > testosterone = estradiol. Binding of [³H]triamcinolone acetonide to cytosol glucocorticoid receptors was maximal at 22 hr of incubation at 0°, and the receptor complex was stable for 48 hr. The receptor complex was not affected by DNase or RNase, but the receptor complex was lost with pronase or heat denaturation. Whole-cell binding assays were also performed, which resulted in similar quantities of maximally bound glucocorticoid receptor as well as the specificity of binding of various hormone analogs as found in the cytosol receptor assays. Transferring the whole cells from 0 to 22° resulted in the appearance in nuclei of approximately 65% bound receptors. However, these translocated receptor complexes do not appear to affect the viability of the L₂C cells as measured by trypan blue exclusion.

¹ Supported by NIH Grant CA 24034 and the McBride-Love Foundation.

² To whom requests for reprints should be addressed.

Received 2/25/80. Accepted 10/ 6/80.