CHARACTERIZATION OF GLUCOCORTICOID-SPECIFIC BINDING IN THE L2C LEUKEMIA

  • 1 January 1981
    • journal article
    • research article
    • Vol. 41  (1) , 138-143
Abstract
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. 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 f[femto]mol/108 cells. A Scatchard plot of specific L2C cytosol binding gave a Kd of 18 nM and an estimate of 2000 binding sites/cell. The specificity of binding in L2C cytosol was triamcinolone acetone > dexamethasone > cortisol > progesterone > testosterone = estradiol. Binding of [3H]triamcinolone acetonide to cytosol glucocorticoid receptors was maximal at 22 h of incubation at 10.degree. and the receptor complex was stable for 48 h. 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 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 0-22.degree. C resulted in the appearance in nuclei of .apprx. 65% bound receptors. These translocated receptor complexes do not appear to affect the viability of the L2C cells as measured by trypan blue exclusion.