Characterization of Cytosolic and Nuclear Glucocorticoid-binding Components in Human Leukemic Lymphocytes¹

Nuclear binding and the physicochemical characteristics of a glucocorticoid-binding component were studied in glucocorticoid-responsive lymphocytes from patients with chronic lymphatic leukemia (CLL). As previously reported from this laboratory for corticoid-sensitive mouse P1798 tumor lymphocytes, 4 mm MgCl₂ and 1 mm spermidine released about 60% of [³H]triamcinolone acetonide ([³H]TA) from nuclei isolated from CLL cells that had been incubated with the radioactive steroid at 37°. Ten mm spermidine extracted 90% of [³H]TA from CLL nuclei and in this respect were as effective as was 0.6 m KCl. Agarose gel filtration and glycerol density gradient centrifugation of 0.6 m KCl, 10 mm spermidine, or 4.5 mm MgCl₂ extracts of [³H]TA-labeled CLL nuclei revealed that most of the radioactivity in the extract was in the form of a large, asymmetrical macromolecule complex when the protease inhibitors, carbobenzoxy-l-phenylalanine or leupeptin, were included in the extraction buffer. Leupeptin was usually more effective than carbobenzoxy-l-phenylalanine and was therefore the protease inhibitor of choice. The [³H]TA complex had an apparent molecular weight of 90,000 to 96,000, a sedimentation coefficient of 3.7S, and a Stokes radius of 56 to 60 Å, corresponding to a frictional ratio of 1.8 and an axial ratio (prolate ellipsoid) of 15. When protection against cleavage was inadequate, a variable amount of 35 Å material was detected by agarose gel filtration, associated with decreased recovery of the 56 to 60 Å complex.

Cytosolic [³H]TA binding components isolated from CLL lymphocytes incubated with [³H]TA at 4° were indistinguishable by agarose gel filtration and glycerol density gradient centrifugation from nuclear-associated [³H]TA macromolecule complexes in cells that were labeled at 37°.

The Stokes radius and sedimentation coefficient of nuclear glucocorticoid-binding components from cells labeled with [³H]dexamethasone were identical to the values characteristic of nuclear-associated [³H]TA-macromolecule complexes. This observation, together with the results of Scatchard analysis of nuclear binding data and of competition experiments, suggested that [³H]TA and [³H]dexamethasone interacted with the same class(es) of binding sites in CLL lymphocytes. However, [³H]dexamethasone complexes appeared to be less stable than [³H]TA complexes, as indicated by the considerable dissociation of [³H]dexamethasone but not of [³H]TA during agarose gel filtration and glycerol density gradient centrifugation.

Our results raise the possibility that a large, asymmetrical glucocorticoid-binding component that can be readily extracted from nuclei with millimolar concentrations of divalent or polyvalent cations may be required for steroid action on human malignant lymphocytes.