High levels of non-activated receptors in glucocorticoid-sensitive S49wt mouse lymphoma cells incubated with dexamethasone.

Upon agonist binding the heteromeric glucocorticoid receptor complex undergoes a conformational change (receptor activation). This event involves the dissociation of a dimer of 90 kDa heat shock proteins. Whereas receptor activation in cytosolic assays is both rapid and irreversible, less is known about the receptor activation and translocation in intact cells during challenge with an agonist. In this paper we report on the receptor status of glucocorticoid-sensitive murine S49 lymphoma cells during dexamethasone exposure. By three different assays, ligand (re)binding, nuclear translocation and hsp90 co-immunoprecipitation, it was found that the majority of the glucocorticoid receptor protein was in a non-activated conformation. Furthermore, prolonged exposure to dexamethasone did not result in increased levels of activated receptors. By assessing receptor activation in situ we found that physiological temperature was less effective in dissociating hsp90 compared to room temperature. These findings indicate that the physiological temperature negatively controls receptor activation, probably due to a thermolabile interaction between the hormone and its cognate receptor.

Temperature dependence of glucocorticoid binding in sensitive and refractory murine leukaemia cells.

The validity of in vitro assays in predicting the susceptibility of leukaemic cells to glucocorticoid-mediated lysis was evaluated in a panel of six murine leukaemia cell lines. In this panel susceptibility to glucocorticoids ranged from highly sensitive to fully resistant. The panel was screened for specific 3H-dexamethasone binding in whole cells and for activation of cytosolic receptors in cell lysates. Specific binding of 3H-dexamethasone was strongly affected by the incubation temperature. In all cell lines, rapid and reversible changes were observed in the stability of agonist-receptor association with a transition temperature of 28 degrees C. Below this temperature, intracellular receptors were found to be in a stable-binding, high-affinity configuration, masking differences in receptor status among the various cell lines. When assayed at 37 degrees C, refractory and fully resistant cells revealed nonsaturating, low-affinity binding of steroid. Saturating, high-affinity binding was, however, restored in these cells by the drug meta-iodobenzylguanidine with concomitant sensitization to dexamethasone-induced lysis. Contrary to observations with intact cells, heat-induced agonist-receptor dissociation in cytosols caused irreversible loss of (re)binding capacity. Activation of cytosolic receptors only recognized fully resistant cell lines as being deficient in the transformation of liganded receptors into a DNA-binding configuration. The assay, however, could not discriminate between three cell lines with highest but varying degrees of sensitivity because of maximal activation. The results indicate that non-physiological temperature and cell disruption strongly and differentially affect steroid binding and receptor activation, respectively. The observations may account for the poor correlation between conventional predictive assays and steroid responsiveness in clinical leukaemia.