Differences in Nuclear Glucocorticoid Binding Between Corticoid-Sensitive and Corticoid-Resistant Lymphocytes of Mouse Lymphoma P1798 and Stabilization of Nuclear Hormone Receptor Complexes With Carbobenzoxy-L-phenylalanine2
Nuclear glucocorticoid binding was compared in the corticoid-sensitive (CS) and corticoid-resistant (CR) strains of mouse lymphoma P1798. Purified nuclei were isolated from tumor cells that had been incubated with [3H]triamcinolone acetonide (TA) for 60 minutes at 37° C. A 0.6-M solution of KCI extracted 80–90% [3H]TA from both CS and CR nuclei, but only 15–20% of [3H]sterold in the salt extract was bound to macromoleculeS as judged by gel filtration (Sephadex G-25). Breakdown of hormone receptor complexes could be prevented by the inclusion of the chymotrypsin inhibitor carbobenzoxy-L-phenylalanine In the 0.8-M KCI extraction buffer. Under these conditions, 70% of [3H]TA extracted from CS nuclei and 80% of [3H]TA extracted from CR nuclei were recovered in the void volume of the Sephadex columns, correspondlng to 6,100±1,560 molecules of [3H]TA per CS nucleus and 3,900±650 molecule. of [3H]TA per CR nucleus. Low concentrations of monovalent (KCI), divalent (Mg2+ or Ca2+), or polyvalent (spermidine) cations consistently extracted a higher percentage of [3H]TA from CS than from CR nuclei, which suggested that interactions of [3H]TA with the nucleus might have been weaker in the sensitive cells. The effects of Mg2+ and spermidine on release of [3H]TA from CS nuclei were neither additive nor temperature-dependent. Furthermore, the magnitude of release was not influenced by the concentration of [3H]TA (1±30 nM) to which the cells were exposed nor by the length of time (10±80 min) they were incubated with the hormone. Experiments in which mixtures of unlabeled CS and labeled CR nuclei (or vice versa) were extracted with 4 mM MgCI2 suggested that CS nuclei did not contain diffusable Mg2+ -dependent [3H)TA-releasing activity nor CR nuclei, an inhibitor of release. Elucidation of the biochemical basis for the differences in nuclear glucocorticoid binding between CS and CR P1798 lymphocytes may shed some light on the mechanism of resistance of the CR cells to glucocorticoid administration