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Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line,BCL1
Authors:Mary F Ruh  Raj K Singh  Clifford J Bellone  Thomas S Ruh
Institution:1. Department of Physiology, St. Louis University School of Medicine, St. Louis, MO 63104, U.S.A.;2. Department of Microbiology, St. Louis University School of Medicine, St. Louis, MO 63104, U.S.A.
Abstract:The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. 3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAF-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated 3H]TA-receptor complexes to chromatin-cellulose extracted with 0–8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocortical receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of 3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6–8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.
Keywords:Triamcinolone acetonide  Glucocorticoid  Steroid receptor  Chromatin binding  (Leukemia cell)  B-cell leukemia line 1  TA  triamcinolone acetonide  Tes
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