Interactions of the Phenylpyrazolo Steroid Cortivazol with Glucocorticoid Receptors in Steroid-Sensitive and -Resistant Human Leukemic Cells1
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[CANCER RESEARCH (SUPPL.) 49, 2253s-2258s, April 15, 1989) Interactions of the Phenylpyrazolo Steroid Cortivazol with Glucocorticoid Receptors in Steroid-sensitive and -resistant Human Leukemic Cells1 E. Brad Thompson,2 Deepak Srivastava, and Betty H. Johnson Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77550 Abstract One of these kinetic sites has an affinity similar to that of dexamethasone, while the other is considerably higher in affin The interactions of glucocorticoids with their receptors somehow de ity (14). Systematic insertional mutagenesis of the human GR termine the cellular responses seen. The high potency glucocorticoid gene followed by tests of ligand binding in an expression system cortivazol differs from the usual glucocorticoids in two ways, structurally have shown that many amino acids participate in the steroid and in binding to receptors. Cortivazol contains a phenylpyrazol fused at carbon atoms 2 and 3 to the A ring of the cyclophenathrene, replacing binding site (7). Covalent binding of two different glucocorti the supposedly essential 3-keto,4,5-double bond pattern of glucocorti coid ligands resulted in attachment at differing amino acids (15, coids. Cortivazol binds to the glucocorticoid receptor in the cytosol from 16). Thus the ligand binding site is complex, and how gluco (KM C7 cells (a human acute lymphoblastic leukemia line) in a fashion corticoids interact with it as agonists or antagonists is still consistent with interaction with at least two sites. Standard glucocorti unclear. coids show only one-site binding. In mutant leukemia cells derived from Since cortivazol shows binding kinetics consistent with two CEM C7, resistant to kill by 10"* M dexamethasone and deficient in or more sites, we have carried out studies to determine whether standard glucocorticoid binding sites, cortivazol still finds a binding site these sites are all on GRs or whether other, distinctive, cortiv and kills the cells. In wild-type leukemia cells, the binding sites of cortivazol, those with both higher (Kt ~5 x 10~'°M)and lower (Kt ~1 X azol specific receptors are involved as well. We have shown that Id"") affinity appear to be on forms of the glucocorticoid receptor itself, dexamethasone mesylate in CEM cells covalently binds to the and not on two different classes of molecules. GR (17). This compound acts, in these and other cells, as a strong antagonist, with weak agonist activity at very high con centrations (18). RU38486 has been extensively documented as Introduction being a strong antiglucocorticoid, with high affinity binding for the GR, including that in lymphoid cells (19-21). The use of The interaction of steroid hormones with their receptors is these two antiglucocorticoids, dexamethasone mesylate and an essential but poorly understood step in their action. In RU38486, has helped to determine whether the high affinity general, the more tightly a steroid ligand binds, the greater is cortivazol site is indeed on the same molecule identified as the its potency in provoking specific cellular responses. As with classical GR. many other steroid hormone receptors, binding kinetics sug gests that for most ligands, glucocorticoid receptors are of a single type. Thus, in most cells a single straight line Scatchard Materials and Methods plot is seen when binding studies are carried out with commonly Cells. Cells of the CEM line cloned and characterized previously used radiolabeled ligands such as cortisol, dexamethasone, or were used in these studies. The properties of these clones have been triamcinolone. The interpretation of this result has been that there is but one type of GR3 molecule and that each GR contains described. They are CEM C7, ICR 27 (receptor deficient mutant of CEM C7), and CEM Cl (22-24). a single binding site for ligand (1). This point of view has been Receptor Binding Assays. Cytosolic receptor binding assays were reinforced by the recent cloning of the receptor and prediction carried out utilizing trinateli dexamethasone or tritiated cortivazol. The of its primary 777 (for the human) amino acid sequence (2-8). experimental procedures used have been described (14). That data and subsequent mutational analysis of the receptor Steroids. Dexamethasone and tritiated dexamethasone and dexa gene (2-8) all fit with the general view that there is but one methasone mesylate were obtained from standard commercial sources. type of glucocorticoid receptor with respect to steroid binding Cortivazol and deacylcortivazol were supplied through the kind offices of J. P. Raynaud, Roussel-UCLAF, Paris, France. Tritiated cortivazol site. Somatic cell genetic analysis in lymphoid cells, selecting was prepared for us by Amersham. The cortivazol and tritiated cortiv for resistance to the lethal effects of glucocorticoids, further azol have been analyzed by us using high performance liquid chroma- supports the one receptor-one site point of view (9-12). tography and were shown to be »99%pure. Certain recent data, however, have complicated this simple Velocity sedimentation analysis was carried out on 5-20% sucrose model. We find that the extremely potent glucocorticoid (Ila, 17/3,21-trihydroxy-6-16a-dimethyl-2'-phenyl-2'-//-preg- gradients as described previously (25). Inhibition of CEM cell growth in mass culture by dexamethasone na-2,4,6-trieno[3,2-c]pyrazol-20-one 21 acetate) known as cor and dexamethasone mesylate was carried out as follows. CEM-C7 cells tivazol (13), a synthetic glucocorticoid with the unusual feature were cultured in RPMI 1640 plus 5% or 10% fetal bovine serum in of a phenylpyrazolo moiety fused to the A ring of the cyclo- humidified 5% CU2-95% air incubators at 37°C.Duplicate assays were conducted in 25-cm2 tissue culture flasks or 6-well tissue culture plates phenanthrene nucleus, shows a complex binding kinetics. When with initial cultures of 2-3 ml at approximately 1 x 10' cells/ml. No equilibrium binding analysis is carried out in cytosols from the more than 1% ethanol was added to controls or to treated cells as line of leukemic lymphoid cells known as CEM C7, cortivazol steroid carrier. On day 4 or 5 hemacytometer counts were conducted binding is consistent with two or more sites being occupied. using trypan blue dye exclusion to determine viability. ' Presented at the Symposium on "Glucocorticoid Receptors: Evolution, Struc ture, Function and Abnormalities," July 14 and IS, 1988, Osaka, Japan. This work was supported in part by National Institutes of Health Grant Results and Discussion CA41407 and the Walls Medical Research Foundation. 2To whom requests for reprints should be addressed, at Rt. F45, Room 601, Effects of Cortivazol on CEM Cells. CEM cells originally Basic Science Building. University of Texas Medical Branch. Galveston, TX 77550-2779. were grown from the blood of a child with acute lymphoblastic 3The abbreviation used is: GR, glucocorticoid receptor. leukemia. From the uncloned line we separated the highly 2253s Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. INTERACTION OF CORTIVAZOL WITH GRs IN LEUKEMIC CELLS 5.0r committed to lyse. From CEM C7 we selected by growth in 10~6 M dexamethasone, a number of glucocorticoid resistant subclones, some of which arose spontaneously and others after chemical mutagenesis (23). All these selected, resistant clones appear to be steroid receptor mutants of one type or another. (We have found no binding sites for progestins, androgens, or estrogens in CEM C7 cells.) The majority of cells isolated from the uncloned CEM cells were glucocorticoid sensitive like C7, but one clone, CEM Cl, was resistant to the lytic and growth inhibitory effects of glucocorticoids. These cells differed from 2x10-'° 5x10-'° 10-3 2x10-3 5x10-9 10-8 2x10-8 5x10-8 1<H 2x10-' 5x10-' 10-« the dexamethasone selected resistant clones by having appar STEROID CONC IM) ently normal glucocorticoid receptors and glutamine synthetase induction, but no c-myc or growth inhibition by high dose Fig. 1. Cultures of CEM-C7 were incubated with various concentrations of dexamethasone (24, 26, and "Note Added in Proof). dexamelhasone or deacylcortivazol for 18 h and then assayed for glutamine synthetase (activity from Ref. 29 with permission). Addition of cortivazol to the various types of dexamethasone resistant CEM cells produced surprising results. To describe these meaningfully, further details of the properties of the resistant cells first are necessary. The dexamethasone selected resistant clones divide into 2 major groups. The first is char acterized by having some residual glucocorticoid binding sites, with affinity for dexamethasone similar to that of wild type, but with a steroid binding lesion such that the receptors lose all ligand when cytosols containing steroid receptor complexes are warmed to 20°Cor greater. As a consequence, the receptor cannot alter its structure to the DNA binding form necessary to provoke further cellular activity. These mutants therefore show the essential participation of the steroid in that process. They are referred to as "activation labile" mutants. All activa tion labile mutants arose spontaneously from CEM C7. Mem bers of the second group all arose after chemical mutagenesis, followed by selection in dexamethasone. Clones of this group showed greatly reduced levels of dexamethasone binding, usu ally to <10% of wild-type (23). Thus these clones are referred to as "receptor deficient." The single clone comprising a third class of resistance is CEM Cl, mentioned above. It has an apparently normal receptor but is not growth inhibited by dexamethasone and is therefore described as "lysis defective." KT" 6 x 10"" 2 x 10"" 10"* 5 x 10"" When cortivazol was applied to clones from all three classes 2 x 10•¿"10' 5 x 10"' 2 x 10-" 10' of dexamethasone resistant cells, strong growth inhibition was STEROID CONC (M) observed (30). In the case of the activation labile and receptor Fig.