Interactions of the Phenylpyrazolo Steroid Cortivazol with Glucocorticoid Receptors in Steroid-Sensitive and -Resistant Human Leukemic Cells1

Home , Cortivazol

[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~'Â°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 Â»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 37Â°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

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-'Â° 5x10-'Â° 10-3 2x10-3 5x10-9 10-8 2x10-8 5x10-8 1

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 20Â°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 10â€¢¿"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. 2. CEM C7 Cells were plated in the presence of various concentrations deficient clones, one could argue that the cortivazol, by virtue of dexamethasone (â€¢)ordeacylcortivazol (A) and colonies which arose after 10- of tighter binding, was able to utilize the residual receptors 14 days were stained and counted (a). Alternatively, cells were exposed to the same concentrations of dexamethasone and deacylcortivazol for 48 h and then more effectively to induce cell kill. In the case of the CEM Cl washed free of steroid and plated in the absence of steroid (e). Reprinted from lysis defective cells, one could argue that the tighter association Ref. 29 with permission. of ligand with receptor allowed a response via a "leaky" lysis mechanism. However, an alternative explanation for all three glucocorticoid sensitive CEM C7 clone (22). The known re classes of dexamethasone resistant cells being sensitive to cor sponses to glucocorticoids in these cells can be arbitrarily tivazol would be that the phenylpyrazolo steroid acted via two divided into early and late. The early responses include c-myc distinct mechanisms, one using the standard glucocorticoid down-regulation (26), cell shrinkage (22), ornithine decarbox- receptor (lost in the receptor mutants) and the other taking a ylase down-regulation,4 and glutamine synthetase induction different pathway, still available in the receptor mutants. Some (27). All these occur within 12 h after applying hormone. The support for this possibility came from experiments in which late (>24 h) responses include inhibition of rRNA synthesis,5 resistance against cortivazol was selected for in the dexameth cessation of growth in the d phase of the cell cycle, increased asone resistant mutants. We found that in these experiments, DNase activity, and cell lysis (22, 28). Cortivazol and its dea- cortivazol resistance was achieved with little further loss of cylated form act as extremely potent agonists in CEM C7 cells, dexamethasone binding sites in the activation labile mutants, as inducers of both glutamine synthetase (early response) and suggesting that a different pathway might be involved (Table growth inhibition/cell death (late response). Figs. 1 and 2 show 1). These results prompted us to examine further the binding dose-response curves for each of these effects. The data indicate of cortivazol in CEM cells. that the cortivazol compounds are 20 to 50 times more potent Binding of Cortivazol to CEM Cells. Competitive binding than dexamethasone in these cells (29). They also show that studies using varied concentrations of cortivazol (or its equally after 48 h of continued exposure to steroids, the cells are potent deacetylated form) against a constant amount of [3H] ' B. Johnson, C. Townsend, and E. B. Thompson, unpublished observations. dexamethasone showed that on average the phenylprazolocor- " E. A. Thompson and E. B. Thompson, unpublished observations. ticoid was about 20 times more tightly bound than dexameth- 2254s

Table 1 Dexamethasone binding sites in clones of dexamethasone resistant CEM cells with activation labile glucocorticoid receptors, sensitive or resistant to deacylcortivazol Clones 3R7 and 4R4 are spontaneous mutants of wild-type CEM C7 cells. Both are resistant to all known effects of up to 10 '' M dexamethasone in these cells and have been shown to contain activation labile glucocorticoid receptors (31, 32). Both are growth inhibited by deacylcortivazol. Subclones R1-R5 from each were selected for growth in the face of 10~6M deacylcortivazol. Parental and BOUND/FREE subclones were assayedfor [3H]dexamethasone binding sites by standard compet itive binding assay. Data, rounded to nearest 100, from Ref. 30.

to binding CloneSubclone3R73R7R1R2R3R44R44R4R1R2R3R4R5ResponsedeacylcortivazolSensitiveResistantResistantResistantResistantSensitiveResistantResistantResistantResistantResistantDexamethasone(sites/cell)81008000740064007300340026003500220027003800

O.I 0.2 O.Ã• 0.4 0.5 0.6 0.8 0.9 BOUND(pmol/mg protein)

Fig. 4. Cortivazol binding (*) in CEM C7 cytosol. Scatchard plot. Residual binding after pretreatment of cytosol with dexamethasone mesylate (O).

When similar binding studies were carried out on a clone of the dexamethasone resistant, receptor deficient cells that never theless responded to cortivazol, binding was again seen, but only of that typical of a straight line, single site glucocorticoid kinetics with a KÂ¿ofapproximately 8 x 10~'Â°M,similar to the higher affinity site seen in wild type, CEM C7 cytosols (14, 33). The cytosol of the mutant cells shows little competible binding of dexamethasone, although competitive binding stud ies in the intact mutant cells can reveal binding of dexametha sone, up to about one-tenth of wild type, but too erratic in nature to permit reliable Scatchard plots for comparison with wild type. Various mixing, somatic cell hybridization, and receptor gene transfection experiments have failed to reveal any B/F inhibitor of dexamethasone binding in the mutant cells (9, 10)'':

.15 the defect they possess seems to reside entirely in the glucocor ticoid receptor. In fact, this conclusion was strongly reinforced by our recent experiments introducing the human glucocorti .10 coid receptor gene into the mutant cells by transfection. Such gene transfer restored cellular responses, both lytic and induc tive, in the mutant cells (34). Thus, steroid binding studies show .05- one dexamethasone, but two cortivazol sites (or negative cooperativity) in wild type CEM C7 cells, whereas in the mutant one finds virtually no dexamethasone sites, but a single intact .2 .3 .4 high affinity cortivazol site. Additional studies on the latter, by B (pm/mg protein) Schlechte and Schmidt (33), confirmed our report that cortiv azol can prevent growth of the receptor-deficient mutant cells Fig. 3. Dexamethasonebinding in CEM C7 cell cytosol. Scatchardplot. Inset, calculated K,

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. INTERACTION OF CORTIVAZOL WITH GRs IN LEUKEMIC CELLS tion of ligand the counts associated with the high affinity site receptor due to a mutation. The data also suggest that RU38486 might be obscured by the far greater amount of radioactivity on in high concentrations binds to the retained site. In work to be the lesser affinity site; so we have repeated the experiments at presented elsewhere, we have shown that these mutant cells 0.5 nM [3H]cortivazol a concentration likely to label almost contain full sized receptor mRNA in quantities only slightly exclusively the high affinity site. Although the total number of reduced from wild type and that they also contain levels of counts is low, a clear-cut competible peak is seen at approxi immunologically reactive receptor in excess of that predicted mately 4s (Fig. 5). Thus, both the higher and lower affinity by measurable dexamethasone binding sites (36, 37). These cortivazol sites run at the typical glucocorticoid receptor loca data favor the interpretation that the mutants contain an altered tion on these gradients. receptor, one capable of binding and retaining cortivazol and We also examined the behavior of the cortivazol-labeled of interacting specifically but weakly with RU38486 and dexa cytosol on DEAE-cellulose. This ion exchange agent can distin methasone. guish the unactivated from the activated glucocorticoid recep Dexamethasone mesylate is a covalent affinity ligand for tor. The unactivated steroid-receptor complex, that which can glucocorticoid receptors. In several cell systems it acts as a not bind DNA, Ã©lÃ»tesfromDEAE-cellulose when the salt in strong antagonist to dexamethasone (21, 38, 39), while at very the eluting buffer reaches about 0.2 M. The activated, DNA- high concentrations it behaves as a partial agonist (18, 38, 39). binding glucocorticoid-receptor complex is eluted by much We confirmed that these properties pertained in wild-type CEM lower salt, about 0.05-0.8 M (35). Cortivazol-labeled cytosols C7 cells. Fig. 7 shows that at lower concentrations dexameth asone mesylate blocks the cytolytic/growth inhibitory effects of of CEM C7 cells showed the same behavior (Fig. 6). dexamethasone in CEM C7 cells. The figure also shows that at Use of Antiglucocorticoids to Help Define the Cortivazol Bind ing Site(s). As mentioned above, a 100-fold excess of the strong very high concentrations, dexamethasone mesylate itself can produce growth inhibition. It is well known that dexamethasone antiglucocorticoid RU38486 blocked the growth inhibitory re binding is entirely blocked by dexamethasone mesylate and the sponse of a receptor deficient clone of CEM cells to cortivazol, mesylated compound labels immunologically identifiable glu suggesting that the single cortivazol binding site they retain is cocorticoid receptor. Dexamethasone mesylate labeled receptor on a mutated receptor or that they have lost one form of the also has been shown to bind specifically to the long terminal repeat of the mouse mammary tumor virus gene, the specific 80 n c/s-active regulatory site for glucocorticoid receptors (40, 41). â€¢¿Uncompeled Thus, there can be little doubt that dexamethasone mesylate o Competed binds to the authentic glucocorticoid receptor. We therefore B 60-1 studied the ability of this compound to interfere with cortivazol binding in CEM C7 cytosols. Cortivazol at a wide range of l concentrations, so as to label both the high and low affinity Ã¶40 sites, was added to the cytosol, with or without prior addition of dexamethasone mesylate. As Fig. 4 shows, the affinity ligand l blocked cortivazol binding virtually completely. The open cir O 20- cles in the figure represent the few residual [3H]cortivazol counts bound in dexamethasone mesylate treated cytosols. There are not sufficient specific counts remaining to allow a 0J reliable curve to be known. We conclude that no significant 20 40 60 cortivazol site remains in the dexamethasone mesylate treated Fraction Number (1 = Top) cytosol. Fig. 5. Velocity sedimentation gradient analysis of high affinity cortivazol Because dexamethasone mesylate can bind to other proteins receptor in CEM C7 cells. The peak of competible radioactivity is calculated to be 4s, by comparison with the location of a bovine serum albumin standard, run containing available thiol groups in addition to the glucocorti on a parallel gradient (not shown). coid receptor, the possibility remained that the block of cortiv-

CVZ-LABELLED CEM CELL PROTEINS CVZ-LABELLED CEM CELL PROTEINS ON DEAE ON DEAE Unactivated Activated 5000-

Fig. 6. DEAE-cellulosechromatogramsde- 40OO 4000- picting unactivated and activated |'H]cortivazol receptor complexes of C7 cytosol. Cytosol was incubated with 10 nM [3H]cortivazol. After activation (30 min at 25Â°C),10 mM sodium Â¿ 30OO 3 molybdate was added, free steroid was re moved by dextran coated charcoal, and bound radioactivity was eluted with a 5-400 mM po tassium phosphate (KP) gradient ( ). Reprinted from Ref. 14 with permission.

1000-

10 15 20 10 15 20 Fraction Number Fraction Number 2256s

quite different structures and modes of binding. Dexametha sone itself did not seem to compete for the higher affinity D Dexamethasone â€¢¿DexamethasoneMesylate cortivazol site (14). The data also strongly suggest that the 75%25%Lethal D Both higher potency of cortivazol is due to its binding with high affinity to a glucocorticoid receptor site not occupied or occu pied with lower affinity by dexamethasone. Is this site on the same receptor molecule as the lower affinity site, or is there a second class of glucocorticoid receptor molecules in CEM C7 cells, not capable of binding dexamethasone and other glucocorticoids that contain a 3-keto group rather than a phenylpyr- azole attached to carbon atoms 2 and 3 of the A ring? Two (9 alternative exclusive explanations could fit the data. Explanation I. A single class of receptors with one site for "standard" glucocorticoid but two sites for cortivazol. The 10"* 2.5x10'" SxlO4 10'7 5X10'7 io* unusual side group of cortivazol would account in this model Steroid Molar Concentrations for the special higher affinity site and would account for the Fig. 7. Effect of increasing concentrations of steroids on cell growth in CEM- high potency of cortivazol. Mutation of this molecule in the C7 cells. Shown are dexamethasone, dexamethasone mesylate, and dexametha- steroid binding region lead to the "receptor deficient" clone sone plus dexamethasone mesylate in equimolar concentration. After 4 to S days of incubation the number of viable treated cells was compared to control cultures. discussed in this paper. We have shown that this clone possesses full sized 7-kilobase glucocorticoid receptor mRNA and im- azol binding to its higher affinity site was an interaction with a munologically identifiable, full sized receptor protein in consid protein other than true glucocorticoid receptor. To rule this erable excess over the quantity of sites recognized by dexameth out, we again used the antiglucocorticoid, RU38486. This ste asone binding. This residual receptor in the mutant can be roid is well known to bind with high affinity to the glucocorti labeled, at least in part, by dexamethasone mesylate (37, 42). coid and progesterone receptors (20, 21). Upon repeating our In this model, therefore, one would postulate a binding site binding experiments with low concentrations of [3H]cortivazol mutation that has altered the physical properties of a single Â±competition with unlabeled RU38486, we confirmed that all class of receptors so as to cause the near complete loss of binding was blocked by simultaneous addition of excess antig affinity for dexamethasone, and of the lower affinity cortivazol lucocorticoid (not shown). Thus both the high affinity and low site. In this case the question would be whether these latter two affinity cortivazol sites appear to be on molecules which exhibit sites are at exactly the same location in the receptor protein, the properties of classical glucocorticoid receptors. since they certainly overlap functionally. The residual cortivazol Proposed Model of Cortivazol Binding in CEM Cells. The site on the mutated receptor still retains high affinity for this curvilinear nature of the Scatchard plot of cortivazol binding ligand at low temperature but is heat labile, so that most in CEM C7 cytosol demands consideration of the nature of the cortivazol binding is lower affinity at higher temperatures. This interactions of this steroid with binding proteins. Cortivazol is phenomenon would explain the described binding and physio logical response properties of the mutant to cortivazol, i.e., the approximately 30 times more potent than dexamethasone in fact that a site with Ka ~8 x IO"10 M is measured at 4Â°Cin provoking cell kill and glutamine synthetase induction in these cytosols, whereas concentrations of the steroid over 10~8M are cells. Apparently these effects involve the interaction of cortiv required to inhibit live, growing cells at 37Â°C. azol with some form or forms of the glucocorticoid receptor. Negative cooperativity appears an unlikely explanation of the Explanation 2. Cl cells have two alÃelesfor the GR gene, one complex binding kinetics, since no difference in the dissociation of which is already mutated to give a form of the receptor that rate of cortivazol when it was prebound to cytosol and diluted no longer binds dexamethasone but still can bind cortivazol. in the presence or absence of excess fresh cortivazol was seen The other alÃeleis normal and binds both steroids. Conse (14). One is thus led to the conclusion that the binding kinetics quently cortivazol displays a two site binding curve due to its are due to multiple sites rather than negative cooperativity at a differing affinities for the two forms of the receptor, while single site. The simplest multiple site model is one of two sites, dexamethasone gives a one site curve because it only binds to and computer resolution of the binding data suggests that this the product of the normal alÃele.Inthe ICR27 receptor deficient is a kinetic possibility. Therefore in this discussion we have mutants the normal alÃelehas been mutated to a state in which considered the possibility that CEM C7 cells have two physical neither steroid binds, but the original mutant alÃelestill ex sites for cortivazol binding, corresponding to two kinetically presses its low affinity receptor. Thus essentially no dexameth distinguishable sites. The questions then become: Are both sites asone binding is seen and the cortivazol binding kinetics display on some form of the glucocorticoid receptor? If that is so, are one site. Some recent data support this model. They come from there two sites on one receptor molecule, or are there two better quantification of the GR mRNA and GR immunologi- different glucocorticoid receptor molecules? If part of the cor cally detectable protein in the receptor deficient mutants. We tivazol binding is on another molecule than the glucocorticoid have found that the quantity of mRNA in basal state cells is receptor, what molecule provides that second site? nearly as much as that in wild type cells and, second, that the Our data support the conclusion that all the cortivazol bind quantity of immunologically detectable protein is approxi ing seen is on actual glucocorticoid receptors. Both the higher mately one-half that of wild type cells (37). These data would and lower affinity cortivazol sites show physical properties with fit with the second of these models. The proposed mutation classic glucocorticoid sites, and cortivazol blocks binding of would be one in which mRNA from both alÃelescontinues to dexamethasone mesylate to standard, M, 97,000 receptor (33). be produced in the receptor defective cells, but only one of the The binding of low concentrations of cortivazol (to examine alÃelesmakes a message that is in fact capable of being trans specifically the higher affinity site) is blocked by dexamethasone lated into protein. That protein, however, is the product of the mesylate or RU38486, two classic antiglucocorticoids with original mutant alÃelecarried from the C7 parent and unable to 2257s

Harmon, J. M. Mechanisms of glucocorticoid function in human leukemic bind well to dexamethasone, only doing so with cortivazol due cells: analysis of receptor gene mutants of the activation-labile type using the to its special structure. Our current preference therefore is for covalent affinity ligand dexamethasone mesylate. J. Steroid Biochem., 30: this second model. Direct isolation of the mutant gene for 63-70, 1988. 19. Bourgeois, S., Pfahl, M., and Baulieu, E-E. DNA binding properties of sequencing to distinguish the models is under way. glucocorticosteroid receptors bound to the steroid antagonist RU-486. EMBO J., J: 751-756, 1985. 20. Schmidt, T. J. In vitro activation and DNA binding affinity of human Note Added in Proof lymphoid (CEM-C7) cytoplasmic receptors labeled with the antiglucocorticoid RU38486. J. Steroid Biochem., 24: 853-863, 1986. Now in press is more complete documentation of the c-myc effects 21. Lazar, G., and Agarwal, M. K. Physiological action and receptor binding of mentioned herein. See Y. S. Yuh and E. B. Thompson, Glucocorticoid a newly synthesized and novel antiglucocorticoid. Biochem. Biophys Res. effect on oncogene/growth gene expression in human T-lymphoblastic Commun., 134: 44-50, 1986. leukemic cell line CCRF-CEM: specific c-myc mRNA suppression by 22. Norman, M. R., and Thompson, E. B. Characterization of a glucocorticoid- sensitive human lymphoid cell line. Cancer Res., 37: 3785-3791, 1977. dexamethasone, J. Biol. Chem., in press, 1989. 23. Harmon, J. M., and Thompson, E. B. Isolation and characterization of dexamethasone-resistant mutants from human lymphoid cell line CEM-C7. Mol. Cell. Biol., /: 512-521, 1981. References 24. Zawydiwski, R., Harmon, J. M., and Thompson, E. B. Glucocorticoid- resistant human acute lymphoblastic leukemia cell line with functional recep 1. Rosseau. G. G., and Baxter J. D. Glucocorticoid receptors. In: J. D. Baxter tor. Cancer Res., 43: 3865-3873, 1983. and G. G. Rousseau (eds.), Glucocorticoid Hormone Action: Monographs 25. Schmidt, T. J., and Thompson, E. B. Glucocorticoid receptors and glutamine on Endocrinology, pp. 49-77. New York: Springer-Verlag, 1979. synthetase in leukemic Sezary cells. Cancer Res., 39: 376-382, 1979. 2. Weinberger, C., Hollenberg, S. M., Ong, E. S., Harmon, J. M., Brower, S. 26. Thompson, E. B., Yuh, Y. S., Harbour, D., Ashraf, J., Johnson, B., and T., Cidlowski, J., Thompson, E. B., and Rosenfeld, M. G. Identification of Harmon, J. M. Growth inhibition of CEM cells by glucocorticoids: C-MYC human glucocorticoid receptor complementary' DNA clones by epitope selec down regulation, and the topology of the glucocorticoid receptor. In: J. A. tion. Science (Wash. DC), 228: 740-742, 1985. Gustafsson, H. Eriksson, and J. Carlstedt-Duke (eds.), The Steroid/Thyroid 3. Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A.. Lebo, Hormone Receptor Family and Gene Regulation. Stockholm: Birkhauser R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. Primary structure Verlag, in press, 1989. and expression of a functional human glucocorticoid receptor cDNA. Nature 27. Harmon, J. M., and Thompson, E. B. Glutamine synthetase induction by (Lond.),3I8:635-641, 1985. glucocorticoids in the glucocorticoid-sensitive human leukemic cell line 4. Miesfeld, R., Okret, S., Wilkstrom, A-C., WrÃ¤nge,O., Gustafsson, J-A., and CEM-C7. J. Cell. Physiol., 110:155-160, 1982. Yamamoto, K. R. Characterization of a steroid hormone receptor gene and 28. Harmon, J. M., Norman, M. R., Fowlkes, B. J., and Thompson, E. B. mRNA in wild-type and mutant cells. Nature (Lond.), 312: 779-781, 1984. Dexamethasone induces irreversible d arrest and death of a human lymphoid 5. Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikstron, cell line. J. Cell. Physiol., 98: 267-278, 1979. A-C., Gustafsson, J-A., and Yamamoto, K. R. Genetic complementation of 29. Harmon, J. M., Schmidt, T. J., and Thompson, E. B. Deacylcortivazol acts a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. through glucocorticoid receptors. J. Steroid Biochem., 14: 273-279, 1981. Cell, 46:389-399, 1986. 30. Harmon, J. M., Schmidt, T. J., and Thompson, E. B. Non-glucocorticoid 6. Danielsen, M., Northrop, J. P., and Ringold, G. M. The mouse glucocorticoid receptor-mediated effects of the potent glucocorticoid deacylcortivazol. Can receptor: mapping of functional domains by cloning, sequencing and expres cer Res., 42: 2110-2114, 1982. sion of wild-type and mutant receptor proteins. EMBO J., 5: 2513-2522, 31. Schmidt, T. J., Harmon, J. M., and Thompson, E. B. "Activation-labile" 1986. glucocorticoid-receptor complexes of a steroid-resistant variant on CEM-C7 7. Gigure, V., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. Func human lymphoid cells. Nature (Lond.), 286: 507-510, 1980. tional domains of the human glucocorticoid receptor. Cell, 46: 645-652, 32. Harmon, J. M., Schmidt, T. J., and Thompson, E. B. Molybdate-sensitive 1986. and molybdate-resistant activation-labile glucocorticoid-receptor mutants of 8. Danielsen, M., Northrup, J. P., Jonklaas, J., and Ringold, G. M. Domains the human lymphoid cell line CEM-C7. J. Steroid Biochem., 21: 227-236, of the glucocorticoid receptor involved in specific and nonspecific deoxyri- 1984. bonucleic acid binding, hormone activation, and transcriptional enhance 33. Schlechte, J. A., and Schmidt, T. J. Use of [3H]cortivazol to characterize ment. Mol. Endocrino!., /: 816-822, 1987. glucocorticoid receptors in a dexamethasone-resistant human leukemic cell 9. Yuh, Y. S., and Thompson, E. B. Complementation between glucocorticoid line. J. Clin. Endocrino!. Metab., 64: 441-446, 1987. receptor and lymphocytolysis in somatic cell hybrids of two glucocorticoid- 34. Harbour, D. V., ChambÃ³n,P., and Thompson, E. B. Glucocorticoid mediated resistant human leukemic clonal cell lines. Somat. Cell Mol. Genet., If: 33- lysis of lymphoblasts maps to the DNA binding region of the glucocorticoid 45, 1987. receptor. In: Program and Abstracts, 70th Annual Meeting of the Endocrine 10. Harmon, J. M., Thompson, E. B., and Baione, K. A. Analysis of glucocorti- Society, A671, p. 188, 1988. coid-resistant human leukemic cells by somatic cell hybridization. Cancer 35. Sakaue, Y., and Thompson, E. B. Characterization of two forms of glucocor Res., 45: 1587-1593, 1985. ticoid hormone-receptor complex separated by DEAE-cellulose column chro- 11. Pfahl, M., and Bourgeois, S. Analysis of steroid resistance in lymphoid cell matography. Biochem. Biophys. Res. Commun., 77: 533-541, 1977. hybrids. Somat. Cell Genet., 6: 63-74, 1980. 36. Thompson, E. B., Yuh, Y-S., Ashruf, J., Gametchu, B., Linder, M., and 12. Gehring. U., and Thompson, E. B. Somatic cell fusion in the study of Harmon, J. M. Molecular genetic analysis of glucocorticoid actions in human glucocorticoid action. In: J. D. Baxter and G. G. Rousseau (eds.), Glucocor leukemic cells. UCLA Symp. Mol. Cell. Biol., 74: 221-237, 1988. ticoid Hormone Action: Monographs on Endocrinology, pp. 219-242. New 37. Harmon, J. M., Elsasser, M. S., Eisen, L. P., Urda, L. A., Ashraf, J., and York: Springer-Verlag, 1979. Thompson, E. B. Glucocorticoid receptor expression in "receptorless" mu 13. Steelman, S. L., Morgan, E. R., and Glitzer, M. S. Heterocyclic corticoster- tants isolated from the human leukemic cell line GEM-C7. Mol. Endocrino!, oidsâ€”I. Biological properties of the 6,16 dimethyl-4,6-pregnadiene- in press, 1989. 11a, 17,21-triol-20 one/3,2c/-2'-phenyl pyrazole-21 -acetate and its 21 desoxy 38. Simons, S. S., Jr., and Thompson, E. B. Dexamethasone-21 -mesylate: an derivative. Steroids, 18: 129-139, 1971. affinity label of glucocorticoid receptors from rat hepatoma tissue culture 14. Schlechte, J. A., Simons, S. S., Jr., Lewis, D. A., and Thompson, E. B. [3H] cells. Proc. Nati. Acad. Sci. USA, 78: 3541-3545, 1981. Cortivazol: a unique high affinity ligand for the glucocorticoid receptor. 39. Simons, S. S., Jr., and Miller, P. A. Affinity labeling steroids as biologically Endocrinology, 117: 1355-1362, 1985. active probes of antiglucocorticoid action. J. Steroid Biochem., 24: 25-32, 15. Simons, S. S., Jr., Pumphrey, J., Rudikoff, S., and Eisen, H. J. Identification 1986. of cysteine 656 as the amino acid of hepatoma tissue culture cell glucocorti 40. Simons, S. S., Jr., and Miller, P. A. Comparison of DNA binding properties coid receptors that is covalently labeled by dexamethasone 21-mesylate. J. of activated, covalent and noncovalent glucocorticoid receptor-steroid com Biol. Chem., 262: 9676-9680. 1987. plexes from HTC cells. Biochemistry, 23: 6876-6882, 1984. 16. Carlstedt-Duke, J., Stromstedt, P-E., Persson, B., Cederlund, E., Gustafsson, 41. Miller, P. A., Ostrowski, M. C., Hager, G. L., and Simons, S. S., Jr. Covalent J-A., and Jornvall, H. Identification of hormone-interacting amino acid and noncovalent receptor-glucocorticoid complexes preferentially bind to the residues within the steroid-binding domain of the glucocorticoid receptor in same regions of the long terminal repeat of muring mammary tumor virus relation to other steroid hormone receptors. J. Biol. Chem., 263:6842-6846, proviral DNA. Biochemistry, 23: 6883-6889, 1984. 1988. 42. Thompson, E. B., Zawydiwski, R., Brower, S. T., Eisen, H. J., Simons, S. 17. Harmon, J. M., Eisen, H. J., Brower, S. T., Simons, S. S., Jr., Langley, C. S., Jr., Schmidt, T. J., Schlechte, J. A., Moore, D. E., Norman, M. R., and L., and Thompson, E. B. Identification of human leukemic glucocorticoid Harmon, J. M. Properties and function of human glucocorticoid receptors receptors using affinity labeling and anti-human glucocorticoid receptor in steroid-sensitive and -resistant leukemic cells. In: H. Eriksson and J. A. antibodies. Cancer Res., 44: 4540-4547. 1984. Gustafsson (eds.), Steroid Hormone Receptors: Structure and Function, pp. 18. Thompson, E. B., Yuh, Y. S., Ashraf, J., Gametchu, B., Johnson, B. H.. and 171-194. Amsterdam: Elsevier Science Publishing Co., 1983.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. Interactions of the Phenylpyrazolo Steroid Cortivazol with Glucocorticoid Receptors in Steroid-sensitive and -resistant Human Leukemic Cells

E. Brad Thompson, Deepak Srivastava and Betty H. Johnson

Cancer Res 1989;49:2253s-2258s.

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