Biomedical subjects

J M Harmon

Publications and source records attributed to J M Harmon.

At least 37 records · Page 2Linked to original sources

PubMed1989

Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7.

The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.

Blotting, Northern↗

PubMed1988

Positive regulation of the glucocorticoid receptor in human T-cells sensitive to the cytolytic effects of glucocorticoids.

Regulation of glucocorticoid receptor (GR) protein and mRNA were examined in the human leukemic T-cell line CEM-C7. Unlike other cells in which GR regulation has been examined, the growth of these cells is inhibited by glucocorticoids, leading to cell death. Treatment of glucocorticoid-sensitive CEM-C7 cells with 1 microM dexamethasone for 18 h resulted in an increase in both cytoplasmic and nuclear GR protein, as determined by immunoblotting with anti-human GR antisera. Analysis of GR mRNA levels by Northern blotting revealed a corresponding increase in mRNA in steroid-treated cells. An increase in GR mRNA was detectable after as little as 3 h of treatment with dexamethasone, and GR mRNA concentration continued to increase for at least 18 h, well before the onset of growth arrest or cell death. GR mRNA concentration was not altered after dexamethasone treatment of the glucocorticoid-resistant mutant cell line ICR27TK.3, which lacks functional GR. Thus, the increase in GR seen in glucocorticoid-sensitive cells is a GR-mediated response. These results are in sharp contrast to the down-regulation of GR reported in other cells and tissues, and suggest that regulation of the GR by its cognate ligand may be tissue-specific.

Affinity Labels↗

PubMed1988

Mechanisms of glucocorticoid function in human leukemic cells: analysis of receptor gene mutants of the activation-labile type using the covalent affinity ligand dexamethasone mesylate.

In the cultured acute lymphoblastic leukemic (ALL) cell line, clones of sensitive cells are killed by receptor-occupying concentrations of glucocorticoids. In addition, several types of resistance have been identified. The types of resistance are r- (glucocorticoid binding site loss), ract/l (activation labile receptors) and r+ly- (defective lysis mechanism). The two types of receptor mutants have been examined for the presence and expression of the glucocorticoid receptor (GR) gene. Southern blot analysis, using a full-length cDNA probe for human GR, shows that the gene in both is grossly intact. Examination of the expression of the gene by Northern blots reveals the presence of normal, 7-kb message in both types of receptor mutants, though in amounts somewhat reduced from wild-type. This report focuses on the activation labile mutants. Since characterization of these mutants suggests that they can bind ligand but not retain it during activation, we hypothesized that they would respond normally to a ligand that could not be lost during activation. This seems to be the case. When the covalent affinity ligand dexamethasone mesylate, itself a partial glucocorticoid agonist/antagonist, is used, the ract/l cells are killed to an extent corresponding to that evoked by a sub-optimal concentration of the full agonist dexamethasone. We conclude: (1) that the ract/l receptors can function to kill cells if provided a ligand that they do not lose during activation; (2) that the partial agonist activity of dexamethasone mesylate for cell killing is not due to release of a small amount of free dexamethasone; (3) that the poor agonist activity of dexamethasone mesylate receptor complexes suggests that the role of steroid is strictly to participate in conversion of the receptor to its DNA binding form, after which presence of the steroid actually interferes with proper receptor action.

Cell Division↗

PubMed1988

Activation of the human glucocorticoid receptor: evidence for a two-step model.

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.

Cell Line↗

PubMed1987

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments.

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.

Affinity Labels↗

PubMed1986

Analysis of glucocorticoid receptor activation by high resolution two-dimensional electrophoresis of affinity-labeled receptor.

To determine if activation of the glucocorticoid receptor involves covalent charge modification of the steroid-binding protein, unactivated and activated IM-9 cell glucocorticoid receptors were examined by high resolution two-dimensional gel electrophoresis. As previously reported (Smith, A. C., and Harmon, J. M. (1985) Biochemistry 24, 4946-4951), two-dimensional electrophoresis of immunopurified, [3H]dexamethasone mesylate-labeled, steroid-binding protein from unactivated receptors resolves two 92-kDa isoforms (pI congruent to 5.7 and 6.0-6.5). After activation, the apparent pI of neither isoform was altered, indicating that there had been no covalent charge modification of the steroid-binding protein. Thus, the physicochemical changes observed after activation of the steroid receptor cannot be explained by dephosphorylation or other models which involve covalent charge modification of the steroid-binding protein. This conclusion was consistent with the observation that treatment of immunopurified, affinity-labeled receptors with calf intestine alkaline phosphatase did not alter the apparent pI values or distribution of the steroid-binding protein isoforms. However, chromatography of activated steroid-receptor complexes on DNA-cellulose revealed that only the more basic of the two steroid-binding protein isoforms bound to DNA. Therefore, the charge heterogeneity of the steroid-binding protein may be important in regulating the ability of the steroid-binding protein to interact with DNA.

Affinity Labels↗

PubMed1986

Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB.

Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.

Animals↗

PubMed1986

Glucocorticoid receptors and glucocorticoid resistance in human leukemia in vivo and in vitro.

Clinical measurements quantitating glucocorticoid receptor sites in leukemic blasts may give useful prognostic information. In childhood ALL, where the most data is available, "high" receptor content in peripheral or marrow blasts correlated with likelihood of remission on therapy, longer durations of remission and better prognosis generally. In lymphomas and CLL as well, high receptor content correlated with likelihood of response to steroid therapy, though the number of studies is less. In AML the correlation with receptor site content is moot, and in other leukemias the reports are less complete. A model system for childhood ALL is provided by CEM cells, a glucocorticoid sensitive human cell line from a patient with the disease. These cells have glucocorticoid receptors which must be filled by hormone for greater than 24 hr for cell lysis to begin. Four types of glucocorticoid resistance have been identified thus far in clones of these cells. Their distinctive properties are described and their relevance to clinical situations briefly discussed.

Animals↗

PubMed1986

Activation of the rat kidney mineralocorticoid receptor.

Activation of the rat kidney mineralocorticoid receptor was investigated using diethylaminoethyl (DEAE)-cellulose, DNA-cellulose, and gel permeation chromatography. Specific labeling of the mineralocorticoid receptor was achieved by labeling with [3H]aldosterone in the presence of the pure glucocorticoid RU28362. The specificity of labeling was confirmed by the lack of immunoreactivity of [3H]aldosterone-labeled material with the monoclonal antiglucocorticoid receptor antibody BURG-1. The unactivated aldosterone-mineralocorticoid receptor complex did not bind to DNA-cellulose, was eluted from DEAE-cellulose at relatively high salt (195 mM KCl) concentration, and had an apparent Stokes radius when chromatographed on Sephacryl S300 of 6.3 nm. After activation, the aldosterone-mineralocorticoid receptor complex had increased affinity for DNA-cellulose and decreased affinity for DEAE-cellulose and appeared as a smaller complex when chromatographed on Sephacryl S300. These changes were blocked by sodium molybdate. Our results indicate that activation of the rat kidney mineralocorticoid receptor is analogous to activation of the glucocorticoid receptor and suggest that activation of the mineralocorticoid receptor involves dissociation of a multimeric receptor form.

Aldosterone↗

PubMed1985

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis.

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.

Affinity Labels↗

PubMed1985

Glucocorticoid receptors in human leukemias and related diseases.

The evidence to date is compelling that steroid initiated cell lysis involves participation of the glucocorticoid receptor. Not only do the concentrations and specificity of hormones for cell lysis and receptor occupancy correspond, but also steroid resistant cells selected with or without prior mutagenesis often have altered receptors. The glucocorticoid receptor protein from humans and other species is a approximately 95,000 d, thiol group-containing monomer, prone to aggregation when "unactivated." After having bound steroid and been "activated," the monomeric steroid-receptor complex is altered in charge and shape so that its binding to chromatin and DNA is greatly enhanced. Simple measurement of numbers of receptor sites in cells from patients with various blood dyscrasias has given, in some disease, good correlations between high numbers of receptor sites and good therapeutic response. These correlations are strongest for childhood acute lymphoblastic leukemia (ALL) and for non Hodgkins' lymphoma. In other diseases, notably acute myelogenous leukemia, such correlations have not been found. The CEM human ALL line has been used in vitro to study mechanisms of glucocorticoid action and resistance. The requirement for "activated" steroid-receptor complex for cell lysis is shown in these cells by the spontaneous occurrence of steroid resistant, activation-labile receptor mutants. A second category of resistant cells with normal receptors has been defined. Treatment of these "lysis defective" resistant cells with compounds which result in DNA demethylation can render them steroid sensitive. Since DNA demethylation can allow formerly silent genes to become transcribed, it is possible that one or more genes specific for lysis has been "opened" in such cells. Alternatively, DNA demethylation may produce a general biochemical effect on the cell which renders it susceptible to lysis. Mutagenized CEM cells selected for steroid resistance give rise to a third class of mutants, which are deficient in receptor quantity. Each of these classes of steroid resistant cells contains information pertinent to understanding the use of glucocorticoids and the role of glucocorticoid receptors in human leukopathic disease.

Animals↗

PubMed1985

Identification of human glucocorticoid receptor complementary DNA clones by epitope selection.

Steroid hormones regulate cellular differentiation and physiologic functions predominantly through gene transcription. Regulation is achieved by the interaction of specific steroid receptor proteins and target genes. Expression cloning techniques were used to select human glucocorticoid receptor complementary DNA clones in order to define the mechanism by which the receptor exerts its transcriptional control. Immobilized fusion proteins from individual clones were used to select epitope-specific antibody which was subsequently eluted and identified by binding to protein blots of cellular extracts. Three cross-hybridizing clones containing inserts expressing antigenic determinants of the human glucocorticoid receptor were isolated.

Cloning, Molecular↗

PubMed1985

Analysis of glucocorticoid-resistant human leukemic cells by somatic cell hybridization.

Glucocorticoid-resistant mutants isolated form the glucocorticoid-sensitive human leukemic cell line CEM-C7 can be divided into three phenotypes: those with almost no glucocorticoid-binding activity (r-); those whose steroid-receptor complexes are unstable during attempted activation but are stabilized by the presence of sodium molybdate (actl:molybdate-sensitive); and those whose steroid-receptor complexes are unstable during attempted activation but are insensitive to the presence of molybdate (actl:molybdate-resistant). To determine if these phenotypes represent different mutations within the glucocorticoid receptor locus itself or reflect alterations in other components modifying receptor function, somatic hybrids were constructed between wild-type cells and all three classes of resistant mutants, as well as between various classes of resistant mutants. Hybrids were analyzed for chromosome content, steroid-induced growth inhibition, induction of the enzyme glutamine synthetase, and glucocorticoid receptor content. Hybrids constructed between wild-type cells and any of the three classes of resistant cells were growth-inhibited in the presence of dexamethasone, displayed normal levels of glutamine synthetase induction, and contained a quantity of glucocorticoid receptor approximately equal to the sum of the glucocorticoid receptor concentrations of the parental cell lines. Hybrids constructed between various classes of resistant cells were not growth-inhibited by dexamethasone, displayed no glutamine synthetase induction, and also contained the sum of the glucocorticoid receptor concentration of the individual parents. Thus, each phenotype is recessive, and there is no complementation between phenotypes. We conclude that the three phenotypes are the result of different mutations within the glucocorticoid receptor locus itself and do not represent the presence of dominant receptor-inactivating factors or the absence of positive regulatory components.

Cell Line↗

PubMed1984

Molybdate-sensitive and molybdate-resistant activation-labile glucocorticoid-receptor mutants of the human lymphoid cell line CEM-C7.

The basis for the glucocorticoid resistance of three (3R7, 3R43 and 4R4) genetically independent mutants derived from the glucocorticoid-sensitive human lymphoid cell line CEM-C7 was examined. Each mutant contained significant, albeit reduced, amounts of steroid binding activity measured in both whole and broken cell assays. These activities were of similar high affinity and specificity to that seen in the sensitive parent, suggesting that the steroid binding portion of the receptors in these mutants was normal. However, nuclear translocation of steroid-receptor (SR) complexes was defective in all three clones. Analysis of SR complex activation by DEAE-cellulose chromatography established that receptors from the mutant clones were extremely labile under conditions which would activate normal SR complexes. Such lability was not exhibited by the unoccupied or occupied but unactivated forms of these receptors, indicating that all three were "activation labile" (act1). SR complexes of clones 3R7 and 4R4 were completely protected by the inclusion of molybdate during attempted activation and were classified as act1:molybdate-sensitive. However, SR complexes of clone 3R43 were unstable during attempted activation, even in the presence of 50 mM molybdate, and were thus classified as act1:molybdate-resistant. Our results suggest that while the act1 phenotype may predominate among spontaneously derived glucocorticoid-resistant mutants derived from CEM-C7, this phenotype may be the consequence of at least two different mutations.

Cell Line↗

PubMed1984

Identification of human leukemic glucocorticoid receptors using affinity labeling and anti-human glucocorticoid receptor antibodies.

Antisera raised against human lymphoid glucocorticoid receptors were used in combination with the glucocorticoid receptor affinity label [3H]dexamethasone 21-mesylate [( 3H]DM) to identify the glucocorticoid receptors of the human B-lymphoblastoid cell line IM-9 and the human T-cell leukemic cell line CEM-C7. Antisera were obtained following immunization of New Zealand White rabbits with [3H]triamcinolone acetonide [( 3H]TA)-glucocorticoid receptor complexes partially purified by two-stage DNA-cellulose chromatography. The presence of anti-human glucocorticoid receptor antibodies was verified by: (a) adsorption of [3H]TA-receptor-antibody complexes to Protein A; (b) a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [3H]TA-receptor complexes incubated with immune serum; and (c) the ability of immune serum to displace [3H]TA-receptor complexes on sucrose gradients. These antibodies also recognized rat liver and murine S49 cell glucocorticoid receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled IM-9 cytosol identified a major competable band with a molecular weight of approximately 90,000, three minor competable components with molecular weights of approximately 78,000, approximately 51,000, and approximately 38,500, and at least 21 other noncompetable components. Following immunoprecipitation of [3H]DM-labeled cytosol with immune serum, only the Mr 90,000 and 78,000 components were seen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled CEM-C7 cytosol revealed a larger number of [3H]DM-labeled components. However, after immunoprecipitation of [3H]DM-labeled CEM-C7 cytosol, a predominant competable component with a molecular weight of 90,000 was easily identified. This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used. Thus, the combination of affinity labeling and anti-human glucocorticoid receptor antibodies is capable of providing direct physical identification of human lymphoid glucocorticoid receptors.

Affinity Labels↗

PubMed1983

Glucocorticoid-resistant human acute lymphoblastic leukemic cell line with functional receptor.

A receptor-containing, steroid-resistant clone of CEM cells, CEM-C1, was isolated without selective pressure from the wild-type population. The biological and physicochemical properties of glucocorticoid receptors in CEM-C1 cells were compared to those from a clone (CEM-C7) sensitive to glucocorticoid-mediated lysis. In a whole-cell binding assay, CEM-C1 cells exhibited high affinity for [3H]dexamethasone (Kd, 22 nM), nuclear translocation of steroid:receptor complex (nt, 43%) and were found to contain, on the average, 12,000 receptor sites/cell (R0). These steroid-binding parameters were similar to those displayed by wild-type CEM-C7 cells: Kd, 19 nM; nt, 47%; and R0, approximately 14,000 sites/cell. The ion-exchange and gel permeation profiles were indistinguishable from those of identically treated CEM-C7 cytosols. Thus, diethylaminoethyl cellulose chromatography of CEM-C1 cytosol showed that [3H]triamcinolone acetonide:receptor complex was eluted at 50 mM phosphate and 220 mM phosphate under "activating" and "nonactivating" conditions, respectively. Receptor complex of activated CEM-C1 cytosol bound to DNA-cellulose and was eluted at 100 mM salt. Filtration of unactivated CEM-C1 cytosol over Sephacryl S-300 generated a single peak of radioactivity for receptor complex with a calculated Stokes' radius of 55 to 59 A. Dexamethasone induced glutamine synthetase in CEM-C1. The dose dependence (50% effective dose, approximately 20 nM) and maximal fold increase (1.9, 1 microM dexamethasone) were comparable to those observed in CEM-C7. Since CEM-C1 cells contain apparently normal, functional cytosolic receptor, the results suggest that resistance to glucocorticoid in these cells involves a defect(s) at another locus.

Cell Membrane↗

PubMed1982

Glutamine synthetase induction by glucocorticoids in the glucocorticoid-sensitive human leukemic cell line CEM-C7.

Treatment of CEM-C7 cells with glucocorticoids produces a 2.5-fold increase in the activity of the enzyme glutamine synthetase (GS). This increase is specific for steroids with glucocorticoid activity adn occurs over a range of steroid concentrations consistent with a receptor-mediated mechanism. Half-maximal and maximal inductions by dexamethasone (dex) occur at 2 X 10(-8) M and 2 X 10(-7) M dex, respectively, concentrations approximately equal to those necessary to produce half and full occupancy of glucocorticoid receptors. GS activity began to increase 1 hour after dex treatment and was complete by 12 hours. This is well before any of the growth inhibitory or cytolytic effects of dex on this cell line occur. This increase was dependent on the presence of glucocorticoid receptors and required both RNA and protein synthesis. Removal of dex following stimulation to maximal levels resulted in a decrease of GS activity to preinduced levels with a half-time of 5 hours. Glutamine deprivation of cells resulted in increased GS activity. However, even in the total absence of glutamine, dex treatment elicited a 2.0-2.5-fold increase in GS activity, ruling out inhibition of glutamine uptake as a mechanism for the dex-induced increase. Experiments with 5'-bromodeoxyuridine (BrdU) demonstrated that GS elevation was sensitive to BrdU substitution of DNA, while dex-induced growth inhibition was not. Therefore GS elevation and growth inhibition in this cell line appear to be independently expressed steroid responses.

Bromodeoxyuridine↗

PubMed1982

Non-glucocorticoid receptor-mediated effects of the potent glucocorticoid deacylcortivazol.

Deacylcortivazol (DAC), a potent glucocorticoid, contains a phenyl-pyrazole moiety fused to the 2--3 position of the traditional steroid nucleus. When incubated with glucocorticoid-resistant mutants derived from the glucocorticoid-sensitive human leukemic cell line CEM-C7, DAC caused significant growth inhibition. However, this effect required 1 microM DAC, a concentration 50 times higher than that necessary for glucocorticoid receptor saturation. Cytotoxicity was observed in both mutants containing high-affinity glucocorticoid receptors defective in nuclear translocation and a mutant completely devoid of receptors. Further, in dexamethasone-resistant clones, DAC elicited only marginal increases in the activity of the glucocorticoid-inducible enzyme glutamine synthetase. Clones resistant to high concentrations of DAC could not be directly isolated from CEM-C7. However, stable DAC-resistant clones could be isolated from dexamethasone-resistant subclones of CEM-C7 with a frequency of 1 to 8 x 10(-4). These data are consistent with resistance to DAC being acquired in a two-step process. Our results suggest that the cytotoxicity of DAC at concentrations higher than necessary for glucocorticoid receptor saturation is not mediated by glucocorticoid receptors. Thus, DAC may be a bifunctional compound having both steroid receptor-mediated and receptor-independent cytotoxicity.

Cell Division↗