Uptake and receptor binding of dexamethasone in cultured 7800 C1 hepatoma cells in relation to regulation of cell growth and peroxisomal beta-oxidation

1. Uptake and binding of dexamethasone to glucocorticoid receptor has been studied in Morris hepatoma 7800 C1 cells in relation to its effect on cell growth and peroxisomal beta-oxidation. 2. Intact cells showed saturable, specific dexamethasone binding of limited capacity and Scatchard analysis revealed one single class of binding sites with equilibrium dissociation constant (Kd) of 0.24 nM similar to other glucocorticoid receptors. However, the binding capacity of 24 fmol/mg cell protein is less than 5% of previously reported values. 3. Uptake of [3H]dexamethasone by intact cells was temperature dependent giving a linear Arrhenius plot with a calculated energy of activation of 58.5 kJ mol-1 x degree-1. 4. Cytosol fractions had specific binding proteins for glucocorticoid hormones with sedimentation coefficient of ca 7S. No specific binding sites for [3H]dexamethasone was demonstrated in purified membrane fractions. 5. Dexamethasone and the synthetic fatty acid analogue tetradecylthio acetic acid (TTA) both inhibited the growth of the 7800 C1 cells and induced the peroxisomal acyl-CoA oxidase activity. A combination of the two compounds gave additive effects. Both these effects of dexamethasone and TTA were counteracted by insulin. 6. We conclude that dexamethasone induces growth inhibition and enzyme induction by binding to functional intracellular glucocorticoid receptors. The action of dexamethasone is consistent with a dissolution in the membrane from where it diffuses passively into the cell and binds to specific receptors in an energy dependent step. 6. The synergistic action of dexamethasone and TTA and the counteraction exerted by insulin are not due to changes in the dexamethasone receptor affinity or binding capacity.     

Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function

Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP-dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein

In order to determine the ratio of phosphates to hormone-binding sites on nonactivated (non-DNA-binding) glucocorticoid receptors in WEHI-7 mouse thymoma cells, we have extracted these receptors from cells grown to a steady state with 32P, labeled them with a saturating concentration of [3H]dexamethasone 21-mesylate, purified them using a monoclonal antibody, and analyzed them by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The complexes contained approximately 5 mol of phosphate/mol of bound steroid. Only half of the phosphates were associated with the approximately 100-kDa protein which is labeled with [3H]dexamethasone 21-mesylate. The remaining phosphates were associated with the approximately 90-kDa non-steroid-binding component of the nonactivated complex. Dual label studies, using [35S]methionine to measure receptor protein and 32P to measure receptor phosphates, have enabled us to determine the phosphate content, relative to receptor protein, of both nonactivated and activated cytosolic complexes generated in intact WEHI-7 cells exposed to triamcinolone acetonide at 37 degrees C. The total amount of phosphate associated with the activated complex is roughly half of that associated with the nonactivated complex, the decrease being accounted for by dissociation of the approximately 90-kDa phosphoprotein which accompanies activation. However, the ratio of 32P to 35S counts associated with the approximately 100-kDa steroid-binding protein is the same for the activated and nonactivated complexes. These results indicate that there is no net change in the phosphorylation of the approximately 100-kDa steroid-binding component of the cytosolic glucocorticoid-receptor complex upon activation in the intact cell.     

Isolation of new types of dexamethasone-resistant variants from a cAMP-resistant lymphoma

We have developed a sequential selection procedure for the isolation of novel steroid-resistant variants of the murine thymoma WEHI-7. The first step involves the isolation of cell lines with an altered cAMP-dependent protein kinase (cAPK) activity by selection for resistance to dibutyryl cAMP (dbcAMP). The second step involves the selection for resistance to dexamethasone (dex) which results in the isolation of variants with decreased receptor function and a cAMPrdexr phenotype. The initial selection, to cAMPr, serves as a permissive step since isolation of spontaneous glucocorticoid resistance from wild-type WEHI-7 does not occur at a measurable frequency. The results demonstrate a potential role for cAPK in regulating the functional levels of glucocorticoid receptor and suggest that mutations in other cellular functions that affect receptor activity could lead to steroid resistance in lymphoid cells.     

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort     

The effects of cell membrane alteration on glucocorticoid uptake by the AtT-20/D-1 target cell

The glucocorticoid-sensitive AtT-20/D-1 cell line was used to study cellular uptake of glucocorticoids. A previous observation that glucocorticoid uptake by these cells was temperature dependent had prompted us to postulate that glucocorticoids entered the cell by a temperature-sensitive transport process located in the cell membrane. Attempts were then made to perturb the membrane mechanism. In some of these experiments, intact cells were treated with neuraminidase or pronase. The release of sialic acid in the case of neuraminidase treatmentand of sialic acid and cell surface peptides in the case of pronase treatment demonstrated that the enzymes were effective. Approx. 60% of total cellular sialic acid was released by a 15 min incubation with 20 μg/ml neuraminidase at 25°C. The treated cells appeared to be viable, in that they continued to produce corticotropin at a normal rate, yet intact cell glucocorticoid binding at both 4 and 25°C was only 20–30% of that of untreated cells. Treatment with pronase also caused steroid uptake at 4 and 25°C to be reduced, although the extent of reduction was less than that seen following neuraminidase treatment.In other experiments, the effect of exposure of AtT-20/D-1 cells to ethanol or dimethyl sulfoxide was determined. The solvent concentrations used (0.5–10%) did not alter cell viability significantly, and the ability of the cytosol receptor to bind steroid in a cell-free preparation was unimpaired. However, incubation of intact cells with 10% (v/v) dimethyl sulfoxide or ethanol resulted in an 80–90% decrease in steroid uptake at 25°C.We conclude that steroid uptake by the intact cell can be perturbed by treatments which do not affect the cytosol receptor or alter cell viability. These results support the postulate that glucocorticoids enter the AtT-20/D-1 cell by a specific membrane-associated mechanism.     

Glucocorticoid Receptors in Murine Erythroleukaemic Cells

Abstract

Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation. Specific binding of dexamethasone was measured. A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified. Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 ⁺- 8.2 pmol/g protein) than in untreated controls (87.9 ⁺- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM). Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.     

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

31P NMR characterization of cellular metabolism during dexamethasone induced apoptosis in human leukemic cell lines

³¹P NMR has been used to study the effects of dexamethasone on phosphorus metabolism in one dexamethasone (dex)-sensitive (CEM-C7) and three different dex-resistant (CEM-C1, CEM-4R4, and CEM-ICR27) human leukemic cell lines. The use of these cell lines, containing widely varying amounts of glucocorticoid receptors, made it possible to evaluate the receptor-mediated contributions to the modes of action of dexamethasone in these cells. To evaluate the effects of dexamethasone without any significant contribution from experimental conditions, all the experiments were done with parallel controls. Results obtained showed: (1) significantly different levels of phosphorylethanolamine (PE) and phosphorylcholine (PC) among cell lines, suggesting significant differences in phospholipid metabolism; (2) the dexamethasone induced reduction of phosphomonoester (PE + PC), ATP, and metabolic rates probably through glucocorticoid receptor mediated mechanisms; (3) the dexamethasone induced stimulation of cellular metabolism in a process which seems to be independent of glucocorticoid receptors; and (4) the dexamethasone induced alkaline shift of intracellular pH in all the cell lines except ICR27. The reduction in PME levels seems to be an earlier step in dexamethasone-induced apoptosis than the reduction in ATP. The degree of alkaline shift was found to correlate with the number of glucocorticoid receptors present. The possible involvement of phospholipid metabolites as second messengers in dexamethasone-induced apoptosis is discussed. © 1994 Wiley-Liss, Inc.     

Effect of salicylic acid on glucucorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma

The binding activity of [³H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [³H]dexamethasone of 3.64 · 10^?8 M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 · 10⁵.Total binding activity to [³H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid at 37°C. The maximum enhancement was seen at the concentration of 10^?3 M and in 3 h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicylic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin, also enhanced the total binding activity to [³H]dexamethasone of the cytoplasmic fraction at the concentration of 2 · 10^?5 M and 2 · 10^?7 M, respectively.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Enhancement of cytokine expression in transiently transfected cells by magnetoliposome mediated hyperthermia

We investigated the enhancement of cytokine expression by heat treatment in transiently transfected glioma cells. The cells were transfected with plasmid bearing the interferon (IFN)-β gene under control of the MMTV promoter, which is inducible by glucocorticoid (dexamethasone). Then magnetite particles (10 nm diameter) as intracellular heating material were incorporated to the cells as the form of magnetoliposome. After 5 hours of incorporation, alternative magnetic field (384Oe, 118kHz) was applied for intracellular heating. IFN-β secreted in the medium was assayed and its concentration was compared to the extracellular heating induced expression, both in the presence and absence of dexamethasone. Higher IFN-β concentration was detected in intracellular heating even at lower temperature, 39 °C, than 43 °C in extracellular heating. The IFN-β expression level reached in the presence of dexamethasone was about three times higher than in the absence of inducer. In intracellular heating of 60 min, the surviving cell number reduced until 20%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of cell density on induction of growth hormone receptors by dexamethasone in primary cultured rat hepatocytes

The effect of cell density on the regulation of growth hormone (GH) receptors was studied by measuring specific binding of [125I]hGH to primary cultured hepatocytes with or without dexamethasone, which induces GH receptors. In cell cultures without dexamethasone, the cell density did not affect the level of binding of labeled GH appreciably. On the other hand, in the presence of dexamethasone, which induced an increase in the level of GH receptors on the cells, GH-binding by cultured cells at low cell density (3.3 x 10(4) cells/cm2) was about one-third of that of cells at high cell density (10(5) cells/cm2). Scatchard plot analysis showed that the cell-density dependent change in induction of GH binding, by dexamethasone was due to change in the number of binding sites without significant change in their affinity. The binding capacity of glucocorticoid receptors, measured as specific binding of [3H]dexamethasone to the hepatocytes, was not significantly affected by cell density. These results suggest that cell density modulates GH receptor induction by dexamethasone via events after glucocorticoid receptor binding.     

Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells

1. A competitive binding assay was adapted for determination of the specific binding of glucocorticoids to cytoplasmic receptors from rat thymus cells. The steroid–receptor complexes prepared by incubation of a cytoplasmic fraction from rat thymus cells with [1,2-³H₂]cortisol or with [1,2,4-³H₃]triamcinolone acetonide had rates of dissociation at 37°C similar to those from intact cells. 2. The cytoplasmic receptor was unstable at 3°C, but the rate of inactivation was decreased in the presence of 2.5mm-EDTA. The steroid–receptor complex was stable. 3. Rate constants for association and for dissociation, and association constants, were determined for the interactions of cortisol, cortexolone, dexamethasone and triamcinolone acetonide with the cytoplasmic receptor at 3°C. Differences in the association constants for different steroids could largely be accounted for by the differences in the rate constants for dissociation, but the rate constants for association did not vary greatly; the implications of these findings for the nature of the steroid-binding site are discussed. 4. A cytoplasmic fraction prepared from cells which had been incubated at 37°C under anaerobic conditions bound much less [1,2-³H₂]cortisol than did a fraction from aerobic cells, but the binding capacity was restored after exposure of the anaerobic cells to O₂. 5. The specific binding of [1,2-³H₂]-cortisol to intact thymus cells incubated aerobically was not affected by the presence of 0.1mm-cycloheximide, nor did this concentration of cycloheximide inhibit the recovery of specific binding observed when anaerobic cells were transferred to an aerobic atmosphere. 6. The energy dependence of specific binding of cortisol to the receptor is discussed with reference to possible mechanisms.     

Glucocorticoid receptors and cell cycle progression in human melanoma cell lines

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole-cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content per cell correlated with the reduction in final plating efficiency of glucocorticoid-treated cells, suggesting a receptor-mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M-5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 microM dexamethasone for 1 h. Evidence is presented of a real arrest of M-5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1-S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by flow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M-5A and NKI-26 cells suggested differentiation of the cells during glucocorticoid-induced arrest.     

Changes in glucocorticoid receptors in different regions of brain of immature and mature male rats

Specific cytosolic binding for synthetic glucocorticoid dexamethasone was studied in several brain regions (hypothalamus, hippocampus, caudate nucleus, cerebellum, cerebral cortex) of immature (3-week) and mature (26-week) male rats, intact and adrenalectomized. A significant regional difference was observed in the concentration of in vitro [3H] dexamethasone binding in the brain of adrenalectomized rats at both ages, with the highest levels in the hippocampus. A marked decrease in specific binding was observed in all brain regions of adrenalectomized mature rats as compared to immature. The dexamethasone binding was significantly lower in all brain regions of normal intact animals as compared to adrenalectomized rats in both ages.     

Direct studies of beta-adrenergic receptors intact frog erythrocytes.

(?) [³H]Dihydroalprenolol, a potent competitive β-adrenergic antagonist can be used to directly study β-adrenergic receptors by ligand binding techniques in an intact cell system, the frog erythrocyte. At 37°, binding reached equilibrium within 1 minute. Upon addition of excess unlabeled propranolol, complete dissociation of receptor bound ligand occurred within 1 minute. The characteristics of (?) [³Hdihydroalprenolol binding to β-adrenergic receptors in intact cells were quite similar to those previously demonstrated with isolated membrane fractions. The equilibrium dissociation constant for (?) [³H]dihydroalprenolol was 1.5 nM. Order of potency of agonists and antagonists in competing for the binding sites was appropriate for the β-adrenergic receptor as was the stereospecificity of binding ((?) isomers more potent than (+) isomers). Saturation studies with these intact cells indicated 1700 binding sites/cell in excellent agreement with the number previously estimated from membrane studies. Preincubation of cells with 10^?5M isoproterenol produced a 36% fall in number of β-adrenergic receptors. It is concluded that (?) [³H]dihydroalprenolol can be used to directly study the properties and regulation of β-adrenergic receptors in intact cell as well as broken cell preparations.