Debunking hCG.
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AIDS-Related Opportunistic Infections↗
Biomedical subjects
T Antakly
Publications and source records attributed to T Antakly.
Low molecular weight components but not dimeric HCG inhibit growth and down-regulate AP-1 transcription factor in Kaposi's sarcoma cells.
Kaposi's sarcoma, a sexually dimorphic disease inflicting high mortality in AIDS, remains at present without effective treatment. A recent report (Nature 375:64, 1995) showed that the placental glycoprotein hormone, human chorionic gonadotropin (HCG), and surprisingly its beta subunit, inhibit tumorigenicity and metastasis of Kaposi's sarcoma cells in mice xenografts. The anti-KS efficacy of a commercial HCG was subsequently demonstrated in clinical trials. Experimental data presented herein confirm that commercial HCG preparations (known to be about 25% pure) display significant inhibitory action in a dose-dependent manner. However, pure and biologically active HCG has no effect on Kaposi's sarcoma growth in culture. In fact, incubation of Kaposi's sarcoma cells with either one of four different well characterized preparations of pure HCG dimer or any of its two subunits did not alter cellular proliferation suggesting that a contaminant (or degradation product) may be the active agent. Commercial HCG preparations were subfractionated based on molecular size and each fraction was tested with respect to inhibition of KS cell growth, HCG radioreceptor binding and steroidogenic bioactivity. Results demonstrate that the anti-KS activity resides among low molecular weight components, and not in bona fide (macromolecular) HCG. Our study indicates that HCG activity and anti-KS action are separable. Interestingly, the active components in the crude HCG markedly down-regulate AP-1, a complex of transcription factors of the immediate-early response genes associated with cell growth. We conclude that, as yet unidentified molecules, present in the commercial HCG preparations, are responsible for the growth inhibitory effects presumably via the AP-1 signalling pathway.
Acquired Immunodeficiency Syndrome↗
Glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing apoptosis.
We have studied the effects of glucocorticoids on the activity and viability of neonatal rat osteoclasts in vitro. In the bone slice assay, glucocorticoids caused a dose-dependent decrease in the amount of bone resorbed, which was accompanied by a parallel decrease in osteoclast number. Loss of osteoclasts was due to their death, which occurred by the process of apoptosis. Evidence for the latter was obtained by a range of techniques, including time-lapse video microscopy, acridine orange staining, DNA fragment detection and transmission electron microscopy. Immunocytochemistry revealed the presence of glucocorticoid receptors in osteoclasts, and glucocorticoid-induced cell death could be prevented by the glucocorticoid receptor antagonist, RU486. These observations suggest that glucocorticoids promote receptor-mediated apoptosis of rat osteoclasts in vitro. This finding may help to explain recent data indicating that, in sharp contrast with their effects on the human skeleton, glucocorticoids inhibit bone resorption and increase bone mass in rats in vivo.
Animals↗
A shift in the ligand responsiveness of thyroid hormone receptor alpha induced by heterodimerization with retinoid X receptor alpha.
Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R alpha forms stable heterodimers with RXR alpha in living cells. Most important, we describe a new role for RXR alpha in modulating ligand-dependent T3R alpha activity: heterodimerization with RXR alpha greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R alpha. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R alpha plus RXR alpha than by T3R alpha alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R alpha homodimers but does not affect DNA binding by T3R alpha:RXR alpha heterodimers. We provide evidence that increased activities of T3R alpha at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR alpha-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.
Amino Acid Sequence↗
Expression and cytokine regulation of glucocorticoid receptors in Kaposi's sarcoma.
Development of Kaposi's sarcoma (KS) after glucocorticoid therapy has been observed in a variety of clinical states including human immunodeficiency virus-1 infection and recent in vitro studies provided evidence for a direct stimulation effect of glucocorticoid hormones on KS cell proliferation. The importance of glucocorticoids in KS pathogenesis is further highlighted by the finding that glucocorticoids synergize with cytokines to promote acquired immune deficiency syndrome (AIDS)-associated KS (AIDS-KS) growth. Furthermore, cytokine effects were abrogated by the glucocorticoid antagonist RU-486. As glucocorticoid action is mediated through activation of their intracellular cognate receptors, we hypothesized that enhanced responsiveness of AIDS-KS cells to glucocorticoids may be due to elevated glucocorticoid receptor (GR) content. Indeed, high expression of GRs in AIDS-KS tumor biopsies was detected both at the level of mRNA and protein. Quantitative measurements of GRs in these specimens by a sensitive immunoassay showed that GR content was significantly elevated in the tumor tissue (4663 fmol/mg protein) compared with the uninvolved skin of the same patients (2777 fmol/mg protein), both of which were markedly above the normal skin of healthy donors (893 fmol/mg protein). Immunocytochemical analysis confirmed the presence of GRs in the cytoplasm and the nucleus of KS cells. Interestingly, four major KS cytokines, namely interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and oncostatin M, all of which are known autocrine growth factors for AIDS-KS cells, significantly increased the expression of functional GRs in cultured AIDS-KS cells. The latter result may explain, at least in part, the synergistic effect of glucocorticoid and oncostatin M on AIDS-KS cell proliferation. Thus, the high levels of GR expression in AIDS-KS and the up-regulation of GRs by KS-growth-promoting factors may confer enhanced and sustained sensitivity to the stimulatory effects of glucocorticoids. The data presented also provide molecular bases for therapeutic interventions targeting GRs in this disease.
Acquired Immunodeficiency Syndrome↗
Inhibition of AIDS-Kaposi's sarcoma cell proliferation following retinoic acid receptor activation.
Retinoids, a group of natural and synthetic vitamin A analogues the receptors of which belong to the superfamily of steroid receptors, can exert profound effects on growth and/or differentiation of embryonic and neoplastic cells. Kaposi's sarcoma (KS), previously a rare multicentric neoplasm, has become epidemic with HIV infection, although the etiology of KS remains obscure. In the present study, the effects of two potent retinoids, all-trans-retinoic acid (RA) and 13-cis-RA, on the expression of retinoic acid receptor alpha and the growth of AIDS-related KS (AIDS-KS) cells were examined. The proliferation of AIDS-KS cells was significantly inhibited by RA and 13-cis-RA in a dose-dependent manner with 50% inhibitory concentration of 1.4 x 10(-10) M and 4.7 x 10(-9) M, respectively, which correlate with their potency. Growth inhibition was time dependent with maximal inhibition of 90% after 3 days of treatment with 10(-8) M RA. Growth inhibition by RA was further potentiated by forskolin (1 microM), an intracellular cyclic AMP-inducing agent. Moreover, RA treatment blocked the proliferative effect of oncostatin M and tumor necrosis factor alpha, two major KS autocrine growth factors. The effects of RA were accompanied by a dramatic increase in nuclear staining for retinoic acid receptor alpha and in the relative number of strongly positive retinoic acid receptor alpha nuclei. Finally, RA induced morphological changes as KS cells became more flattened, better spread, and more adhesive to the substrate. These results suggest that retinoids inhibit proliferation of AIDS-KS cells and further support their utility as therapeutic agents in AIDS-KS.
Acquired Immunodeficiency Syndrome↗
Inhibition of transcription factor GATA-4 expression blocks in vitro cardiac muscle differentiation.
Commitment of mesodermal cells to the cardiac lineage is a very early event that occurs during gastrulation, and differentiation of cardiac muscle cells begins in the presomite stage prior to formation of the beating heart tube. However, the molecular events, including gene products that are required for differentiation of cardiac muscle cells, remain essentially unknown. GATA-4 is a recently characterized cardiac muscle-restricted transcription factor whose properties suggest an important regulatory role in heart development. We tested the role of GATA-4 in cardiac differentiation, using the pluripotent P19 embryonal carcinoma cells, which can be differentiated into beating cardiac muscle cells. In this system, GATA-4 transcripts and protein are restricted to cells committed to the cardiac lineage, and induction of GATA-4 precedes expression of cardiac marker genes and appearance of beating cells. Inhibition of GATA-4 expression by antisense transcripts blocks development of beating cardiac muscle cells and interferes with expression of cardiac muscle markers. These data indicate that GATA-4 is necessary for development of cardiac muscle cells and identify for the first time a tissue-specific transcription factor that may be crucial for early steps of mammalian cardiogenesis.
Antisense Elements (Genetics)↗
Differential regulation of glucocorticoid receptor expression by ligand in fetal rat lung cells.
The glucocorticoid receptor (GR) mediates glucocorticoid stimulation of surfactant production by fetal mammalian lung. In many other tissues, glucorticoids decrease expression of GR, thereby reducing responsiveness to these hormones. We therefore determined whether there is a similar effect of exogenous glucocorticoids on GR in fetal rat whole lung, and in the principal cell types involved in the stimulation of surfactant, the fibroblasts and the epithelial cells. The ontogeny of GR in late gestation lung differed between the two cell types, with maximal levels occurring in fibroblasts on gestational d 19, and on d 20 in epithelial cells. Administration of dexamethasone (1 mg/kg) to the mother on gestational d 18 or 19 (term = 22 d) increased specific GR binding activity in whole lung 24 h later. Furthermore, in vitro, incubation of cultured fibroblasts of gestational d 20 with 10(-7) M cortisol increased GR immunoreactive protein and binding activity in a dose- and time-dependent manner, without affecting cellular levels of GR mRNA. However, identical treatment of d 20 distal airway epithelial cells was followed by decreased GR protein without significant change in cellular GR mRNA. Surfactant protein-A protein levels, taken as assessments of lung maturation, were increased in response to the same treatment. Our findings suggest that hormonal regulation of GR in fetal lung cells occurs at a posttranscriptional level, and is cell-specific. In the context of substantial increases in circulating glucocorticoid concentrations during late gestation, these findings may be of physiologic importance to the biochemical maturation of the antenatal lung.
Animals↗
AIDS-related Kaposi's sarcoma: evidence for direct stimulatory effect of glucocorticoid on cell proliferation.
Glucocorticoid therapy has been linked to increased risk of development of Kaposi's sarcoma (KS), which has become epidemic among HIV-infected individuals. However, no experimental evidence is available to explain the role of glucocorticoid in KS biopathology. We investigated the direct effect of dexamethasone (Dex) on the growth of cultured KS cells derived from acquired immune deficiency syndrome (AIDS) patients (AIDS-KS). Dex significantly stimulated the proliferation of AIDS-KS cells. Moreover, simultaneous exposure to Dex and oncostatin M, a KS major cytokine, produced a dramatic synergistic effect on proliferation of AIDS-KS cell. This suggests an interaction between glucocorticoid and growth factor intracellular pathways in KS cells. The expression of glucocorticoid receptor protein and mRNA in AIDS-KS cell cultures was examined by radioimmunoassay and in situ hybridization, respectively. Compared with other well studied cell lines, AIDS-KS cells contain an unusually high level of glucocorticoid receptor protein, which is further upregulated by glucocorticoid treatment. RU-486, a glucocorticoid receptor antagonist, completely abolished the stimulatory effect of Dex and reduced the synergistic effect of Dex and oncostatin M on proliferation of AIDS-KS. These findings demonstrate that glucocorticoid stimulates directly the proliferation of AIDS-KS cells via the modulation of glucocorticoid receptor expression.
Acquired Immunodeficiency Syndrome↗
A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription.
In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.
Amino Acid Sequence↗
Immunocytochemical localization of glucocorticoid receptors in the spinal cord: effects of adrenalectomy, glucocorticoid treatment, and spinal cord transection.
1. Studies were performed to determine the changes in immunoreactive (IR) type II glucocorticoid receptors of the ventral horn of the spinal cord produced by adrenalectomy (ADX), dexamethasone (DEX) treatment, and spinal cord transection in rats. 2. These treatments did not significantly affect the number of IR neurons of the ventral horn; however, staining intensity was enhanced after ADX and decreased following 4 days of DEX. A similar response pattern was observed for glial-type cells. 3. In control rats, about half of the ventral horn motoneurons were surrounded by immunoreactive glial perineuronal cells. These perineuronal cells increased after ADX (77% of counted neurons) and decreased following DEX treatment (32%; P < 0.05). 4. Two days after transection, staining was intensified in ventral horn motoneurons and glial cells located in the spinal cord below the lesion. Immunoreactive perineuronal cells increased to 85% of counted neurons, from a value of 66% in sham-operated rats (P < 0.05). 5. These findings suggest considerable plasticity of the spinal cord GCR in response to changes in hormonal levels and experimental lesions. It is possible that factors involved in cell to cell communication with transfer of hypothetical regulatory molecules may play roles in GCR regulation and the increased immunoreaction of glia associated with neurons following transection and ADX.
Adrenalectomy↗
The atrial natriuretic peptide system in rat ovaries.
The atrial natriuretic peptide (ANP) gene is expressed in several extracardiac tissues where ANP is thought to be involved in autocrine or paracrine regulation. The current studies were designed to characterize the ANP system in rat ovaries. ANP content in rat ovaries was estimated by RIA to be 240 +/- 70 pg/mg protein. HPLC revealed the presence of the 28-amino acid circulating peptide as well as the 126-amino acid prohormone, suggesting that the ovaries are a site of ANP synthesis. Indeed, ANP messenger RNA was detected in this tissue by RNase mapping. ANP present in ovarian extracts displaced [125I]ANP from bovine adrenal receptors (R1 class) in a dose-dependent manner and in parallel to the synthetic peptide, indicating that it possesses biological activity. Immunocytochemical studies localized ANP to interstitial cells surrounding the follicles; weaker but specific staining was also observed in the ovum. High affinity ANP receptors (dissociation constant, 0.30 +/- 0.06 nM; maximum binding capacity, 160 +/- 40 fmol/mg protein) were identified in ovarian membranes. Unlabeled ANP but not c-atrial natriuretic factor (a specific agonist of ANP clearance receptors) competed with binding of [125I]ANP to ovarian membranes in a dose-dependent manner, suggesting that ovarian ANP receptors are predominantly of the R1 class. This was confirmed by cross-linking studies with [125I]ANP, which detected a single protein band with a molecular size of about 120 kilodaltons, corresponding to that of the guanylate cyclase-coupled R1 class of receptor. Consistent with the presence of biologically active receptors, ANP markedly enhanced cGMP accumulation (by 15-fold) in ovarian cells. The presence of both local ANP synthesis and high affinity transducing receptors in the ovaries indicates that the peptide plays a local role in ovarian growth or steroidogenesis.
Adrenal Glands↗
Reduced expression of glucocorticoid receptor levels in human osteoarthritic chondrocytes. Role in the suppression of metalloprotease synthesis.
Glucocorticoid occupancy of a large percentage of glucocorticoid receptor (GR) is necessary for the suppression of matrix metalloprotease synthesis by human articular chondrocytes. In this study, we evaluated the levels of GR binding, cellular GR protein, and messenger RNA expression in both normal and osteoarthritic (OA) human articular chondrocytes and compared the degree of suppression of collagenase synthesis by glucocorticoids in cultures of the two cell types in order to investigate whether or not the GR system played an important role in the pathophysiology of OA. By radioreceptor binding assay, we recorded 56,320 +/- 8,230 sites per cell (mean +/- SE, n = 9) in primary cultures of normal chondrocytes and 27,480 +/- 14,240 sites in OA cells (n = 10, P < 0.0001). Equilibrium dissociation constant (Kd) values did not vary between normal (12.4 +/- 1.4 nmol/L) and OA (13.0 +/- 1.8 nmol/L). Subculturing of primary OA chondrocytes resulted in the up-regulation of the number of GR binding sites per cell to values comparable to those obtained in normal chondrocytes. Analysis of protein-immuno dot-blots of cytosolic extracts from normal (n = 4) and OA chondrocytes (n = 4) revealed that the former cytosols contained a 1.9 +/- 0.2 (P < 0.05) higher relative density of GR protein than the latter. By comparing the optical densities of GR-polymerase chain reaction products generated from normal (n = 6) and OA (n = 9) chondrocyte total RNA (normalized using an internal standard, glyceraldehyde phosphate dehydrogenase), we established a relative ratio, normal/OA, of 1.4. Experiments comparing the biological responsiveness of normal and OA chondrocytes to glucocorticoid suppression of interleukin-1-stimulated metalloprotease synthesis showed that dexamethasone inhibited collagenase synthesis in a dose-dependent manner with an IC50 of 6.3 +/- 1.2 x 10(-10) mol/L (n = 5) in normal cells while an IC50 of 5.0 +/- 0.4 x 10(-9) mol/L (P < 0.05) was recorded using OA (n = 5) chondrocytes. The results suggest that OA chondrocytes express fewer GR than normal cells as a result of a decrease in specific gene expression. The decreased responsiveness of OA cells to circulating glucocorticoids may be among the factors responsible for an increased level of metalloprotease synthesis by chondrocytes in OA cartilage.
Base Sequence↗
Subcellular distribution of [3H]-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography.
The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively. Cytosol (exclusively of all organelles) and nucleus showed comparatively weak labeling, at 9% and 7%, respectively. This study thus clearly establishes with the electron microscope, the localization of glucocorticoid binding sites using DM. It remains to be determined whether or not these DM binding sites represent bona fide glucocorticoid receptors or nonreceptor proteins that bind DM. Whereas the functional significance of the subcellular distribution of DM is not known, the labeling of the cytosol may represent localization of the steroid and GR in their traditional compartment. The steroid antagonistic properties of DM may have prevented the DM-GR complexes from translocating to the nucleus. However, the significant labeling of the sER-rich cytosol and mitochondria was unexpected and raises intriguing questions that are being addressed in current studies.
Animals↗
Corticostatic peptides.
In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14-18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and "supporting" endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.
Adrenal Glands↗
Cell-specific expression of the glucocorticoid receptor within granular convoluted tubules of the rat submaxillary gland.
The submaxillary gland, a heterogeneous tissue composed essentially of two functionally distinct cell types (tubular epithelial and acinar), offers an interesting system in which to study the mechanisms of steroid-dependent growth and differentiation. One cell type, the granular convoluted tubular (GCT) cell, secretes a large number of physiologically important polypeptides, including epidermal and nerve growth factors. Two steroids, androgens and glucocorticoids, greatly influence the growth, differentiation, and secretory activity of GCT cells. Because glucocorticoids can partially mimic or potentiate androgen effects, it has been thought that glucocorticoids act via androgen receptors. Since the presence of glucocorticoid receptors is a prerequisite for glucocorticoid action, we have investigated the presence and cellular distribution of glucocorticoid receptors within the rat submaxillary gland. Binding experiments using [3H]dexamethasone revealed the presence of high affinity binding sites in rat submaxillary tissue homogenates. Most of these sites were specifically competed by dexamethasone, corticosterone, and a pure glucocorticoid agonist RU 28362. Neither testosterone nor dihydrotestosterone competed for glucocorticoid binding. The cellular distribution of glucocorticoid receptors within the submaxillary gland was investigated by immunocytochemistry, using two highly specific glucocorticoid receptor antibodies. The receptor was localized in the GCT cells, but not in the acinar cells of rat and mouse submaxillary tissue sections. In GCT cells, the glucocorticoid receptor colocalized with several secretory polypeptides, including epidermal growth factor, nerve growth factor, alpha 2u-globulin, and atrial natriuretic factor. These data suggest that the submaxillary gland is a target for glucocorticoid action and that at least part of the glucocorticoid effects on this tissue are mediated by bona fide glucocorticoid receptors.
Adrenalectomy↗
Glucocorticoid receptor mediated inhibition of interleukin-1 stimulated neutral metalloprotease synthesis in normal human chondrocytes.
Glucocorticoids play an important role in the therapy of arthritic diseases. We sought, firstly, to identify, characterize and localize glucocorticoid receptors (GR) in normal human chondrocytes and, secondly, to determine whether glucocorticoid suppression of human recombinant interleukin-1 beta (rhIL-1 beta)-stimulated metalloproteases (MPs) synthesis by chondrocytes requires GR occupancy. Radioligand binding studies with cultured chondrocytes revealed the presence of high affinity-low capacity [3H]dexamethasone (DEX) binding sites with the following kinetic parameters: Kd = 12.5 +/- 1.4 nmol/L, Nmax = 57,560 +/- 3,960 sites per cell. Competition studies indicated that the DEX binding site was glucocorticoid specific and the competitive hierarchy established was: DEX greater than RU-26988 greater than RU-486 greater than cortisol greater than progesterone much greater than testosterone greater than estradiol-17 beta. Immunocytochemical studies using a specific anti-human GR antiserum identified immunoreactive material primarily in the cytoplasm with cells cultured in the absence of glucocorticoids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western immunoblotting analysis of chondrocyte cytosol detected the presence of a macromolecular species comigrating with a standard protein possessing a molecular weight of 94 kilodalton. rhIL-1 beta provoked the synthesis and secretion of the MPs stromelysin and collagenase from human chondrocytes in a saturable, coordinate, and dose-dependent fashion. DEX and cortisol inhibited the cytokine-stimulated MP synthesis in similar dose-dependent fashions: DEX, IC50 for stromelysin and collagenase suppression was 1.12 X 10(-8) mol/L and 1.26 X 10(-9) mol/L, respectively and the IC50 for cortisol was 6.3 X 10(-7) mol/L and 4.9 X 10(-8) mol/L, respectively. rhIL-1 beta failed to stimulate metalloprotease synthesis and release from chondrocytes pretreated with 10 nmol/L DEX, even after 20 days of incubation. The antiglucocorticoid, RU-486 completely reversed the DEX induced suppression of MP synthesis at 10(-7) mol/L. RU-486 alone had no effect on MP synthesis. We believe there is a biochemical rationale for the therapeutic efficacy of glucocorticoid administration in the management of arthritic diseases such as osteoarthritis and rheumatoid arthritis, and cytokines such as IL-1 are likely to be involved in the increase in MP synthesis.
Adolescent↗
Immunocytochemical localization of the glucocorticoid receptor in steroid-sensitive and -resistant human leukemic cells.
Because of their lympholytic action, glucocorticoids are included in most therapeutic regimens for the treatment of lymphomas and leukemias. The presence of functional glucocorticoid receptor may be predictive of the response to hormonal therapy in these diseases. We have developed an immunocytochemical procedure for human glucocorticoid receptor (GR) in order to assess its level and subcellular distribution in a well-studied system of childhood lymphoblastic leukemia cells (CEM), where sensitive and resistant subclones have been established. Several fixation and cell permeabilization protocols were compared. The most sensitive and reproducible one for light microscopy was prefixation in Bouin's solution followed by cytocentrifugation. Using various polyclonal and monoclonal antibodies, the GR was consistently localized predominantly in the cell cytoplasm in the absence of steroid. We compared localization of GR following glucocorticoid treatment in the glucocorticoid-sensitive clone CEM C7 with resistant subclones (4R4, 3R43, ICR27). Upon incubation with glucocorticoid, an increase in nuclear staining was clearly observed in the steroid-sensitive C7 cells. Although the resistant cell lines contain immunoreactive GR, they failed to show nuclear translocation following glucocorticoid treatment. This outlines the importance of the immunocytochemical procedure to distinguish between sensitive and resistant leukemic cells. Whether this test could be used prospectively to help select glucocorticoid therapy in human leukemias and lymphomas can now be examined.
Antibodies, Monoclonal↗