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Biomedical subjects

E B Thompson

Publications and source records attributed to E B Thompson.

At least 91 records · Page 5Linked to original sources
PubMed1990

Two glucocorticoid binding sites on the human glucocorticoid receptor.

Glucocorticoids are known to have a lytic effect in leukemic cells via interactions with the glucocorticoid receptor (GR). Cortisol and various synthetic glucocorticoids bind to the GR with one-site kinetics. Cortivazol (CVZ) is a unique, high potency synthetic glucocorticoid, which has a phenylpyrazol fused to the A-ring of the steroid nucleus and displays binding consistent with two or more sites in the cytosol from CEM C7 cells (a human acute lymphoblastic T-cell line). It has previously been shown that the lower affinity class of sites are similar in affinity and site molarity to those recognized by dexamethasone. The higher affinity sites bind CVZ with 20- to 50-fold greater affinity, consistent with CVZ's enhanced biological effects. In mutant leukemic cells resistant to the lytic effects of dexamethasone, CVZ both lyses the cells and recognizes a single class of sites similar to the high affinity site in CEM C7 cells. We have carried out experiments to define the nature of the higher affinity CVZ binding site. We now show that: 1) CVZ has more than one binding site in a second, independent, B-cell line, IM-9; 2) the antiglucocorticoid RU 38486 is able to block both CVZ's higher and lower affinity sites; 3) all of CVZ's binding sites are on a protein immunologically indistinguishable from the human GR; and 4) freshly isolated clones of CVZ-resistant cells have lost all binding sites for CVZ. These data indicate that CVZ is recognizing two glucocorticoid binding sites on the human GR or a protein very similar to it.

B-Lymphocytes↗
PubMed1990

Human oxysterol-binding protein. I. Identification and characterization in liver.

The function of cellular oxysterol-binding proteins is not known. Because of the proposed receptor-like role of these proteins in regulating cholesterol synthesis and the importance of the liver in the cholesterol metabolism of the body, we have studied the oxysterol-binding sites found in specimens of human liver. A protein that binds with high affinity to certain oxygenated derivatives of cholesterol has been identified and characterized in several ways. When 25-hydroxy-[3H]cholesterol is used as ligand, specific binding components can be identified in cytosolic liver extracts fractionated on sucrose density gradients in the fractions sedimenting at 4S and 7S. Cholesterol and steroid hormones do not compete for these binding components, but several oxysterols do. In cytosolic extracts, all binding appears to be a single class kinetically, with an apparent Kd of 28 x 10(-9) mol/L. We found the protein responsible to have a pI of about 4.8. Purification of the protein allowed preparation of an antiserum and subsequent immunoprecipitation of a photoaffinity-labeled component from crude cytosolic extracts. Electrophoresis of the immunoprecipitates revealed a single specifically labeled protein band, with a mol wt of about 57,000. The protein described here may have a regulatory role in the synthesis of cholesterol in the human.

Ammonium Sulfate↗
PubMed1990

Overexpression of full-length human glucocorticoid receptor in Spodoptera frugiperda cells using the baculovirus expression vector system.

We have expressed a full-length human glucocorticoid receptor (hGR) in Spodoptera frugiperda (Sf9) cells using the baculovirus expression vector system (BEVS). The level of expression is approximately 100-fold greater than in CEM-C7 cells. Between 0.5-1.0 mg hGR can be generated per liter of Sf9 cell culture. The expressed hGR is capable of binding glucocorticoids with specificity and high affinity. Covalent labeling with 3H-dexamethasone mesylate and Western blot analysis using a polyclonal antibody indicate that the molecular weight of the expressed protein is approximately 94 k. The nonactivated receptor sediments as a 8-9S complex in sucrose gradients and can be heat activated to a 4S form. The activated receptor is capable of retarding the migration of a 23 base-pair DNA fragment containing the glucocorticoid response element from the tyrosine aminotransferase gene. These data indicate that the expressed GR displays characteristics identical to those of GR from mammalian cells. By scaling up this culture we can, for the first time, obtain enough purified full-length receptor for crystallographic and functional studies which could provide new insight into exactly how hGR works.

Animals↗
PubMed1990

Capacity of state health agencies to meet nutrition objectives in maternal and child health.

Public health nutritionists in 54 official state health agencies were surveyed in 1987 to determine to what extent they were prepared to implement the Model State Nutrition Objectives developed by the Association of State and Territorial Public Health Nutrition Directors. Objectives related to services to the maternal and child health (MCH) population were the focus of one part of the survey. One half of all states have plans for nutrition services integrated into their state MCH plans. More than 75% of state agencies collect data on the nutritional status of pregnant and lactating women, infants, and preschool children. Fewer than half collect data on dietary intake patterns or nutrition knowledge. Thirty-one agencies reported a formal quality assurance program for one or more subsets of the MCH population. At least 75% of all states provide dietary intake recommendations, screening and assessment protocols, and policies concerning referrals to maternal and infant health programs. State health agencies are already involved in activities that will facilitate adoption of the model state nutrition objectives.

Adult↗
PubMed1989

Purification, subunit structure, and DNA binding properties of the mouse oxysterol receptor.

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, has been purified from mouse L cell cytosol greater than 3,600-fold in its undenatured form and to apparent homogeneity upon further electrophoresis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified 7.5 S receptor appears to be a dimer with similar or identical subunits of Mr 95,000. Proteolytic cleavage by an endogenous factor(s) gives rise to a 4.2 S form of the receptor which is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a heterogeneous mixture of ligand binding fragments of Mr 30,000-60,000. This 4.2 S form of the receptor retains high affinity for the oxysterol ligand and exhibits a more rapid oxysterol binding rate than the 7.5 S form. The 7.5 S form of the receptor binds to DNA-cellulose at low salt concentrations at neutral pH, and its affinity increases at low pH or in the presence of Zn2+. Receptor preparations from mouse liver were purified approximately 900-fold by the same purification procedure, but this was accompanied by conversion of the 7.5 S liver receptor to a approximately 4 S form, Mr approximately 55,000.

Affinity Labels↗
PubMed1989

Glucocorticoid effect on oncogene/growth gene expression in human T lymphoblastic leukemic cell line CCRF-CEM. Specific c-myc mRNA suppression by dexamethasone.

Glucocorticoids induce growth inhibition and eventually cause cell lysis in certain sensitive leukemic cells. To investigate how glucocorticoids interact with cell growth pathways, we studied the expression of 14 growth-related genes in dexamethasone-treated CEM-C7A cells, a steroid-sensitive clone of the CCRF-CEM cell line, and in several closely related clones. The 14 genes studied were chosen to represent four different levels of mitogenic signal transduction. Detectable mRNA levels were found for 8 of the 14 genes, but among these only c-myc expression was obviously suppressed by dexamethasone. The c-myc mRNA levels declined abruptly during the first 12 h after addition of 1 microM dexamethasone, and maximal suppression occurred by 18 h. This change was not seen in the C7A controls, in the glucocorticoid-resistant, receptor-deficient clone ICR-27, or in the glucocorticoid-resistant, receptor-positive clone C1. H.10, a hybrid clone between C1 and ICR-27, showed restoration of the sensitive phenotype, and in H.10 cells the c-myc mRNA was also suppressed by dexamethasone. Our results suggest that: 1) functional glucocorticoid receptor is required for inducing c-myc suppression. 2) In dexamethasone-resistant cells with functional receptors c-myc is not suppressed. 3) The growth arrest induced by glucocorticoids correlates with, and may be regulated via, suppression of c-myc expression.

Blotting, Northern↗
PubMed1989

Differential modulation of gene induction by glucocorticoids and antiglucocorticoids in rat hepatoma tissue culture cells.

Studies of glucocorticoid and antiglucocorticoid induction of tyrosine aminotransferase (TAT) in two rat hepatoma cell lines (Fu5-5 and HTC) are described. These studies revealed several phenomena that are not consistent with the current models of steroid hormone action: (a) TAT induction occurred at glucocorticoid levels below those required for comparable receptor occupancy in Fu5-5, but not in HTC, cells; (b) the ability of antiglucocorticoids to induce TAT is higher in Fu5-5 than in HTC cells; (c) the values of the amount of TAT agonist activity with the antiglucocorticoid dexamethasone 21-mesylate and of log10 of the dexamethasone concentration required for half-maximal induction of TAT were not constant over time but varied in a linear, reciprocal manner. This modulation was seen for several glucocorticoids and antiglucocorticoids at the level of both TAT enzyme and mRNA but not for two other glucocorticoid inducible genes in the same cells. These results, plus the fact that a similar difference in the concentration required for half-maximal TAT induction in Fu5-5 cells was seen for both glucocorticoids and cyclic AMP, argue that the modulation occurs at some point distal to receptor-steroid complex binding to the biologically active nuclear sites but proximal to translation of TAT mRNA. In order to explain these results, it is pointed out that models involving second messengers are entirely appropriate for steroid hormone action. The participation of a modulated trans-acting factor in such a model may explain the above results.

Animals↗
PubMed1989

Demonstration of the intracellular localization and up-regulation of glucocorticoid receptor by in situ hybridization and immunocytochemistry.

Glucocorticoid response in target cells closely correlates with glucocorticoid receptor (GR) level. We have compared the localization of GR (by immunocytochemistry) and GR mRNA levels (by in situ hybridization) in steroid-sensitive and steroid-resistant subclones derived from the human CEM lymphoid cell line. In addition, GR was localized in lymphoid cells from patients with various hematological malignancies. In the absence of preincubation with the steroid, GR was localized at the light and electron microscopic level predominantly in the cytoplasm and to a weaker extent in the nucleus. In the presence of steroid, the GR was shown to translocate to the nucleus in the steroid-sensitive but not in the steroid-resistant cell lines. Furthermore, GR and GR mRNA levels were increased following glucocorticoid treatment of the sensitive cell line but not the resistant one. These data support a role for receptor regulation and translocation in the overall mechanism of glucocorticoid hormone action.

Base Sequence↗
PubMed1989

Interactions of the phenylpyrazolo steroid cortivazol with glucocorticoid receptors in steroid-sensitive and -resistant human leukemic cells.

The interactions of glucocorticoids with their receptors somehow determine the cellular responses seen. The high potency glucocorticoid cortivazol differs from the usual glucocorticoids in two ways, structurally and in binding to receptors. Cortivazol contains a phenylpyrazol fused at carbon atoms 2 and 3 to the A ring of the cyclophenathrene, replacing the supposedly essential 3-keto,4,5-double bond pattern of glucocorticoids. Cortivazol binds to the glucocorticoid receptor in the cytosol from CEM C7 cells (a human acute lymphoblastic leukemia line) in a fashion consistent with interaction with at least two sites. Standard glucocorticoids show only one-site binding. In mutant leukemia cells derived from CEM C7, resistant to kill by 10(-6) M dexamethasone and deficient in standard glucocorticoid binding sites, cortivazol still finds a binding site and kills the cells. In wild-type leukemia cells, the binding sites of cortivazol, those with both higher (Kd approximately 5 x 10(-10) M) and lower (Kd approximately 1 x 10(-8] affinity appear to be on forms of the glucocorticoid receptor itself, and not on two different classes of molecules.

Alleles↗
PubMed1989

Flow cytometric analysis of glucocorticoid receptor using monoclonal antibody and fluoresceinated ligand probes.

Conditions were established for single cell analysis of glucocorticoid receptor (GR) content by flow cytometry using several clones of a human leukemic cell line (CCRF-CEM). These included CEM-7A, 7R, C1, and ICR 27 Tk.3 cells which were examined both by standard [3H]dexamethasone radiometric binding and by two independent flow cytometry assays. The latter involved either mouse monoclonal antibody against GR (GR-MoAb) or fluoresceinated cortisol ligand probes. For CEM-7A, 7R, and C1 cells, there was a correlation between GR-MoAb and radiometrically defined GR values. However, clone ICR-27 Tk.3 with low [3H]dexamethasone binding exhibited the highest GR-MoAb fluorescence. The fluoresceinated cortisol assay correlated with dexamethasone binding values in all four clones. Thus, GR-MoAb identifies the total immunologically reactive GR present, while the fluoresceinated cortisol assay quantifies only the functionally intact GR in terms of its initial binding. Their combined use may reveal the cellular heterogeneity of GR expression and function also in human tumor samples, to which they have been successfully applied. When coupled with DNA counterstaining, GR expression can be related directly to frequently DNA-aneuploid tumor cells and cell cycle distribution.

Antibodies, Monoclonal↗
PubMed1989

Abnormal glucocorticoid receptor gene and mRNA in primary cortisol resistance.

Abnormal steroid hormone receptors have been implicated as causing several forms of primary steroid hormone resistance in humans, but as yet no abnormality has been described at the gene level. We describe the analysis of the mRNA and genomic DNA from the Epstein-Barr (EB) virus transformed cells of two siblings with Primary Cortisol Resistance. The cells of the propositus and his brother show a decreased level of glucocorticoid receptor (GR) mRNA, and the genomic DNA of both individuals shows an altered restriction enzyme pattern with the restriction enzyme Bgl II, one of eleven restriction enzymes tested. The genomic differences could be detected with a probe specific for the putative steroid binding domain of the human GR gene.

Cell Transformation, Viral↗
PubMed1989

Dexamethasone control of growth hormone mRNA levels in GH3 pituitary cells is cycloheximide-sensitive and primarily posttranscriptional.

To clarify the mechanism of growth hormone (GH) gene activation by glucocorticoids in GH3 pituitary cells, GH mRNA accumulation in nuclear and cytoplasmic compartments was measured in the presence and absence of cycloheximide. In dexamethasone-treated cells, levels of GH mRNA were increased in the nucleus by 6 h and in the cytoplasm by 12 h. Dexamethasone treatment caused a 5- to 24-fold rise in total GH mRNA levels by 48-72 h. The differential elevation of nuclear levels of GH mRNA relative to the amount of cytoplasmic GH mRNA persisted for 48 h. A transient accumulation of GH mRNA in the nucleus was followed by a brief rise in cytoplasmic GH mRNA levels in GH3 cells treated simultaneously with dexamethasone and cycloheximide. In GH3 cells pretreated for 2 h with cycloheximide, the rise in nuclear and cytoplasmic GH mRNA levels mediated by dexamethasone was blocked completely. Levels of glucocorticoid receptor were unaffected by cycloheximide. These data suggest that the stimulation of GH mRNA levels by glucocorticoids is initiated within the nucleus and that cycloheximide-sensitive events are essential for this stimulation to occur. To assess the importance of GH gene transcriptional activation by glucocorticoids, nuclear transcription run-on reactions and assays of GH promoter activity in an aminoglycoside 3'-phosphotransferase (Neo) fusion gene within stably transformed GH3 cells were performed. Evidence for a weak, transient transcriptional activation of the GH gene by dexamethasone in nuclear run-on assays was obtained. Consistent with this idea, a 30-72 h exposure to dexamethasone raised levels of Neo mRNA in GH-Neo GH3 cell transformants by less than or equal to 2-fold. We conclude that glucocorticoid stimulation of GH mRNA in GH3 cells requires ongoing protein synthesis and can occur largely independently of GH gene transcriptional activation.

Animals↗
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

Extinction of growth hormone expression in somatic cell hybrids involves repression of the specific trans-activator GHF-1.

Growth hormone (GH) expression in pituitary-derived cells has been attributed to the presence of a positive trans-activator, GHF-1, which binds to two sites on the GH promoter. Somatic cell hybridization of non-GH-expressing L cells with pituitary-derived GH3 cells usually results in extinction of GH production. While previous studies showed that extinction occurs at the level of GH transcription, the exact mechanism remained elusive. We therefore characterized two parental cell lines and three hybrids, two of which extinguish GH expression and one in which GH is reexpressed after loss of mouse chromosomal material. Using in vivo transfections, in vitro transcription, DNAase I footprints, and immunoblotting experiments, no evidence for a direct repressor of GH transcription was found. Rather, extinction of GH expression in fibroblast x pituitary hybrids was accompanied by loss of GHF-1 protein and mRNA expression, suggesting that extinction occurs by repression of this trans-activator.

Animals↗
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

Unlinked regulation of the sensitivity of primary glucocorticoid-inducible responses in mouse mammary tumor virus infected Fu5-5 rat hepatoma cells.

The enzyme tyrosine aminotransferase (TAT) is induced by unusually low concentrations of glucocorticoids in Fu5-5 cells. We have isolated clones of Fu5-5 cells infected with mouse mammary tumor virus (MMTV) in order to simultaneously compare the glucocorticoid regulation of the host cell gene, TAT, with that of another primary inducible gene, MMTV. In the two clones that were examined in detail, MMTV RNA induction occurred at 4- to 11-fold higher concentrations of dexamethasone than those needed for induction of TAT mRNA. Furthermore, the amount of agonist activity displayed by the irreversible antiglucocorticoid dexamethasone 21-mesylate was greater for the induction of TAT mRNA than for MMTV RNA. These results extend our previous observations of unequal sensitivity of induction of TAT enzyme activity in two hepatoma cell lines and show that differential glucocorticoid regulation of gene induction within the same cell can occur at a pretranslational step. The present data also indicate that the unusual properties of TAT gene induction are not shared by all primary, glucocorticoid-inducible responses of the same cell and imply that additional factors mediating differential regulation of glucocorticoid-responsive genes are involved.

Animals↗
PubMed1987

Complementation between glucocorticoid receptor and lymphocytolysis in somatic cell hybrids of two glucocorticoid-resistant human leukemic clonal cell lines.

Somatic hybrids between two glucocorticoid-resistant clonal cell lines, CEM C1 and ICR-27, isolated independently from the CCRF-CEM human lymphoblastoid cell line, were constructed to investigate the complementation effect between the glucocorticoid receptor and the gene product(s) for inducing receptor-mediated lymphocytolysis. CEM C1 (r+ly-) has a normal amount of functional glucocorticoid receptor as compared to the steroid-sensitive clonal line CEM C7. Clone ICR-27 (r-ly?), which was originally isolated following mutagenesis of CEM C7 with the mutagen ICR 191, has few glucocorticoid receptors as determined by whole-cell receptor assay. The eight randomly selected CEM C1 X ICR-27 hybrid clones all showed sensitivity to 10(-6) M dexamethasone (ly+). The receptor site content of two near-tetraploid hybrids chosen for analysis was close to that of CEM C1 (r+). Hybrids constructed between CEM C1 and the receptor-bearing, steroid-sensitive clone, CEM C7 (r+ly+) also showed glucocorticoid sensitivity, and their receptor sites corresponded to the sum of those of CEM C1 and CEM C7 (r+r+ly+). These results indicate: that CEM C1 has no trans-active inhibitor of lysis; that CEM C1 has intact glucocorticoid receptor; and that ICR-27 and CEM C1 complement one another to restore lymphocytolysis. Therefore, CEM C1 cells can serve as a donor of human glucocorticoid receptors.

Cell Line↗