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E B Thompson

Publications and source records attributed to E B Thompson.

At least 73 records · Page 4Linked to original sources
PubMed1993

Identification of the activation-labile gene: a single point mutation in the human glucocorticoid receptor presents as two distinct receptor phenotypes.

CCRF-CEM-C7 is a well characterized human leukemic clonal cell line which is lysed by dexamethasone (dex). Originating from the wild-type CEM-C7 cells are two dex-resistant clones which are not lysed by 1 microM dex and have functionally defective glucocorticoid receptors (GR). They are receptorless ICR27TK.3 and activation-labile 4R4 cells. ICR27TK.3 and 4R4 cells have distinct cellular phenotypes, as indicated by dissimilar numbers of dex-binding sites despite similar levels of GR mRNA and immunochemically detectable GR. We have now investigated the molecular defects in the GR of ICR27TK.3 and 4R4 cells by determining the nucleotide sequence of their GR. Our results support the biochemical evidence previously reported by others for the presence of both a normal (GR+) and a mutant (GR*) allele in CEM-C7 cells. We clearly show that the wild-type CEM-C7 cells express two alleles of GR, the normal GR+ and the abnormal GR*, which has a Leu753--> Phe753 mutation. We demonstrate that both ICR27TK.3 and 4R4 cells contain only the abnormal GR* and that the normal GR+ gene is deleted in both of these GR defective clones. Our results further show that the GR* is basically an activation-labile receptor and has diminished functional capability in a transfection assay measuring GR-driven transcription. Thus, these two phenotypically different cell lines express similar amounts of an identical GR* containing a single point mutation at amino acid 753. A single point mutation in the steroid-binding domain of the GR, therefore, may behave differently, depending on the cellular milieu in which it is expressed.

Amino Acid Sequence↗
PubMed1993

Characterization of the human oxysterol receptor overexpressed in the baculovirus system.

Oxysterols are potent regulators of enzymes of the de novo cholesterol biosynthetic pathway and do not require the LDL (low density lipoprotein):LDL receptor system for their regulatory actions. The search for an alternate transduction system led to the identification of an oxysterol binding protein. This cytosolic protein has been extensively characterized, purified, and cloned. Although it fulfills the pharmacologic criteria for an oxysterol receptor by binding to oxysterols with affinities corresponding to their regulatory potencies, its function in maintaining cholesterol homeostasis has not been determined. We have overexpressed the human oxysterol receptor in Spodoptera frugiperda cells using the Baculovirus system. The overexpressed protein binds oxysterols, but not cholesterol. The affinity for 25-hydroxycholesterol determined by competitive binding assay was 7.3 +/- 4.4 nM (mean +/- SD), and the relative affinities of several other oxysterols approximately corresponded to their potencies in cell systems. The expressed protein migrated as a single immunoreactive band on denaturing polyacrylamide gels with a molecular mass of 94 kDa. The molecular mass calculated from sucrose gradient centrifugation and gel filtration was 273 kDa for the 9.8S form, 217 kDa for the 7.8S form, and 184 kDa for the 6.6S form. However, velocity gradient centrifugation and heparin-sepharose chromatography each indicated that there were at least two fractions containing specific oxysterol binding. We conclude that we have successfully overexpressed the human oxysterol receptor and that biochemical analysis of the overexpressed protein provides evidence of interactions with other proteins. Further analysis of the overexpressed protein should provide clues regarding its role in maintaining cholesterol homeostasis.

Animals↗
PubMed1993

Actions and interactions of glucocorticoids and transforming growth factor beta on two related human myeloma cell lines.

To evaluate possible involvement of a paracrine/autocrine inhibitory growth factor in myeloma cell growth, we studied the expression and actions of two forms of transforming growth factor beta (TGF-beta 1 and TGF-beta 2) on two closely related myeloma cell lines, OPM-1 and OPM-2. Earlier studies showed that both cell lines contain glucocorticoid receptors, but only OPM-2 cells are growth inhibited by dexamethasone (Dex). We found that OPM-2 growth was inhibited by TGF-beta, with TGF-beta 1 exerting a greater effect than TGF-beta 2, and Dex plus TGF-beta 1 acting synergistically. In OPM-1 (Dex insensitive), TGF-beta mRNA was not expressed, whereas it was induced by Dex in OPM-2. It was also possible to block partially the growth inhibition of Dex in OPM-2 cells by the addition of anti-TGF-beta 1 antibodies. These data suggest that the glucocorticoid effect(s) on myeloma cells may be mediated at least in part through modulation of internal and/or external levels of TGF-beta 1.

Dexamethasone↗
PubMed1992

Glucocorticoid, interleukin-2, and prostaglandin interactions in a clonal human leukemic T-cell line.

We have studied the growth effects of conditioned media, interleukin-2 and PGE prostaglandin analogs on the glucocorticoid-sensitive human leukemic T-cell clone, CEM-C7. After 4 days, the glucocorticoid dexamethasone at approximately 10 nM kills 50% of CEM-C7 cells. To test the hypothesis that glucocorticoid-mediated lymphocytolysis was due to suppression of lymphokine expression only, we attempted to protect CEM-C7 cells from lysis by provision of lymphokine(s). Conditioned media from interleukin-2 secreting Jurkat T-cells as well as the glucocorticoid-insensitive, but receptor positive clone, CEM-C1, failed to prevent lymphocytolysis; exogenous interleukin-2 also did not provide protection. There were complex, biphasic interactions between dexamethasone and the synthetic PGEs, enisoprost and enisoprost free acid. Low doses of enisoprost alone (0.01 to 1 microgram/ml) stimulated growth, and in combinations completely reversed the growth inhibitory effects of 10 nM dexamethasone. Higher concentrations of enisoprost were inherently lethal and were additive to the steroid effect. Thus the glucocorticoid-induced lymphocytolysis in this human leukemic T-cell line may be modified biphasically by PGE prostaglandins, depending on their concentration. However, interleukin-2 or components in the conditioned media assayed had no effect in ameliorating the lethal response to glucocorticoid.

Alprostadil↗
PubMed1992

Glucocorticoids in malignant lymphoid cells: gene regulation and the minimum receptor fragment for lysis.

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocorticoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocorticoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half "Zn fingers" of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, preceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.

Cell Line↗
PubMed1992

Extinction of prolactin gene expression in somatic cell hybrids is correlated with the repression of the pituitary-specific trans-activator GHF-1/Pit-1.

The tissue-specific expression of the PRL and GH genes is dependent on the presence of a pituitary-specific trans-activator, GHF-1/Pit-1. Previous studies indicate that somatic cell hybridization of rat pituitary GH3 cells with LB82 mouse fibroblasts frequently results in the extinction of GH and PRL expression. The extinction of the GH gene occurs at the transcriptional level, and is accompanied by repression of GHF-1/Pit-1 synthesis. To elucidate the mechanism of PRL extinction we further characterized these same somatic cell hybrid lines as well as the parental GH3 and LB82 cells. The pattern of PRL extinction and reexpression paralleled that of GH in the three hybrid lines that were examined. Two of these lines extinguished both GH and PRL synthesis, while a third displayed reexpression of both genes, apparently due to the loss of mouse chromosomal material. These studies revealed that the extinction of PRL expression in these hybrid lines occurs at the level of mRNA accumulation and is strongly correlated with the loss of GHF-1/Pit-1 mRNA and protein synthesis. These data suggest that in pituitary x fibroblast hybrids repression of the trans-activator GHF-1/Pit-1 is a primary mechanism for the extinction of PRL and GH gene expression.

Animals↗
PubMed1991

Mapping the human glucocorticoid receptor for leukemic cell death.

We have mapped the regions of the glucocorticoid receptor important for killing the cells of a line of human leukemic lymphoblasts. The results show that the glucocorticoid response element-specific DNA binding domain is essential, and that only the sequence including the amino acids that subsume the first zinc finger through about half of the second zinc finger are absolutely necessary. Furthermore, in contrast to assays of receptor mutants for ability to increase gene transcription, deletion of the steroid binding domain results in a functionally constitutive receptor fully as active for cell kill as is the holoreceptor after addition of ligand. Deletion of most known transcription-activation, dimerization, ligand-binding, and nuclear translocation regions still leaves a receptor fragment highly potent for cell lethality. The results suggest that delivery systems for such a fragment could result in effective new therapies for certain malignancies.

Animals↗
PubMed1991

Synergism between steroid response and promoter elements during cell-free transcription.

We have analyzed quantitatively the influence of distal promoter elements on steroid-responsive gene expression in vitro. Functional synergism between enhancer and distal promoter elements was examined using two model promoters, one containing a natural promoter (mouse mammary tumor virus long terminal repeat) and one constructed artificially. Human glucocorticoid receptor (GR) expressed in baculovirus induces transcription from a mouse mammary tumor virus long terminal repeat-containing DNA template. Transcription is diminished by oligonucleotides containing a nuclear factor 1 (NF-1)-binding site or a glucocorticoid/progesterone response element. Quantitative analysis indicates that NF-1 and GR act synergistically during transcriptional activation. In contrast, efficient activation by GR or purified chick progesterone receptor of a glucocorticoid/progesterone response element-linked ovalbumin promoter does not require interaction with the chicken ovalbumin upstream promoter (COUP) element in the distal promoter. Lack of synergism is not related to enhancer strength, since the glucocorticoid/progesterone response elements can be moved further from the promoter or reduced to a single copy response element without increasing the dependence upon COUP. Strong synergism is restored following substitution of an NF-1 distal promoter element for the COUP element in this construct. Our results suggest that synergism between steroid response and distal promoter elements is dependent upon the identity of the promoter element rather than upon the inherent strength of the enhancer element.

Animals↗
PubMed1991

Cortivazol mediated induction of glucocorticoid receptor messenger ribonucleic acid in wild-type and dexamethasone-resistant human leukemic (CEM) cells.

Cortivazol is a phenylpyrazolo glucocorticoid of high potency and unusual structure. In both wild-type and highly dexamethasone(dex)-resistant clones of the human leukemic cell line CEM, exposure to cortivazol leads to cell death. It has been shown recently that in wild-type CEM cells but not in a dex-resistant, glucocorticoid receptor(GR)-defective clone ICR-27 TK-3, dex induces GR mRNA. To test the hypothesis that cortivazol acts in dex-resistant cells by making use of the residual GR found there, wild-type and dex-resistant clones were treated with various concentrations of cortivazol and induction of GR mRNA was studied. Cortivazol significantly induced GR mRNA in the normal CEM-C7 as well as in two classes of dex-resistant clones, although the dex-resistant clones needed at least 10 times more cortivazol than the normal cells for significant GR mRNA induction. Increased levels of GR mRNA were noticed as early as 3 h after treatment. A general correlation between induction of GR mRNA and lysis of the normal and dex-resistant cells was found. Positive induction of GR mRNA might be one of the earliest crucial steps in the lysis of normal and dex-resistant CEM cells, or might serve as a marker for the process. However, the lysis pathway in the dex-resistant cells is defective in that dex-resistant clones needed significantly more cortivazol than the normal cells for lysis of the cells.

Cell Survival↗
PubMed1991

Molecular modification of anticholinergics as probes for muscarinic receptors. Part 4. Ileal selective muscarinic antagonists.

Systematic studies of the structure-activity relationships of atropine-like anticholinergic drugs have provided valuable information about the nature of the muscarinic receptor. In this study, the pharmacological activities of the (Z) and (E)-isomers of 2-phenylcyclohexyl diethylaminoethyl ether (1 and 2, respectively) in the isolated rat left atrium were investigated and compared with their activities in the isolated rat ileum preparation. Compound 1 was found to be one of the most ileal selective muscarinic antagonists reported to date. Other data concerning possible differences in the receptor-bound conformations of tropate- versus benzilate-derived muscarinic antagonists are also presented.

Animals↗
PubMed1990

Recombinant human glucocorticoid receptor induces transcription of hormone response genes in vitro.

A recombinant full length human glucocorticoid receptor stimulates transcription in vitro of test genes containing synthetic glucocorticoid and progesterone response elements or murine mammary tumor virus promoter. The receptor expressed in a baculoviral vector is highly active, enhancing transcription of hormone response genes greater than 30-fold even at a receptor concentration of 1.2 nM. The enhancement of transcription is glucocorticoid and progesterone response element-dependent, suggesting that it is a receptor mediated event. In vitro and in vivo treatment with the agonist dexamethasone or with the antagonist Ru486 did not alter significantly the functional activity of partially purified receptor. Kinetic studies suggest that both glucocorticoid receptor and HeLa cell extracts are required for formation of a stable committed transcriptional complex. Our results indicate that the action of glucocorticoid receptor on gene transcription is similar to that defined recently for the progesterone receptor and may be a general mechanism for all steroid receptors.

Animals↗
PubMed1990

Glucocorticoid effects on myeloma cells in culture: correlation of growth inhibition with induction of glucocorticoid receptor messenger RNA.

Glucocorticoids are widely used for the treatment of multiple myeloma. To investigate the direct actions of glucocorticoids on myeloma cells, we have used three cell lines of human multiple myeloma, OPM-1, OPM-2, and RPMI 8226. When growth curves of these cells were examined, OPM-1 cells were resistant, while OPM-2 were sensitive to dexamethasone (DEX). In cultures of OPM-2 cells, addition of DEX led to virtual cessation of growth, with only 16% of the residual cells viable after 4 days. RPMI 8226 appeared to be slightly sensitive, showing some slowing of growth for several days in DEX, with later recovery. Viabilities of OPM-1 and RPMI 8226 cells were not affected. Secretion of immunoglobulin (Ig-lambda) was also partially suppressed, by 30% in OPM-2 and 14% in OPM-1. No significant suppression was observed in RPMI 8226. To explore the mechanism of these differential responses to the steroid, glucocorticoid receptor (GR) was examined. Binding assays showed high affinity binding sites in all three cell lines: 64 +/- 11 fmol/10(6) cells in OPM-1, 78 +/- 14 in OPM-2, and 62 +/- 16 in RPMI 8226. Nuclear transfer of GR and DNA-cellulose binding after heat activation appeared similar in all three cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol proteins labeled with [3H]dexamethasone mesylate showed a GR of Mr 95,000 in all three. When GR mRNA was studied in these cells, all of them had GR mRNA of approximately 7 kilobases, but OPM-2 and RPMI 8226 had 3 times more GR mRNA than OPM-1. OPM-2 GR mRNA was induced 2-fold by DEX treatment at 5 x 10(-9) M or greater. OPM-1 GR mRNA was much less sensitive, with no response at less than 10(-6) M DEX and only 1.5-fold induction at that concentration. These results demonstrate that some myeloma cells can be killed by a direct action of glucocorticoids. The quantity and affinity of GR in the cells were not predictive of this response. Therefore, we propose that the resistance of OPM-1 and the relative resistance of RPMI 8226 to glucocorticoid inhibition of cell growth is by post-receptor mechanisms. The high sensitivity of induction of GR mRNA in OPM-2 may correlate with glucocorticoid-evoked cell kill.

Blotting, Northern↗
PubMed1990

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↗
PubMed1990

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media.

CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 micrograms/ml each transferrin and insulin + 5 ng/ml sodium selenite +/- 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo. with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability greater than or equal to 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium.

CD4 Antigens↗
PubMed1990

Steroid mediated lysis of lymphoblasts requires the DNA binding region of the steroid hormone receptor.

Glucocorticoids kill certain types of lymphoblasts, but the mechanisms are unknown. It is clear that sufficient numbers of functional glucocorticoid receptors are required to mediate lysis, but whether they do so through the classical model of steroid hormone activation and modulation of gene expression has not been established. In this report we have asked which region(s) of the steroid receptor are important for mediating lysis in leukemic T lymphoblasts. CEM-ICR 27 leukemic lymphoblasts, a clone of CEM cells which lack functional glucocorticoid receptors and therefore are neither lysed by dexamethasone nor capable of showing glutamine synthetase induction, were provided with steroid receptors by DNA transfections of various receptor gene constructs. We measured steroid mediated lysis, receptor number and induction of glutamine synthetase in the transfected cells. Our results provide evidence that the lysis mechanism in the ICR27 lymphoblasts is restored when functional receptor number is restored. The DNA binding region specifying high affinity for GRE sites is required. Lysis is mediated by any steroid that allows for activation of the receptor containing such a region. Our data support the view that steroid-mediated cell death occurs by a process requiring direct interaction of steroid-receptor complexes with the genome.

Binding Sites↗
PubMed1990

Hemoglobin S antigelation agents based on 5-bromotryptophan with potential for sickle cell anemia.

5-Bromotryptophan (5-BrTrp) is the most potent amino acid derivative reported in the literature to inhibit the gelation of hemoglobin S (from sickle cell anemia patients). Trp-Trp is also more potent than Trp as an antigelation agent. Therefore, we have prepared a series of dipeptides containing 5-BrTrp and evaluated the antigelation activity. 5-BrTrp-5-BrTrp is the most potent, i.e., 5.9 times the activity of Trp, followed by 5-BrTrp-Trp and then Trp-5-BrTrp. This improved antigelation potency for 5-BrTrp-5-BrTrp and 5-BrTrp-Trp is very significant and will be pursued further as lead compounds with potential for sickle cell anemia.

Anemia, Sickle Cell↗
PubMed1990

The promoter and first, untranslated exon of the human glucocorticoid receptor gene are GC rich but lack consensus glucocorticoid receptor element sites.

Glucocorticoid receptor mRNA is regulated by glucocorticoids. We found no consensus glucocorticoid response element, TATA box, or CAAT box but many GC boxes in approximately 3 kilobases of the 5'-flanking sequence of the human glucocorticoid receptor gene. We identified several transcription start sites, an untranslated exon 1, and the coding content of exon 2.

Base Sequence↗