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Biomedical subjects

E B Thompson

Publications and source records attributed to E B Thompson.

At least 109 records · Page 6Linked to original sources
PubMed1987

The structure of the human glucocorticoid receptor and its gene.

The human glucocorticoid receptor (hGR) is a ubiquitous, phosphorylated protein of Mr about 95,000, as determined by PAGE or other physical methods. It is found intracellularly, and current immunocytochemical studies suggest that, in the absence of ligand, it is predominantly cytoplasmic. Limited proteolysis shows that the protein can be divided into steroid-binding, DNA-binding and strongly antigenic regions, roughly one-third each of the molecule. Recently, the hGR gene has been cloned and sequenced, and analysis of the gene has confirmed and extended this general description of the protein. The DNA-binding region is cysteine-rich, and the carboxy-terminal end of the molecule is the steroid-binding region. Analysis of CEM cells, a line of human leukemic lymphoblasts, has resulted in isolation of three classes of resistant cells: receptor-site deficient (r-), receptor activation labile (r act/l), and wild-type receptor but lysis-defective (r+ly-). Monoclonal antibodies to the hGR have been produced and used to show that the r- clone does contain immunoreactive receptor. Northern blots of the RNA of these cells, probed with the full-length hGR cDNA, reveal that the r- cells have mRNA for hGR of normal size but somewhat less in amount than normal r+ cells. Previous somatic cell hybrids had shown failure to complement in crosses between r- and r act/l mutants, but dominance of the sensitive phenotype in r+ x r- and r+ x r act/l hybrids. We now show, however, that the r+ly- clone does complement the receptor-site deficient (r-) mutant. Hybrids between the two are completely steroid sensitive.

Antibodies, Monoclonal↗
PubMed1987

Molecular modification of anticholinergics as probes for muscarinic receptors. 1. Amino esters of alpha-substituted phenylacetic acid and related analogues.

Two series of compounds having the general structure of C6H5CRR'COOCH2CH2NEt2 were synthesized and examined for their antispasmodic activities. These compounds were selected as structural probes for exploring the nature of muscarinic cholinergic receptor binding sites that interact with atropine-like anticholinergics. These studies indicate a rather strict size limitation for the hydrophobic region of the receptor and suggest intramolecular hydrogen bonding as a possible means to explain the observed stereoselectivity.

Alkylation↗
PubMed1987

Molecular modification of anticholinergics as probes for muscarinic receptors. 3. Conformationally restricted analogues of benactyzine.

The synthesis and pharmacological evaluation of conformationally restricted analogues of certain anticholinergic agents is a powerful method for probing the topography of the muscarinic receptor. In the present study, clues as to the binding conformation of structurally flexible anticholinergics are provided by approximating certain conformations of benactyzine by synthetic analogues 1-6, which are structurally locked into desired conformations. The pharmacological activity of each analogue is an indication of how well particular conformational models are accommodated by the receptor. The conformation of benactyzine in which an intramolecular hydrogen bond may exist between the hydroxyl group and the carbonyl oxygen of the ester group (conformation I) is approximated by the synthetic analogue 2,2-diphenyl-3-tetrahydrofuranyl (diethylamino)ethyl ether (1) and related analogues. Pharmacological evaluation using dose-response experiments on isolated rat ileum tissue demonstrated that these compounds noncompetitively inhibited acetylcholine-induced ileum contractions. Restriction of the conformational freedom of the amino side chain of 1 by synthesis of the hexahydro[3,4-b]furan derivative 3 provided a weak but competitive inhibitor at low concentration. The conformation of benactyzine in which an intramolecular hydrogen bond may exist between the hydroxyl group and the ether oxygen of the ester group (conformation II) is approximated by 2,2-diphenyl-4-[2-(diethylamino)ethyl]-3-tetrahydrofuranone (4). Pharmacological studies showed that this compound competitively inhibited acetylcholine-induced ileum contractions. These experiments provide evidence that receptor-bound conformation II for benactyzine is preferred over conformation I in providing competitive binding with the muscarinic receptor.

Acetylcholine↗
PubMed1987

Molecular modification of anticholinergics as probes for muscarinic receptors. 2. Amino esters of alpha-methyltropic acid.

As a continuation of our goals to study molecular probes for muscarinic cholinergic receptors, a series of 3-substituted 2-methyl-2-phenylpropanoates with the general structure of C6H5C(CH2X)(CH3)COOCH2CH2NEt2 where X = OH, OTs, F, Cl, Br, I, and OAc were prepared and their antispasmodic activities examined on isolated rat ileum preparations. Structure-activity relationship studies with these compounds provide further evidence suggesting that binding of an aromatic moiety in a specific location within the hydrophobic region of the receptor is important for anticholinergic potency. A nucleophilic displacement of chloride by "naked" fluoride under mild conditions is also reported.

Animals↗
PubMed1987

Establishment and characterization of a human myeloid cell line from Philadelphia chromosome-negative myeloblastic leukemia arising in a patient with myelodysplastic syndrome.

A new hematopoietic cell line derived from a patient with Philadelphia chromosome (Ph1)-negative myeloblastic leukemia arising from a form of myelodysplastic syndrome (MDS) is described. This cell line, designated TMM, consists of immature cells with the morphological characteristics of young myeloblasts and grows in suspension culture with a doubling time of about 30 hours. By cytochemical analysis the cultured cells were positive for acid phosphatase. They were free of the Epstein-Barr virus-associated nuclear antigen as well as terminal deoxynucleotidyl transferase. Further phenotypic analysis revealed the expression of the myelomonocytic-specific antigen Leu-M1 and receptors for the Fc portion of IgG. Partial differentiation of these cells could be induced by dimethyl sulfoxide, tetradecanoyl phorbol acetate, or hypoxanthine and resulted in cells of the myeloid series expressing lysozyme and receptors for the C3b complement protein. The karyotype was 46,XY, lacked the Ph1 chromosome, and displayed no abnormalities at the light microscopic level. No rearrangement of the bcr-c-abl gene complex was found. This cell line should be useful for studying an important type of the heterogeneous population constituting Ph1-negative myeloblastic leukemia, arising in this instance from MDS, as well as for studying differentiation and proliferation of human pluripotent stem cells.

Antigens, Neoplasm↗
PubMed1986

Rat growth hormone gene expression is correlated with an unmethylated CGCG sequence near the transcription initiation site.

The methylation status of the rat growth hormone (GH) gene was compared in DNA obtained from GH-producing and GH-nonproducing sources by digestion with three methylation-sensitive restriction enzymes. GH gene expression was correlated with an unmethylated ThaI site (CGCG) 144-bp upstream of the GH RNA transcription initiation site. This ThaI site was unmethylated in nine GH-producing subclones of the rat pituitary tissue culture cell line GH3 and in greater than 50% of the DNA isolated from rat anterior pituitary, a gland containing GH-producing somatotroph cells as well as GH-nonproducing cells. In DNA prepared from GH-nonproducing tissues, e.g., rat spleen, kidney, liver, and brain, this ThaI site was entirely methylated. Furthermore, this site was entirely methylated in hybrid cells formed by the fusion of GH3 cells with mouse fibroblasts in which GH production has been extinguished. DNA methylation at 10 other restriction sites located throughout the rat GH gene region failed to correlate with GH expression in GH-producing subclones of GH3 cells as well as in GH-nonproducing GH3 X LB82 hybrid cells. We suggest that the conserved absence of methylation 144 bp 5' of the RNA transcription initiation site of transcribed GH genes identifies a potential GH gene control region.

Adenoma↗
PubMed1986

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

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

Animals↗
PubMed1986

A time-course study of hypothyroidism-induced hypotension: its relation to hypothermia.

Dietary administration of propylthiouracil (0.2%) to male Sprague-Dawley rats for a period of 8 weeks induced hypothyroidism and hypotension in these animals. Resting heart rate, body weight, colonic temperature and serum thyroxine (T4) were significantly lower in the treated hypothyroid rats than in the controls. Time-course study indicates that the maximal depression of these parameters occurred about the eighth week of PTU treatment; except T4 level which declined abruptly about the second week and remained steady thereafter. The mechanism(s) of hypotension is (are) unknown but there seems to be an interrelationship between hypothyroidism-induced hypotension and the parameters monitored, since discontinuation of PTU treatment reversed that trend of the response toward control levels.

Animals↗
PubMed1985

Primary structure and expression of a functional human glucocorticoid receptor cDNA.

Identification of complementary DNAs encoding the human glucocorticoid receptor predicts two protein forms, of 777 (alpha) and 742 (beta) amino acids, which differ at their carboxy termini. The proteins contain a cysteine/lysine/arginine-rich region which may define the DNA-binding domain. Pure radiolabelled glucocorticoid receptor, synthesized in vitro, is immunoreactive and possesses intrinsic steroid-binding activity characteristic of the native glucocorticoid receptor.

Amino Acid Sequence↗
PubMed1985

Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain.

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA.

Animals↗
PubMed1985

Glucocorticoid receptors in human leukemias and related diseases.

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

Animals↗
PubMed1985

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

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

Cloning, Molecular↗
PubMed1985

The karyotype of the glucocorticoid-sensitive, lymphoblastic human T-cell line CCRF-CEM shows a unique deleted and inverted chromosome 9.

Chromosome karyotypes were prepared from the highly glucocorticoid-sensitive clone C7 of the human acute lymphoblastic leukemia T-cell line CCRF-CEM. The modal number of chromosomes is 47, and one chromosome #9 has a pericentric inversion of the heterochromatin region (9qh) plus a deletion of the short arm. In most cells, there is an extra chromosome #20. All other chromosomes appear to be normal. Examination of the uncloned line CCRF-CEM (ATCC CCL 119), which was frozen away shortly after the line was originated and has undergone fewer passages than CEM C7, also revealed the same abnormality of chromosome #9. Each of 10 other clones isolated from CCRF-CEM in this laboratory also contained the abnormal chromosome #9.

Cell Line↗
PubMed1985

Formation of a fluorescent glucocorticoid receptor-steroid complex in HTC cell cytosol.

An intensely fluorescent rhodamine derivative of dexamethasone (i.e. Dex-C2-Rho) was synthesized. Dex-C2-Rho possessed high affinity for HTC cell glucocorticoid receptors in cell-free systems. Whole cell activity and receptor affinity of Dex-C2-Rho were both much lower, apparently due to problems with cell permeability and/or metabolism. A specific, fluorescent receptor-steroid complex at concentrations as low as 1 X 10(-9) M could readily be observed with crude HTC cell receptors after removal of the free Dex-C2-Rho. This appears to be the first report of a fluorescent glucocorticoid receptor-steroid complex.

Animals↗
PubMed1985

[3H]cortivazol: a unique high affinity ligand for the glucocorticoid receptor.

Cortivazol (CVZ) and deacylcortivazol (DAC) are pyrazolosteroids with potent glucocorticoid activity. In previous work we showed that DAC is 40-fold more potent than dexamethasone (DEX) in lysing leukemic lymphoblasts. To assess the interaction between these atypical steroids and the glucocorticoid receptor, we examined the binding of [3H]CVZ to cytosol from glucocorticoid-sensitive and -resistant variants of the human leukemic cell line CEM C7. In glucocorticoid-sensitive cells [3H]CVZ causes a 2-fold induction of glutamine synthetase and binds to a protein in the 4.6 S region of high salt sucrose gradients. On DEAE-cellulose chromatography, [3H]CVZ-receptor complexes show a shift from high (0.25 M KP) to low salt (0.09 M KP) eluting forms upon activation. CVZ competes for a 97,000-dalton protein labeled by [3H]dexamethasone mesylate. Scatchard analysis of the binding of [3H]CVZ in glucocorticoid-sensitive cells revealed a curvilinear plot which resolved into high (0.4 nM) and low (11 nM) affinity components. The receptor concentration of the low affinity site (0.30 pmol/mg protein) was approximately twice that of the high affinity site (0.14 pmol/mg protein). Dissociation experiments with dilution and/or excess unlabeled CVZ supported the presence of independent sites. In contrast, the binding of [3H]DEX to C7 cytosol revealed a single class of binding sites (Kd = 1.9 nM; receptor concentration, 0.46 pmol/mg protein). Examination of the binding of [3H]CVZ using 10(-5) M DEX as the competing ligand showed that DEX binds only to the low affinity site detected by [3H]CVZ. In cytosol from a glucocorticoid-resistant cell line with virtually no [3H]DEX binding, [3H]CVZ detected a single high affinity binding site that was similar in dissociation constant (0.8 nM) and receptor concentration (0.13 pmol/mg protein) to the high affinity site detected in the glucocorticoid-sensitive cell line C7.

Binding, Competitive↗
PubMed1985

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

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

Cell Line↗