cyclic-gmp and Leukemia--Erythroblastic--Acute

Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the beta-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the gamma-globin gene. sGC, an obligate heterodimer of alpha- and beta-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different beta-like globin genes showed that, whereas the beta-subunit is expressed at similar levels, high-level expression of the alpha-subunit is preferentially observed in erythroid cells expressing gamma-globin but not those expressing beta-globin. Also, the levels of expression of the gamma-globin gene correlate to those of the alpha-subunit. sGC activators or cGMP analogs increased expression of the gamma-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with beta-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the gamma-globin gene. Furthermore, increased expression of the gamma-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC-PKG pathway constitutes a mechanism that regulates expression of the gamma-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the beta-globin disorders.

Topics: Butyrates; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression; Globins; Guanylate Cyclase; Hemin; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Signal Transduction; Solubility; Tumor Cells, Cultured

Differentiation of murine erythroleukemia (MEL) cells induced by hexamethylene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-releasing agents and two membrane-permeable cGMP analogues. Since the effect of the NO-releasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosolic guanylate cyclase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced beta-globin and delta-aminolevulinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited the HMBA-induced changes in beta-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited HMBA-inducible enhancer function of the alpha-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothelial cells and macrophages, i.e. during acute or chronic inflammation.

Topics: 5-Aminolevulinate Synthetase; Acetamides; Animals; Base Sequence; Cell Differentiation; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Dimethyl Sulfoxide; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; GATA1 Transcription Factor; Gene Expression Regulation, Neoplastic; Globins; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Molsidomine; NF-E2 Transcription Factor; NF-E2 Transcription Factor, p45 Subunit; Nitric Oxide; Nuclear Proteins; Oligonucleotide Probes; Oncogenes; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Purinones; Recombinant Proteins; RNA, Messenger; Thionucleotides; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

1. The changes in electrophoretic distribution of cytosolic protein kinases have been studied in relation to proliferation, differentiation and transformation in murine erythroleukaemic cells, using a non-denaturing polyacrylamide gel electrophoresis system. 2. Native molecular masses of the major forms were determined by Ferguson plots. 3. A two dimensional electrophoresis method was developed for determination of the subunit molecular masses. 4. These studies suggest that the major bands of activity contain components which may correspond to cGMP dependent protein kinase, cAMP dependent protein kinase and protein kinase C. 5. On hexamethylene bisacetamide induced differentiation of the cells, changes in proportions of the different forms were observed.

Topics: Acetamides; Animals; Cell Differentiation; Cell Division; Cyclic AMP; Cyclic GMP; Cytosol; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Histones; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Phosphorylation; Protein Kinase C; Protein Kinases

Immediate changes in protein phosphorylation in Friend erythroleukaemia cells (FELC) were examined in response to stimulation with dimethylsulphoxide (DMSO), which triggers cell differentiation, 12-O-tetra-decanoyl-phorbol 13-acetate (TPA), a tumour promoter, and the Ca++ ionophore A23187. The effects of the cyclic nucleotides, cAMP and cGMP, on the patterns of phosphoproteins were also analysed. Autoradiographs of two-dimensional gels reveal that within 30 min of treatment by the various agents differences in the patterns of protein phosphorylation can be seen. Treatment with DMSO and TPA altered the phosphoprotein patterns in different ways. These patterns were distinct from those observed in untreated and cyclic nucleotide treated cells. Treatment with the calcium ionophore caused a unique phosphorylation pattern unlike any of the others. These data suggest that the very early changes seen in the patterns of protein phosphorylation in FELC may be related to the induction of differentiation.

Topics: Animals; Calcimycin; Cell Differentiation; Cyclic AMP; Cyclic GMP; Dimethyl Sulfoxide; Friend murine leukemia virus; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Phosphoproteins; Phosphorylation; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.

Topics: Adrenergic alpha-Agonists; Calcium; Carrier Proteins; Cell Line; Cyclic AMP; Cyclic GMP; Cytoplasm; Extracellular Space; Humans; Inositol Phosphates; Leukemia, Erythroblastic, Acute; Receptors, Adrenergic, alpha; Sodium-Hydrogen Exchangers

Various types of normal and neoplastic lymphoid cells in culture respond to interferon with elevated cyclic GMP levels. Human pre-T and natural killer cells are more sensitive to this effect than B lymphocytes. Cultured neurons and erythroid cells also show the effect. However, fibroblasts from different origin, although responsive to the antiviral effect of interferon, do not elevate their levels of cyclic GMP. Only fibroblasts from patients with Down's syndrome which are trisomic for chromosome 21 and more sensitive than their normal disomic counterparts to the antiviral action of interferon exhibit enhanced cyclic GMP levels following interferon treatment. These results suggest than an increase in cyclic GMP does not mediate the principal biologic actions of interferon. Further, the inability of certain cell types to increase their cyclic GMP content in response to interferon may reflect differences in their degree of interferon sensitivity rather than an inherent inability to respond to this action of interferon.

Topics: Animals; Antiviral Agents; Azides; B-Lymphocytes; Cats; Cell Line; Cells, Cultured; Chick Embryo; Cyclic GMP; Erythrocytes; Fibroblasts; Humans; Interferons; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Lymphocytes; Mice; Mice, Inbred Strains; Neurons; Sodium Azide; T-Lymphocytes

Topics: Animals; Cell Differentiation; Cell Line; Cyclic AMP; Cyclic GMP; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental