cytochalasin-b and Leukemia--Myeloid

Leukemic cells are arrested in less differentiated stages of development. However, there are many reports that various leukemic cells can be induced to differentiate into mature cells with attenuation of proliferative and leukemogenic activity. Mouse myeloid leukemia (Ml) cells both in vitro and in vivo can be induced to differentiate into macrophages and granulocytes by various compounds. During long-term culture of Ml cells, however, some populations of the cells spontaneously become resistant to induces of cell differentiation. The isolated resistant variant cell lines have higher leukemogenicity than parent cells and produce differentiation inhibitory protein factors (I-factor). The variant cell lines are useful to studies on how leukemic cells are arrested in less differentiated stages even in the presence of differentiation inducers both in vitro and in vivo. We have purified and characterized the I-factor. We review herein the properties of the I-factor and other inhibitory factors of differentiation of myeloid leukemia cells.

Topics: Animals; Cell Differentiation; Cytochalasin B; Cytokines; Humans; Leukemia, Myeloid; Prostaglandins; Proto-Oncogenes; Tetradecanoylphorbol Acetate

We investigated the mechanism of beta 1 integrin-mediated adherence of stimulated granulocytic HL60 cells to fibronectin using a monoclonal antibody (15/7) that recognizes beta 1 integrins only when the receptors are active for ligand binding. Phorbol myristate acetate (PMA) stimulated expression of the 15/7 epitope on granulocytic HL60 by nearly fivefold but had an insignificant effect on the expression of the epitope on undifferentiated HL60 cells. These results paralleled the effect of PMA on HL60 and granulocytic HL60 adhesion to fibronectin, indicating that activation of beta 1 integrins is important for beta 1-mediated adherence of granulocytic HL60 cells to fibronectin. Agonists that stimulate alpha 5 beta 1-dependent human polymorphonuclear leukocyte (PMN) adhesion to fibronectin (C5a and PMA) also upregulated the 15/7 epitope on purified human PMNs. Although PMA rapidly induces increased levels of filamentous actin (F-actin) in granulocytic HL60 cells and a decrease in F-actin levels in undifferentiated HL60 cells, depolymerization of the actin cytoskeleton with cytochalasin B did not affect increased expression of the 15/7 epitope on granulocytic HL60 cells. Cytochalasin B did, however, inhibit granulocytic HL60 adherence to fibronectin by 50%, demonstrating that actin polymerization is important for optimal beta 1-dependent granulocytic adherence.

Topics: Actins; Adult; Cell Adhesion; Cell Differentiation; Complement C5a; Cytochalasin B; Cytoskeleton; Epitopes; Fibronectins; Granulocytes; Humans; Integrin beta1; Integrins; Leukemia, Myeloid; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation

While the primary targets for granulocyte-macrophage colony-stimulating factor (GM-CSF) are hematopoietic precursors and mature myeloid cells, GM-CSF receptors (GMR) are also found on normal tissues including placenta, endothelium, and oligodendrocytes as well as certain malignant cells. The function of GMR in these nonhematopoietic cells is unknown. We studied the function of GMR in human melanoma cell lines. Six of seven cell lines tested (clones 1-5 and 3.44 of SK-MEL-131, SK-MEL-188, SK-MEL-23, SK-MEL-22, and SK-MEL-22A) expressed mRNA encoding the membrane-bound and soluble isoforms of the alpha subunit of the GMR. Melanoma cell lines in early stages of differentiation expressed the largest quantities of alpha-subunit mRNA. Although five of these lines expressed trace levels of mRNA encoding the beta subunit of the GMR, Scatchard analysis of equilibrium binding data derived from three of the cell lines showed that they expressed only low-affinity GMR. Clones 3.44 and 1-5 of SK-MEL-131, and SK-MEL-188 cells expressed receptors with a dissociation constant (kd) for GM-CSF in the following ranges: 0.7 to 0.8, 1.2 to 1.8, and 0.4 to 0.8 nmol/L, respectively. GM-CSF stimulated glucose uptake in four of the melanoma cell lines expressing the alpha subunit, presumably through facilitative glucose transporters, as uptake was blocked by cytochalasin B but not cytochalasin E. Stimulation of glucose uptake was transient, with maximum stimulation occurring at approximately 30 minutes in the presence of 1 nmol/L GM-CSF. GM-CSF stimulated glucose uptake 1.4- to 2.0-fold but did not stimulate cell proliferation. These results suggest a metabolic role for the low-affinity GMR in melanoma cell lines and indicate that the alpha subunit of the GMR can signal for increased glucose uptake in nonhematopoietic tumor cells.

Topics: Base Sequence; Biological Transport, Active; Cell Line; Cytochalasin B; Cytochalasins; Deoxyglucose; DNA Primers; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kinetics; Leukemia, Myeloid; Macromolecular Substances; Melanoma; Molecular Sequence Data; Monosaccharide Transport Proteins; Polymerase Chain Reaction; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Topics: Acute Disease; Antigens, CD; Azacitidine; Cell Differentiation; Cell Division; Cell Line; Cytochalasin B; Cytokines; Fibroblast Growth Factor 2; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Humans; Leukemia, Myeloid; Megakaryocytes; Microscopy, Immunoelectron; Models, Biological; Receptors, Cytokine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Crocidolite, one of the most carcinogenic asbestos fibers, induces the release of reactive oxygen species (ROS) from neutrophils and macrophages. Using HPLC combined with electrochemical detection, we determined that 8-hydroxydeoxyguanosine (8OHdG), a molecule typical of mutagenic oxidative DNA damage, was induced in the cellular DNA of a human promyelocytic leukemia cell line, HL60, incubated with crocidolite. Crocidolite increased 8OHdG in the cellular DNA of phorbol myristate acetate (PMA)-differentiated HL60, which phagocytosed crocidolite. PMA-differentiated HL60 released ROS spontaneously, as determined by ESR with 5,5-dimethylpyrrolone-N-oxide as a spin trap. However, the release of ROS from the cell line did not increase after the addition of crocidolite. The addition of superoxide dismutase at a sufficient concentration to scavenge ROS released from the cell did not inhibit the 8OHdG increase induced by crocidolite. Cytochalasin B, which inhibited phagocytosis, did not inhibit the release of ROS. However, it inhibited the crocidolite-induced 8OHdG increase by 48.3%. Contrary to PMA-differentiated HL60, undifferentiated HL60 neither phagocytosed crocidolite nor showed a crocidolite-induced increase in 8OHdG formation. The 8OHdG increase induced by crocidolite was not correlated with ROS release, but with the internalization of crocidolite, suggesting that the increase was not due to an increase in ROS release from the cell but was due to the conversion of relatively inert ROS to highly reactive ROS, such as hydroxyl radicals, by crocidolite that was internalized and close to DNA.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Asbestos, Crocidolite; Catalase; Cell Differentiation; Cytochalasin B; Deoxyguanosine; DNA Damage; DNA, Neoplasm; Humans; In Vitro Techniques; Leukemia, Myeloid; Reactive Oxygen Species; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Isolated granulocytes from normal individuals and patients suffering from chronic myeloid leukemia (CML) displayed different fluorescent patterns on treatment with fluorescein isothiocyanate concanavalin A (Fl-Con A). The ligand was internalized by 86% of the normal granulocytes, while 80% of the leukemic granulocytes exhibited Fl-Con A localized on the cell periphery. In further experiments, pretreatment of the normal granulocytes with cytochalasin B, iodoacetamide, 2-deoxyglucose and sodium fluoride (but not with sodium azide or dinitrophenol) was found to drastically inhibit internalization of the ligand. However, pretreatment of granulocytes from CML patients with cytochalasin B and 2-deoxyglucose, caused only a little alteration in the pattern of Fl-Con A labelling relative to untreated cells. These results indicate that CML granulocytes are defective in their ability to endocytose Fl-Con A. We suggest that this differential interaction between Fl-Con A and normal and leukemic granulocytes is a convenient system to study the initial steps in receptor mediated endocytosis of Concanavalin A.

Topics: Azides; Cell Differentiation; Cell Membrane; Colchicine; Concanavalin A; Cytochalasin B; Endocytosis; Fluorescein-5-isothiocyanate; Fluoresceins; Granulocytes; Histocytochemistry; Humans; Iodoacetamide; Kinetics; Leukemia, Myeloid; Sodium Azide; Sodium Fluoride; Time Factors

D-glucose carrier protein in K562 cell membrane was studied by photoaffinity labeling with cytochalasin B. The saturable cytochalasin B binding in purified K562 cell membranes was 90 pmol/mg and 200 pmol/mg protein in the presence of D-glucose and D-sorbitol, respectively. More than half of the total cytochalasin B binding could be depressed by D-glucose. The results of SDS-PAGE analysis of K562 cell membranes after photoaffinity labeling at 0.1 microM cytochalasin B showed that the main peak of covalently bound [3H]-cytochalasin B was in the Mr range of 46-65 KDa. The label found in the peak was reduced by more than 50% in the presence of 0.5 M D-glucose, the inhibition similar being to that obtained in the binding experiment. This polypeptide has a slightly higher molecular weight than that of the human erythrocyte cell membrane.

Topics: Affinity Labels; Cell Fractionation; Cell Line; Cell Membrane; Cytochalasin B; Electrophoresis, Polyacrylamide Gel; Glucose; Humans; Kinetics; Leukemia, Myeloid; Monosaccharide Transport Proteins

A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI-3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor-dependent cells (FDC-P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism. We have investigated the effect of HCGF on FDC-P cells further and found that: (i) HCGF markedly and rapidly increases lactate production; (ii) high extracellular glucose or glycolytic intermediate concentrations can maintain FDC-P cell viability to some extent whilst stimulating lactate production; (iii) the uptake of 2-deoxyglucose by FDC-P2 cells is stimulated by HCGF in a dose-dependent fashion. This uptake is inhibited by cytochalasin B; (iv) HCGF does not stimulate L-glucose uptake by FDC-P cells. These results suggest that HCGF acts to maintain FDC-P cells via its action on glucose transport. The significance of these results to haemopoiesis and leukaemogenesis is discussed.

Topics: Adenosine Triphosphate; Biological Transport, Active; Cell Survival; Cytochalasin B; Energy Metabolism; Glucose; Glycolysis; Growth Substances; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Lactates; Leukemia, Myeloid

Topics: Animals; Bromodeoxyuridine; Cell Division; Cell Line; Cell Movement; Culture Media; Cytarabine; Cytochalasin B; Depression, Chemical; Floxuridine; Latex; Leukemia, Myeloid; Mice; Mice, Inbred Strains; Microspheres; Mitosis; Phagocytosis; Polystyrenes; Radiation Effects; Trypsin; X-Rays