monensin and Leukemia--Erythroblastic--Acute

Topics: Amiloride; Animals; Calcimycin; Calcium; Carrier Proteins; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dimethyl Sulfoxide; Electrolytes; Erythropoiesis; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lipopolysaccharides; Lymphocytes; Lymphoma; Mice; Monensin; Ouabain; Protons; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase

Wheat Germ Agglutinin (WGA) cytotoxicity has been studied using two human leukemia cell lines, Molt3 and K562, and human peripheral blood mononuclear cells (PBMC). In spite of similar binding at the cell surface, WGA was found to promote cell death to a different extent in Molt3, K562 and PBMC and to induce different death events leading to apoptosis in Molt3 and either apoptosis and necrosis in K562 cells and PBMC. In Molt3 but not in K562 cells, WGA cytotoxicity could be potentiated 66-200 fold by 50 nM monensin, a carboxylic ionophore that perturbs the intracellular trafficking of endocytosed molecules. Synergism between the cytotoxic activities of WGA and monensin was demonstrated in Molt3 cells by comparing non toxic, or slightly toxic, doses of WGA and monensin alone or in combination. These results show that the cytotoxic effect of WGA is dependent on internalisation events which may differ among the cell lines used. WGA and monensin can enter the human diet being a component of wheat germ and an antibiotic used for zootechnic reasons in the bioindustry, respectively. These data reveal the synergistic effect between two dietary molecules, otherwise per se toxic at much higher concentrations, with possible implications for human and animal health.

Topics: Antifungal Agents; Apoptosis; Drug Interactions; Humans; Leukemia, Erythroblastic, Acute; Leukemia, T-Cell; Leukocytes, Mononuclear; Monensin; Tumor Cells, Cultured; Wheat Germ Agglutinins

The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37 degrees C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (< 25 degrees C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron-(II) chelators such as 2,2'-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 microM cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2'-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-55Fe, followed by addition of iron(II) and/or iron(III) chelators and extraction of chelator-55Fe complexes into organic solvent. Virtually all of the accumulated 55Fe in the chelatable pool could be complexed by iron(II) chelators. The cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5 microM, and its mean transit time through the chelatable pool was 1-2 h. We conclude that after iron is translocated from the endosomes, it is maintained in the cytosol as a transit pool of chelatable iron(II). The ostensible absence of chelatable iron(III) implicates the intracellular operation of vigorous reductive mechanisms.

Topics: 2,2'-Dipyridyl; Ammonium Chloride; Azides; Biological Transport; Cell Line; Chloroquine; Cytoplasm; Endocytosis; Fluoresceins; Humans; Iron; Iron Chelating Agents; Kinetics; Leukemia, Erythroblastic, Acute; Monensin; Receptors, Transferrin; Transferrin; Tumor Cells, Cultured

Because a major limitation of ODN (oligodeoxynucleotide) use is inefficient cellular uptake, methods to improve ODN uptake could have important implications in the investigational and possibly therapeutic use of ODNs. In this study, antisense c-myc ODN cellular uptake in elevated extracellular calcium was increased up to 48-fold in the four cell lines examined. The role of calcium in ODN cellular uptake was examined using a 21-base ODN complementary to the c-myc proto-oncogene and the Rauscher cells. Cells were pretreated with uptake inhibitors in either 1.8 (physiologic) or 5.4 mM calcium prior to addition of (32P) labelled ODN. In physiologic calcium conditions, ODN cellular uptake was partially dependent on cellular energy and a trypsin-sensitive surface protein. In contrast, in the presence of elevated (5.4 mM) extracellular calcium, trypsinization and metabolic inhibition had a reduced and no effect, respectively, on uptake. Endocytosis and lysosomotropic inhibitors did not decrease uptake in either calcium concentrations. Therefore, the mechanism of ODN uptake may depend on the level of extracellular calcium. Furthermore, surface binding accounted for approximately 60% of total uptake in both physiologic and elevated calcium concentrations, suggesting that the increased uptake was not due exclusively to increased surface binding. Thus, the predominant mechanism of ODN uptake may depend on the extracellular calcium concentration.

Topics: Animals; Arsenicals; Base Sequence; Biological Transport; Calcium; Cell Line; Chloroquine; Deoxyglucose; Genes, myc; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Macrophages; Molecular Sequence Data; Monensin; Oligonucleotides, Antisense; Proto-Oncogene Mas; Trypsin; Tumor Cells, Cultured

The O-glycosylation of leukosialin, a major sialoglycoprotein found on leukocytes, has been studied in the human erythroleukemic cell line K562. The appearance of its O-linked chains has been followed in pulse-chase experiments with [35S]methionine by immunoprecipitation with an anti-peptide antiserum as well as with a lectin from Salvia sclarea seeds (SSA) specific for GalNAc-Ser/Thr and the peanut (Arachis hypogaea) agglutinin (PNA) which recognizes Gal beta 1----3GalNAc-Ser/Thr structures. An O-glycan-free precursor was converted into the fully O-glycosylated mature form within the 10-min labeling period and no intermediates carrying only GalNAc-Ser/Thr structures could be detected. The ionophore monensin was used in order to slow down intracellular traffic and thus O-glycan synthesis. The drug partly inhibited the transport from rough endoplasmic reticulum (RER) to the Golgi and also the cell-surface expression of leukosialin. It was found to have a marked effect on the synthesis of O-linked carbohydrate structures of leukosialin since the amount of O-glycans containing only GalNAc or NeuNAc alpha 2----6GalNAc was significantly increased after monensin treatment. Under these conditions the biosynthesis of the N-glycan on leukosialin was completely arrested in an endoglycosidase-H-sensitive step of processing, whereas the O-glycans already contained galactose and sialic acid although at a reduced level. On the other hand, the small amounts of leukosialin expressed on the cell surface of monensin-treated cells carried the same glycans as those remaining blocked inside the cell. In addition, immunocytochemical studies using SSA and PNA on untreated K562 cells suggested the absence of detectable amounts of GalNAc-Ser/Thr-bearing glycoproteins in the RER as well as in the Golgi. In contrast Gal beta 1----3GalNAc structures could be detected on intracellular membranes which were tentatively identified as the cis-Golgi. Together these results lead us to the following conclusions: N-glycan transfer occurs in the RER before the initiation of O-glycans which takes place at the entrance of the protein into the Golgi; further elongation of O-glycans with galactose and sialic acid follows very rapidly, probably before the final processing of N-glycans to complex-type structures.

Topics: Antigens, CD; Biological Transport; Cell Line; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Glycoside Hydrolases; Glycosylation; Golgi Apparatus; Humans; Lectins; Leukemia, Erythroblastic, Acute; Leukosialin; Microscopy, Electron; Monensin; Oligosaccharides; Polysaccharides; Sialoglycoproteins

The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells. The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only. The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids. Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin. In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of [3H]TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions. On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of [3H]TPP+-accumulation. These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential.

Topics: Amino Acids; Cell Line; Culture Media; Humans; Leukemia, Erythroblastic, Acute; Membrane Potentials; Monensin; Onium Compounds; Organophosphorus Compounds; Ouabain; Rubidium Radioisotopes; Tumor Cells, Cultured

The endocytosis and recycling of the human transferrin receptor were evaluated by several experimental modalities in K562 cells perturbed with 10(-5) M monensin. The work presented is an extension of a previous study demonstrating both complete inhibition of release of internalized human transferrin and a 50% reduction in the number of cell surface transferrin binding sites in K562 cells treated with monensin (Stein, B. S., Bensch, K. G., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772). The data directly reveal the existence of two distinct transferrin receptor recycling pathways. One pathway is monensin-sensitive and is felt to represent recycling of transferrin receptors through the Golgi apparatus, and the other pathway is monensin-resistant and most likely represents non-Golgi-mediated transferrin receptor recycling. A transferrin-free K562 cell culture system was developed and used to demonstrate that cell surface transferrin receptors can be endocytosed without antecedent ligand binding, indicating that there are factors other than transferrin binding which regulate receptor internalization. Evidence is presented suggesting that two transferrin receptor recycling pathways are also operant in K562 cells under ligand-free conditions, signifying that trafficking of receptor into either recycling pathway is not highly ligand-dependent.

Topics: Antibodies; Cell Line; Cell Membrane; Cycloheximide; Endocytosis; Fluorescent Antibody Technique; Golgi Apparatus; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Monensin; Receptors, Cell Surface; Receptors, Transferrin; Transferrin

Human monocytes (M phi) show high cytolytic activity towards antibody-coated tumor cells (AbK562). In this report, the relationship between the cytoskeleton in the M phi and the M phi cytolytic activity has been investigated. The actin filament inhibitors cytochalasin B and dihydrocytochalasin B (H2CB) both reduced M phi-mediated lysis of AbK562 cells by approximately 50% at a concentration of 1 microM. This concentration of H2CB did not inhibit the number of target cells bound to M phi. Dihydrocytochalasin B did not inhibit the M phi ability to release cytotoxic protein factors, suggesting that H2CB does not inhibit lysis by inhibiting release of cytotoxic protein factors. Immunofluorescence microscopy showed a rapid accumulation of actin filaments towards the contact area in more than 80% of the examined M phi-AbK562 conjugates. Exposure to H2CB did not prevent this accumulation, but caused aggregation of the accumulated actin filaments in the contact area with the target cell. Accumulation of actin filaments did not occur toward tumor cells not coated with antibodies. Scanning and thin section electron microscopy demonstrated large M phi pseudopodia directed toward the AbK562 cells, with close apposition of the effector and target cell membranes with interdigitations. The formation of the M phi pseudopodia was inhibited by exposure to H2CB. These observations indicate that M phi membrane motility toward AbK562 cells is closely related to M phi-mediated lysis of AbK562 cells. Immunofluorescence microscopy of the microtubule-organizing center (MTOC) and the Golgi apparatus revealed that both the MTOC and the Golgi apparatus in M phi reoriented towards the bound AbK562 cells in approximately 45% of the examined M phi-AbK562 conjugates. The microtubule-depolymerizing drugs colchicine and vinblastine did not inhibit M phi-mediated lysis of AbK562 cells at concentrations which disrupted the microtubule arrays in the M phi. The carboxylic ionophore monensin, which blocks Golgi-derived secretion, inhibited M phi-mediated lysis of AbK562 to a lesser extent as compared to H2CB. These results suggest that microtubule functions are of less importance in M phi-mediated lysis of AbK562 cells as compared to actin filament functions. However, the MTOC and the Golgi apparatus could participate in M phi-mediated lysis of AbK562 cells by mechanisms related to secretion of cytotoxic molecules.

Topics: Actins; Adult; Animals; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Cytoskeleton; Fluorescent Antibody Technique; Golgi Apparatus; Humans; Leukemia, Erythroblastic, Acute; Microtubules; Monensin; Monocytes; Rabbits

The highly selective cytotoxicity of site-directed ricin A chain conjugates can be potentiated by membrane-active carboxylic ionophores. The combined use of the two agents results in much faster inactivation of ribosomes and subsequent cell death and lysis. The potency of A chain cytotoxins is correspondingly increased by several orders of magnitude and cells that sparsely express the target antigen or receptor can be killed.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cytotoxins; Disulfides; Furans; Humans; Ionophores; Leukemia, Erythroblastic, Acute; Mice; Monensin; Receptors, Cell Surface; Receptors, Transferrin; Ricin; Transferrin