Increased endocytosis and formation of multivesicular bodies in phorbol ester-stimulated human monoblastic U-937 cells

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to arrest mitotic activity and induce macrophage differentiation in the U-937 monoblastic cell line. The acute effect of TPA on ultrastructural morphology and endocytic activity of U-937 cells was studied. TPA induced within 15 min a marked enlargement of multivesicular bodies (MVBs), comprising both volume and number of inclusion vesicles (other organelles appeared unchanged). At this stage the MBVs frequently showed tubular cytoplasmic extensions. Inclusion vesicles accumulated in MBVs with prolonged incubation (60 min). Horseradish peroxidase (HRP) and cationized ferritin (CF) added to the medium were routed preferentially to MVBs in TPA-stimulated cells. In contrast to MVBs of unstimulated cells many of the TPA-induced MVBs showed a positive cytochemical reaction to acid phosphatase. The MVBs in cells incubated with ionomycin, a calcium ionophore, did not differ from those of unstimulated cells. Cellular uptake of 125I-HRP was increased five times the control values already after 5 min of TPA stimulation. The uptake increased further with prolonged incubation (60 min), but at a slower rate. Together these indicate a TPA-induced transfer by endocytosis of portions of the plasma membrane to the lysosomal system via MVBs. Consideration of MVBs as part of the receptor-mediated endocytic pathway suggests that this effect of TPA might involve down-regulation of cell-surface receptors. The possibility of MVBs as a proton-sequestrating compartment, responsible for the cytoplasmic alkalinization previously reported for TPA-stimulated U-937 monoblastic cells, is discussed.     

Geldanamycin induces cell cycle arrest in K562 erythroleukemic cells

Geldanamycin (GA), a benzoquinone ansamycin, is one of the specific inhibitors of 90-kDa heat shock protein and induces growth inhibition and apoptosis in certain cancer cell lines. We have investigated the mechanism of GA-induced growth inhibition in K562 erythroleukemic cells. DNA flow-cytometric analysis indicated that GA-induced growth arrest was associated with G2/M phase arrest of the cell cycle. GA treatment down-regulated the expression of cyclin B1 and inhibited phosphorylation of Cdc2 protein, both key regulatory proteins at the G2/M boundary. GA also markedly inhibited the Cdc2 kinase activity, which may be in part a result of up-regulation of p27KIP1 by GA. The present results suggest a novel mechanism that p27KIP1 could be involved in the regulation of G2 to M phase transition.     

TPA induces cytoplasmic alkalinization in human monoblastic U-937 cells without activation of Na+/H+ exchange

The effect of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on cytoplasmic pH (pHi) and H+ extrusion was studied in the human monoblastic cell line U-937. About 2 min after addition of TPA, pHi started to increase and reached a steady state 10-15 min later. The resulting alkalinization corresponded to 0.03 and 0.09 pH units at 10(-10) and 10(-7) M TPA, respectively. The TPA-induced increase in pHi was independent of the presence of extracellular Na+. Moreover, TPA did not affect the H+ extrusion from the U-937 cells. Together these observations indicate the presence of a novel mechanism for TPA-induced cytoplasmic alkalinization. This mechanism is independent of Na+/H+ exchange across the plasma membrane, but may involve organelle sequestration of H+.     

Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na⁺-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroin-denyl]oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. © 1996 Wiley-Liss, Inc.     

The time-course of cyclic AMP signaling is critical for leukemia U-937 cell differentiation

The regulation of the cAMP signaling is intimately involved in several cellular processes, including cell differentiation. Here, we provide strong evidence supporting that the time-course of cAMP signal is critical for leukemia U-937 cell differentiation. Three stimulating-cAMP agents were used to analyze the correlation between cAMP time-course and cell differentiation. All three agents denoted similar cAMP maximal responses in dose-response experiments. The kinetic of desensitization showed differential characteristics, while H2 receptor desensitized homologously without affecting PGE2 or forskolin effect, PGE2 response showed mixed desensitization characterized by a homologous initial phase followed by a heterologous phase. Regarding forskolin, long-term stimuli attenuated PGE2 and H2 agonist response without affecting adenylyl cyclase activity. In the absence of phosphodiesterase inhibitors, the three agents induced similar maximal cAMP levels after 5 min, but only that induced by the H2 agonist returned to basal levels. Consistent with this observation, H2 agonist was not able to induce U-937 cell maturation in contrast to PGE2 and forskolin, supporting the importance of time-course signaling in the determination of cell behavior.     

Cell kinetics of the K-562 cell line in microculture

The cell kinetic parameters of K-562 leukemia cells were studied using microwell cultures in which growth was initiated from a single cell. Total population growth was studied by direct enumeration, 3H-thymidine labelling, and flow cytometry. Clonogenic cell growth was studied by replating and 3H-thymidine suicide. In 7-day clones of K-562 cells, durations of the total cell cycle, G1, S, G2, and M phases were 20.8 h, 3.5 h, 12.9 h, 3.3 h, and 1.1 h, respectively; the growth fraction was 0.92 and the cell loss factor was 0.084. Study of colony-forming cells by replating indicated that clonogenic cells comprised 40% of total cells. 3H-Thymidine suicide showed that cell-cycle duration for these cells was 22.5 h and that S-phase duration was 11.7 h.     

Ion channels in human macrophages compared with the U-937 cell line

Summary The human cell line U-937 has been used extensively to model many macrophage functions. We have examined the cell membranes of human monocyte-derived macrophages (HMDM) and U-937 cells to compare membrane properties as expressed by single ion channel currents. The patch-clamp technique was applied to isolated, nonactivated, inside-out patches of cell membranes obtained from HMDM and from the U-937 cell line. Voltage-gated potassium channels of similar conductance but different kinetics are present in both types of cells, and a calcium-activated potassium channel is present only in the HMDM. These differences in ion channel properties suggest fundamentally different behavior between these two cell types at the level of the cell membrane.     

Cytoplasmic pH is regulated in isolated avian osteoclasts by a Cl-/HCO3- exchanger

Osteoclast resorb bone in an acid compartment formed by the bone-attachment site. The low pH of the resorption compartment provides a lysosome-like milieu suitable for acid proteases to degrade collagen. Solubilization of the hydroxyapatite that makes up bone mineral consumes about 2 moles of protons per moles of calcium dissolved, requiring a massive proton flux to maintain a low pH in the resorption compartment. In order to determine how the osteoclast maintains a physiological cytoplasmic pH while secreting massive amounts of acid, we studied the intracellular pH of osteoclasts using esterified fluorescein derivatives while controlling the electrolyte composition of the medium. The principal finding is that osteoclasts have a high capacity for chloride/bicarbonate exchange which enables them to maintain normal intracellular pH in the face of a large loading of base equivalents. Thus, the overall process of proton secretion during bone resorption is similar to the polarized acid elimination by renal epithelia, involving a proton pump on one surface of the cell, and a Cl-/HCO3- exchange to maintain cytoplasmic pH.     

Iron metabolism is modified by the copper status of a human erythroleukemic (K562) cell line.

Copper deficiency is known to result in a microcytic, hypochromic anemia. Red cells of copper-deficient animals have less hemoglobin than their copper-adequate counterparts. The objective of this work was to determine what role copper plays in maintaining hemoglobin levels. It was hypothesized that the primary defect lies in intracellular iron metabolism. The influence of copper supplementation on iron uptake and storage was examined in a cell line capable of hemoglobin synthesis. The results demonstrated that copper supplementation of human K562 cells was associated with higher cytosolic iron levels and ferritin levels. Copper supplementation of the cell culture altered the initial rate of iron uptake from transferrin and enhanced iron uptake in noninduced cells; however, in hemin-induced K562 cells, which express fewer transferrin receptors on the cell surface, copper appeared to reduce iron uptake. Subsequent studies showed that the cells were able to take up the same amount of iron from transferrin when incubated over a longer period of time (24 hr). In the noninduced (non-hemoglobin synthesizing) cells, proportionally more iron was associated with the ferritin. We concluded from these studies that copper affects both uptake and storage of iron and that copper supplementation reduces cellular iron turnover.     

Cytoplasmic pH and glycolysis in the Dictyostelium discoideum cell cycle

Lactate production measurements during the cell cycle of synchronized populations of Dictyostelium discoideum cells reveal cyclic variations in glycolysis which correspond with pH_i oscillations which were discovered by us previously [(1985) Cell, in press]. Aerobic lactate production varies about 6-fold during the cell cycle and the lactate maxima correlate with (~ 0.25 pH unit) cyclic increases in pH. However, artificially altering pH_i using weak acids or bases does not influence the rate of lactate production in asynchronous cell populations. This result suggests that the cyclic variations in pH_i and those in glycolytic rate are not causally related events.     

Synergistic effect of phospholipid-based liposomes and propylthiouracil on U-937 cell growth

Scientific evidence indicates that exogenous phospholipids in the form of liposomes can affect cell growth. Effects of liposomes on cell growth depend on several factors including composition of liposomes, lipid concentration, and type of cells studied. Because phagocytic cells such as monocytes and macrophages are natural targets of liposomes, intracellular delivery of drugs to modulate cellular activity of these cells is of interest. We explored the effects of phospholipid-based liposomes composed of soy bean phosphatidylcholine (PC) as the main lipid component on U-937 cell growth. Effects of charge-imposing lipids and cholesterol were also studied. In addition, we investigated whether phospholipid-based liposomes would exert any interaction on cell growth with propylthiouracil, a drug with known antiproliferative activity. We found that PC in the form of extruded liposomes had intrinsic antiproliferative activity on U-937 cells at concentrations of 200 microM and up without any appreciable cytotoxicity. Phosphatidylserine and phosphatidylglycerol, but not dicetlylphosphate, at 10 mol% increased growth retardation activity of PC liposomes. Cholesterol at 30 mol% did not have any effect on cell growth, except for liposomes composed of PC and phosphatidylserine, where growth retardation was negated in the presence of cholesterol. Synergistic effect on cell growth was seen with certain liposome compositions when 5.5 microg/mL of propylthiouracil was coincubated. The results of this study suggest that the effects of exogenous lipids on cell growth should be taken into consideration when PC-based liposomes are to be used as drug delivery systems, especially when the targets are cells with phagocytic activity.     

Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines.

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.     

Mitochondrial damage and metabolic compensatory mechanisms induced by hyperoxia in the U-937 cell line

Experimental hyperoxia represents a suitable in vitro model to study some pathogenic mechanisms related to oxidative stress. Moreover, it allows the investigation of the molecular pathophysiology underlying oxygen therapy and toxicity. In this study, a modified experimental set up was adopted to accomplish a model of moderate hyperoxia (50% O(2), 96 h culture) to induce oxidative stress in the human leukemia cell line, U-937. Spectrophotometric measurements of mitochondrial respiratory enzyme activities, NMR spectroscopy of culture media, determination of antioxidant enzyme activities, and cell proliferation and differentiation assays were performed. The data showed that moderate hyperoxia in this myeloid cell line causes: i) intriguing alterations in the mitochondrial activities at the levels of succinate dehydrogenase and succinate-cytochrome c reductase; ii) induction of metabolic compensatory adaptations, with significant shift to glycolysis; iii) induction of different antioxidant enzyme activities; iv) significant cell growth inhibition and v) no significant apoptosis. This work will permit better characterization the mitochondrial damage induced by hyperoxia. In particular, the data showed a large increase in the succinate cytochrome c reductase activity, which could be a fundamental pathogenic mechanism at the basis of oxygen toxicity.     

Modulation of Fc gamma receptors on the human macrophage cell line U-937

Macrophage receptors for the Fc portion of IgG play an important role in host defense, inflammation, and the pathophysiology of autoimmune disorders. We studied one important function of Fc gamma receptors--the ability to bind IgG ligand. Direct binding experiments analyzed by nonlinear regression were consistent with monomeric and trimeric IgG binding to a single class of receptors. Indirect binding experiments were also consistent with this interpretation and revealed that both IgG ligands completely inhibited the binding of the other. In addition, we used an anti-Fc gamma RII monoclonal antibody known to compete for the Fc gamma RII ligand binding site and known to inhibit IgG trimer binding to other cells. At concentrations of antibody which saturated all Fc gamma RII sites, no inhibition of IgG trimer binding to U-937 was observed. This was evident despite the observation that the numbers of Fc gamma RI and Fc gamma RII, determined by equilibrium binding of monomeric IgG and anti-Fc gamma RII antibody, respectively, were similar on U-937. Monoclonal antibodies were used to compare the expression and modulation of Fc gamma receptor proteins with their ability to bind monomeric and trimeric IgG ligands. Dexamethasone and gamma-interferon regulated U-937 Fc gamma RI protein expression and IgG ligand binding to a similar degree. In contrast, the expression of Fc gamma RII was not altered by dexamethasone. Interferon-gamma primarily stimulated Fc gamma RI, as determined both by reactivity with monoclonal antibody (227 +/- 26%) and by monomeric IgG ligand binding (350 +/- 151%). In addition, dexamethasone inhibited by 33% the gamma-interferon effect on Fc gamma RI protein and by 56% the effect on Fc gamma RI binding of monomeric IgG. Preincubation of U-937 with anti-Fc gamma RII antibody did not alter the effect of dexamethasone or gamma-interferon on IgG trimer binding. These data indicate that on U-937 cells Fc gamma RII does not function in the recognition of small molecular weight immune complexes and that Fc gamma RI is the Fc gamma receptor responsible for the binding of both monomeric and trimeric human IgG. Furthermore, Fc gamma RI is the major Fc gamma receptor on U-937 that is modulated by both gamma-interferon and glucocorticoids.     

Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein

Royal jelly was fractionated by ion-exchange chromatography and a protein (DIII protein) that had growth stimulating activity to the U-937 human myeloid cell line was obtained. The molecular weight of the DIII protein was 58 kDa on SDS-PAGE. The growth stimulating activity of the DIII protein was shown to be relatively heat and pH stable.     

Patterns of glycosidases in the histiocytic cell line U-937. Effects of agents inducing cell differentiation

1. The cell bound glycosidases in sublines and clones of the histiocytic cell line U-937 have previously been shown to display characteristic patterns. 2. In this paper the effects of differentiation inducing agents upon glycosidase patterns of one subline, U-937 GTB, are presented. 3. Teleocidin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), dimethyl-sulfoxide (DMSO), dihydroxyvitamin D3 and supernatants from mixed lymphocyte culture (MCL) all induce cellular differentiation of U-937 GTB. 4. Significant changes of the levels of cell bound glycosidases were seen after addition of inducing agents. 5. Alterations have been monitored as relative effects upon the absolute glycosidase activities and as effects upon selected ratios of different glycosidases. 6. The separate inducing agents show distinct enzyme patterns.     

Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons

The acquisition of neuronal type-specific morphogenesis is a central feature of neuronal differentiation and has important consequences for region-specific nervous system functions. Here, we report that the cell type-specific cholesterol profile determines the differential modulation of axon and dendrite outgrowths in hippocampal and cerebral cortical neurons in culture. The extent of axon and dendrite outgrowths is greater and the polarity formation occurs earlier in cortical neurons than in hippocampal neurons. The cholesterol concentrations in total homogenate and the lipid rafts from hippocampal neurons are significantly higher than those from cortical neurons. Cholesterol depletion by beta-cyclodextrin markedly enhanced the neurite outgrowth and accelerated the establishment of neuronal polarity in hippocampal neurons, which were similarly observed in nontreated cortical neurons, whereas cholesterol loading had no effects. In contrast, both depletion and loading of cholesterol decreased the neurite outgrowths in cortical neurons. The stimulation of neurite outgrowth and polarity formation induced by cholesterol depletion was accompanied by an enhanced localization of Fyn, a Src kinase, in the lipid rafts of hippocampal neurons. A concomitant treatment with beta-cyclodextrin and a Src family kinase inhibitor, PP2, specifically blocked axon outgrowth but not dendrite outgrowth (both of which were enhanced by beta-cyclodextrin) in hippocampal neurons, suggesting that axon outgrowth modulated by cholesterol is induced in a Fyn-dependent manner. These results suggest that cellular cholesterol modulates axon and dendrite outgrowths and neuronal polarization under culture conditions and also that the difference in cholesterol profile between hippocampal and cortical neurons underlies the difference in neurite outgrowth between these two types of neurons.