calcimycin 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

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

Topics: Animals; Bacterial Proteins; Blotting, Western; Calcimycin; Cell Survival; Complement C9; Dose-Response Relationship, Drug; HCT116 Cells; Hemolysis; HSP70 Heat-Shock Proteins; Humans; Ionophores; K562 Cells; Leukemia, Erythroblastic, Acute; Pyridines; Rabbits; Recombinant Proteins; RNA Interference; Streptolysins; Thiazoles

The Ca2+-induced expression of the primary response genes egr-1 and c-fos was investigated in the murine erythroleukaemia cell line ELM-I-1. Exposure of the cells to the Ca2+-ionophore A23187 led to a rapid transient rise in egr-1 and c-fos mRNA production followed by an increase in Egr-1 and c-Fos protein levels as well as an increase in Egr-1 and activator protein 1 (AP-1) DNA-binding activity. Preincubation of the cells with KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinases, strongly decreased the Ca2+-mediated expression of egr-1 and c-fos. In contrast, treatment with cyclosporin A, which inhibits the Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin, increased both egr-1 and c-fos mRNA production and the DNA-binding activity of the Egr-1 and AP-1 transcription factors in response to the intracellular Ca+ concentration ([Ca2+]i)-increasing agents A23187 or cyclopiazonic acid. Enhancement of the Ca2+-induced c-fos and egr-1 expression by cyclosporin A was correlated with the capability of this agent to inhibit calcineurin phosphatase activity in ELM-I-1 cells. Studies on the phosphorylation state and DNA-binding activity of the cAMP response element-binding protein (CREB) did not demonstrate an early Ca2+-dependent activation of this transcription factor, suggesting that the regulation of c-fos and egr-1 expression by Ca2+ is not linked to CREB in the haematopoietic ELM-I-1 cells. The results indicate that calcineurin exerts negative regulatory effects on both egr-1 and c-fos expression in murine erythroleukaemia cells, in addition to the calcineurin-mediated down-regulation of c-myb expression observed previously in this cell system. This study therefore emphasizes the important role of calcineurin as a negative modulator of gene expression in certain cell types.

Topics: Animals; Calcimycin; Calcineurin Inhibitors; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cyclic AMP Response Element-Binding Protein; Cyclosporine; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Genes, fos; Immediate-Early Proteins; Leukemia, Erythroblastic, Acute; Mice; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-fos; RNA, Messenger; Tacrolimus; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca2+)-activated 86Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of 125I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell KCa2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca2+ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca2+. The extent of inhibition of whole cell MEL KCa2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single KCa2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca2+ currents in PC12 cells >> Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single KCa2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells.

Topics: Animals; Animals, Newborn; Antifungal Agents; Calcimycin; Calcium; Clotrimazole; Ionophores; Leukemia, Erythroblastic, Acute; Mice; Patch-Clamp Techniques; PC12 Cells; Potassium Channels; Rats; Rats, Wistar; Superior Cervical Ganglion; Tumor Cells, Cultured

The human immature K562 erythroid cell line was studied for its capacity to produce and to metabolize the phospholipid molecule platelet-activating factor (PAF). K562 cells produced PAF under calcium ionophore stimulation. Lyso PAF and acetyl-CoA (the acetate donor molecule for the acetylation of lyso PAF into PAF) had no effect on the amounts of PAF produced by ionophore-stimulated cells. The metabolism of PAF and lyso PAF by K562 cells was compared to that of freshly-isolated human bone marrow erythroblasts and blood erythrocytes. K562 cells rapidly metabolized [3H]PAF and [3H]lyso PAF with 1-alkyl analogue of phosphatidylcholine as the major metabolic product. In contrast, blood erythrocytes did not. PAF acetylhydrolase activity levels in K562 cells and bone marrow erythroblasts were similar and higher than in blood erythrocytes. PAF (1-100 nM) stimulated [3H]thymidine incorporation in K562 cells grown in low serum concentration, a non-metabolizable PAF agonist being more potent than PAF to stimulate thymidine incorporation. PAF receptor mRNA was detected in K562 cells by polymerase chain reaction on reverse transcripts. The present study demonstrates that K562 cells produce and metabolize PAF and underlines the putative role of erythroid precursors in the modulation of bone marrow PAF concentrations. The effect of PAF on the growth of K562 cells might be mediated through PAF receptors suggesting a potential role of PAF on the proliferation and functions of human erythroid marrow precursors.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Acetyl Coenzyme A; Calcimycin; Cell Division; Cells, Cultured; Erythroblasts; Erythrocytes; Humans; Ionophores; Leukemia, Erythroblastic, Acute; Phospholipases A; Platelet Activating Factor; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; RNA, Messenger; Tumor Cells, Cultured

Heat shock proteins (HSPs) are induced in mammalian cells in a variety of pathophysiological states and have an important role in cytoprotection in vitro and in vivo. In this study, we report that the calcium ionophore A23187, a glucose-regulated protein (GRP) inducer, dramatically inhibits HSP70 synthesis and HSP70 mRNA transcription after induction by heat shock, sodium arsenite, or prostaglandin A1 treatment in human K562 cells. A23187 does not suppress, and it actually prolongs, the DNA-binding activity of the human heat shock transcription factor (HSF), while it alters HSF1 phosphorylation in heat shock-treated cells. To inhibit HSP70 expression, A23187 needs to be present during heat shock, while treatment before or after heat shock does not affect HSP70 mRNA transcription. The GRP inducer thapsigargin, which specifically inhibits the endoplasmic reticulum Ca2+-ATPase, has no effect on heat-induced HSP70 synthesis, indicating that A23187 inhibitory activity is not due to depletion of intracellular calcium stores and is independent of the concomitant induction of GRP genes. Inhibition of HSP70 expression is correlated with alterations in HSF1 phosphorylation in heat-shocked cells, but not in sodium arsenite-treated cells, indicating that different mechanisms may be involved in mediating A23187 inhibitory activity.

Topics: Calcimycin; Calcium-Transporting ATPases; Cell Line; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression; Heat Shock Transcription Factors; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Ionophores; Leukemia, Erythroblastic, Acute; Membrane Proteins; Methionine; Molecular Chaperones; Phosphorylation; Protein Biosynthesis; RNA, Messenger; Terpenes; Thapsigargin; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Translation has an established role in the regulation of cell growth. Posttranslational modification of translation initiation and elongation factors or regulation of mRNA polyadenylation represent common means of regulating translation in response to mitogenic or developmental signals. Induced differentiation of Friend virus-transformed erythroleukemia cells is accompanied by a rapid decrease in the translation rate of these cells. Although inducers do not alter initiation factor modifications, characterization of their effect on mRNA translation provides evidence that this is mediated by the poly(A)-binding protein (PABP). Inducer exposure results in an increase in the amount of mRNA that sediments at 80 S and a decrease in the amount in polysomes. Although these 80 S ribosomes have characteristics previously attributed to "vacant ribosomal couples," including lability in 500 mM KCl and an inability to incorporate amino acids into protein, we provide evidence that these 80 S complexes are not vacant but contain mRNA that is stably bound to the 40 S subunit, whereas the 60 S subunit is dissociated from the complex by high salt. The absence of eukaryotic initiation factor 2 from these complexes suggests that translation has proceeded through subunit joining. Immunoblotting demonstrates that the mRNAs in these 80 S ribosomal complexes do not contain bound PABP and that this protein is found to be almost exclusively associated with translating polysomes. These data suggest that the PABP plays a role in the accumulation of these 80 S ribosomal.mRNA complexes and may facilitate the formation of translationally active salt-stable ribosomes.

Topics: Amino Acids; Animals; Calcimycin; Cell Differentiation; Dimethyl Sulfoxide; Eukaryotic Initiation Factor-2; Gene Expression Regulation, Leukemic; Leukemia, Erythroblastic, Acute; Mice; Poly(A)-Binding Proteins; Polyribosomes; Potassium Chloride; Protein Biosynthesis; Ribosomes; RNA-Binding Proteins; RNA, Messenger; RNA, Neoplasm; Subcellular Fractions; Tumor Cells, Cultured

A high-mobility group 1 (HMG1) protein type isolated from murine erythroleukaemia (MEL) cells promotes acceleration of the differentiation process when added to a MEL cell culture together with the inducer hexamethylene bisacetamide. We now provide direct evidence that the presence of HMG1 protein in the extracellular medium is essential for terminal erythroid differentiation. An extracellular function for HMG1 protein in MEL cell is further supported by a demonstration that this protein is released from MEL cells exposed to the chemical inducer and that the addition of an anti-(HMG1 protein) monoclonal antibody to the cell culture inhibits the differentiation process almost completely. The release of HMG1 protein from MEL cells is modulated by compounds affecting cell calcium homoeostasis, such as a calcium ionophore or verapamil. In fact, in the presence of the ionophore an increased rate of differentiation is accompanied by an enhanced extracellular release of HMG1 protein, whereas in the presence of verapamil both phenomena are significantly decreased.

Topics: Acetamides; Animals; Antineoplastic Agents; Calcimycin; Cell Differentiation; High Mobility Group Proteins; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured

This study was conducted on human K562 lymphocytes to investigate the mechanisms implicated in the regulation of the serotonin transport process. The uptake of serotonin in these cells was saturable (Km, 3.37 microM; Vmax, 2.03 nmol/10(6) cells) and Na+ dependent; isoosmotic replacement of Na+ with choline chloride in the assay medium resulted in the decreased uptake process. Augmentation of intracellular free calcium, [Ca2+]i, by thapsigargin decreased the uptake of serotonin in these cells. Similarly, addition of calcium ionophores (A23187) and ionomycin also inhibited serotonin transport. In Fura-2-loaded cells, these agents increased the [Ca2+]i contents. These results suggest that an increase in [Ca2+]i is implicated with a decrease in serotonin transport. Since an increase in [Ca2+]i is known to activate calmodulin (CaM), we employed CaM antagonists. Calmodulin antagonists W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalene-sulfonamide) and mellitin inhibited serotonin uptake in K562 cells, suggesting that CaM is involved in serotonin transport regulation. Furthermore, acute exposure of K562 cells to known protein kinase C (PKC) activators, phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoylglycerol (DiC8), curtailed serotonin uptake by these cells. However, staurosporine (a PKC inhibitor) failed to abolish the inhibitory effects of PMA on serotonin transport in these cells, indicating that the target of PMA is not PKC. Nonetheless, the PMA-induced inhibitory effects are specific as 4 alpha-phorbol-12,13,didecanoate (a phorbol ester known not to activate PKC) failed to mimic PMA-like actions on serotonin transport in K562 cells. DiC8 not only exerted higher inhibitory effects than PMA but also had additive effects in the presence of the latter on serotonin transport. These results suggest that in addition to PKC, there are other cellular targets (of PMA) implicated in serotonin transport regulation.

Topics: Biological Transport; Calcimycin; Calcium; Calmodulin; Enzyme Activation; Humans; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Lymphocytes; Protein Kinase C; Serotonin; Serotonin Antagonists; Thapsigargin; Tumor Cells, Cultured

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Animals; Base Sequence; Calcimycin; Cell Line; Conserved Sequence; Gene Expression; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Leukemia, Erythroblastic, Acute; Microsomes; Models, Molecular; Molecular Sequence Data; Oligodeoxyribonucleotides; Protein Conformation; Rats; RNA, Messenger; Sequence Homology, Amino Acid; Tumor Cells, Cultured

It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.

Topics: Actin Cytoskeleton; Calcimycin; Calcium Channel Blockers; Cytochalasin B; Edetic Acid; Endocytosis; Humans; Iron; Leukemia, Erythroblastic, Acute; Leukemia, Promyelocytic, Acute; Microscopy, Electron; Microtubules; Oxidation-Reduction; Phalloidine; Reactive Oxygen Species; Receptors, Transferrin; Stress, Physiological; Tumor Cells, Cultured; Vitamin K

The effects of calcium ionophores A23187 and ionomycin on the c-myb and c-myc mRNA levels have been investigated in the Friend erythroleukemia cell line F4-6 using Northern blot analysis. Treatment of the cells with 0.5-4 microM A23187 or 1-4 microM ionomycin induced a concentration-dependent decrease in c-myb mRNA; this decrease was abolished by EGTA. c-myc mRNA levels were only moderately affected. After 12-24 h of calcium ionophore exposure, c-myb mRNA returned to pretreatment levels. No similar decrease in c-myb mRNA was seen with the sodium ionophore monensin (up to 16 microM). The dimethyl sulfoxide-induced suppression of c-myb and also of c-myc mRNA levels was not prevented in Ca(2+)-free medium and thus appeared Ca(2+)-independent. A23187 and ionomycin were capable of inducing beta-globin mRNA synthesis in F4-6 cells. Prolonged calcium ionophore exposure, however, strongly reduced cell viability and resulted only in a slight hemoglobin increase at lower concentrations. These results suggest that a rise in [Ca2+]i may be a signal leading to a transient decrease in c-myb mRNA and the initiation of erythroid differentiation in Friend cells. The transient suppression of c-myb mRNA levels represents a common feature of the action of dimethyl sulfoxide and calcium ionophores.

Topics: Actins; Animals; Blotting, Northern; Calcimycin; Cell Survival; Dimethyl Sulfoxide; Egtazic Acid; Friend murine leukemia virus; Gene Expression Regulation, Neoplastic; Genes, myc; Hemoglobins; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; RNA, Messenger; Tumor Cells, Cultured

Human erythroleukemia cells transformed arachidonic acid and prostaglandin endoperoxide H2 into thromboxane A2. Stimulation of these cells with A23187 or thrombin, however, produced no thromboxane. Similarly, cells labeled with [3H]-arachidonic acid released no detectable label upon stimulation. Data suggest that human erythroleukemia cells contain the enzymatic capacity for thromboxane formation from exogenous precursors, but lack the endogenous mechanisms for arachidonate release. The presence of thromboxane synthase messenger RNA was verified using the polymerase chain reaction. Amplification and sequence analysis of a 528 bp cDNA demonstrated virtually 100% identity to a published thromboxane synthase cDNA fragment.

Topics: Amino Acid Sequence; Arachidonic Acid; Base Sequence; Calcimycin; Electrophoresis, Polyacrylamide Gel; Gene Expression; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Polymerase Chain Reaction; Prostaglandins H; RNA, Messenger; Sequence Homology, Nucleic Acid; Thrombin; Thromboxane A2; Thromboxane-A Synthase; Tumor Cells, Cultured

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid-specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Calcimycin; Calcium; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Erythroid Precursor Cells; Gene Expression; Globins; Ionomycin; Leukemia, Erythroblastic, Acute; Mice

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 erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.

Topics: Adenosine Diphosphate; Calcimycin; Calcium; Cell Line; Enzyme Activation; Epinephrine; Glycerophospholipids; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Myristic Acid; Myristic Acids; Phosphatidic Acids; Phosphatidylinositols; Phospholipase D; Phospholipases; Platelet Activating Factor; Tetradecanoylphorbol Acetate; Thrombin; Tumor Cells, Cultured

This study deals with the modulation of the plasma membrane potential (delta psi p) of murine erythroleukemia (MEL) cells by cell-substratum or cell-cell contact. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane; it appeared strongly, and inversely, influenced by the two types of cell contacts. Contact with the culture surface produced a delta psi p hyperpolarization directly proportional to average distance among the ideal centers of the cells on this surface (d) within the range 10-80 microns. A detailed mathematical analysis of the function delta psi p = f(d) is presented, as well as experiments involving the use of ionophores (valinomycin and A23187) and the conditioning of the culture surface. We concluded that the d-dependent hyperpolarization (dDH) was the result of a complex interplay between the activating properties of substratum on Ca2+-dependent K+ channels (KCa) and some substratum-adherent factors that are shed by MEL cells and antagonize KCa activation (substratum-attached cellular factors = SACF). By contrast, contact of the cells with each other, obtained by incubating MEL cells at d smaller than the average cell diameter (phi = 10 microns), produced a marked delta psi p depolarization. This intercellular contact-dependent depolarization (ICDD) was unaffected by valinomycin; it was abolished by substituting Na+ in the external medium with a nondiffusible cation (choline), which shows that ICDD was sustained by Na+ influxes, probably mediated by stretch-activated (s.a.) cation channels.

Topics: Animals; Biomechanical Phenomena; Calcimycin; Calcium; Cations; Cell Communication; Cell Membrane; Cells, Cultured; Electrophysiology; Extracellular Space; Ion Channels; Ions; Leukemia, Erythroblastic, Acute; Mice; Potassium Channels; Sodium; Valinomycin

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetamides; Animals; Calcimycin; Calcium; Cell Differentiation; Cell Membrane Permeability; Chlorides; Diazepam; Dimethyl Sulfoxide; Electrochemistry; Ion Channels; Leukemia, Erythroblastic, Acute; Mathematics; Membrane Potentials; Mice; Potassium; Sodium; Tetraethylammonium Compounds; Tetrodotoxin; Time Factors; Valinomycin

Since the involvement of calcium ions in the regulation of cell division and differentiation has been proposed, in this study we have examined the effect of extracellular calcium and of calcium-modulating agents on the DMSO-induced differentiation of murine erythroleukaemia cells. Neither proliferation nor differentiation of these cells was affected by calcium deprivation in the culture medium. Moreover, calcium-chelating agents or agents blocking intracellular calcium uptake induced a marked inhibition of cell differentiation. Intracellular calcium antagonists induced inhibition when cells were grown in a calcium-deprived medium. In contrast, murine erythroleukaemia cell differentiation was unaffected by agents that increased intracellular concentration of calcium. Our results indicate that a mobilization of calcium is indispensable for eliciting full cellular response, but the increase in intracellular level of this cation is not sufficient for complete signal transduction. It is likely that a marked alteration of the intracellular calcium system and availability could be responsible for the independence of our cell system from calcium modulation.

Topics: 4-Aminopyridine; Aminopyridines; Amrinone; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Egtazic Acid; Erythrocytes; Erythropoiesis; Friend murine leukemia virus; Gallic Acid; Leukemia, Erythroblastic, Acute; Mice; Razoxane; Verapamil

An alteration in the rate of calcium transport appears to be the rate-limiting event for the commitment of murine erythroleukemia (MEL) cells to initiate a program of terminal erythroid differentiation. The dimethyl sulfoxide (DMSO)-induced commitment of MEL cells to erythroid differentiation can be inhibited by treatment of cells with the calcium-chelating agent EGTA. Upon removal of EGTA, cells initiate commitment without the 12-h lag normally observed after treatment with DMSO alone. Treatment of cells with DMSO in the presence of calcium ionophore A23187 causes cells to initiate commitment from time zero with no lag. These results suggest that the lag is the time required for DMSO to alter the calcium transport properties of the cell.

Topics: Animals; Biological Transport; Calcimycin; Calcium; Cell Line; Dimethyl Sulfoxide; Egtazic Acid; Erythropoiesis; Leukemia, Erythroblastic, Acute; Mice

The action of procaine on the terminal erythroid differentiation of murine erythroleukemia (MEL) cells has been investigated at the level of individual cells. At concentrations (7 X 10-4 M) which had no inhibitory effect on cell growth, pretreatment of these cells with procaine for 12-24 hr caused a pronounced inhibition ( greater than 90%) of commitment of terminal erythroid differentiation of dimethyl sulfoxide (DMSO)-treated cells. Simultaneous treatment of MEL cells with DMSO and procaine, however, resulted to only slight inhibition (less than 20%) of commitment. Blockade of commitment by procaine pretreatment appears to be general since it was observed in cells treated with other inducers (6-thioguanine, dimethylformamide). Procaine pretreatment did not abolish the ability of MEL cells to complete the "latent period" and commit upon the removal of the block. Reversal of procaine inhibition of commitment was obtained by the addition of either CaCl2 (1.0 mM), calcium ionophore A23817 (1 microgram/ml), but not of MgCl2 (1.0 mM). From these data we conclude that procaine inhibits the terminal erythroid differentiation of MEL cells by blocking an event or process required for commitment which occurs prior to commitment itself. Our results suggest that this process involves calcium metabolism.

Topics: Animals; Calcimycin; Calcium; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Erythropoiesis; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Procaine