sq-23377 and Leukemia--Promyelocytic--Acute

Tissue factor (TF) production is under strict control in mature monocytic cells. However, constitutive expression of TF can be found in myelomonocytic cells and in haematopoietic cells arrested at an early stage of differentiation. In this paper we show that TF expression is down-regulated during the monocyte/granulocyte differentiation process, using the human monoblastic U-937 and the acute promyelocytic leukaemia NB4 cell lines as models. Expression of TF mRNA, protein and procoagulant activity (PCA) was constitutively high in untreated cells. Exposure of U-937 cells to 1alpha,25-dihydroxycholecalciferol (VitD3) and all-trans retinoic acid (ATRA) resulted in down-regulation of TF expression and PCA. In NB4 cells induction by ATRA, but not VitD3, resulted in the down-regulation of TF expression and PCA. Consistent with this, induction of terminal differentiation, as confirmed by the expression of differentiation associated antigens and cell cycle arrest, was inversely correlated to TF expression in U-937 and NB4 cells. Moreover, terminally differentiated U-937 cells retained the capacity to respond to inflammatory mediators, i.e. lipopolysaccharide and interferon-gamma, by a rapid increase in TF expression. In conclusion, we show that not only ATRA but also VitD3 is a potent suppressor of monocytic TF expression and thus might have potential clinical use for the treatment of coagulopathies.

Topics: Cell Cycle; Cell Differentiation; Cholecalciferol; Gene Expression Regulation, Leukemic; Humans; Interferon-alpha; Interferon-gamma; Interleukin-1; Ionomycin; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Thromboplastin; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; U937 Cells

The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all-trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.

Topics: Antigens, CD; Base Sequence; Calcium; Cell Culture Techniques; Cell Differentiation; Cell Division; Collagen; Drug Synergism; Fibronectins; Gene Expression Regulation, Leukemic; Humans; Integrin alpha6; Integrin beta1; Ionomycin; Ionophores; Laminin; Leukemia, Promyelocytic, Acute; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Oxidation-Reduction; Polymerase Chain Reaction; Receptors, Laminin; Tretinoin; Tumor Cells, Cultured

The aim was to investigate in detail the influence of intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i) on apoptosis in HL-60 human promyelocytic leukaemia cells. The pHi was controlled by changing the pH of media as well as by interfering with the pHi regulatory mechanisms with 3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazincarboxamide (HMA; an inhibitor of Na+/H+ antiport), 4-diiosothiocyanatostilbene-2,2'disulfonic acid, (DIDS; an inhibitor of Na(+)-dependent HCO3-/Cl- exchange) and nigericin (a K+ ionophore). The [Ca2+]i was increased with ionomycin, a Ca2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of 3H from 3H-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and 3H release at different pHi, it was shown that apoptosis occurred in HL-60 cells when the pHi was lowered from normal pHi of 7.4 to about 7.2-6.7 with a peak increase at pHi 6.8-6.9. Addition of 4 microM ionomycin to RPMI 1640 medium, which contained 615 microM Ca2+, elevated the apoptosis in the cells. Such an increase in apoptosis by ionomycin in HL-60 cells appeared to result from both an increase in [Ca2+]i and from a decline in pHi. The results indicate that the acidic intratumour environment may greatly affect the response of neoplastic tissues to hyperthermia, radiation and chemotherapeutic drugs which cause apoptosis.

Topics: Apoptosis; Calcium; DNA Damage; HL-60 Cells; Humans; Hydrogen-Ion Concentration; Ionomycin; Ionophores; Leukemia, Promyelocytic, Acute

We have selected calcium ionophore (A23187)-resistant cells (AR-7) in a human promyelocytic leukemia cell line, HL-60. AR-7 showed approximately 8.5-fold resistance to A23187 compared with the wild type cells after continuous exposure for 3 days. AR-7 had cross resistance to ionomycin (4.6-fold) and thapsigargin (340-fold) which can also increase intracellular Ca++. Similar magnitude of resistance to apoptosis, as defined by characteristic morphology and internucleosomal DNA fragmentation, induced by these agents was observed after 4 hr of incubation. However, both the elevation of intracellular Ca++ following stimulation with various concentrations of A23187 and the sensitivity to anti-cancer agents including etoposide, 1-beta-D arabinofuranosylcytosine, and hydroxyurea were comparable between the two cell types. This cell line is considered to be useful for exploring the mechanism(s) of cell death, especially apoptosis, induced by calcium ionophore.

Topics: Antineoplastic Agents; Apoptosis; Calcimycin; Calcium; Cell Division; Cytarabine; DNA; Drug Resistance; Etoposide; Flow Cytometry; Humans; Ionomycin; Leukemia, Promyelocytic, Acute; Terpenes; Thapsigargin; Tumor Cells, Cultured; Ultraviolet Rays

We examined the kinetics of polyamine uptake by human myeloid cells at different stages of maturity. The Km values of putrescine, spermidine and spermine transport by HL-60 cells were 52, 7.9 and 8.1 microM, respectively. These values decreased to 5.1, 1.7 and 0.77 microM, respectively, in HL-60 cells induced to mature past the promyelocyte stage by DMSO. In human PMNs, the respective Km values were 501, 479 and 381 microM. Transport by HL-60 cells was enhanced when intracellular polyamine levels were reduced with difluoromethylornithine. Thus, HL-60 cell maturation is accompanied by an increase in the affinity of their polyamine transport system. This system is much more efficient than that found in end-stage PMNs, suggesting that it plays a more important role in supporting the metabolic requirements of HL-60 cells. Alternatively, the low affinity of the PMN polyamine transport system could represent an adaptation to the high polyamine concentrations found at infection sites.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Biological Transport; Cell Line; Genistein; Gramicidin; Humans; In Vitro Techniques; Ionomycin; Isoflavones; Isoquinolines; Kinetics; Leukemia, Promyelocytic, Acute; Neutrophils; Piperazines; Protein Kinase Inhibitors; Putrescine; Spermidine; Spermine; Sulfonamides; Temperature; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

An axiom of apoptosis is that increases in cytosolic Ca2+ activate a Ca2+/Mg(2+)-dependent endonuclease. However, when HL-60 human promyelocytic leukemia cells were incubated with the Ca2+ ionophore ionomycin in varied extracellular Ca2+, DNA digestion was independent of extracellular Ca2+. Under these conditions, intracellular Ca2+ concentrations did not correlate with the observed DNA digestion. In contrast, intracellular acidification correlated well with DNA digestion. These data indicate that increased intracellular Ca2+ is not the primary signal for endonuclease activation in all forms of apoptosis, but that intracellular acidification may be involved. The observed intracellular acidification is consistent with the involvement of deoxyribonuclease II in apoptosis.

Topics: Calcium; Cell Death; Cell Line; Cytosol; DNA, Neoplasm; Endonucleases; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Ionomycin; Kinetics; Leukemia, Promyelocytic, Acute

The inositol trisphosphate (InsP3) receptor is an essential regulator of intracellular calcium in many cells including chemoattractant- and cytokine-stimulated neutrophils and differentiated promyelocytic leukemic (HL-60) cells. We examined the expression and function of the InsP3 receptor and the transcriptional regulation of the InsP3 receptor gene in HL-60 cells and in HL-60 cells treated for 1-5 days with 1 microM retinoic acid. Radioligand binding studies using membranes from control and retinoic acid-treated HL-60 cells showed that the Bmax of InsP3 receptor increased progressively from 0.24 to 0.69 pmol/mg protein during 5 days retinoic acid treatment with no change in KD (19 nM). During this period, maximal InsP3-stimulated Ca2+ mobilization increased 2-3-fold. InsP3 receptor mRNA was present at low levels in HL-60 cells but was increased significantly after treatment with retinoic acid, reaching maximal levels of approximately 4-fold greater than untreated cells after 4 days treatment with retinoic acid. Nuclear run-on assays indicated that the elevated steady state level of InsP3 receptor mRNA in retinoic acid-treated HL-60 cells was primarily the result of enhanced transcription of the InsP3 receptor gene. Furthermore, the transcriptional enhancing effect of retinoic acid was seen in the presence of cycloheximide, suggesting that the InsP3 receptor gene is directly regulated by retinoic acid. The studies also demonstrate that the InsP3 receptor mRNA is rapidly degraded in HL-60 cells by a mechanism that also requires protein synthesis.

Topics: Actins; Blotting, Northern; Calcium; Calcium Channels; Cycloheximide; Dactinomycin; Gene Expression Regulation, Neoplastic; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ionomycin; Kinetics; Leukemia, Promyelocytic, Acute; Poly A; Proto-Oncogenes; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; RNA; RNA, Messenger; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.

Topics: Calcium; Cell Line; Cell Membrane; Cytosol; Dimethyl Sulfoxide; Egtazic Acid; Gramicidin; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Kinetics; Leukemia, Promyelocytic, Acute; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Ouabain; Potassium Chloride; Second Messenger Systems; Tretinoin; Tumor Cells, Cultured

The marine toxin maitotoxin (MTX) and the chemotactic peptide fMet-Leu-Phe (fMLP) induce the formation of inositol phosphates in HL-60 cells differentiated with dibutyryl cyclic AMP. The increase in [3H]inositol(1,4,5)-trisphosphate is rapid but transient after fMLP stimulation, whereas MTX-induced increase in [3H]inositol(1,4,5)-trisphosphate occurs at a slower rate and is sustained over time. In both cases increases in [Ca++]i, measured with fura-2, parallel the formation of inositol trisphosphate. MTX-mediated stimulation of inositol phosphate formation is inhibited in the absence of calcium, whereas the response to fMLP is not. The calcium ionophore ionomycin stimulates the formation of inositol phosphates in differentiated HL-60 cells. The magnitude of the response is smaller than that obtained with MTX. Ionomycin also induces a rapid but sustained increase of [Ca++]i. In undifferentiated HL-60 cells, neither fMLP nor ionomycin induce significant inositol phosphate formation, and the increase in [Ca++]i elicited by ionomycin is transient. In contrast, the effects of MTX on phosphoinositide breakdown and on [Ca++]i in undifferentiated cells are nearly identical to those elicited by MTX in differentiated cells. In the presence of the intracellular calcium chelator BAPTA, fMLP, ionomycin and MTX still stimulate the generation of inositol phosphates. Guanyl nucleotides and calcium stimulate phospholipase C activity in membrane preparations from differentiated HL-60 cells. fMLP stimulates the enzyme only in the presence of GTP. MTX has no effect on membrane phospholipase C activity.

Topics: Calcium; Egtazic Acid; Guanosine Triphosphate; Humans; Ionomycin; Leukemia, Promyelocytic, Acute; Marine Toxins; N-Formylmethionine Leucyl-Phenylalanine; Oxocins; Phosphatidylinositols; Tumor Cells, Cultured; Type C Phospholipases