Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca2+ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m2) on the agonist-induced Ca2+ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 microM ATP or by 100 microM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2-100 microM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20-30% decrease in the magnitude of [Ca2+]i elevation induced by a low concentration of ATP (<1 microM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20-40% increase of ATP-induced elevation of [Ca2+]i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, Km, value in ATP-treated cells and of the maximal [Ca2+]i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca2+ and/or ion channels.     

Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis

The concentrations of ammonia in the plasma of the mudskipper Boleophthalmus boddaerti exposed to cyanide for 1-6 days were significantly greater than the respective values of the controls. This was due to an increase in the production of NH3 in the muscle and an increase in the retention of NH3 and/or NH4+ in the blood of the cyanide-exposed fish when compared to controls. Cyanide exposure significantly increased the specific activity of muscle AMP deaminase. Since adenylosuccinate synthetase and lyase were also present in the muscle, exposure to cyanide might increase the production of NH3 from the catabolism of purine nucleotides. B. boddaerti exposed to cyanide excreted significantly less ammonia than the control fish. Results indicate changes in the permeability of the epithelial surfaces of the cyanide-exposed fish to NH3 and/or NH4+. Since the tissues and organs needed time to activate the inducible cyanide detoxification mechanisms, the increase in the production of NH3 might be an important defensive mechanism for B. boddaerti during the early phase of cyanide exposure.     

Stimulation by histamine of TPA-dependent hepatocyte growth factor production in promyelocytic leukemia HL-60 cells

Hepatocyte growth factor (HGF) is most likely a physiological hepatotrophic factor that triggers regeneration of the injured liver. Histamine may also be important in the pathophysiology of the injured liver. Previously we showed that histamine production was increased in liver macrophages of mice injected with CCI4, a well-known hepatotoxin. Therefore, it is likely that the biological actions of histamine in repairing processes of the injured liver are mediated by HGF. This study was aimed at examining the effects of histamine on production of HGF using, as a model, the human promyelocytic leukemia cells, HL-60. 12-o-Tetradecanoylphorbol-13-acetate (TPA) markedly stimulated HGF production and release from the cells; the maximal amount of HGF was released at a concentration of 3 ng/ml of TPA. Histamine significantly stimulated the TPA-induced HGF production and release in these cells, depending on incubation time and its dose. These actions of histamine were abrogated by a H2 receptor antagonist, ranitidine.     

A case of superior vena cava syndrome caused by Klebsiella pneumoniae

A 27 yr old man presented with productive cough, fever and manifestations of superior vena cava syndrome. He was an alcoholic but had been in good health until 3 days prior to admission. The physical examination, the chest radiograph and the results of the sputum culture were compatible with Klebsiella pneumoniae pneumonia of the right upper lobe. The superior vena cava scintigram using technetium-99m showed near total occlusion of the superior vena cava, while sputum cytology, chest computed tomography, and bronchoscopy were all negative for malignant aetiology. Antibiotic therapy brought about slow resolution of the pneumonia and also of the superior vena caval obstruction. The follow-up scintigram showed normalized venous flow of the superior vena cava. To our knowledge, this is the first case of superior vena cava syndrome developed in probable association with Klebsiella pneumoniae pneumonia.     

Production and activation of matrix metalloprotease-9 (MMP-9) by HL-60 promyelocytic leukemia cells

Human promyelocytic HL-60 cells have been used as a model of acute leukemia to investigate the expression and the regulation of matrix metalloproteases (MMPs), known to contribute to the degradation of extracellular matrix components. As shown by gelatin zymography, HL-60 cells constitutively released significant amounts of proMMP-9 (92 kDa) and moderate amounts of proMMP-2 (72 kDa). Furthermore, casein zymography confirmed the presence of serine proteases in the form of pro-urokinase. Activation of proMMP-9 was dependent on the plasminogen activator/plasmin (PA/plasmin) system and was inhibited by aprotinin. MMP-9 was only detected in cellular extracts or conditioned media incubated with HL-60 cells, indicating that cells are essential to the activation process. Addition of plasminogen increased by 3-fold the basal invasive rate of these cells across a matrigel layer (2.1% versus 0.7% in control cells after 4 h of incubation). Taken together, these results indicate that HL-60 cells exhibit an autocrine activation mechanism of proMMP-9 via the PA/plasmin system and that activation of proMMP-9 increases their invasive potential.     

Induction of thrombospondin-1 by all-trans retinoic acid modulates growth and differentiation of HL-60 myeloid leukemia cells

Thrombospondin-1 (TSP), a multifunctional extracellular matrix protein, modulates human hematopoietic stem cell adherence and thus may play a role in blood cell proliferation and/or differentiation. The expression of TSP was studied in the human myeloid leukemia cell line, HL-60, upon differentiation into monocytes by phorbol-13-monoacetate (PMA) or into granulocytes by all-trans retinoic acid (RA). HL-60 cells cultured under serum-free conditions constitutively secreted low amounts of TSP into the cultured medium, approximately 13 ng/10(6) cells/24 h. PMA used at 4 x 10(-8) M did not significantly modulate TSP secretion over a 24 h period. In contrast, RA at 10(-7) M induced a 5- to 10-fold increase in TSP secreted by HL-60 cells during their differentiation into granulocytes over a 5 day period. The role of secreted TSP in RA-dependent cessation of growth and differentiation was examined using blocking anti-TSP antibodies. In the presence of the polyclonal anti-TSP antibody R5 (25 microg/ml), growth of RA-treated HL-60 cells was maintained at control levels for up to 3 days and a concomitant delay in granulocytic differentiation was observed. Moreover, the addition of soluble TSP (0.5-5 microg/ml) to untreated HL-60 cells decreased their growth and promoted their differentiation in a dose-dependent manner. Using a neutralizing antibody to transforming growth factor beta (TGF-beta) or purified TGF-beta1 we further demonstrated that the effects of TSP were not mediated through activation of latent TGF-beta. These studies indicate that TSP decreases the proliferation and promotes the differentiation of HL-60 cells.     

Role of serine and ICE-like proteases in induction of apoptosis by etoposide in human leukemia HL-60 cells

This study was undertaken to examine the role of proteases in etoposide-induced apoptosis of human leukemia HL-60 cells. We found the potent activity to produce internucleosomal DNA fragmentation in a 150 000 g supernatant of cell lysate which was prepared from etoposide-treated HL-60 cells undergoing apoptosis. This nuclear-DNA fragmenting activity could be detected when the supernatant was incubated with isolated nuclei under Mg2+-dependent conditions. On the other hand, we could not detect such activity in the supernatant of cell lysate from non-treated HL-60 cells. Treatment of the supernatant with a serine protease inhibitor, N-tosyl-L-phenylala-nylchloromethyl ketone (TPCK), abolished the DNA fragmenting activity. An inhibitor of interleukin 1-beta-converting enzyme (ICE), Z-Val-Ala-Asp-fluoromethyl ketone (VAD-FMK), had no effect on this DNA fragmenting activity in vitro. However, when the cells were incubated with etoposide in the presence of VAD-FMK, the formation of TPCK-sensitive DNA fragmenting activity was blocked. Our data indicate that serine and ICE-like proteases may be involved in etoposide-induced apoptosis at the different stages, and especially a serine protease may be closely associated with the final step for induction of internucleosomal DNA fragmentation during apoptosis in HL-60 cells.     

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.     

Induction of apoptosis in HL-60 human promyelocytic leukemia cells by adenosine A(3) receptor agonists

The effects of adenosine (ADO) analogs on cells of the human promyelocytic HL-60 line were examined. ADO A(3) receptor agonists, N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA, 30-60 microM) and 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA, 10-30 microM) induced apoptotic cell death. In contrast, neither an A(1)/A(2) antagonist (XAC) nor other selective ADO receptor agonists (CPA, NECA and CGS21680) induced apoptosis at concentrations of <30 microM. Both IB-MECA and CI-IB-MECA significantly induced Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx, suggesting the presence of phospholipase C-coupled ADO A(3) receptors on HL-60 cells. This was further supported by the presence of mRNA of ADO A3 receptor in the cells. These results suggest that activation of ADO A(3) receptors is responsible for the ADO-induced apoptosis in HL-60 cells and could be of potential therapeutic value in the treatment of leukemia.     

Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells

Dopamine (DA) has been reported to depolarize neurons in the prefrontal cortex (PFC). To further characterize this effect of DA, we made whole cell recordings from PFC pyramidal cells in rat brain slices. As reported previously, DA depolarized most PFC cells tested. This effect of DA was concentration-dependent and persisted in the presence of synaptic blockade, indicating a direct effect of DA on the recorded cell. During DA-induced depolarization, PFC neurons consistently showed an increase in excitability, suggesting that the depolarization is not directly related to DA-induced inhibition of PFC neurons previously observed in vivo. Surprisingly, the effect of DA was not mimicked or blocked by several commonly used DA agonists and DA antagonists. The alpha and beta antagonists phentolamine and alprenolol and the atypical antipsychotic drug clozapine also showed no significant effect on DA-induced depolarization. These results suggest that DA-induced depolarization may be mediated by a nonspecific mechanism. However, it remains possible that there exists a new type of DA receptors in the PFC not sensitive to classical DA agonists and antagonists, particularly given the fact that DA applied in the same manner depolarized only PFC neurons but not those in the striatum or the substantia nigra.     

Effect of intracellular acidity and ionomycin on apoptosis in HL-60 cells

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.     

Taxol-enhanced cytotoxic effect of radiation in human promyelocytic leukemia cells: relative resistance of multidrug-resistant HL-60 cells in vitro

Cytotoxic effects of sequential taxol (paclitaxel) and X-irradiation on drug-sensitive human cultured promyelocytic leukemia (HL-60) cell line and its multidrug-resistant sublines were examined using photometric MTT test and flow cytometry. Paclitaxel (at concentrations 1-10 nmol) stimulated the cytotoxic effect of irradiation in HL-60 and to a lesser extent also in HL-60/ADR, but not in HL-60/VCR cells. The stimulation of radiation-induced cytotoxic effect by paclitaxel correlated with its potential to induce cell cycle and viability alterations identified with flow cytometric analysis (i.e. increased propidium iodide staining, increased side scatter, decreased forward angle scatter, accumulation of necrotic cell detritus, apoptotic pre-G0 cells and cells in the G2/M phase of the cell cycle).     

N-Alkylphthalimides: structural requirement of thalidomidal action on 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor alpha production by human leukemia HL-60 cells

Phthalimide analogs N-substituted with n-butyl, tert-butyl, hexyl and adamantyl groups were designed and prepared as simplified analogs of thalidomide and methylthalidomide. All the compounds prepared except N-n-butylphthalimide showed thalidomidal activity on 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor (TNF)-alpha production by human leukemia HL-60 cells. Among the investigated compounds, including thalidomide and methylthalidomide, N-adamantylphthalimide showed the most potent TNF-alpha production-enhancing activity.     

A lignan and four terpenoids from Brucea javanica that induce differentiation with cultured HL-60 promyelocytic leukemia cells

A novel lignan, guaiacylglycerol-beta-O-6'-(2-methoxy)cinnamyl alcohol either, three known simaroubolides, brusatol, dehydrobrusatol, yadanziolide C, and the known terpenoid, blumenol A, were obtained as active compounds from an ethyl acetate-soluble extract of Brucea javanica, using a bioassay based on the induction of cell differentiation with human promyelocytic leukemia (HL-60) cells. Also obtained were the known coumarinolignan, cleomiscosin A, and the known quassinoid glycoside, bruceoside B, which were inactive in the HL-60 cell test system. The structure of the new lignan was determined by a combination of 1D and 2D NMR techniques.     

环氧合酶-2抑制剂对白血病细胞HL-60的化疗增敏作用及机制

目的 观察环氧合酶-2(COX-2)抑制剂塞来昔布对白血病细胞株HL-60的化疗增敏作用,并对其机制进行初步探讨.方法 MTT法评估塞来昔布、多柔比星及二者联合对HL-60细胞的生长抑制效应;流式细胞术(FCM)检测细胞的凋亡;反转录聚合酶链反应(RT-PCR)检测Survivin基因的表达;Western blotting检测Survivin蛋白的表达.结果 多柔比星联合塞来昔布5和10μmol/L对HL-60细胞的半数抑制浓度(IC50)分别为0.25及0.16μg/ml,明显低于多柔比星单用的IC50(0.48μg/ml);多柔比星0.10μg/ml联合10 μmol/L塞来昔布下调Survivin基因mRNA及蛋白的表达;联合塞来昔布5和10 μmol/L的凋亡率[分别为(13.07±1.66)%及(22.36±1.84)%]较多柔比星0.10μg/ml单用[(5.72±1.25)%]明显增加(P<0.01).结论 COX-2抑制剂塞来昔布对白血病细胞株HL-60具有明显的化疗增敏作用,其初步机制涉及下调Survivin的表达,增加细胞凋亡.     

(Sp)-8-chloroadenosine 3'',5''-cyclophosphate induced differentiation on human leukemia HL-60 cells

The structure and effects of sulfonylurea derivatives were described. Today the second generation of sulfonylureas rather is prescribed because of its lower dosage and better peripheral activity. The non-insulin-dependent diabetes after weight reduction and failure of other oral therapy is the main indication to introduction of sulfonylurea derivates.     

Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded homodimeric mucin-like glycoprotein on leukocytes that interacts with both P- and E-selectin. In this report we describe the structures of the Ser/Thr-linked O-glycans of PSGL-1 synthesized by HL-60 cells metabolically radiolabeled with 3H-sugar precursors. In control studies, the O-glycans on CD43 (leukosialin), a mucin-like glycoprotein also expressed by HL-60 cells, were analyzed and compared to those of PSGL-1. O-Glycans were released from Ser/Thr residues by mild base/borohydride treatment of purified glycoproteins, and glycan structures were determined by a combination of techniques. In contrast to expectations, PSGL-1 is not heavily fucosylated; a majority of the O-glycans are disialylated or neutral forms of the core-2 tetrasaccharide Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAcOH++ +. A minority of the O-glycans are alpha-1,3-fucosylated that occur as two major species containing the sialyl Lewis x antigen; one species is a disialylated, monofucosylated glycan, and the other is a monosialylated, trifucosylated glycan having a polylactosamine backbone. CD43 lacks the fucosylated glycans found on PSGL-1 and is enriched for the nonfucosylated, disialylated core-2 hexasaccharide. These results demonstrate that PSGL-1 contains unique fucosylated O-glycans that are predicted to be critical for high affinity interactions between PSGL-1 and selectins.