氟苯达唑对人急性髓系白血病HL-60细胞的增殖抑制作用研究

目的:研究氟苯达唑对人急性髓系白血病HL-60细胞增殖的抑制作用,明确氟苯达唑对HL-60细胞周期,凋亡发生的作用机制。方法:噻唑蓝法(MTT)检测氟苯达唑对人急性髓系白血病HL-60细胞的生长抑制作用,流式细胞术检测氟苯达唑对HL-60细胞周期,DNA片段化的影响,免疫印迹法检测Caspase, Raf, Bcl-2家族蛋白表达。结果:氟苯达唑抑制人急性髓系白血病HL-60细胞生长,HL-60细胞G2/M期增加,与阴性对照组相比,在一定的剂量和时间内,差别具有显著统计学意义;DNA片段化上升,0.25,0.5,1μM组与对照组相比差别具有显著统计学意义,促使Cleaved PARP,Cleaved-caspase 3,Cleaved-caspase 9蛋白表达量趋势增加;Bag-1和Bcl-2蛋白表达量降低;b-raf,c-raf磷酸化蛋白表达水平逐渐降低。结论:氟苯达唑通过诱导HL-60细胞阻滞于G2/M期,增加DNA片段化水平,激活Caspase, Raf, Bcl-2家族介导的凋亡相关通路抑制人急性髓系白血病HL-60细胞增殖,诱导人急性髓系白血病HL-60细胞发生凋亡而发挥抗肿瘤作用。     

Farnesylpyridinium, an analog of isoprenoid farnesol, induces apoptosis but suppresses apoptotic body formation in human promyelocytic leukemia cells

1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, initially induced morphological changes similar to those of typical apoptosis in human leukemia HL-60 cells but FPy-treated cells were characterized by the absolute absence of final apoptotic events such as fragmentation into apoptotic bodies. FPy-induced cell death was considered to be apoptotic on the basis of the induction of DNA fragmentation and the protection against these events by the coaddition of a pan-caspase inhibitor. The increase in the cytoplasmic cytochrome c level supported the possibility that FPy-treated cells should have the ability to complete the entire apoptotic process ending in cell fragmentation and apoptotic body formation. At concentrations too low to induce apoptosis, FPy could suppress the induction of apoptotic body formation in HL-60 cells by typical inducers of apoptosis such as actinomycin D or anisomycin. FPy exhibited a cytochalasin-like effect on spatial arrangement of actin filament independent of its apoptosis-inducing activity.     

A critical role of redox state in determining HL-60 cell granulocytic differentiation and apoptosis via involvement of PKC and NF-κB

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl-l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-κB.     

Granulocyte colony-stimulating factor-induced activation of protein kinase-C in myeloid cells

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, differentiation, and activation of myeloid cells. It binds to a high affinity receptor (G-CSF-R) expressed on myeloid cells, for which the signal transduction mechanisms other than protein tyrosine kinase (PTK) activation have not been completely identified. We explored the potential involvement of protein kinase-C (PKC) in G-CSF-R signal transduction. In this report, we provide direct evidence of PKC activation by G-CSF-R. G-CSF treatment of peripheral blood neutrophils, granulocytic cell lines (HL-60, NFS-60, KG-1), and monocytic cell lines (WEHI-3B,U-937) resulted in PKC activation. Chelerythrine chloride and HA-100, an isoquinolinesulfonamide derivative, the specific inhibitors of PKC, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), a chelator of intracellular calcium, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), an inhibitor of intracellular calcium release, blocked G-CSF-induced PKC activation in HL-60 cells, and reduced CD11b upregulation in neutrophils, but did not affect ligand-binding or down-modulation of G-CSF-R. Methyl 2,5-dihydroxycinnamate (MDHC), a potent inhibitor of protein tyrosine kinases (PTK), also inhibited PKC activation in response to G-CSF treatment, suggesting that PKC activation may occur downstream of PTK activation. Our results demonstrate the involvement of PKC in G-CSF-R signal transduction, and suggest a common signaling pathway in myeloid cells of granulocytic and monocytic lineages. J. Cell. Biochem. 66:286–296, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

A new endogenous differentiating factor (myelopeptide-4) for myeloid cells

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.     

Phyllanthus urinaria induces the Fas receptor/ligand expression and ceramide-mediated apoptosis in HL-60 cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect on human myeloid leukemia cells in this study. The water extract of P. urinaria induced the apoptosis of HL-60 cells as demonstrated by morphological change, DNA fragmentation and increased caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The P. urinaria-induced apoptosis of HL-60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expressions of Fas receptor and Fas ligand, but not p53, were also induced in HL-60 cells dose- and time-dependently. The inhibitor of ceramide synthase, fumonisin B1, completely suppressed the apoptosis induced by P. urinaria and this inhibitory effect of fumonisin B1 could be eliminated by the addition of ceramide. It indicated that the activity of ceramide synthase is critical for the P. urinaria-induced apoptosis in HL-60 cells. The P. urinaria-induced apoptosis in HL-60 cells is mediated through a ceramide-related pathway.     

Involvement of protein kinase C-beta and ceramide in tumor necrosis factor-alpha-induced but not Fas-induced apoptosis of human myeloid leukemia cells.

The role of protein kinase C-beta (PKC-beta) in apoptosis induced by tumor necrosis factor (TNF)-alpha and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-beta. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-alpha-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-beta reestablished their susceptibility to TNF-alpha-induced apoptosis. The apoptotic effect of TNF-alpha in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-beta deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-alpha-induced apoptosis involves PKC-beta and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.     

Regulation of myeloid-specific calcium binding protein synthesis by cytosolic protein kinase C.

Two calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) is shown to enhance the inductive effect of RA and 1 alpha,25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1 alpha,25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1 alpha,25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.     

12-o-Tetradecanoylphorbol 13-acetate prevents baicalein-induced apoptosis via activation of protein kinase C and JNKs in human leukemia cells

In the present study, we found that baicalein (BE), but not its glycoside baicalin (BI), induced apoptosis in human leukemia HL-60 and Jurkat cells, but not in primary murine peritoneal macrophages (PMs) or human polymorphonuclear (PMN) cells, by the MTT assay, LDH release assay, and flow cytometric analysis. Activation of the caspase 3, but not caspase 1, enzyme via inducing protein processing was detected in BE-induced apoptosis. The ROS-scavenging activity of BE was identified by the anti-DPPH radical, DCHF-DA, and in vitro plasmid digestion assay, and none of chemical antioxidants including allpurinol (ALL), N-acetyl-cystein (NAC), and diphenylene iodonium (DPI) affected BE-induced apoptosis in HL-60 cells. This suggests that apoptosis induced by BE is independent of the production of ROS in HL-60 cells. Interestingly, the apoptotic events such as DNA ladders formation and activation of the caspase 3 cascade were significantly blocked by TPA addition in the presence of membrane translocation of PKCα, and TPA-induced protection was reduced by adding the PKC inhibitors, GF-109203X and staurosporin. TPA addition induces the phosphorylation of JNKs and ERKs, but not p38, protein in HL-60 cells, and incubation of HL-60 cells with JNKs inhibitor SP600125, but not ERKs inhibitor, PD98059 or the p38 inhibitor SB203580, suppressed the protective effect of TPA against BE-induced apoptotic events including DNA ladders, apoptotic bodies, caspase 3 and D4-GDI protein cleavage in according with blocking JNKs protein phosphorylation. In addition, PKC inhibitor GF-109203X treatment blocks TPA-induced ERKs and JNKs protein phosphorylation, which indicates that activation of PKC locates at upstream of MAPKs activation in TPA-treated HL-60 cells. Additionally, a loss in mitochondrial membrane potential with a reduction in Bcl-2 protein expression, the induction of Bad protein phosphorylation, and translocation of cytochrome c from mitochondria to the cytosol were observed in BE-treated HL-60 cells, and these events were prevented by the addition of TPA. GF-109203X and SP600125 suppression of TPA against cytochrome c release induced by BE was identified. This suggests that activation of PKC and JNKs participate in TPA's prevention of BE-induced apoptosis via suppressing mitochondrial dysfunction in HL-60 cells.     

Apoptosis and Its Modulation in Human Promyelocytic HL-60 Cells Treated with DNA Topoisomerase I and II Inhibitors

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca²⁺/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosomesized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.     

Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin

Anthrax lethal toxin (LT) is comprised of protective antigen and lethal factor. Lethal factor enters mammalian cells in a protective antigen-dependent process and cleaves mitogen-activated protein kinase kinases. Although LT has no observable effect on many cell types, it causes necrosis in macrophages derived from certain mouse strains and apoptosis in activated mouse macrophages. In this study, we observed that LT treatment of three different human monocytic cell lines U-937, HL-60 and THP-1 did not induce cell death. Cells did become susceptible to the toxin, however, after differentiation into a macrophage-like state. Treatment with LT resulted in decreased phosphorylation of p38, ERK1/2 and JNK in both undifferentiated and differentiated HL-60 cells, suggesting that the change in susceptibility does not result from differences in toxin delivery or substrate cleavage. Death of differentiated HL-60 cells was accompanied by chromosome condensation and DNA fragmentation, but was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. In addition, we observed that the macrophage differentiation process could be inhibited by LT. Our results indicate that LT-mediated death of mouse and human macrophages may occur through distinct processes and that the differentiation state of human cells can determine susceptibility or resistance to LT.     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ     

Curcumin abolishes apoptosis resistance of calcitriol-differentiated HL-60 cells

Exposure of HL-60 cells to 1,25-dihydroxyvitamin D(3) (calcitriol) induces their differentiation into monocytes. This terminal differentiation is associated with acquired resistance to many proapoptotic stimuli. Here we show that differentiated HL-60 cells undergo apoptosis upon curcumin treatment although they retain resistance to apoptosis induced by a topoisomerase poison - etoposide. Curcumin induced changes of nuclear morphology, DNA fragmentation, release of cytochrome c as well as caspase activation in both differentiated and undifferentiated cells. Experiments performed in other laboratories suggested that curcumin initiates apoptosis by DNA damage that results from topoisomerase II poisoning. We measured gammaH2AX expression, a marker of DNA double strand breaks, in both undifferentiated and differentiated HL-60 cells treated with curcumin or etoposide. In etoposide-treated undifferentiated cells early gammaH2AX expression correlated with initiation phase of apoptosis. In contrast, in curcumin-treated cells gammaH2AX expression correlated with apoptotic DNA fragmentation, which is characteristic for the execution phase of apoptosis. Our experiments show that curcumin overcomes the resistance of calcitriol-differentiated HL-60 cells to DNA-damage-induced apoptosis by activating other cell signaling pathways leading to cell death of HL-60.     

Magnesium deprivation inhibits the expression of differentiation-related phenotypes in human promyelocytic leukemia HL-60 cells

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.     

3-Deazauridine triggers dose-dependent apoptosis in myeloid leukemia cells and enhances retinoic acid-induced granulocytic differentiation of HL-60 cells

Therapeutic nucleoside analogue 3-deazauridine (DU) exerts cytotoxic activity against cancer cells by disruption of DNA synthesis resulting in cell death. The present study evaluates whether DU alone at doses 2.5-15 microM or in combination with all trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) is effective against myelogenous leukemia. The data of this study indicate that DU induces dose-dependent cell death by apoptosis in myeloid leukemia cell lines HL-60, NB4, HEL and K562 as demonstrated by cell staining or flow cytometry and agarose gel electrophoresis. 24h-treatment with DU produced dose-dependent HL-60 cell growth inhibition and dose-independent S phase arrest that was not reversed upon removal of higher doses of DU (10-15 microM). Exposition to nontoxic dose of DU (2.5 microM) for 24h followed by RA or dbcAMP and 96 h-cotreatment with DU significantly enhanced RA- but not dbcAMP-mediated granulocytic differentiation. Cell maturation was paralleled with an increase in the proportion of cells in G1 or G2+M phase. We conclude that, depending on the dose or the sequence of administration with RA, an inhibitor of DNA replication, DU triggers a process of either differentiation or apoptosis in myeloid leukemia cells.