Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells

Survivin, a member of inhibitor of apoptosis family of proteins, plays important roles in both cell proliferation and cell death. We previously observed that Survivin is overexpressed in leukemic cell lines and blasts from patients with acute myelogenous leukemia (AML). To understand the roles of Survivin in AML and search for new approaches to the treatment of AML, we inhibited Survivin expression in HL-60 cells with a Survivin anti-sense oligonucleotide (sur-AS-ODN) (ISIS 23722). This blocked significant numbers of HL-60 cells in G2/M phase, and halted cell proliferation at 24 hrs and progressing over time. There was only a slight increase in the number of apoptotic cells at 24 hrs compared with cells treated with nonsense oligonucleotide (NS-ODN). At 48 hrs, however, there were significant increases in sub-G1 phase and annexin V+ cells, suggesting that cell division defects caused cell death. This was supported by the finding that a reduction in the Survivin protein by sur-AS-ODN in cells under serum-free medium did not induce G2/M block and cell death compared to cells treated with NS-ODN. The formation of polyploid cells was observed 48 hrs after sur-AS-ODN treatment, as was the activation of caspase 3, which suggested that apoptotic cell death had occurred. The mitochondrial release of cytochrome C and Smac and the nuclear translocation of the apoptosis-inducing factor were also detected. Our results suggest that Survivin is essential for cell cycle progression in leukemic cells. Reduced Survivin expression causes a cell-cycle defect that leads to cell death through a mitochondrial pathway. This finding has potential utility for therapy of patients with AML.     

Microinjection of macromolecules into leukemic cells by cell fusion technique: Search for intracellular growth-suppressive factors

To investigate the intracellular molecular events during leukemic cell proliferation, we have examined the method of ghost-mediated microinjection of macromolecules into leukemic cell line cells (HL-60). Samples were packed into red cell ghosts. Microinjection was performed by the fusion of ghosts and HL-60 cells using the hemagglutinating virus of Japan (HVJ). Fusion rate was about 80–90%, when determined by the injection of FITC-labeled globulins (IgG) or diphtheria toxin fragment A into HL-60 cells. When the nuclear protein extract from normal granulocytes was injected into HL-60 cells, their growth was significantly suppressed. The injection of the nuclear protein extract from HL-60 itself into HL-60 cells did not inhibit their growth. This finding suggests that leukemic cells may be deficient in intracellular regulatory factors which have suppressive activity on cell growth.     

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.     

Platonin induces autophagy-associated cell death in human leukemia cells

Platonin is a photosensitizer used for photodynamic therapy. In this study, we tested the effect of platonin on human leukemic cells. Treatment with platonin in the dark markedly reduced cell membrane integrity, and induced significant G₀/G₁ arrest of a panel of human leukemic cell lines, including U937, HL-60, K562, NB4 and THP-1. Development of hypodiploid cells was not evident in these cell lines within 24 h, but was noted in U937, HL-60 and NB4 cells after 24 h. No myeloid differentiation of these cells was noted after 5-day treatment. Intriguingly, exposure of monoblastic U937 cells to platonin caused changes characteristic of autophagy, including appearance of cytoplasmic membranous vacuoles and formation of acidic vesicular organelles (AVO) in more than 95% of cells. The platonin-induced autophagy was accompanied by localization of microtubule-associated protein 1 light chain 3 to autophagosomes. Pretreatment with pancaspase inhibitor Z-VAD-fmk abrogated the platonin-induced hypodiploidity, but had no effect on growth inhibition and formation of AVO, indicating a caspase-independent autophagy-associated cell death. Pretreatment of cells with 3-methyladenine attenuated platonin-mediated growth inhibition and formation of AVO. Platonin augmented the expression of BNIP3 in both U937 and K562 cells, whereas had an opposite effect on phosphorylation of mTOR downstream molecule p70S6K. Platonin, at the condition inducing autophagy, induced the mitochondrial membrane permeation. These results suggest that the platonin is capable of inhibiting growth as well as inducing cell death, mainly autophagy-associated, in leukemic cells via a mitochondria-mediated and caspase-independent pathway. A markedly less viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. Platonin, other than a photodynamic agent, may offer significant promise as a therapeutic agent against leukemia.     

Enhanced Expression of αV Integrin Subunit and Osteopontin during Differentiation of HL-60 Cells along the Monocytic Pathway

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for α_V integrin subunit was observed. The α_Vβ₃ integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation.     

Independent regulation of Fuc-TIV and Fuc-TVII genes leading to modulation of cell surface antigen expression in developing myeloid cells

FucosyLated antigen expression, fucosyltransferase activitiesand expression of Fuc-TIV and Fuc-TVII genes have been measuredin the human leukemic cell lines KG1a, arrested at the undifferentiatedmyeloblast stage of maturation and KG1, arrested at the myeloblastand early promyelocytic stage. The results are compared withthose we earlier found for the later promyelocytic cell lineHL-60 and the myelocyte form into which HL-60 cells can be inducedto differentiate. These leukemic cell lines, and the differentiatedHL-60 cells, are believed to correspond to four successive stagesof myeloid maturation in the bone marrow. Fuc-TVII mRNA wasstrongly expressed in the myeloblastic KG1a cells but expressionwas less in KG1 and HL-60 cells. In contrast to the sharp fallin Fuc-TIV expression observed on differentiation of HL-60 cells,the expression of Fuc-TIV mRNA showed a progressive increasefrom KG1a to H1-60 cells; thus the peak of expression of thisgene was at the HL-60 promyelocyte stage. This peak correlatedwith an increase in fucosyltransferase activity with nonsialylatedacceptors and the transitory downregulation of cell surfacesialyl-Le^x expression and upregulation of Le^x, VIM-2 and Le^yexpression. The variations in levels of expression of the fucosylatedantigens on the surface of the developing myelold cells thereforecorrelate with variations in mRNA expression arising from theindependent regulation of Fuc-TIV and Fuc-TVII genes.     

Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation.

The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells' resistance to apoptosis may be due to the acquisition of expression of bcl-XL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-XL can block this process.     

Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin

Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells.     

GPI-PLD调节CEA的释放对白血病HL-60细胞的增殖和凋亡的影响

糖基化磷脂酰肌醇特异性磷脂酶D(glycosyl phosphatidyl inositol specific phospholipase D,GPI-PLD)是人体内唯一可水解细胞膜表面GPI结构、调节GPI锚定蛋白释放的酶.将GPI-PLD转染入急性粒细胞白血病(AGL)的HL-60细胞株,采用实时荧光定量PCR法和Western blot法确定转染后HL-60细胞内GPI-PLD的表达水平;并检测GPI-PLD活性;噻唑蓝(MTT)检测HL-60细胞的增殖;流式细胞仪检测HL-60细胞的凋亡.ELISA检测GPI锚定癌胚抗原(CEA)的表达和释放情况.转染GPI-PLD后,HL-60细胞株中GPI-PLD表达量与活性增加;MTT检测显示,GPI-PLD过表达后HL-60细胞株增殖生长受到抑制;流式检测证实HL-60细胞凋亡增加;且GPI锚定的蛋白质CEA释放增加.该结果提示GPI-PLD基因有抗肿瘤的作用,过表达GPI-PLD后能抑制HL-60细胞增殖且促进其凋亡,所涉机制可能与GPI-PLD释放GPI锚定蛋白,增强白血病细胞对补体杀伤的敏感性有关.     

Specific processing of poly(ADP-ribose) polymerase, accompanied by activation of caspase-3 and elevation/reduction of ceramide/hydrogen peroxide levels, during induction of apoptosis in host HL-60 cells infected by the human granulocytic ehrlichiosis (HGE) agent

We studied the mechanism by which the human granulocytic ehrlichiosis (HGE) agent induces programmed cell death (apoptosis) in human promyelocytic HL-60 leukemia cells. Using several New York HGE isolates, we show that the HGE agent-elicited apoptosis is accompanied by increased processing of nuclear enzyme poly(ADP-ribose) polymerase (PARP), concurrent with a noticeable increase in caspase 3 activities. A marked increase in the amounts of the signaling molecule ceramide but not of diacylglycerol was also observed in HGE agent-infected HL-60 cells, compared with the amounts in uninfected controls. Simultaneous or prior treatment of infected HL-60 cells with the ceramide synthase inhibitor fumonisin B1 did not affect the magnitude of infection by the intracellular pathogen, as determined by both the presence of morulae and the expression of its outer surface membrane protein, p44. These results suggest that the observed changes in ceramide are generated through the sphingomyelinase pathway and not by way of de novo synthesis of ceramide. We also assayed for changes in intracellular hydrogen peroxide and show that the HGE agent causes a decrease in its concentrations in infected cells.     

Topotecan and methotrexate alter expression of the apoptosis-related genes BCL2, FAS and BCL2L12 in leukemic HL-60 cells

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12, which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12, BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS, with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.     

Characterization of a serine protease-mediated cell death program activated in human leukemia cells

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

Inhibition of mTOR-Dependent Autophagy Sensitizes Leukemic Cells to Cytarabine-Induced Apoptotic Death

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.     

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.     

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.     

Disruption of microtubules induces an endogenous suicide pathway in human leukaemia HL-60 cells

Abstract. Terminally differentiated HL-60 cells undergoing programmed cell death (apoptosis) in culture were found to have a disrupted microtubular network. Treatment of undifferentiated HL-60 cells with microtubule-disrupting agents alone was found to induce apoptosis en masse in these cells. In contrast, disruption of microfilaments did not induce apoptosis; instead these cells underwent necrosis, the pathological mode of cell death. Apoptosis in response to microtubule disruption in HL-60 cells was characterized by cell shape changes, nuclear condensation followed by fragmentation and the separation of the cell into numerous intact fragments, termed apoptotic bodies. Apoptosis of these cells was further confirmed by DNA analysis, which demonstrated the activation of an endogenous endonuclease which cleaved the DNA of these cells into oligonucleosomal fragments. Microtubule disrupting agents were found to exert these effects over a wide range of doses. Apoptosis was also inducible in HL-60 cells, in a dose-dependant manner, by the calcium ionophore A23187. Since microtubules are known to be highly sensitive to intracellular calcium fluctuations, this suggests that calcium influx could act at the microtubule level in efTftctino annntnsis