Redistribution of cytochrome c is not an essential requirement in C2-ceramide induced apoptosis in HL-60 cells

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis. The present study investigates the potential role of bcl-2 on apoptosis in HL-60 cells induced by different agents. HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Staurosporine (STS) promoted DNA fragmentation dose-dependently in the 6 h exposure assay while C2-ceramide was relatively slow in the induction of apoptosis (approximately 40% after 24 h) and required higher concentrations (> 20 microM). Caspases inhibitors, Ac-YVAD-cmk (100 microM) and zVAD-fmk (20 microM) had no effect on DNA fragmentation themselves. However, they blocked C2-ceramide-induced caspase-3 cleavage and apoptosis, but not the release of cytochrome c from the mitochondria. In addition, we found that both Ac-YVAD-cmk and zVAD-fmk failed to protect STS-induced apoptosis in HL-60 cells. Overexpression of bcl-2 inhibited STS and C2-ceramide induced cytochrome c redistribution, caspase-3 activation and apoptosis. These results suggest a protective role of bcl-2 in the regulation of apoptosis and cytochrome c release is unlikely to be involved in the final common pathway in apoptosis.     

Synthesis of non-natural C2-homo-ceramide and its apoptotic activity against HL-60 cells

Non-natural ceramide analogues, C2-homo-ceramide and C2-homo-dihydroceramide, were prepared from L-aspartic acid via L-homo-serine. The apoptotic activities of the synthesized ceramide analogues were examined in HL-60 human leukemia cells. C2-homo- and C2-bishomo-ceramide indicate low but considerable apoptotic activities in comparison with C2-ceramide.     

Characterization of C2-ceramide-resistant HL-60 subline (HL-CR): involvement of PKC delta in C2-ceramide resistance

We have established a C2-ceramide-resistant HL-60 subline (HL-CR). HL-CR cells were resistant not only to C2-ceramide but also to various anticancer drugs. HL-CR cells did not respond to differentiation-inducing reagents including 1alpha,25-dihydroxyvitamin D(3), retinoic acid, and 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA induced apoptosis in HL-CR cells much slower than in parental HL-60 cells. As it was reported that PKC isozymes were involved in C2-ceramide-induced apoptosis, we investigated the role of PKC isozymes in C2-ceramide resistance in HL-CR cells. The protein level of PKC delta was lower in HL-CR cells than in parental HL-60 cells, whereas the levels of PKC alpha, betaI, epsilon, and zeta were rather higher in HL-CR cells than in parental cells. Translocation of PKC delta from membrane to cytosol was induced by C2-ceramide in HL-CR cells as well as in wild-type HL-60 cells. Furthermore, overexpression of PKC delta in HL-CR cells potentiated C2-ceramide- and TPA-induced apoptosis and growth inhibition. These results suggest a role for ceramide in apoptosis and differentiation in HL-60 cells, and also suggest that PKC delta might be involved in ceramide- and TPA-induced apoptosis.     

Apoptotic mechanisms of myricitrin isolated from Madhuca longifolia leaves in HL-60 leukemia cells

Molecular Biology Reports - Myricitrin, a naturally occurring flavonoid in Madhuca longifolia, possesses several medicinal properties. Even though our earlier work revealed its role against the...     

Synthesis and anticancer activity of benzyloxybenzaldehyde derivatives against HL-60 cells

A series of benzyloxybenzaldehyde derivatives were prepared and tested against the HL-60 cell line for anticancer activity. Preliminary structure-activity relationships were established. It was discovered that 2-(benzyloxy)benzaldehyde (17), 2-(benzyloxy)-4-methoxybenzaldehyde (26), 2-(benzyloxy)-5-methoxybenzaldehyde (27), 2-(benzyloxy)-5-chlorobenzaldehyde (28), 2-[(3-methoxybenzyl)oxy]benzaldehyde (29), 2-[(2-chlorobenzyl)oxy]benzaldehyde (30), and 2-[(4-chlorobenzyl)oxy]benzaldehyde (31) exhibited significant activity at 1-10 microM. Among them, compound 29 was the most potent one. The morphological assessment and DNA fragmentation analysis indicated that these compounds arrested cell cycle progression at G2/M phase and induced cell apoptosis. They resulted in the loss of mitochondrial membrane potential after 12h of treatment.     

2,2'-Pyridoin derivatives protect HL-60 cells against oxidative stress

Focusing on 2,2'-pyridoin (1, 1,2-di(2-pyridyl)-1,2-ethenediol) and its synthetic derivatives as the lead compound of the potent antioxidative enediol, their protective effect against oxidative stress was evaluated on the HL-60 cell system. 2,2'-Pyridoins showed no remarkable cytotoxic effect on HL-60 cells. The derivatives 1, 2, 3, 5, and 6 inhibited H(2)O(2)-induced cell death and intracellular oxidative stress more significantly than ascorbic acid. Since 2,2'-pyridoins are oxidized to the diketones, 2,2'-pyridils, in a protic solvent, the antioxidant activity of 2,2'-pyridils was also investigated. 2,2'-Pyridils showed antioxidant activity in the cell; however, the activity was lower than that of 2,2'-pyridoins. These results suggested that 2,2'-pyrdoin derivatives can be good cytoprotective agents against oxidative stress.     

Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2

Prostaglandin (PG) A2 has been reported to inhibit the growth or induce apoptosis of various tumor cells. In the present study, PGA2 inhibited the growth of HL-60 cells and concomitantly-induced nuclear condensation and DNA fragmentation, characteristics of apoptosis. Down-regulation of c-myc mRNA, and activation of caspase-3 were observed in the PGA2 -treated cells. PGA2-induced DNA fragmentation was completely abolished in the presence of zVAD-Fmk or zDEVD-Fmk. But, relative cell survival was not improved up to that of untreated cells by pretreatment of caspase inhibitors, and c-myc down-regulation was not recovered by caspase inhibitors, either. Moreover, cytochrome c release and activation of caspase-9 was also observed in apoptotic cells and a specific inhibitor of caspase-9 (zLEHD-Fmk) prevented both DNA fragmentation and activation of caspase-3, but not relative cell survival, implying the upstream mitochondrial event of caspase-3 activation. In addition, antagonistic Fas antibody (ZB4) exerted no effect on the apoptosis. Taken together, these results suggest that PGA2 may induce the apoptosis as well as growth inhibition in HL-60 cells, and cytochrome c release and caspase activation seem to play a critical role in this apoptosis which might be independent or downstream of growth inhibition associated with c-myc down-regulation.     

Enlargement and contracture of C2-ceramide channels

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that ceramide, but not dihydroceramide, forms large and stable channels in phospholipid membranes and outer membranes of isolated mitochondria. C(2)-ceramide channel formation is characterized by conductance increments ranging from <1 to >200 nS. These conductance increments often represent the enlargement and contracture of channels rather than the opening and closure of independent channels. Enlargement is supported by the observation that many small conductance increments can lead to a large decrement. Also the initial conductances favor cations, but this selectivity drops dramatically with increasing total conductance. La(+3) causes rapid ceramide channel disassembly in a manner indicative of large conducting structures. These channels have a propensity to contract by a defined size (often multiples of 4 nS) indicating the formation of cylindrical channels with preferred diameters rather than a continuum of sizes. The results are consistent with ceramides forming barrel-stave channels whose size can change by loss or insertion of multiple ceramide columns.     

Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells

The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar Km and Vmax (approximately 0.2 mM and approximately 3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37 degrees C; (2) the aSMase is much less sensitive to Triton X-100 ( > 5 mM), compared with < or = 0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFalpha, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect Km or Vmax of the nSMase in HL-60 sonicates. This suggests that TNFalpha may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed.     

Effects of inducers of differentiation on protein kinase C and CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase activities of HL-60 leukemia cells

Exposure of HL-60 leukemia cells to either 12-O-tetradecanoylphorbol-13-acetate (TPA), dimethylsulfoxide (DMSO), exogenous gangliosides GM3, GM1, or bovine brain ganglioside mixture (BBG) resulted in a marked inhibition of the growth of cells. The order of the inhibitory potency was TPA greater than GM3 greater than DMSO greater than BBG greater than GM1. In contrast, sulfatides were without effect on cellular replication. Treatment of HL-60 cells with TPA or GM3 induced differentiation along the monocyte/macrophage lineage, while treatment with DMSO induced maturation along the granulocytic pathway. These effects were accompanied by more than a twofold increase in protein kinase C (PKC) activity. In contrast, treatment with GM1, BBG, or sulfatides caused only a relatively small increase in PKC activity. The activity of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase (ST1), a key enzyme for membrane gangliosides synthesis, in HL-60 cells was also influenced by the exposure to TPA, GM3, DMSO, GM1, or sulfatides. The inducers of differentiation, TPA and DMSO, caused an increase in ST1 activity, whereas GM3, which also induced cellular differentiation, inhibited ST1 activity, perhaps through the action of end-product inhibition. The non-inducers of differentiation, GM1 and sulfatides, also increased the activity of ST1, but to a much lesser extent. The findings suggest that the direct or indirect modulation of PKC activity by some of these agents may be involved, at least in part, in the regulation of cellular growth and differentiation. Furthermore, it is conceivable that differences in PKC activity may be responsible for the changes in ST1 activity associated with cell differentiation and proliferation.     

The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.     

Action of 2-nonenal and 4-hydroxynonenal on phosphoinositide-specific phosopholipase C in undifferentiated and DMSO-differentiated HL-60 cells

The promyelocytic cell line HL-60 has been used as an in vitro model to study the mechanism of action of two chemotactic aldehydes, 2-nonenal and 4-hydroxynonenal. Increasing aldehyde concentrations have been added to undifferentiated and DMSO-differentiated cells incubated at 37 degrees C and their effect on phosphoinositide-specific phospholipase C has been analysed by using a specific inositol-1,4,5-tris-phosphate assay system. Concentrations of 2-nonenal between 10(-9) and 10(-7) M significantly increased the enzymatic-activity in DMSO-differentiated HL-60 cells, while 10(-9) and 10(-8) M concentrations were active in the undifferentiated cells. 4-Hydroxynonenal was able to activate phospholipase C both in undifferentiated and DMSO-differentiated cells at concentrations ranging from 10(-8) to 10(-6) M. The concentrations of both compounds active on phospholipase C displayed a good correspondence with those which had been reported to be chemotactic towards rat neutrophils. In the case of 4-hydroxynonenal, the present results confirm its ability to activate phospholipase C, which we had previously shown in isolated neutrophil plasma membranes. The comparison of the effects of 2-nonenal and 4-hydroxynonenal on chemotaxis and phospholipase C activation suggests a common mechanism of action for both aldehydes, for which the presence of the double bond seems to be required.     

microRNA-181b targets MLK2 in HL-60 cells

microRNAs(miRNAs) play critical roles in many different cellular processes,including metabolism,apoptosis,differentiation and development.We showed miR-181b to be highly expressed in acute myeloid leukemia(AML).Furthermore,miR-181b contributed to proliferation of AML cells by targeting MLK2.Our results demonstrated that miR-181b plays an important role in the biology of AML and may be useful in developing therapies targeting miRNAs.     

P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.     

C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation

We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.     

Short-chain 3-ketoceramides, strong apoptosis inducers against human leukemia HL-60 cells

Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3, C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochondria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apoptotic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain 3-ketoceramides, that is, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B), and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more powerful than the corresponding alcoholic analogue, N-acetyl-D-erythro-sphingosine. Observations of DNA fragmentation, caspase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and the effects of exogenous glutathione on these phenomena are also discussed.     

Antimycin A-induced apoptosis of HL-60 cells

BACKGROUND: Previous experiments in our laboratory investigating apoptosis induced in HL-60 cells by camptothecin (CAM) have revealed that the sequence and rapidity of the apoptotic phenomena in an individual cell depend on the proliferative state of that cell when it encounters CAM. The role of mitochondria in HL-60 apoptosis was explored using an inhibitor of oxidative phosphorylation, antimycin A (AMA). METHODS: Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC), uptake of propidium iodide (PI), and DNA content after membrane fixation/permeabilization were monitored by flow cytometry. Z-VAD-FMK was used to inhibit caspases. Fluorescence microscopy was used to examine cell morphology. RESULTS: Cells in the G1 phase of the cell cycle were the first to exhibit signs of apoptosis in response to 100 microM AMA and some of these cells disintegrated without exposing to phosphatidylserine (PS). Caspase inhibition prevented fragmentation of DNA, the nucleus, and the cell, but only delayed PS exposure and loss of plasma membrane integrity. CONCLUSIONS: The highly active mitochondria of G1-phase HL-60 cells make them particularly sensitive to AMA. PS exposure and plasma membrane damage are mediated by noncaspase molecules released from mitochondria. We hypothesize that if mitochondria are subjected to a sufficiently severe insult, whether indirectly as a result of extensive CAM-induced DNA damage or directly by the effect of AMA on electron transport, the nature and quantities of the proapoptotic molecules released are such that apoptosis proceeds to the point of cell disintegration before the PS exposure pathway is complete.     

Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 microm in both HL-60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 microm for HL-60 cells and 60 microm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.