Sphingosine-induced c-jun expression: differences between sphingosine- and C2-ceramide-mediated signaling pathways

Sphingolipids such as ceramide and sphingosine are putative intracellular signal mediators in cell differentiation, growth inhibition and apoptosis. Previously, we reported that C2-ceramide induced c-jun expression in apoptosis of human leukemia HL-60 cells. Here we report that sphingosine also induced c-jun expression in apoptosis of HL-60 cells. Sphingosine-induced c-jun expression was stimulated by H-89, a protein kinase A inhibitor, whereas C2-ceramide-induced c-jun expression was inhibited by protein kinase C inhibitors. Furthermore, H-89 potentiated sphingosine-induced but not C2-ceramide-induced growth inhibition. These results suggest that sphingosine and C2-ceramide might induce c-jun expression and apoptosis in distinct signaling pathways.     

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.     

Inhibition of trifluoperazine-induced DNA fragmentation by cyclic AMP mediated signaling

Trifluoperazine (TFP), a phenothiazine antipsychotic agent with calmodulin antagonist property, induces DNA fragmentation in a dose- and time-dependent manner in PC12 cells. Various agents affecting calcium mediated intracellular signal transduction such as calcium chelators, calcium ionopores, inhibitors of phospholipase C, and activators/inhibitors of protein kinase C did not block TFP-induced DNA fragmentation. Some of these agents themselves induced DNA fragmentation in the conditions under which they were examined. However, cholera toxin (selective Gs activator), forskolin (adenylate cyclase activator) or dibutyryl cyclic AMP (cyclic AMP analogue) inhibited TFP-induced DNA fragmentation in a dose-dependent manner. These results suggest that it is not the calcium but the Gs and adenylate cyclase pathways that play an important role in TFP-induced DNA fragmentation in PC12 cells.     

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

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-dependent 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 effecting apoptosis.     

Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways.

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such properties. Thapsigargin, which raises intracellular Ca2+ levels by antagonizing its sequestration, induced immunophenotypic and morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2+ chelating compound EGTA and by calmodulin antagonists trifluoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antagonists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, strongly blocked CD14 down-regulation. These results are consistent with a Ca2+-dependent mechanism for CI-induced differentiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process.     

Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex

Protein kinase C (PKC) is an important constituent of the signaling pathways involved in apoptosis. We report here that like staurosporine, withaferin A is a potent inhibitor of PKC. In Leishmania donovani, the inhibition of PKC by withaferin A causes depolarization of DeltaPsim and generates ROS inside cells. Loss of DeltaPsim leads to the release of cytochrome c into the cytosol and subsequently activates caspase-like proteases and oligonucleosomal DNA cleavage. Moreover, in treated cells, oxidative DNA lesions facilitate the stabilization of topoisomerase I-mediated cleavable complexes, which also contribute to DNA fragmentation. However, withaferin A and staurosporine cannot induce cleavable complex formation in vitro with recombinant topoisomerase I nor with nuclear extracts from control cells. Taken together, our results indicate that inhibition of PKC by withaferin A is a central event for the induction of apoptosis and that the stabilization of topoisomerase I-DNA complex is necessary to amplify apoptotic process.     

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     

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.     

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.     

Differential activation of c-Jun NH2-terminal kinase and p38 pathways during FTY720-induced apoptosis of T lymphocytes that is suppressed by the extracellular signal-regulated kinase pathway

FTY720 is a novel immunosuppressive drug derived from a metabolite from Isaria sinclairii that is known to induce apoptosis of rat splenic T cells. In this study, we examined the intracellular signaling pathway triggered by FTY720. Treatment of human Jurkat T lymphocytes with FTY720-induced apoptosis characterized by DNA fragmentation. The same treatment induced activation of protein kinases such as c-Jun NH2-terminal kinase (JNK), p38/CSBP (CSAID-binding protein), and a novel 36-kDa myelin basic protein (MBP) kinase, but not extracellular signal-regulated kinase (ERK). Pretreatment of Jurkat cells with DEVD-CHO blocked FTY720-induced DNA fragmentation as well as the activation of p38/CSBP. However, DEVD-CHO treatment failed to inhibit FTY720-induced activation of JNK and the 36-kDa MBP kinase. We have also demonstrated that activation of the ERK signaling pathway completely suppressed the FTY720-induced apoptotic process including activation of caspase 3 and activation of JNK and the 36-kDa MBP kinase. Furthermore, transient expression of constitutively active mitogen-activated protein kinase/ERK kinase (MEK) protected the cells from FTY720-induced cell death. The effect of MEK was canceled by coexpression of a mitogen-activated protein kinase phosphatase, CL100. These results indicate that JNK and p38 pathways are differentially regulated during FTY720-induced apoptosis and that activation of ERK pathway alone is sufficient to cancel the FTY720-induced death signal.     

Cordyceps sinensis mycelium extract induces human premyelocytic leukemia cell apoptosis through mitochondrion pathway

This study was to identify the signaling pathways for the induction of HL-60 cell apoptosis by Cordyceps sinensis mycelium extract (CSME). CSME at 25 mug/ml induced nuclear fragmentation and DNA degradation, two hallmark events of apoptosis, in the HL-60 cells within 12-24 hrs of treatment. Concomitantly, several major events in the mitochondrial signal pathway occurred, including the loss of MTP (DeltaPsi(m)), cytochrome c release into the cytoplasm, the decrease in Bcl-2 protein level, the translocation of Bax protein from cytoplasm into mitochondria, and the activation of caspase-2, -3, and -9, but caspase-8, the initiator caspase in the death receptor pathway, was not activated. These results suggest that CSME induces apoptosis in HL-60 cell through the mitochondrial pathway rather than the death receptor pathway.     

Caspase-dependent activation of calpain during drug-induced apoptosis

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.     

Vaticanol C-induced cell death is associated with inhibition of pro-survival signaling in HL60 human leukemia cell line

Recently, we found that vaticanol C (a resveratrol tetramer), which was isolated from stem bark of Dipterocarpaceae, exhibited growth suppression and induction of apoptosis via the loss of mitochondrial membrane potential and consequent caspases activation. The detailed mechanisms are not clearly understood. We decided to attempt to gain further insight into the mechanisms underlying vaticanol C-induced apoptosis in HL-60 cells. Treatment of HL-60 cells with vaticanol C was found to cause a marked decrease in the level of phosphorylated extracellular signal-regulated kinase (ERK) concurrent with inhibited phosphorylation of its upstream kinase mitogen-activates protein kinase kinase (MEK). Moreover, exposure to vaticanol C led to a significant reduction in the level of phosphorylated Akt. Thus, vaticanol C induced inhibition of both ERK and Akt phosphorylation, resulting in reduced phosphorylation of Bad. These results suggest that vaticanol C might induce apoptosis via a mechanism involving activation of Bad through disruption of pro-survival signaling pathways.     

Imidazole-induced cell death,associated with intracellular acidification,caspase-3 activation,DFF-45 cleavage,but not oligonucleosomal DNA fragmentation

Intracellular acidification is known to be involved in the initiation phase of apoptosis. However, the necessity of intracellular acidic conditions in the execution phase of apoptosis remains unknown. In this study, we found that in HL-60 cells imidazole induces cell death, associated with intracellular acidification, caspase-3 activation and DFF-45 cleavage, but not oligonucleosomal DNA fragmentation. A caspase inhibitor prevented cell death but not intracellular acidification. When pHi was neutralized by changing from imidazole-containing medium to fresh medium, oligonucleosomal DNA fragmentation and increased caspase-3 activity was observed in the imidazole-treated HL-60 cells. Furthermore, the DNA fragmentation induced by intracellular neutralization was inhibited by caspase inhibitor treatment. These results indicate that imidazole induces caspase-dependent cell death, and suggest that maintaining pHi in the neutral range is essential for the induction of oligonucleosomal DNA fragmentation in the execution phase of apoptosis.     

Regulation of DNA fragmentation: the role of caspases and phosphorylation

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation.     

Extracellular ATP as a trigger for apoptosis or programmed cell death

Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.     

TLQP-21, a neuroendocrine VGF-derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c- jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.