ONO-AE-248诱发中性粒细胞非凋亡性程序化细胞死亡的生化信号转导初探

目的：研究ONO-AE-248诱发中性粒细胞（PMN）非凋亡性程序化细胞死亡过程中，细胞死亡相关的蛋白分子caspase-3、caspase-8、p-38MAPK和PKC的作用，探讨这种细胞死亡的分子机制。方法：运用光镜和DNA电泳对PMN形态学变化和生化特征进行观察和评估；通过Western blot显示PMN中caspase-3、caspase-8活性改变和p-38MAPK磷酸化改变；运用MTS法检测PKC抑制剂对PMN活性的影响。结果：PMN经ONO-AE-248刺激，绝大多数分叶核融合成单叶核（12小时），DNA琼脂糖电泳结果显示缺乏梯状条带（24小时），而且ONO-AE-248对caspase-3、caspase-8活性和p-38MAPK磷酸化水平无明显影响。然而，PKC抑制剂STS和H-7能显著抑制ONO-AE-248对PMN的促死亡效应。结论：ONO-AE-248所诱导的PMN死亡可能是一种非凋亡性的主动性细胞死亡，即非凋亡性程序化细胞死亡。该死亡过程不依赖caspase-3、caspase-8的活化和p-38MAPK的磷酸化，但是PKC可能发挥正向的调控作用。     

Induction of neutrophil death resembling neither apoptosis nor necrosis by ONO-AE-248, a selective agonist for PGE2 receptor subtype 3

An increase of intracellular cAMP mediated by prostaglandin E2 (PGE2) has been shown to delay spontaneous apoptosis of neutrophils. It has been demonstrated that a selective agonist for PGE2 receptor subtype 3 (the EP3 receptor) is capable of decreasing cAMP and stimulating phosphoinositide turnover in various types of cells. We investigated the effect of a selective EP3 receptor agonist, ONO-AE-248, on neutrophil viability. ONO-AE-248 rapidly caused a unique form of neutrophil death. The agonist primarily induced morphological changes of the nucleus, including fusion of the lobules, decreased compactness of the chromatin, and blebbing and rupture of the nuclear membrane. This was followed by an increase of plasma membrane permeability and cell lysis. During these processes, neither apoptotic changes such as nuclear condensation, DNA fragmentation, and expression of phospholipid phosphatidylserine on the plasma membrane nor necrotic changes such as chromatin clumping and organelle destruction were apparent in the treated neutrophils. The fatal effect of the agonist night be specific for neutrophils because it failed to promote the rapid death of other types of cells. Although activation of neutrophils by ONO-AE-248 was not evident, experiments using metabolic inhibitors demonstrated that the agonist caused neutrophil death via the activation of protein kinase C in the presence of intracellular ATP. These findings indicated that EP3 receptor-mediated signals might promote a novel form of neutrophil death, which differs from typical apoptosis or necrosis.     

ONO-AE-248诱导的中性粒细胞非凋亡性程序化细胞死亡的形态学变化

目的研究前列腺素E受体亚型3(EP3)激动剂ONO-AE-248诱导的中性粒细胞(PMN)非凋亡性程序化细胞死亡的形态学变化,探讨PMN死亡形式的多样性和复杂性。方法运用透射电镜、荧光显微镜及激光共聚焦扫描显微镜观察PMN在自发性凋亡与ONO-AE-248刺激下亚细胞微结构的变化。结果以5mmol/L的ONO-AE-248刺激PMN12h后,电镜观察绝大多数PMN的多叶核融合为单叶核,核质疏松,核膜分层、起泡甚至破裂,核质溢出,但细胞膜较完整,细胞器无明显改变。以荧光染料DAPI标染活细胞核,ONO-AE-248刺激后,PMN表现为多叶核融合成大而模糊的球形,核染色密度较低;ONO-AE-248刺激的PMN死亡仍有细胞膜磷脂酰丝氨酸(PS)的外翻。ONO-AE-248刺激组与自发性凋亡组PMN在线粒体的形态结构、分布和细胞质内的染色性均有明显差异。结论ONO-AE-248在体外能刺激人PMN发生非凋亡性程序化细胞死亡,这一刺激过程不影响自发性凋亡的PMN细胞膜PS的外翻,并且细胞核和线粒体的改变是其早期最为显著的形态学改变,提示二者可能是ONO-AE-248刺激的靶标和早期的药物反应中心。     

ONO-AE-248诱发的中性粒细胞非凋亡、非坏死性死亡

目的:观察前列腺素E2受体亚型3(EP3)激动剂ONO-AE-248对中性粒细胞的影响。方法:运用电子显微镜、MTS法、RT-PCR和流式细胞术等技术,对中性粒细胞的形态变化、生物学活性及凋亡进行观察和评估,并对EP3受体进行分析。结果:中性粒细胞存在EP3受体基因的表达,以5mmol/L的ONO-AE-248作用12h后,其多叶核融合为单叶核,核膜起泡、破裂,核质溢出,细胞死亡;但其快速死亡不具有细胞凋亡和坏死的形态特征且需要能量(ATP)供应。结论:ONO-AE-248所致这种新型细胞主动死亡的形式,可能为“非凋亡性程序化细胞死亡”的一种,这对于重新认识和定位中性粒细胞在非特异性免疫中的作用和地位提出了新的思路。     

ONO-AE-248诱导的中性粒细胞非凋亡、非坏死性死亡中未出现DNA小片断化的分子机制初探

目的探讨ONO-AE-248诱导的中性粒细胞非凋亡非坏死性死亡中没有出现DNA小片段的分子机制，为进一步确立这种新型细胞死亡形式提供实验室依据。方法体外新鲜分离人外周血中性粒细胞与ONO—AE-248共同培养，TUNEL法结合激光共聚焦扫描显微镜检测凋亡细胞并观察细胞核形态；Westernblotting检测caspase-3的活性；RT—PCR检测DNA断裂因子（DFF40）的表达水平。实验中同时设立空白组、LPS抑制凋亡组和TNF—α．促进凋亡组为对照。结果ONO—AE-248刺激培养的中性粒细胞经TUNEL法检测未见阳性细胞，细胞核染色密度较低，边界不清，分叶不明显，核形较大；Western blotting结果显示ONO—AE-248对caspase一3前体裂解无显著影响；RT—PCR结果显示ONO—AE-248诱导了DFF40表达明显下调。结论ONO-AE-248诱导的中性粒细胞死亡过程中细胞核DNA断裂方式明显不同于自发性凋亡，DFF40表达下调可能是其没有出现DNA小片段化的主要原因之一。     

TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO.

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. In the present study, we report that mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. We show that the Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. Our results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis.     

Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes

Mitochondria play a central role in many apoptotic reactions. Although mitochondrial apoptotic changes and caspase activation have been demonstrated in the apoptotic thymocytes, cell death signal through mitochondria in TCR-stimulated thymocytes has not been fully understood. In this study, we show that TCR stimulation induced disruption of mitochondrial transmembrane potential (Delta Psi(m)), the cytochrome c release from mitochondira, capase-3 activation, and the cell death of thymocytes. Bongkrekic acid, an inhibitor of Delta Psi(m) disruption, blocked the cytochrome c release from mitochondria and the following caspase-3-mediated cell death. Furthermore, a pro-apoptotic Bcl-2 family protein, Bax, but not Bad or Bid, was translocated from cytosol to mitochondria in TCR-stimulated thymocytes. This translocation and the following apoptotic changes were inhibited by SB203580, a p38 kinase inhibitor, in a specific manner. These results suggest that activated p38 kinase pathway by TCR stimulation induces translocation of Bax to mitochondria, causing Delta Psi(m) disruption, and the release of cytochrome c, which finally induces caspase-3-mediated apoptosis in thymocytes.     

Protection against CD95-induced apoptosis by chlamydial infection at a mitochondrial step

Chlamydiae are obligate intracellular bacteria that infect human epithelial and myeloid cells. Previous work has established that chlamydiae are able to protect a cell against apoptosis induced by certain experimentally applied stimuli. Here we provide an analysis of this protective activity against the signal transduction during CD95-induced apoptosis. In HeLa cells overexpressing CD95, infection with Chlamydia trachomatis inhibited the appearance of apoptotic morphology, effector caspase activity, the activation of caspase-9 and -3, and the release of cytochrome c from mitochondria. However, caspase-8-processing and activity (measured as cleavage of Bid) were unaffected by the chlamydial infection. Similarly, infection with the species C. pneumoniae did not prevent the activation of caspase-8 but inhibited the appearance of effector caspase activity upon signaling through CD95. Furthermore, infection with C. trachomatis was able to inhibit CD95-induced apoptosis in Jurkat lymphoid cells, where a mitochondrial contribution is required, but not in SKW6.4 lymphoid cells, where caspase-8 directly activates caspase-3. Taken together, these data show that chlamydial infection can protect cells against CD95-induced apoptosis but only where a mitochondrial signaling step is necessary for apoptotic signal transduction.     

The protection of Bcl-2 overexpression on rat cortical neuronal injury caused by analogous ischemia/reperfusion in vitro

Recent studies have suggested that neuronal apoptosis in cerebral ischemia could arise from dysfunction of endoplasmic reticulum (ER) and mitochondria. B-cell lymphoma/leukemia-2 gene (Bcl-2) has been described as an inhibitor both in programmed cell death (PCD) and ER dysfunction during apoptosis, and the Bcl-2 family play a key role in regulating the PCD, both locally at the ER and from a distance at the mitochondrial membrane. However, its signal pathways and concrete mechanisms in endoplasmic reticulum-initiated apoptosis remain incompletely understood. We therefore investigate whether ischemia/reperfusion (I/R) causes neuronal apoptosis in part via cross-talk between ER and mitochondria or not, and how the overexpression of Bcl-2 prevents this form of cell death. Here we show that analogous I/R-induced cell death occurs consequent to interactions of ER stress and mitochondrial death pathways. The participation of the mitochondrial pathway was demonstrated by the release of cytochrome C (cyt C) from mitochondrial into cytoplasmic fractions and caspase-9 cleavage. The involvement of ER stress was further supported by the observable increase of glucose-regulated protein 78(GRP78)/BiP expression and caspase-12 activity. Furthermore, prior to these changes, swelling of the ER lumen and dissociation of ribosomes from rough ER were detected by electron microscopy. Bcl-2 overexpression inhibits the release of cyt C and the activation of caspase-9/-8/-3 but not caspase-12 based on the results of Western blot. These suggest that cross-talk between ER and mitochondria participate in neuronal damage after ischemia/reperfusion. Bcl-2 overexpression could suppress I/R-induced neuronal apoptosis via influencing mitochondrial integrity.     

The roles of endogenous reactive oxygen species and nitric oxide in triptolide-induced apoptotic cell death in macrophages

Triptolide, a major active component extracted from the root of Tripterygium wilfordii Hook f, has been shown to possess potent immunosuppressive and anti-inflammatory properties. In the present report, we reported that triptolide increased the generation of reactive oxygen species (ROS) and nitric oxide (NO) and induced apoptosis of RAW 264.7 cells in a dose-dependent manner (5-25 ng/ml). The antioxidant, reduced glutathione (GSH), significantly inhibited triptolide-induced apoptosis and inhibited the degradation of Bcl-2 protein, disruption of mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, and cleavage of poly-(ADP-ribose)-polymerase. The inducible nitric oxide synthase-specific inhibitor 1400w blocked triptolide-induced apoptosis, but did not alter mitochondria disruption and caspase-3 activation. These results, for the first time, implicated that the increased endogenous ROS and NO co-mediated triptolide-induced apoptosis in macrophages. ROS initiated triptolide-induced apoptosis by the mitochondria signal pathway, while the apoptotic cell death mediated by NO was not via mitochondria collapse and caspase-3 activation. In addition, combining mathematical calculation and computer simulation based on our conventional experimental results, we set and validated the apoptotic model and provided more dynamic processes of triptolide-induced apoptotic cascade in macrophages.     

Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3

Nitric oxide (NO) is elevated in the synovial fluids and sera of patients with rheumatoid arthritis (RA) and is thought to be an important proinflammatory mediator in the rheumatoid synovium. To test the hypothesis that NO might modulate the apoptosis-inducing signal pathway, we investigated the effects of NO on rheumatoid synovial-cell apoptosis induced by Fas ligation with anti-Fas antibody. Pretreatment of synovial cells with the NO donor S-nitro-N-acetylpenicillamine (SNAP) prevented the Fas-mediated induction of apoptosis. The activation of caspase-3 was required to mediate Fas-induced synovial cell apoptosis. The NO donor SNAP inhibited Fas-induced caspase-3 activation in rheumatoid synovial cells. However, NO did not interrupt Fas-induced caspase-8 cleavage or subsequent cytochrome c release into the cytosol in rheumatoid synovial cells. These data indicate that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase-3 activation. Thus, we propose that NO interferes with cell death signal transduction and may contribute to rheumatoid synovial cell proliferation by inhibiting induction of apoptosis.     

谷氨酸诱导海马神经元凋亡涉及线粒体释放细胞色素C

目的：建立体外谷氨酸诱导神经元兴奋损伤模型，探索其凋亡发生是否通过线粒体信号转导途径介导的细胞色素C（Cyt C）释放而实现，为今后干预性使用神经保护剂提供依据。方法:分离及培养新生Wistar大鼠海马神经元，选用合适谷氨酸浓度建立神经元损伤模型；利用LDH测定及流式细胞仪Annexin V/PI双染色法检测谷氨酸暴露后不同时点神经元凋亡及坏死的动态改变；采用Western blotting法检测caspase-3活性及线粒体内和胞浆内Cyt C水平动态变化。结果:谷氨酸诱导神经元损伤呈明显浓度及时间依赖性，50 μmol/L浓度可使LDH释放量明显增加 (18.4%,P<0.05)，暴露后6 h凋亡率显著增加；凋亡发生前，神经元caspase-3活性已明显增高（3 h），6 h达高峰；线粒体Cyt C释放发生在caspase-3增高前，30 min时胞浆内Cyt C水平即明显增加（P<0.05），3 h胞浆内Cyt C水平超过线粒体内，而线粒体内Cyt C水平进行性减少。结论:50 μmol/L谷氨酸可诱导海马神经元凋亡，凋亡机制可能是通过损伤线粒体膜，使Cyt C易位释放入胞浆激活caspase级联反应而致。     

Leptospira interrogans Induces Apoptosis in Macrophages via Caspase-8- and Caspase-3-Dependent Pathways

Apoptosis of host cells plays an important role in modulating the pathogenesis of many infectious diseases. It has been reported that Leptospira interrogans, the causal agent of leptospirosis, induces apoptosis in macrophages and hepatocytes. However, the molecular mechanisms responsible for host cell death remained largely unknown. Here we demonstrate that L. interrogans induced apoptosis in a macrophage-like cell line, J774A.1, and primary murine macrophages in a time- and dose-dependent manner. Apoptosis was associated with the activation of cysteine aspartic acid-specific proteases (caspase-3, caspase-6, and caspase-8), the increased expression of Fas-associated death domain (FADD), and the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP) and nuclear lamina protein (lamin A and lamin C). Caspase-9 was activated to a lesser extent, whereas no release of cytochrome c from mitochondria was detectable. Inhibition of caspase-8 impaired L. interrogans-induced caspase-3 and -6 activation, as well as PARP and lamin A/C cleavage and apoptosis, suggesting that apoptosis is initiated via caspase-8 activation. Furthermore, caspase-3 was required for the activation of caspase-6 and seemed to be involved in caspase-9 activation through a feedback amplification loop. These data indicate that L. interrogans-induced apoptosis in macrophages is mediated by caspase-3 and -6 activation through a FADD-caspase-8-dependent pathway, independently of mitochondrial cytochrome c-caspase-9-dependent signaling.     

Evidence for a Novel, Caspase-8-Independent, Fas Death Domain-Mediated Apoptotic Pathway

Certain caspase-8 null cell lines demonstrate resistance toFas-induced apoptosis, indicating that the Fas/FasL apoptoticpathway may be caspase-8-dependent. Some reports, however, haveshown that Fas induces cell death independent of caspase-8. Herewe provide evidence for an alternative, caspase-8-independent,Fas death domain-mediated apoptotic pathway. Murine 12B1-D1 cellsexpress procaspase-3, -8, and -9, which were activated upon thedimerization of Fas death domain. Bid was cleaved andmitochondrial transmembrane potential was disrupted in thisapoptotic process. All apoptotic events were completely blockedby the broad-spectrum caspase inhibitor Z-VAD-FMK, but not byother peptide caspase inhibitors. Cyclosporin A (CsA), whichinhibits mitochondrial transition pore permeability, blockedneither pore permeability disruption nor caspase activation.However, CsA plus caspase-8 inhibitor blocked all apoptoticevents of 12B1-D1 induced by Fas death domain dimerization. Ourdata therefore suggest that there is a novel,caspase-8-independent, Z-VAD-FMK-inhibitable, apoptotic pathway in12B1-D1 cells that targets mitochondria directly.     

Brain-derived neurotrophic factor activation of TrkB protects neurons from HIV-1/gp120-induced cell death

Patients with the human immunodeficiency virus type 1 (HIV-1) develop in the late phase of infection a complex of neurological signs termed Acquired Immune Deficiency Syndrome-Related Dementia (ADC). These patients exhibit cortical and subcortical atrophy. Considerable experimental data indicate that the HIV-1 envelope glycoprotein gp120 may be one of the agents causing neuronal cell death. Gp120 causes neuronal cell death both in vitro and in vivo by activating a caspase-dependent apoptotic pathway, and in particular caspase-3. The neurotrophin brain-derived neurotrophic factor (BDNF) has been shown to prevent gp120-mediated apoptosis of cerebellar granule cells by inhibiting caspase-3 activation. However, the signal transduction pathway that contributes to the neuroprotective effects of BDNF has not been determined. BDNF binds with high affinity to the tyrosine kinase receptor TrkB and activates different intracellular signaling cascade including the extracellular signal-related kinases (ERK) and the phosphatidylinositol 3-kinase (PI3-K). Pharmacological inhibition of TrkB or ERK1/2, but not PI3-K, greatly reduced the ability of BDNF to block gp120-mediated apoptosis of cerebellar granule cells. These findings suggest that TrkB-mediated activation of ERK1/2 is the main signaling pathway that contributes to neuroprotection against gp120.     

Lidocaine-induced apoptosis and necrosis in U937 cells depending on its dosage

Local anesthetics are known to affect a variety of cellular responses other than the action of anesthetics through the Na(+) channel blockade. In this study, we examined the effect of a common local anesthetic lidocaine on the cellular activity and viability of human histiocytic lymphoma U937 cells. The cellular activity and viability were assessed by WST-1 reduction activity and trypan blue exclusion test, respectively. Induction of apoptosis was monitored by DNA ladder formation, reduction of mitochondrial transmembrane potential (DeltaPsim), caspase-3 activity and nuclear morphology. Lidocaine at concentrations below 12 mM induced apoptosis characterized by DNA fragmentation and chromatin condensation dose- and time-dependently. A pan-caspase inhibitor and a caspase-3 inhibitor blocked DNA ladder formation followed by the reduction of cell death. However, the caspase inhibitors did not affect the DeltaPsim, but cyclosporin A inhibited the collapse of DeltaPsim followed by a reduction of cell death. Lidocaine-induced apoptosis was mitochondria- and caspase-dependent, but the collapse of DeltaPsim was independent of caspase activation. At concentrations above 15 mM, lidocaine induced necrosis with early disruption of membrane integrity. These results indicate that lidocaine induced apoptosis and necrosis in U937 cells depending on its dosage.     

A comparison of the signal pathways between the TNF alpha- and oridonin-induced murine L929 fibrosarcoma cell death

Oridonin, an active component isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, we compared the signal transduction pathways between TNFalpha-and oridonin-induced L929 cell death. Oridonin and TNFalpha initiated apoptotic morphologic changes, but DNA fragmentation was found in TNFalpha-treated L929 cells but not in oridonin-treated ones. The pan-caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) augmented oridonin-and TNFalpha-induced cell death. However, the caspase-9 inhibitor (z-LEHD-fmk) only increased oridonin-induced L929 cell death. Moreover, poly (ADPribose) polymerase (PARP) was cleaved in oridonin-treated L929 cells but not in the TNFalpha-treated groups, and the caspase-3 inhibitor (z-DEVD-fmk) failed to inhibit PARP cleavage. These results showed that only oridonin-induced L929 cell death required PARP degradation in a caspase-3 independent manner. In addition, oridonin increased the ratio of Bax/Bcl-2 protein expression, but TNFalpha did not. TNFalpha induced p38 and ERK activation, whereas oridonin triggered only ERK activation. We also investigated the effect of oridonin on intracellular TNFalpha expression, and found that oridonin augmented endogenous pro-TNFalpha expression and its upstream protein IkB phosphorylation. These results indicated that although oridonin promoted endogenous pro-TNFalpha expression, a great difference existed between the signal pathways through which TNFalpha-and oridonin-induced cell death.     

Activation of voltage-sensitive sodium channels during oxygen deprivation leads to apoptotic neuronal death

Sodium (Na(+)) entry into neurons during hypoxia is known to be associated with cell death. However, it is not clear whether Na(+) entry causes cell death and by what mechanisms this increased Na(+) entry induces death. In this study we used cultures of rat neocortical neurons to show that an increase in intracellular sodium (Na(i)(+)) through voltage-sensitive sodium channels (VSSCs), during hypoxia contributes to apoptosis. Hypoxia increased Na(i)(+) and induced neuronal apoptosis, as assessed by electron microscopy, annexin V staining, and terminal UDP nick end labeling staining. Reducing Na(+) entry with the VSSC blocker, tetrodotoxin (TTX), attenuated apoptotic neuronal death via a reduction in caspase-3 activation. Since the attenuation of apoptosis by TTX during hypoxia suggested that the activation of VSSCs and Na(+) entry are crucial events in hypoxia-induced cell death, we also determined whether the activation of VSSCs per se could lead to apoptosis under resting conditions. Increasing Na(+) entry with the VSSC activator veratridine also induced neuronal apoptosis and caspase-3 activation. These data indicate that a) Na(+) entry via VSSCs during hypoxia leads to apoptotic cell death which is mediated, in part, by caspase-3 and b) activation of VSSCs during oxygen deprivation is a major event by which hypoxia induces cell death.     

Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases

Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% +/- 1.8% and 81.7% +/- 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% +/- 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.