Early and late apoptotic events induced in human glioblastoma cells by Hypericin PDT

During photodynamic therapy (PDT) the interaction between light and photosensitizers induces oxidative stress and the production of reactive oxygen species (ROS). ROS formation influences ion channels and causes changes in the Ca²⁺ concentration which may induce further reactions leading to cell stimulation or cell death. In this study we analysed the influence of irradiation conditions on the cell death mechanisms during Hypericin-induced PDT. The process of apoptosis is characterized by morphological and physiological changes of the cell membrane and of the whole intracellular chain reactions. The translocation of the phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane is one of the earliest indications of apoptotic cell death. The translocation was traced by the binding of fluorescein isothiocyanate (FITC)-conjugated Annexin-V to phosphatidylserine and fluorescence activated cell sorting (FACS) analysis.     

Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death

Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.     

Specific Activation of the Cysteine Protease CPP32 during the Negative Selection of T Cells in the Thymus

Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide–MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti–CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.     

Spontaneous apoptosis and highly active antiretroviral therapy (HAART).

Increased programmed cell death (PCD) or apoptosis has been detected in the T cells of HIV-infected subjects; it is held partially responsible for the continuous loss of CD4+ T cells during the natural course of HIV infection. Highly active antiretroviral therapy (HAART) decreases the viral load and leads to an increase of CD4+ count in vivo. In this study we evaluated PCD in total peripheral blood mononuclear cells, CD8+ and CD4+ lymphocytes before and four weeks after initiation of HAART. Seven HIV-1-infected patients were investigated. Viral load was assessed by RT-polymerase chain reaction and PCD by flow cytometry using apoptosis by 7 amino actinomycin D (7AAD) and propidium iodide (PI). After four weeks of HAART, CD4+ T and CD8+ T cell levels were stable, and plasma HIV-RNA copies were significantly decreased. In four of the patients (4/7), HIV-RNA levels were reduced to undetectable levels (fewer than 400 copies per milliliter). A statistically significant reduction of apoptosis among CD4+ cells was observed (P < 0.03), though neither in the CD8+ T cell population nor in peripheral blood mononuclear cells (PBMCS). These results demonstrate the beneficial effect of HAART on apoptosis of CD4+ cells in the early treatment stage.     

膜联蛋白在肿瘤凋亡显像中的研究进展

目前的影像学检查方法尚不能在肿瘤治疗早期即对疗效进行有效判断,而活体凋亡显像能在细胞水平对肿瘤组织的改变进行早期、无创、动态监测,从而准确判断肿瘤细胞对化疗药物的反应。磷脂酰丝氨酸(PS)由细胞膜内侧翻转到细胞膜表面是细胞凋亡早期最具特征性的改变之一,这使得PS成为细胞凋亡显像的有效靶点。放射性核素标记的膜联蛋白可选择性与翻转到细胞膜表面的PS结合,从而可在体内对早期疗效进行判断。由于在有效的抗肿瘤治疗开始24 h后即可观察到大量的肿瘤细胞发生凋亡,因此活体凋亡显像有助于为临床个体化治疗方案的制订提供指导性意见。本文在临床前研究和临床试验两方面对膜联蛋白凋亡显像在抗肿瘤治疗早期疗效评价中的机制、应用、局限性及未来发展前景作一综述。     

Antimycoplasmal activity of dimethylphenols in a tracheal explant culture system.

Mycoplasma pneumoniae induces pneumonia-like symptoms in hamsters and causes ciliostasis and cytonecrosis in hamster tracheal explants. 2,4-Dimethylphenol and, to a lesser extent, its 2,3-, 2,5-, and 2,6-dimethylphenol isomers protected tracheal explants from these changes after exposure to virulent M. pneumoniae strain PI 1428. The effect was concentration, time, and isomer dependent. At concentrations of 10(-9) M or greater, 2,4-dimethylphenol completely prevented the morphological (loss of ciliated cells) and biochemical (decreased dehydrogenase activity) changes normally observed after exposure to M. pneumoniae. Apparently, 2,4-dimethylphenol interfered with an early event in the infection process. Complete protection required that it be present during the first 2 h of exposure of the explants to the infecting mycoplasmas. These xylenols may prove to be useful tools for helping to define the mechanisms of pathogenesis in certain respiratory infections.     

Human erythrocyte protein 4.1 is a phosphatidylserine binding protein.

The aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS) are the major phospholipids contained in the cytoplasmic leaflet of the human erythrocyte (RBC) plasma membrane and are largely confined to that leaflet over the entire RBC lifespan. In particular, PS, which comprises approximately 13% of total RBC membrane phospholipids, is normally restricted entirely to the cytoplasmic leaflet. However, molecular mechanisms that regulate this asymmetric distribution of phospholipids are largely unknown. We examined elliptocytic RBCs that completely lacked protein 4.1 (HE [4.1 degrees]), but contained normal amounts of all other peripheral membrane proteins, and found approximately 10% of total membrane PS was accessible in the exoplasmic leaflet of these membranes. Inside out vesicles (IOVs) derived from HE [4.1 degrees] RBCs bound fewer PS liposomes than did IOVs derived from normal RBCs. Normal IOVs that were depleted of proteins 2.1 (ankyrin), 4.1, and 4.2 bound fewer PS liposomes similar to HE [4.1 degrees] IOVs, and repletion with protein 4.1 restored PS liposome binding to control levels. Addition of purified protein 4.1 to PS liposomes resulted in saturable binding with the extent of binding being proportional to the liposome PS content. Our data suggests that human RBC protein 4.1 is a PS binding protein and may be involved in the molecular mechanisms that stabilize PS in the cytoplasmic leaflet of the human RBC plasma membrane.     

Review: The role of glutathione in the regulation of apoptosis

Recent advances in the study of the regulation of cell death by apoptosis suggest that changes in mitochondrial permeability frequently precede the development of morphological features such as chromatin condensation, phosphatidylserine inversion of the outer cell membrane and the activation of endonucleases. There is evidence that this permeability transition is associated with, and may be regulated by, changes in the intracellular redox potential. The role of the tripeptide thiol glutathione in the regulation of apoptosis-associated redox changes and the control of mitochondrial membrane permeability is reviewed in this article.     

Mitochondrial control of nuclear apoptosis

Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro- oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.     

Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.

Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of neutrophils and their potentially histotoxic contents is one prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the senescent neutrophil that are associated with their recognition by macrophages are the subject of this investigation. Over 24 h in culture an increasing proportion of human neutrophils from peripheral blood or acutely inflamed joints underwent morphological changes characteristic of programmed cell death or apoptosis. Time-related chromatin cleavage in an internucleosomal pattern indicative of the endogenous endonuclease activation associated with programmed cell death was also demonstrated. A close correlation was observed between the increasing properties of apoptosis in neutrophils and the degree of macrophage recognition of the aging neutrophil population, and a direct relationship between these parameters was confirmed within aged neutrophil populations separated by counterflow centrifugation into fractions with varying proportions of apoptosis. Macrophages from acutely inflamed joints preferentially ingested apoptotic neutrophils and histological evidence was presented for occurrence of the process in situ. Programmed cell death is a phenomenon of widespread biological importance and has not previously been described in a cell of the myeloid line. Because it leads to recognition of intact senescent neutrophils that have not necessarily disgorged their granule contents, these processes may represent a mechanism for the removal of neutrophils during inflammation that also serves to limit the degree of tissue injury.     

Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis.

Increases in mesangial cell number may herald glomerular scarring, but they are not irreversible. This study sought mechanisms by which surplus glomerular mesangial cells can be cleared. A small proportion of cultured mesangial cells exhibited typical morphological features of apoptosis (programmed cell death), which was increased by growth factor deprivation or exposure to cycloheximide, stimuli known to increase apoptosis in other cell types. Apoptosis was confirmed by typical internucleosomal chromatin cleavage. In vivo, clear morphological evidence of mesangial apoptosis leading to phagocytosis by neighboring mesangial cells was obtained in self-limited mesangial proliferation induced in rats by Thy1.1 antibody, apoptosis occurring approximately 10-fold more frequently than in the healthy rat glomerulus. Indeed, changes in glomerular cell number in Thy1.1 nephritis strongly suggested that apoptosis is the major cell clearance mechanism counterbalancing cell division, thereby mediating resolution of glomerular hypercellularity in experimental mesangial proliferation.     

Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways

Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H2O2 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis.     

Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals.

In immature thymocytes, T cell receptor for antigen (TCR) mobilization leads to an active T cell suicide process, apoptosis, which is involved in the selection of the T cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals (Ameisen, J.C., and A. Capron. 1991. Immunol. Today. 4:102). Here, we report that the selective failure of CD4+ T cells from 59 clinically asymptomatic HIV-infected individuals to proliferate in vitro to TCR mobilization by major histocompatibility complex class II-dependent superantigens and to pokeweed mitogen (PWM) is due to an active CD4+ T cell death process, with the biochemical and ultrastructural features of apoptosis. Activation-induced cell death occurred only in the CD4+ T cell population from HIV-infected asymptomatic individuals and was not observed in T cells from any of 58 HIV-seronegative controls, including nine patients with other acute or chronic infectious diseases. Activation-induced CD4+ T cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody (mAb). The CD28 mAb not only prevented apoptosis but also restored T cell proliferation to stimuli, including PWM, superantigens, and the tetanus and influenza recall antigens. These findings may have implications for the understanding of the pathogenesis of acquired immune deficiency syndrome and for the design of specific therapeutic strategies.     

Caspase-3 apoptotic signaling following injury to the central nervous system.

Apoptotic cell death is a fundamental and highly regulated biological process in which a cell is instructed to participate actively in its own demise. This process of cellular suicide is activated by developmental and environmental cues and normally plays an essential role in eliminating superfluous, damaged, and senescent cells of many tissue types. In recent years, a number of experimental studies have provided evidence of widespread neuronal and glial apoptosis following injury to the central nervous system (CNS). These studies indicate that injury-induced apoptosis can be detected from hours to days following injury and may contribute to neurological dysfunction. Given these findings, understanding the biochemical signaling events controlling apoptosis is a first step towards developing therapeutic agents which would target this cell death process. This review will focus on the molecular cell death pathways responsible for generating the apoptotic phenotype, summarize what is currently known about apoptotic signals activated in the injured CNS, and what potential strategies might be pursued to reduce this cell death process as a means to promote functional recovery.     

番荔枝内酯类聚醚类似物AA005抗白血病细胞体外增殖效应及其可能的机制

本研究探讨番荔枝内酯类聚醚类似物AA005广泛的体外抗白血病细胞增殖效应及对急性早幼粒白血病细胞系NB4的可能作用机制。采用CCK-8试剂盒检测AA005对多种白血病细胞系增殖的影响;台盼蓝染色法检测细胞活率的改变;瑞氏染色后显微镜观察NB4细胞形态学结构的变化;流式细胞术分析细胞死亡形式;WesternBlot检测caspase-3的活化情况及其底物PARP-1的剪切情况;流式细胞术检测低浓度AA005(<200 nmol/L)对细胞周期的影响。结果表明,高浓度AA005(>200 nmol/L)能够抑制多种白血病细胞的增殖,呈浓度依赖性;不同浓度的AA005作用于NB4、U937、K562白血病细胞48 h后,绝大多数细胞死亡;以NB4细胞为例,AA005作用后出现类似凋亡形态特征;AA005几乎可同时诱导细胞发生早期凋亡及晚期凋亡;AA005诱导细胞死亡过程中仅有微弱的caspase-3活化及其底物PARP-1的剪切,caspase广谱抑制剂Z-VAD-fmk不能抑制AA005所致的细胞死亡;非毒性浓度的AA005(<200 nmol/L)引起细胞G2/M期阻滞。结论:番荔枝内酯类聚醚类似物AA005具有广泛的抗白血病细胞增殖效应,其机制可能与AA005诱导白血病细胞死亡及引起细胞G2/M期阻滞有关。     

Apoptotic cells suppress mast cell inflammatory responses via the CD300a immunoreceptor

When a cell undergoes apoptosis, phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. PS acts as an "eat-me" signal to direct phagocytes expressing PS receptors to engulf the apoptotic cell. We recently reported that the immunoreceptor CD300a, which is expressed on myeloid cells, is a PS receptor. We show that CD300a does not facilitate macrophage phagocytosis of apoptotic cells. Instead, CD300a delivers an inhibitory signal in mast cells to suppress production of LPS-induced inflammatory cytokines and chemokines. After cecal ligation and puncture (CLP), when a large number of cells undergo apoptosis in the peritoneal cavity, CD300a-deficient peritoneal mast cells produced more chemoattractant and recruited more neutrophils than did wild-type (WT) mast cells. As a result, CD300a-deficient mice showed increased neutrophil recruitment and improved bacterial clearance in the peritoneal cavity, and survived longer than WT mice. Antibody blockade of CD300a-PS interactions improved bacterial clearance and extended survival of WT mice subjected to CLP. These results indicated that CD300a is a nonphagocytic PS receptor that regulates mast cell inflammatory responses to microbial infections.     

Tubular cell apoptosis and cidofovir-induced acute renal failure

Cidofovir is an antiviral drug with activity against a wide array of DNA viruses including poxvirus. The therapeutic use of cidofovir is marred by a dose-limiting side effect, nephrotoxicity, leading to proximal tubular cell injury and acute renal failure. Treatment with cidofovir requires the routine use of prophylactic measures. A correct knowledge of the cellular and molecular mechanisms of cidofovir toxicity may lead to the development of alternative prophylactic strategies. We recently cared for a patient with irreversible acute renal failure due to cidofovir. Renal biopsy showed tubular cell apoptosis. Cidofovir induced apoptosis in primary cultures of human proximal tubular cells in a temporal (peak apoptosis at 7 days) and concentration (10-40 microg/ml) pattern consistent with that of clinical toxicity. Apoptosis was identified by the presence of hypodiploid cells, by the exposure of annexin V binding sites and by morphological features and was associated with the appearance of active caspase-3 fragments. Cell death was specific as it was also present in a human proximal tubular epithelial cell line (HK-2), but not in a human kidney fibroblast cell line, and was prevented by probenecid. An inhibitor of caspase-3 (DEVD) prevented cidofovir apoptosis. The survival factors present in serum, insulin-like growth factor-1 and hepatocyte growth factor, were also protective. The present data suggest that apoptosis induction is a mechanism contributing to cidofovir nephrotoxicity. The prophylactic administration of factors with survival activity for tubular epithelium should be further explored in cidofovir renal injury.     

In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1.

Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.     

Pertussis toxin inhibits activation-induced cell death of human thymocytes, pre-B leukemia cells and monocytes

Activation of human thymocytes and pre-B cells via the CD3/T cell receptor (TCR) complex or the IgM/B cell receptor complex, respectively, results in apoptotic cell death. Similarly, cross-linking of the activation marker CD69, which belongs to the natural killer complex, causes apoptosis of lipopolysaccharide-preactivated monocytes. Here we show that pertussis toxin (PTX) inhibits the activation-induced apoptosis of these three cell types, though it fails to prevent the programmed cell death that follows exposure of cells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin 4 (monocytes). The capacity of pertussis toxin to suppress activation-induced death is not due to quenching of the activation signal, because thymocytes exposed to PTX are still capable of mobilizing Ca2+ after TCR-alpha/beta cross-linking and proliferate in response to costimulation with PTX and CD3/TCR ligation. The apoptosis- inhibitory effect of PTX depends on the presence of an intact adenosine diphosphate (ADP)-ribosylating moiety, since a mutant pertussis toxin molecule that lacks enzymatic activity, but still possesses the membrane translocating activity, fails to interfere with activation- induced cell death. A toxin that induces a different spectrum of ADP ribosylation than PTX, cholera toxin, fails to inhibit apoptosis. To suppress apoptosis, the intact PTX holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis. These data unravel a PTX sensitive signal transduction event that intervenes during an early step of activation-induced cell death of immune cells.