Suppression of ERK signaling evokes autocrine Fas‐mediated death in arachidonic acid‐treated human chronic myeloid leukemia K562 cells

Arachidonic acid (AA)‐induced apoptotic death of K562 cells (human chronic myeloid leukemic cells) was characteristic of reactive oxygen species (ROS) generation and mitochondrial depolarization. N‐Acetylcysteine pretreatment rescued viability of AA‐treated cells and abolished mitochondrial depolarization. In contrast to no significant changes in phospho‐JNK and phospho‐ERK levels, AA evoked notable activation of p38 MAPK. Unlike that of JNK and p38 MAPK, ERK suppression further reduced the viability of AA‐treated cells. Increases in Fas/FasL protein expression, caspase‐8 activation, the production of tBid and the loss of mitochondrial membrane potential were noted with K562 cells that were treated with a combination of U0126 and AA. Down‐regulation of FADD attenuated U0126‐evoked degradation of procaspase‐8 and Bid. Abolition of p38 MAPK activation abrogated U0126‐elicited Fas/FasL up‐regulation in AA‐treated cells. U0126 pretreatment suppressed c‐Fos phosphorylation but increased p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun protein expression by siRNA suggested that c‐Fos counteracted the effect of c‐Jun on Fas/FasL up‐regulation. Taken together, our data indicate that AA induces the ROS/mitochondria‐dependent death pathway and blocks the ERK pathway which enhances the cytotoxicity of AA through additionally evoking an autocrine Fas‐mediated apoptotic mechanism in K562 cells. J. Cell. Physiol. 222: 625–634, 2010. © 2009 Wiley‐Liss, Inc.     

BBR induces apoptosis in HepG2 cell through an Akt‐ASK1‐ROS‐p38MAPKs‐linked cascade

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase‐3. The expressions of Bcl‐2 protein and pro‐caspase‐3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR‐induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR‐induced apoptosis effects via inhibition of Bax activation and Bcl‐2 inactivation. BBR‐induced, dose‐dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1‐mediated activation of JNK and p38 pathways. J. Cell. Biochem. 109: 329–338, 2010. © 2009 Wiley‐Liss, Inc.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid

Human lung neoplasms frequently express mutations that down‐regulate expression of various tumor suppressor molecules, including mitogen‐activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti‐tumor therapies. We now report that 4‐phenyl‐3‐butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non‐cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell‐cell communication. H2009 human lung carcinoma cells and ras‐transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras‐transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell–cell communication and connexin 43 phosphorylation in ras‐transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up‐regulation of p38 MAPK activity, altered connexin 43 expression, and down‐regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down‐regulated p38 MAPK activity and/or activated JNK and altered cell–cell communication. J. Cell. Biochem. 113: 269–281, 2012. © 2011 Wiley Periodicals, Inc.     

Reactive oxygen species are involved in the IFN‐γ‐stimulated production of Th2 chemokines in HaCaT keratinocytes

The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation‐regulated chemokine (TARC/CCL17) and macrophage‐derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon‐gamma (IFN‐γ)‐treated HaCaT keratinocytes. We found that IFN‐γ‐induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N‐acetyl cysteine (NAC), significantly inhibited the IFN‐γ‐induced production of chemokines as well as the activation of I kappa‐B (IκB)–nuclear factor‐kappa B (NF‐κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen‐activated kinase (MAPK), and NF‐κB suppressed IFN‐γ‐induced production of TARC and MDC. NF‐κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN‐γ‐stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L ‐buthionine sulfoximine (L‐BSO). However, IFN‐γ‐stimulated activation of IκB and NF‐κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN‐γ‐stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN‐γ‐stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF‐κB activation. J. Cell. Physiol. 226: 58–65, 2010. © 2010 Wiley‐Liss, Inc.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

Lentinan exerts synergistic apoptotic effects with paclitaxel in A549 cells via activating ROS‐TXNIP‐NLRP3 inflammasome

Paclitaxel is generally used to treat cancers in clinic as an inhibitor of cell division. However, the acquired resistance in tumours limits its clinical efficacy. Therefore, the aim of this study was to detect whether co‐treatment with lentinan enhanced the anti‐cancer effects of paclitaxel in A549 cells. We found that the combination of paclitaxel and lentinan resulted in a significantly stronger inhibition on A549 cell proliferation than paclitaxel treatment alone. Co‐treatment with paclitaxel and lentinan enhanced cell apoptosis rate by inducing caspase‐3 activation. Furthermore, co‐treatment with paclitaxel and lentinan significantly triggered reactive oxygen species (ROS) production, and increased thioredoxin‐interacting protein (TXNIP) expression. Moreover, co‐treatment with paclitaxel and lentinan enhanced TXNIP‐NLRP3 interaction, and activated NLRP3 inflammasome whereat interleukin‐1β levels were increased and cell apoptosis was induced. In addition, combination of paclitaxel and lentinan could activate apoptosis signal regulating kinase‐1 (ASK1)/p38 mitogen‐activated protein kinase (MAPK) signal which also contributed to cell apoptosis. Taken together, co‐treatment with paclitaxel and lentinan exerts synergistic apoptotic effects in A549 cells through inducing ROS production, and activating NLRP3 inflammasome and ASK1/p38 MAPK signal pathway.     

Reactive oxygen species-mediated mitochondrial pathway is involved in Baohuoside I-induced apoptosis in human non-small cell lung cancer

Baohuoside I (also known as Icariside II) is a flavonoid isolated from Epimedium koreanum Nakai. Although Baohuoside I exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human lung cancer cells are poorly understood. Therefore, in the present study, we investigated the usefulness of Baohuoside I as a potential apoptosis-inducing cytotoxic agent using human adenocarcinoma alveolar basal epithelial A549 cells as in vitro model. The apoptosis induced by Baohuoside I in A549 cells was confirmed by annexin V/propidium iodide double staining, cell cycle analysis and dUTP nick end labeling. Further research revealed that Baohuoside I accelerated apoptosis through the mitochondrial apoptotic pathway, involving the increment of BAX/Bcl-2 ratio, dissipation of mitochondrial membrane potential, transposition of cytochrome c, caspase 3 and caspase 9 activation, degradation of poly (ADP-ribose) polymerase and the over-production of reactive oxygen species (ROS). A pan-caspase inhibitor, Z-VAD-FMK, only partially prevented apoptosis induced by Baohuoside I, while NAC, a scavenger of ROS, diminished its effect more potently. In addition, the apoptotic effect of Baohuoside I was dependent on the activation of ROS downstream effectors, JNK and p38(MAPK), which could be almost abrogated by using inhibitors SB203580 (an inhibitor of p38(MAPK)) and SP600125 (an inhibitor of JNK). These findings suggested that Baohuoside I might exert its cytotoxic effect via the ROS/MAPK pathway.     

Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

Evodiamine and rutaecarpine inhibit migration by LIGHT via suppression of NADPH oxidase activation

LIGHT acted as a new player in the atherogenesis. The dried, unripe fruit of Evodia Fructus (EF) has long been used as a traditional Chinese herbal medicine, and is currently widely used for the treatment of headache, abdominal pain, vomiting, colds and reduced blood circulation. Evodiamine and rutaecarpine are active components of EF. In this study, we investigated the inhibitory effect of evodiamine and rutaecarpine on LIGHT‐induced migration in human monocytes. Evodiamine and rutaecarpine decreased the LIGHT‐induced production of ROS, IL‐8, monocyte chemoattractant protein‐1 (MCP‐1), TNF‐α, and IL‐6, as well as the expression of chemokine receptor (CCR) 1, CCR2 and ICAM‐1 and the phosphorylation of the ERK 1/2 and p38 MAPK. Furthermore, NADPH oxidase assembly inhibitor, AEBSF, blocked LIGHT‐induced migration and activation of CCR1, CCR2, ICAM‐1, and MAPK such as ERK and p38 in a manner similar to evodiamine and rutaecarpine. These findings indicate that the inhibitory effects of evodiamine and rutaecarpine on LIGHT‐induced migration and the activation of CCR1, CCR2, ICAM‐1, ERK, and p38 MAPK occurs via decreased ROS production and NADPH oxidase activation. Taken together, these results indicate that evodiamine and rutaecarpine have the potential for use as an anti‐atherosclerosis agent. J. Cell. Biochem. 107: 123–133, 2009. © 2009 Wiley‐Liss, Inc.     

Phosphorylation of a p38‐like MAPK is involved in sensing cellular redox state and drives atypical tubulin polymer assembly in angiosperms

Reactive oxygen species (ROS) imbalance is a stressful condition for plant cells accompanied by dramatic changes in tubulin cytoskeleton. Here, evidence is provided that alterations in ROS levels directly interfere with the phosphorylation state of a p38‐like MAPK in the angiosperms Triticum turgidum and Arabidopsis thaliana. Both oxidative stress generators and chemicals inducing ROS scavenging or decreasing ROS production resulted in the accumulation of a phospho‐p46 protein similar to p38‐MAPK. Importantly, the rhd2 A. thaliana mutants exhibited a remarkable increase in levels of phospho‐p46. The presence of the p38‐MAPK inhibitor SB203580 attenuated the response to ROS disturbance, prevented microtubule disappearance and resulted in a dramatic decrease in the number of atypical tubulin polymers. Moreover, in roots treated simultaneously with substances inducing ROS overproduction and others resulting in low ROS levels, phospho‐p46 levels and the organization of tubulin cytoskeleton were similar to controls. Collectively, our experimental data suggest, for the first time in plants, that p46 functions as a putative sensor of redox state, the activation of which initiates downstream signalling events leading to microtubule disruption and subsequent assembly of atypical tubulin polymers. Thus, p46 seems to participate in perception of ROS homeostasis disturbance as well as in cellular responses to redox imbalance.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

Vibrio vulnificus-induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.     

Differential activation of mitogen-activated protein kinases following high and low LET radiation in murine macrophage cell line

Mitogen-activated protein kinases have been shown to respond to various stimuli including cytokines, mitogens and gamma irradiation, leading to cell proliferation, differentiation, or death. The duration of their activation determines the specificity of response to each stimulus in various cells. In this study, the crucial intracellular kinases, ERK, JNK, and p38 kinase involved in cell survival, death, or damage and repair were examined for their activity in RAW 264.7 cells at various time points after irradiation with 2 Gy doses of proton ions or X-rays. This is the first report that shows that the MAPK signaling induced after heavy ion or X-ray exposure is not the same. Unlike gamma irradiation, there was prolonged but marginal activation of prosurvival ERK pathway and significant activation of proapoptotic p38 pathway in response to high LET radiation.     

Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in gamma irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5 Gy and 20 Gy gamma irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5 Gy gamma irradiation of PBMCs mainly causes apoptosis, whereas a 20 Gy gamma irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.