Scythe cleavage during Fas (APO-1)-and staurosporine-mediated apoptosis

Scythe is a nuclear protein that has been implicated in the apoptotic process in Drosophila melanogaster; however, its role in apoptosis of mammalian cells is not fully elucidated. Here we show that cleavage of Scythe by caspase-3 occurs after activation of both the extrinsic (i.e. Fas/APO-1-mediated) and the intrinsic (i.e. staurosporine-induced) apoptosis pathway. Moreover, this caspase-dependent cleavage correlates with Scythe translocation from the nucleus to the cytosol. We also show that cytosolic re-localization of Scythe is required for Fas/APO-1-triggered phosphatidylserine (PS) exposure, a signal for macrophage clearance of apoptotic cells. Our data suggest that Scythe cleavage may represent a marker for caspase-3 activation and implicate cytosolic re-localization of Scythe in the pathway of PS exposure.     

Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria

Apoptosis-inducing factor (AIF) is a phylogenetically ancient mitochondrial intermembrane flavoprotein endowed with the unique capacity to induce caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation when added to purified nuclei. In addition to its apoptogenic activity on nuclei, AIF can also participate in the regulation of apoptotic mitochondrial membrane permeabilization and exhibits an NADH oxidase activity. Under normal circumstances, AIF is secluded behind the outer mitochondrial membrane. However, upon apoptosis induction AIF translocates to the cytosol and the nucleus. Injection of anti-AIF antibodies or knockout of the AIF gene have demonstrated that AIF may be required for cell death occurring in response to some stimuli. In particular, inactivation of AIF renders embryonic stem cells resistant to cell death following growth factor withdrawal. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies, the very first wave of (caspase-independent) cell death indispensable for mouse morphogenesis. We have recently found that AIF is neutralized by heat-shock protein (HSP) 70, in a reaction that appears to be independent of ATP or the ATP-binding domain (ABD) of HSP70 and thus differs from the previously described Apaf-1/HSP70 interaction (which requires ATP and the HSP70 ABD). Intriguingly, HSP70 lacking ABD (HSP70 Delta ABD) inhibits apoptosis induced by serum withdrawal, staurosporin, and menadione, three models of apoptosis which are also affected by micro-injection of anti-AIF antibody or genetic ablation of AIF. Altogether, these data suggest that AIF plays a role in the regulation of caspase-independent cell death.     

Apoptosis-inducing factor contributes to epithelial cell apoptosis induced by enteropathogenic Escherichia coli

The mechanisms by which enteropathogenic Escherichia coli (EPEC) causes intestinal epithelial cell apoptosis remain unclear. We tested the hypothesis that apoptosis-inducing factor (AIF) is involved in apoptosis induced by EPEC. Infection of intestinal epithelial cells in vitro with EPEC led to the mitochondrial and cytosolic accumulation of AIF. This effect was partially dependent on caspase activity. Knockdown of AIF with siRNA blocked cellular apoptosis in response to EPEC infection, as assessed by poly(ADP-ribose) polymerase cleavage and oligonucleosome formation. Taken together, these data suggest that caspase-dependent mobilization of AIF contributes to EPEC-induced epithelial cell apoptosis.     

c-Jun NH2-terminal kinase (JNK)-dependent nuclear translocation of apoptosis-inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI-231 B lymphoma cells

WEHI-231 B lymphoma cells have been employed for analysis of antigen-induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis-inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI-231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan-caspase inhibitor BD-fmk blocked mIg-mediated increase in cells with sub-G1 DNA content, whereas it did not affect mIg-mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant-negative form of c-Jun NH2-terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI-231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl-xL, but not by BD-fmk. Moreover, AIF-deficient clones via small interfering RNA (siRNA)-mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF-deficient clones displayed an enhanced sensitivity to mIg-mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti-apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction.     

Stimulation of suicidal erythrocyte death by methylglyoxal.

Diabetes increases the percentage of circulating erythrocytes exposing phosphatidylserine (PS) at the cell surface. PS-exposing erythrocytes are recognized, bound, engulfed and degraded by macrophages. Thus, PS exposure, a feature of suicidal erythrocyte death or eryptosis, accelerates clearance of affected erythrocytes from circulating blood. Moreover, PS-exposing erythrocytes bind to the vascular wall thus interfering with microcirculation. The present study explored mechanisms involved in the triggering of PS exposure by methylgloxal, an extra- and intracellular metabolite which is enhanced in diabetes. PS exposure, cell size and cytosolic Ca(2+)-activity after methylglyoxal treatment were measured by FACS analysis of annexin V binding, forward scatter and Fluo-3-fluorescence, respectively, and it was shown that the treatment significantly enhanced the percentage of PS-exposing erythrocytes at concentrations (0.3 microM) encountered in diabetic patients. Surprisingly, methylglyoxal did not significantly increase cytosolic Ca(2+) concentration, and at concentrations up to 3 microM, did not decrease the forward scatter. Instead, exposure to methylglyoxal inhibited glycolysis thus decreasing ATP and GSH concentrations. In conclusion, methylglyoxal impairs energy production and anti-oxidative defense, effects contributing to the enhanced PS exposure of circulating erythrocytes and eventually resulting in anemia and deranged microcirculation.     

Intracellular localization of Herpes simplex virus type 1 thymidine kinase fused to different fluorescent proteins depends on choice of fluorescent tag

Recognition signals are displayed on the cell surface during apoptosis that enable macrophages to engulf and dispose of the dying cell. A common signal is the externalization of phosphatidylserine (PS). Studies in erythrocytes and platelets have suggested that PS exposure requires the concomitant activation of a phospholipid scramblase (PLS) and inhibition of an adenosine triphosphate (ATP)-dependent aminophospholipid translocase. However, the molecular mechanism underlying PS exposure during apoptosis remains poorly understood. In this study, we provide evidence that expression of PLS is neither necessary nor sufficient for PS exposure during Fas-triggered apoptosis. On the other hand, egress of PS is shown to correlate with a decline in intracellular ATP and inhibition of aminophospholipid translocase activity upon Fas stimulation. Moreover, suppression of intracellular ATP levels by the glucose anti-metabolite, 2-deoxyglucose, alone or in combination with glucose-free medium, potentiates Fas-induced PS exposure in the PLS-expressing Jurkat cell line and enables PLS-defective Raji cells to externalize PS in response to Fas ligation. These studies suggest that intracellular ATP levels can modulate the externalization of PS during apoptosis, and implicate the ATP-dependent aminophospholipid translocase in this process.     

Human Scythe contains a functional nuclear localization sequence and remains in the nucleus during staurosporine-induced apoptosis

Human Scythe (also known as BAT3) has been implicated in the control of apoptosis and regulating heat shock protein (HSP) 70 activity. We have attempted to further characterize the role of human Scythe in HeLa cells by studying the cellular localization and functional domains of a hemagglutinin (HA) epitope-tagged Scythe protein. Several HA-Scythe deletion mutant proteins were expressed in HeLa cells and their localization was detected using indirect immunofluorescence. Our data demonstrate that full-length human Scythe is a nuclear protein that contains an active C-terminal nuclear localization sequence (NLS). Site-directed mutagenesis of the NLS leads to complete nuclear exclusion of full-length Scythe. Furthermore, induction of apoptosis by staurosporine does not cause redistribution or cleavage of Scythe, suggesting that Scythe remains localized in the nucleus during apoptosis. These results provide evidence that Scythe is a nuclear protein that probably does not interact with elements of the apoptotic machinery in the cytosol.     

Aminophospholipid translocase TAT-1 promotes phosphatidylserine exposure during C. elegans apoptosis

Phospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an "eat-me" signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals.     

Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor.

The complete AIF cDNA comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-c-microinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF Delta 1-100) results in the primary cytosolic accumulation of AIF. AIF Delta 1-100-induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.     

Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion

Although apoptosis contributes to myocardial cell death in the ischemia-reperfused heart, the molecular basis of apoptosis is poorly understood. Apoptosis-inducing factor (AIF) has been characterized as a caspase-independent death effector. Upon the induction of apoptosis, mitochondrial AIF is released to the cytoplasm and then enters the nucleus, in which it induces chromatin condensation and 50 kbp DNA fragmentation. In the present study, we examined the role of AIF in ischemia-reperfusion injury in isolated rat hearts. AIF was detected in the cytosolic and nuclear fractions of hearts subjected to ischemia-reperfusion, whereas it was detected only in the mitochondria of control hearts. Moreover, AIF release increased in a reperfusion time-dependent manner. Pulse field gel electrophoresis revealed that 50 kbp DNA fragments were produced by ischemia/reperfusion. In contrast, cytochrome c release and the activation of caspase-3 did not occur to a significant extent. Moreover, ischemic preconditioning attenuated the AIF release and the 50 kbp DNA fragmentation. These results suggest that AIF-dependent apoptosis is likely to attribute to myocardial cell death in the ischemia-reperfused heart and that it is related with the protective effect of ischemic preconditioning.     

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.     

Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22muM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.     

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.     

Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death.

Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of approximately 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed.     

A Mechanism of Release of Calreticulin from Cells During Apoptosis

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca²⁺ homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca²⁺-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.     

2-(4-Methylphenyl)-1,3-selenazol-4-one induces apoptosis by different mechanisms in SKOV3 and HL 60 cells

We examined the ability of the synthetic selenium compound, 2-(4-methylphenyl)-1,3-selenazol-4-one (hereafter designated 3a), to induce apoptosis in a human ovarian cancer cell line (SKOV3) and a human leukemia cell line (HL-60). Flow cytometry showed that 3a treatment induced apoptosis in both cell lines to degrees comparable to that of the positive control, paclitaxel. Apoptosis was measured by PS externalization, DNA fragmentation and decreased mitochondrial membrane potential (MMP). However, analysis of the mechanism of action revealed differences between the responses of the two cell lines. Treatment with 3a arrested the cell cycle and induced caspase-3 activation in HL-60 cells, but not in SKOV3 cells. In contrast, 3a treatment induced apoptosis through translocation of AIF, a novel pro-apoptotic protein, in SKOV3 cells, but not in HL-60 cells. Collectively, our data demonstrated that 3a induced apoptosis in both cell lines, but via different action mechanisms.     

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and β-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEβ-thalassemia.