Showing papers by "Guido Kroemer published in 2001"

Organelle-specific initiation of cell death pathways

Abstract: Nuclear DNA damage and ligation of plasma-membrane death receptors have long been recognized as initial triggers of apoptosis that induce mitochondrial membrane permeabilization (MMP) and/or the direct activation of caspases. Accumulating evidence suggests that other organelles, including the endoplasmic reticulum (ER), lysosomes and the Golgi apparatus, are also major points of integration of pro-apoptotic signalling or damage sensing. Each organelle possesses sensors that detect specific alterations, locally activates signal transduction pathways and emits signals that ensure inter-organellar cross-talk. The ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+ release channels, which might transmit ER Ca2+ responses to mitochondria. The ER also contains several Bcl-2-binding proteins, and Bcl-2 has been reported to exert part of its cytoprotective effect within the ER. Upon membrane destabilization, lysosomes release cathepsins that are endowed with the capacity of triggering MMP. The Golgi apparatus constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside GD3, facilitates local caspase-2 activation and might serve as a storage organelle for latent death receptors. Intriguingly, most organelle-specific death responses finally lead to either MMP or caspase activation, both of which might function as central integrators of the death pathway, thereby streamlining lysosome-, Golgi- or ER-elicited responses into a common pathway.

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death

Abstract: Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

The mitochondrion in apoptosis: how pandora's box opens

Abstract: There is widespread agreement that mitochondria have a function in apoptosis, but the mechanisms behind their involvement remain controversial Here we suggest that opening of a multiprotein complex called the mitochondrial permeability transition pore complex is sufficient (and, usually, necessary) for triggering apoptosis

Heat-shock protein 70 antagonizes apoptosis-inducing factor.

Abstract: Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

Control of Mitochondrial Membrane Permeabilization by Adenine Nucleotide Translocator Interacting with HIV-1 Viral Protein R and Bcl-2

Abstract: Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to Konig's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor

Abstract: 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 ef...

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal

Abstract: Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis

Abstract: Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-XL only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells.

Mitochondria--the suicide organelles.

Abstract: One of the near-to-invariant hallmarks of early apoptosis (programmed cell death) is mitochondrial membrane permeabilization (MMP). It appears that mitochondria fulfill a dual role during the apoptotic process. On the one hand, they integrate multiple different pro-apoptotic signal transducing cascades into a common pathway initiated by MMP. On the other hand, they coordinate the catabolic reactions accompanying late apoptosis by releasing soluble proteins that are normally sequestered within the intermembrane space. In a recent study, Li et al. described a nuclear transcription factor (Nur77/TR1/NGFI-B) that can translocate to mitochondrial membranes to induce MMP. Moreover, two groups identified a novel intermembrane protein (Smac/DIABLO) that specifically neutralizes the inhibitor of apoptosis (IAP) proteins, thereby facilitating the activation of caspases, a class of proteases activated during apoptosis. These findings refine our knowledge how MMP connects to the cellular suicide machinery.

Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437

Abstract: An increasing number of experimental chemotherapeutic agents induce apoptosis by directly triggering mitochondrial membrane permeabilization (MMP). Here we examined MMP induced by lonidamine, arsenite, and the retinoid derivative CD437. Cells overexpressing the cytomegalovirus-encoded protein vMIA, a protein which interacts with the adenine nucleotide translocator, were strongly protected against the MMP-inducing and apoptogenic effects of lonidamine, arsenite, and CD437. In a cell-free system, lonidamine, arsenite, and CD437 induced the permeabilization of ANT proteoliposomes, yet had no effect on protein-free liposomes. The ANT-dependent membrane permeabilization was inhibited by the two ANT ligands ATP and ADP, as well as by recombinant Bcl-2 protein. Lonidamine, arsenite, and CD437, added to synthetic planar lipid bilayers containing ANT, elicited ANT channel activities with clearly distinct conductance levels of 20+/-7, 100+/-30, and 47+/-7 pS, respectively. Altering the ATP/ADP gradient built up on the inner mitochondrial membrane by inhibition of glycolysis and/or oxidative phosphorylation differentially modulated the cytocidal potential of lonidamine, arsenite, and CD437. Inhibition of F(0)F(1)ATPase without glycolysis inhibition sensitized to lonidamine-induced cell death. In contrast, only the combined inhibition of glycolysis plus F(0)F(1)ATPase sensitized to arsenite-induced cell death. No sensitization to cell death induction by CD437 was achieved by glucose depletion and/or oligomycin addition. These results indicate that ANT is a target of lonidamine, arsenite, and CD437 and unravel an unexpected heterogeneity in the mode of action of these three compounds.

Resveratrol, a tumor-suppressive compound from grapes, induces apoptosis via a novel mitochondrial pathway controlled by Bcl-2.

Abstract: SPECIFIC AIMSThe clinical application of the tumor-suppressive agent resveratrol in cancer treatment requires an exact understanding of which molecules sensitize cells to, or antagonize them against, its specific signaling cascade. Bcl-2 is frequently expressed in hematological tumors, is associated with an unfavorable prognosis in some of them, and inhibits the efficacy of cytotoxic agents. Bcl-2 functions may be diverse, but predominantly involve mitochondria. We therefore focused on the mitochondrial signaling pathways induced by resveratrol and potentially counteracted by Bcl-2.PRINCIPAL FINDINGS1. Resveratrol-induced apoptosis is inhibited by Bcl-2To study the role of Bcl-2 in resveratrol-induced apoptosis, we used subclones of the CEM-C7H2 T-ALL cell line, which stably overexpressed Bcl-2. After resveratrol treatment of the vector control cell line, apoptosis was induced in a time-dependent manner. Bcl-2 overexpression protected cells from resveratrol-induced apoptosis even over the extended incubat...

Relocalization of apoptosis-inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo.

Abstract: Apoptosis-inducing factor (AIF) is a novel mediator in apoptosis. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, yet translocates to the nucleus in several in vitro models of apoptosis. To investigate the role of AIF in the apoptotic process in vivo, we induced retinal detachment (RD) by subretinal injection of sodium hyaluronate, either in Brown Norway rats or in C3H mice. Apoptotic DNA fragmentation, as determined by terminal nick-end labeling, was most prominent 3 days after RD. The subcellular localization of AIF was examined by immunohistochemistry and immunoelectron microscopy. In normal photoreceptor cells, AIF was present in the mitochondrion-rich inner segment. However, AIF was found in the nucleus after RD. Photoreceptor apoptosis developed similarly in C3H control mice, and in mice bearing the gld or lpr mutations, indicating that cell death occurs independently from the CD95/CD95 ligand system. Both the mitochondrio-nuclear transition of AIF localization and the nuclear DNA fragmentation were inhibited by subretinal application of brain-derived neurotrophic factor. To our knowledge, this is the first description of AIF relocalization occurring in a clinically relevant, in vivo model of apoptosis.

Erythrocytes: Death of a mummy

Abstract: Simple spirits consider mammalian erythrocytes as membrane-surrounded hemoglobin-containing vesicles floating in circulation. Indeed, they lack organelles including nuclei and mitochondria and, in a sense, they cannot be considered as true cells. Nonetheless, erythrocytes have been intriguing researchers interested in apoptosis. The red blood cell (RBC) life cycle can be divided in two phases, the first one, erythopoiesis consisting in the production of mature erythrocytes in hematopoietic organs, the second one concerning the proper existence of the mature eythrocyte in circulation. Erythropoiesis results from the progressive differentiation of erythroid progenitors, from proerythroblasts through polychromatophylic erythroblasts, then orthochromatic erythroblasts to mature erythrocytes. It is now well established that the hormone erythropoetin stimulates erythropoiesis, at least in part, by protecting erythroblasts from apoptosis presumably by upregulating the anti-apoptotic protein Bcl-XL. 1,2 Indeed, conditional knock-out of Bcl-XL suppresses erythropoiesis at a late stage resulting in hemolytic anemia, and erythropoietin withdrawal triggers erythroblast death and caspase activation. A CD95and caspase-dependent negative regulation of erythropoiesis has been described at the local level. Orthochromatic erythroblasts expressing CD95 ligand would stimulate the CD95 death receptor expressed on immature erythroblasts, thereby creating a negative feedback loop controlling erythropoiesis. Erythopoietin would antagonize this negative control. Thus, not surprisingly, erythrocyte precursors, which are true organelle-containing cells, are susceptible to apoptosis induction, including in pathological stages such as b-thalassemia major, idiopathic acquired sideroblastic anemia and parvovirus B19 infection. Surprisingly, caspase activation has not only negative effects on erythropoiesis. Rather, it appears that the activation of effector caspases is required for RBC formation. In vitro erythropoiesis reportedly is blocked by the pan-caspase inhibitor Z-VAD.fmk. During erythroblast differentiation caspases-3, -6 and -7 are proteolytically activated and cleave nuclear proteins (lamin B and acinus) yet leave intact the transcription factor GATA-1 and fail to trigger the exposure of phosphatidylserine on the plasma membrane surface. This suggests that a sort of abortive apoptosis participates in the differentiation and enucleation of erythroid cells. However, caspase-3 mice do not exhibit any obvious defect in erythropoiesis, and transgenetargeted overexpression of Bcl-2 in the erythroid lineage does not induce any erythropoietic disorder in vivo. Further studies will thus have to confirm the implication of caspase activation and abortive apoptosis in erythroid differentiation. Intriguingly, Nagata and co-workers recently reported that DNAse II is required for erythropoiesis in the fetal liver. According to these authors, DNAse II present in macrophages would be required for efficient enucleation of eythrocyte precursors. DNAse II thus would not participate in a cell-autonomous process and rather act in the heterophagic removal of the nucleus of the differentiating erythroblast, much in the way as this has been described for the macrophage-mediated removal of apoptotic thymocytes. Mammalian fetal erythrocytes as well as erythrocytes from amphibians, reptiles and birds do contain a nucleus, and these cells can undergo normal apoptosis. Thus, fetal human erythrocytes circulating in maternal blood undergo apoptosis in situ. Moreover, chicken erythrocytes can be driven into apoptosis (nuclear and cellular shrinkage, chromatin condensation, TUNEL positivity, staining with Annexin-V) by incubating them either in the absence of serum or in the presence of staurosporin plus cycloheximide. This process is not prevented by Z-VAD.fmk and is not accompanied by caspase activation, suggesting that chicken erythrocyte apoptosis occurs in a caspaseindependent fashion. Two fascinating papers published in this issue of CDD now tackle the question whether mature human erythrocytes can undergo apoptosis-like cell death. In contrast to true cells, human erythrocytes fail to die in the absence of serum or upon addition of staurosporin+cycloheximide, in vitro. However, RBC life span is limited to approximately 120 days in vivo. It has been widely assumed that RBC aging would lead to an increase in intracellular calcium, modifications of the erythrocyte shape, protein degradation and externalization of phosphatidylserine, thereby activating a clearance mechanism involving heterophagic removal in the reticuloendothelial system. Indeed, splenectomy is accompanied by the occurrence of aged erythrocytes in peripheral blood that have failed to be cleared. Here, the groups headed by Klaus Schulze-Osthoff and the tandem formed by Jean-Claude Ameisen and Jean Montreuil report the intriguing finding that Ca ionophores (A23187 or ionomycin) induce an in vitro erythrocyte senescence process characterized by cell shrinkage, membrane microvesiculation, and phosphatidylserine externalization. This process culminates in erythrocyte disintegration, phagocytosis in the presence of macrophages in vitro, and in clearance from the circulation in vivo. Cell Death and Differentiation (2001) 8, 1131 ± 1133 ã 2001 Nature Publishing Group All rights reserved 1350-9047/01 $15.00

Human Immunodeficiency Virus 1 Envelope Glycoprotein Complex-Induced Apoptosis Involves Mammalian Target of Rapamycin/Fkbp12-Rapamycin–Associated Protein–Mediated P53 Phosphorylation

Abstract: Syncytia arising from the fusion of cells expressing a lymphotropic human immunodeficiency virus (HIV)-1-encoded envelope glycoprotein complex (Env) gene with cells expressing the CD4/CXCR4 complex undergo apoptosis through a mitochondrion-controlled pathway initiated by the upregulation of Bax. In syncytial apoptosis, phosphorylation of p53 on serine 15 (p53S15) precedes Bax upregulation, the apoptosis-linked conformational change of Bax, the insertion of Bax in mitochondrial membranes, subsequent release of cytochrome c, caspase activation, and apoptosis. p53S15 phosphorylation also occurs in vivo, in HIV-1(+) donors, where it can be detected in preapoptotic and apoptotic syncytia in lymph nodes, as well as in peripheral blood mononuclear cells, correlating with viral load. Syncytium-induced p53S15 phosphorylation is mediated by the upregulation/activation of mammalian target of rapamycin (mTOR), also called FKBP12-rapamycin-associated protein (FRAP), which coimmunoprecipitates with p53. Inhibition of mTOR/FRAP by rapamycin reduces apoptosis in several paradigms of syncytium-dependent death, including in primary CD4(+) lymphoblasts infected by HIV-1. Concomitantly, rapamycin inhibits p53S15 phosphorylation, mitochondrial translocation of Bax, loss of the mitochondrial transmembrane potential, mitochondrial release of cytochrome c, and nuclear chromatin condensation. Transfection with dominant negative p53 has a similar antiapoptotic action as rapamycin, upstream of the Bax upregulation/translocation. In summary, we demonstrate that phosphorylation of p53S15 by mTOR/FRAP plays a critical role in syncytial apoptosis driven by HIV-1 Env.

Apoptosis-inducing factor mediates microglial and neuronal apoptosis caused by pneumococcus.

Abstract: Streptococcus pneumoniae is the major cause of bacterial meningitis and it damages the hippocampus by inducing neuronal apoptosis. The blocking of caspases provides only partial protection in experimental meningitis, which suggests that there is an additional apoptotic pathway. A trigger of this pathway is the bacterium itself, as exposure of microglia or neurons to live pneumococci induces rapid apoptosis. In this study, apoptosis was not associated with the activation of caspases-1-10 and was not inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor. Rather, apoptosis was attributed to damage to mitochondria, which was followed by the release of apoptosis-inducing factor (AIF) from the mitochondria, large-scale DNA fragmentation, and hypodiploidy. Furthermore, intracytoplasmatic microinjection of AIF-specific antiserum markedly impaired pneumococcus-induced apoptosis. These findings indicate that AIF may play a central role in brain cell apoptosis and bacterial pathogenesis.

Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.

Abstract: The disruption of mitochondrial function is a key component of apoptosis in most cell types. Localization of Bcl-2 to the outer mitochondrial and endoplasmic reticulum membranes is consistent with a role in the inhibition of many forms of apoptosis. In Rat-1 cells, a Bcl-2 mutant targeted exclusively to the endoplasmic reticulum (Bcl-cb5) was effective at inhibiting apoptosis induced by serum starvation/myc, or ceramide but not apoptosis induced by etoposide. The former conditions cause a decrease in mitochondrial transmembrane potential (Δψm) as an early event that precedes the release of cytochrome c from mitochondria. By contrast, when cells are exposed to etoposide, a situation in which cytochrome c release and membrane localization of the pro-apoptotic protein Bax precede loss of Δψm, wild type Bcl-2 but not Bcl-cb5 prevents apoptosis. Therefore, Bcl-2 functions in spatially distinct pathways of apoptosis distinguished by the order of cytochrome c release and loss of Δψm.

Viral and bacterial proteins regulating apoptosis at the mitochondrial level

Abstract: Mitochondrial membrane permeabilization (MMP) is a critical step of several apoptotic pathways. Some infectious intracellular pathogens can regulate (induce or inhibit) apoptosis of their host cells at the mitochondrial level, by targeting proteins to mitochondrial membranes that either induce or inhibit MMP. Pathogen-encoded mitochondrion-targeted proteins may or may not show amino acid sequence homology to Bcl-2-like proteins. Among the Bcl-2-unrelated, mitochondrion-targeted proteins, several interact with the voltage-dependent anion channel (VDAC) or with the adenine nucleotide translocator (ANT). While VDAC-targeted proteins show homology to VDAC/porin, ANT-targeted proteins possess relatively short cationic binding domains, which may facilitate insertion into the negatively charged inner mitochondrial membrane. It may be speculated that such proteins employ pre-existing host-intrinsic mechanisms of MMP control.

A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis.

Abstract: Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1–5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C18) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.

HIV induces lymphocyte apoptosis by a p53-initiated, mitochondrial-mediated mechanism

Abstract: SPECIFIC AIMSHIV-1 induces apoptosis and leads to CD4+ T lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. In this study we provide a mechanistic view on how HIV-1 induces apoptotic death of infected primary human CD4+ T lymphocytes.PRINCIPAL FINDINGS1. HIV damages mitochondria, leading to cytochrome c release and caspase activationThe time course of activation of the extrinsic and intrinsic pathways of apoptosis was examined after HIV-1 infection of primary CD4+ T lymphocytes. The percentage of cells undergoing apoptosis was quantified by flow cytometry and measuring the proportion of cells with sub-G1 DNA content and correlating it with HIV p24 production. Caspase activities were measured at multiple time points after infection by using specific fluorometric substrates (Fig. 1⤻ ). The pattern of caspase activation strongly suggested that the intrinsic pathway of apoptosis induction was operational. This suggestion was borne out by the followin...

Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation.

Abstract: Jurkat T cells showed a major, early decrease in blue autofluorescence in response to Fas/Apo-1/CD95 cross-linking or stimulation with cell-permeant ceramide. This indicates the oxidation/depletion of NADH or NADPH before the onset of apoptosis. Kinetic studies, cytofluorimetric multiparameter analyses and cell sorting experiments indicated a close temporal relationship between NAD(P)H oxidation/depletion and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). In contrast, NAD(P)H depletion was detected well before several other changes associated with late apoptosis, including enhanced superoxide generation, phosphatidylserine exposure on the cell surface, loss of cytosolic K(+), decreased cytoplasmic pH, nuclear DNA fragmentation, cell shrinkage, loss of viability and the appearance of the mitochondrial antigen APO2.7. Full activation of caspase 9 and caspase 3 appeared to be correlated with the appearance of superoxide anions in the mitochondria, and followed the drop in NADPH. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which encodes an inhibitor of the mitochondrial permeability transition (PT) pore, delayed both the DeltaPsi(m) disruption and the depletion of NAD(P)H. Similar effects were observed with the pharmacological PT pore inhibitors, bongkrekic acid and cyclosporin A. Thus there appears to be a close functional relationship between mitochondrial and cellular redox changes during early apoptosis; events that are inhibited by Bcl-2.

Mitochondrial release of apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis.

Abstract: Photodynamic therapy (PDT) is under investigation for the treatment of intimal hyperplastia in conditions such as atherosclerosis and restenosis. Although smooth muscle cells (SMCs) may be a key target for treatment, the effects of PDT on these cells are poorly characterized. In the present study, apoptosis was induced in primary human aortic SMCs by the combination of the photosensitizer verteporfin and visible light. After PDT, an increase in mitochondrial cytochrome c (cyt c) and apoptosis-inducing factor (AIF) levels were detected in the cytosol immediately and their levels increased steadily up to 2 hours. Cytosolic levels of the pro-apoptotic Bcl-2 family member Bax decreased reciprocally throughout this period, but this change did not occur before cyt c release. Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells as compared to a distinct mitochondrial staining in normal cells. AIF translocated from mitochondria to the nucleus during the progression of apoptosis. After cyt c release, caspase-9 and caspase-3 processing was visible by 1 hour and caspase-6, -7, and -8 processing was apparent by 2 hours after PDT. In summary, these results demonstrate for the first time the cellular redistribution of mitochondrial AIF during SMC apoptosis, as well as the early release of cyt c and the subsequent activation of multiple caspases during PDT-induced SMC apoptosis.

Apoptosis induction by the photosensitizer verteporfin: identification of mitochondrial adenine nucleotide translocator as a critical target.

Abstract: We report that the photosensitizer verteporfin kills lymphoma cells by an apoptotic process involving a dissipation of the mitochondrial inner transmembrane potential (deltapsim). Light-activated verteporfin-induced apoptosis was abolished by transfection with Bcl-2, a procedure reported to inhibit the mitochondrial permeability transition pore complex (PTPC). Verteporfin triggered the deltapsim loss in isolated mitochondria in vitro, and this effect was suppressed by bongrekic acid and cyclosporin A. Verteporfin plus light also permeabilized proteoliposomes containing the semipurified PTPC or the purified PTPC component adenine nucleotide translocator (ANT), yet had no effect on protein-free control liposomes. Verteporfin phototoxicity on ANT proteoliposomes was mediated by reactive oxygen species and was prevented by recombinant Bcl-2 or the adenine nucleotides ATP and ADP. In conclusion, verteporfin belongs to a class of clinically used chemotherapeutic agents acting on PTPC and ANT.

Chapter 8 Measurement of membrane permeability and permeability transition of mitochondria

Abstract: Publisher Summary This chapter presents experimental procedures designed to measure apoptosis associated mitochondrial membrane permeabilization, both in intact cells and in isolated mitochondria. Mitochondria are essential for the evolution of complex animals in aerobic conditions. They carry out most cellular oxidations and produce the bulk of an animal cell's ATP. The permeability transition (PT) pore, also called the mitochondrial megachannel or multiple conductance channel, is a dynamic multiprotein complex located at the contact site between the inner and the outer mitochondrial membranes, one of the critical sites of metabolic coordination among the cytosol and the mitochondrial intermembrane and matrix spaces. In isolated mitochondria, opening of the PT pore causes matrix swelling with consequent distension and local disruption of the mitochondrial outer membrane, release of soluble products from the intermembrane space, dissipation of the mitochondrial inner transmembrane potential, and release of small molecules up to 1500 Da from the matrix, through the inner membrane. In several models of apoptosis, pharmacological inhibition of the PT pore is cytoprotective, suggesting that opening of the PT pore can be rate limiting for the death process. Irrespective of this possibility, mitochondrial membrane permeabilization is a general feature of apoptosis.

Mitochondrial control of apoptosis

Abstract: The dysregulation of programmed cell death (apoptosis) is involved in different pathologies including cancer, which is frequently associated with an increase resistance to apoptosis induction. We discovered in 1994 the implication of a specific organelle, the mitochondrion, in apoptosis. Our result have demonstrated that mitochondrial membrane permeabilization (MMP) constitutes a decisive step of the apoptotic process. MMP is regulated by numerous effectors, including the proteins from the Bcl-2/Bax family (oncogenes or tumor suppressor genes which modulate apoptosis), which interact with sessile proteins of mitochondria. MMP can be induced by a large number of pro-apoptotic second messengers, as well as by some experimental anti-cancer agents, suggesting that MMP constitutes a point of integration of the apoptotic response. As a result of MMP, several apoptogenic proteins normally confined to mitochondria are released in the extra-mitochondrial space and participate in the suicidal dismantling of the cell. We have identified several mitochondrial apoptogenic proteins, one of which, the apoptosis inducing factor (AIF) has been cloned. AIF appears to be one of the principal effectors of the apoptotic machinery. Genetic inactivation of AIF abolishes the first wave of apoptosis indispensable for early embryonic morphogenesis. In contrast, its presence in the extra-mitochondrial compartment suffices to kill cells. Altogether, these results allow for the development of new strategies aiming at inducing apoptosis in cancer cells.

Heat Shock Protein 70 Neutralizes Apoptosis-Inducing Factor

Abstract: Programmed cell death (apoptosis) is the physiological process responsible for the demise of superfluous, aged, damaged, mutated, and ectopic cells. Its normal function is essential both for embryonic development and for maintenance of adult tissue homeostasis. Deficient apoptosis participates in cancerogenesis, whereas excessive apoptosis leads to unwarranted cell loss accounting for disparate diseases including neurodegeneration and AIDS. One critical step in the process of apoptosis consists in the permeabilization of mitochondrial membranes, leading to the release of proteins which normally are secluded behind the outer mitochondrial membrane. For example, cytochrome c, which is normally confined to the mitochondrial intermembrane space, is liberated from mitochondria and interacts with a cytosolic protein, Apaf-1, causing its oligomerization and constitution of the so-called apoptosome, a protein complex which activates a specific class of cysteine proteases, the caspases[2]. Another example concerns the so-called apoptosis-inducing factor (AIF), another mitochondrial intermembrane protein which can translocate to the nucleus where it induces chromatin condensation and DNA fragmentation.

B709 Mitochondrial Control of Cell Death.

Abstract: INTRODUCTION. Mitochondrial membrane permeabilization (MMP) is a critical event of early apoptosis. MMP is regulated by proand anti-apoptotic members of the Bax/Bcl-2 family of proteins, via a process which may involve sessile mitochondrial proteins organized in the two membrane-spanning permeability transition pore complex (PTPC). Apoptotic MMP differentially affects the outer and the inner mitochondrial membranes (1-3). The inner mitochondrial membrane becomes permeable to solutes up to 1500 Da, yet retains matrix proteins in their normal localization. In contrast, the outer mitochondrial membrane becomes completely permeabilized to proteins, leading to the release of soluble proteins from the mitochondrial intermembrane space to an ectopic (extra mitochondrial) localization. Several intermembrane proteins can activate catabolic hydrolases involved in the apoptotic process (4). One such protein is cytochrome c, which participates in the caspase activation cascade. Another functionally important intermembrane protein is apoptosis inducing factor (AIF), which functions as a caspase-independent death effector (5).

Contrôle mitochondrial de l’apoptose

Abstract: RESUME La deregulation de l’apoptose (ou « mort cellulaire programmee ») est impliquee dans de nombreuses maladies, notamment les cancers, frequemment resistants a l’induction de l’apoptose. Mon equipe a decouvert en 1994 l’implication d’un organite, la mitochondrie, dans l’apoptose. Nos travaux ont devoile que la permeabilisation des membranes mitochondriales (PMM) constitue une etape decisive du processus apoptotique. La PMM est regulee par de nombreux effecteurs, entre autres les proteines de la famille Bcl-2/Bax (des oncogenes et anti-oncogenes regulateurs de l’apoptose), qui interagissent avec des proteines sessiles mitochondriales. La PMM peut etre induite par un grand nombre de messagers secondaires pro-apoptotiques, ainsi que par certains agents anticancereux experimentaux, ce qui suggere qu’elle constitue un point d’integration de la reponse apoptotique. Suite a la PMM, plusieurs proteines apoptogenes qui normalement sont confinees dans la mitochondrie sont relarguees dans l’espace extra-mitochondrial et participent a l’autodestruction de la cellule. Nous avons reussi a identifier differentes proteines apoptogenes mitochondriales et a cloner le gene de l’une d’entre elles, le facteur inducteur de l’apoptose (apoptosis inducing factor ; AIF). L’AIF semble etre un des effecteurs principaux de la machinerie apoptotique. L’invalidation du gene codant pour AIF abolit la premiere vague d’apoptose necessaire pour la morphogenese embryonnaire. Sa presence dans le compartiment extramitochondrial suffit a tuer les cellules. L’ensemble de ces resultats permet de mettre au point de nouvelles strategies visant a induire l’apoptose dans les cellules cancereuses.