Showing papers by "Guido Kroemer published in 2002"
TL;DR: The crystal structures of both human and mouse AIF have been determined, and the fine mechanisms accounting for its oxidoreductase activity and its electrostatic interaction with double-stranded DNA have been elucidated, indicating the central role of mitochondria in the control of physiological and pathological cell demise.
Abstract: Numerous pro-apoptotic signal transducing molecules act on mitochondria and provoke the permeabilization of the outer mitochondrial membrane, thereby triggering the release of potentially toxic mitochondrial proteins. One of these proteins, apoptosis-inducing factor (AIF), is a phylogenetically old flavoprotein which, in healthy cells, is confined to the mitochondrial intermembrane space. Upon lethal signaling, AIF translocates, via the cytosol, to the nucleus where it binds to DNA and provokes caspase-independent chromatin condensation. The crystal structures of both human and mouse AIF have been determined, and the fine mechanisms accounting for its oxidoreductase activity and its electrostatic interaction with double-stranded DNA have been elucidated. Importantly, the apoptogenic and oxidoreductase functions of AIF can be dissociated. Thus, mutations that abolish the AIF-DNA interaction suppress AIF-induced chromatin condensation, yet have no effect on the NADH oxidase activity. Recent studies suggest AIF to be a major factor determining caspase-independent neuronal death, emphasizing the central role of mitochondria in the control of physiological and pathological cell demise.
500 citations
TL;DR: Data suggest that AIF plays a role in the regulation of caspase-independent cell death, which is essential for programmed cell death during cavitation of embryoid bodies.
490 citations
TL;DR: It is demonstrated that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset casp enzyme-independent mechanism, and that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspases-independent neuronal cellDeath.
Abstract: Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.
489 citations
TL;DR: The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptotic control and unravels novel mechanisms of cell death execution.
Abstract: Apoptosis is a cell-autonomous mode of death that is activated to eradicate superfluous, damaged, mutated, or aged cells. In addition to their role as the cell's powerhouse, mitochondria play a central role in the control of apoptosis. Thus, numerous pro-apoptotic molecules act on mitochondria and provoke the permeabilization of mitochondrial membranes. Soluble proteins contained in the mitochondrial intermembrane space are released through the outer membrane and participate in the organized destruction of the cell. Several among these lethal proteins can activate caspases, a class of cysteine proteases specifically activated in apoptosis, whereas others act in a caspase-independent fashion, by acting as nucleases (e.g., endonuclease G), nuclease activators (e.g., apoptosis-inducing factor), or serine proteases (e.g., Omi/HtrA2). In addition, mitochondria can generate reactive oxygen species, following uncoupling and/or inhibition of the respiratory chain. The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptosis control and unravels novel mechanisms of cell death execution.
475 citations
TL;DR: Mitochondria is subject to a complex regulation, and local alterations in the composition of mitochondrial membranes, as well as alterations in pre-mitochondrial signal-transducing events, can determine chemotherapy resistance in cancer cells, and detecting MMP might thus be useful for detecting chemotherapy responses in vivo.
Abstract: One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis. Apoptosis can be activated through several different signalling pathways, but these all appear to converge at a single event - mitochondrial membrane permeabilization (MMP). This 'point-of-no-return' in the cell death program is a complex process that is regulated by the composition of the mitochondrial membrane and pre-mitochondrial signal-transduction events. MMP is subject to a complex regulation, and local alterations in the composition of mitochondrial membranes, as well as alterations in pre-mitochondrial signal-transducing events, can determine chemotherapy resistance in cancer cells. Detecting MMP might thus be useful for detecting chemotherapy responses in vivo. Several cytotoxic drugs induce MMP by a direct action on mitochondria. This type of agents can enforce death in cells in which upstream signals normally leading to apoptosis have been disabled. Cytotoxic components acting on mitochondria can specifically target proteins from the Bcl-2 family, the peripheral benzodiazepin receptor, or the adenine nucleotide translocase, and/or act by virtue of their physicochemical properties as steroid analogues, cationic ampholytes, redox-active compounds or photosensitizers. Some compounds acting on mitochondria can overcome the cytoprotective effect of Bcl-2-like proteins. Several agents which are already used in anti-cancer chemotherapy can induce MMP, and new drugs specifically designed to target mitochondria are being developed.
399 citations
TL;DR: The crystal structure of human AIF is reported at 1.8 Å resolution and it is shown that recombinant AIF interacts with DNA in a sequence-independent manner and that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation.
Abstract: The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.
345 citations
TL;DR: It is concluded that proplatelet formation is regulated by a caspase activation limited to only some cellular compartments.
342 citations
TL;DR: It is suggested that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA, and ANT can be a critical target in SCFA-induced apoptosis.
Abstract: The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.
321 citations
TL;DR: A cell type-specific modulation of Cdk1 might be taken advantage of for the therapeutic correction of pathogenic imbalances in apoptosis control.
Abstract: The cyclin-dependent kinase 1 (Cdk1), formerly called Cdc2 (or p34(Cdc2)), interacts with cyclin B1 to form an active heterodimer. The activity of Cdk1 is subjected to a complex spatiotemporary regulation, required to guarantee its scheduled contribution to the mitotic prophase and metaphase. Moreover, the activation of Cdk1 may be required for apoptosis induction in some particular pathways of cell killing. This applies to several clinically important settings, for instance to paclitaxel-induced killing of breast cancer cells, in which the ErbB2 receptor kinase can mediate apoptosis inhibition through inactivation of Cdk1. The activation of Cdk1 participates also in HIV-1-induced apoptosis, upstream of the p53-dependent mitochondrial permeabilization step. An unscheduled Cdk1 activation may contribute to neuronal apoptosis occurring in neurodegenerative diseases. Finally, the premature activation of Cdk1 can lead to mitotic catastrophe, for instance after irradiation-induced DNA damage. Thus, a cell type-specific modulation of Cdk1 might be taken advantage of for the therapeutic correction of pathogenic imbalances in apoptosis control.
320 citations
TL;DR: Methods for the detection of alterations in the DeltaPsi(m), based on the incubation of cells with cationic lipophilic fluorochromes, are delineated, which can be taken advantage of to detect apoptosis at an early stage.
271 citations
TL;DR: Cumulative evidence supports the hypothesis that mTOR acts as a master switch of cellular catabolism and anabolism, and has recently found to have profound effects on the control of apoptosis.
Abstract: The intracellular receptor of rapamycin, a macrolide antibiotic produced by Streptomyces hygroscopicus, is FKBP12 (FK506-binding protein). The rapamycin-FKBP12 complex, in turn, specifically interacts with the mammalian target of rapamycin (mTOR), to potently inhibit mTOR signaling to downstream targets. Cumulative evidence supports the hypothesis that mTOR acts as a master switch of cellular catabolism and anabolism. In addition, mTOR has been recently found to have profound effects on the control of apoptosis. MTOR is a serine/threonine kinase which signals to downstream effectors, either through direct phosphorylation or via the inhibition of the phosphatase PP2A. Under normal circumstances, in the presence of growth factor and nutrients, mTOR is constitutively activated. This activation is achieved, in part, through the insulin receptor or insulin-like growth factor receptor pathways, via a cascade that involves the activation of phosphatidylinositide-3-kinase (PI3K), then phosphatidyl inositol-3,4,5 phosphate-mediated act ivat ion of Akt/PKB-mediated phosphorylation of mTOR. Deacetylated tRNA species accumulating as a result of amino acid shortage may act as negative regulators of mTOR, through a pathway that remains to be elucidated. Moreover, the c-Abl protein tyrosine kinase phosphorylates mTOR and inhibits its action (Figure 1).
TL;DR: Its active site structure and redox properties suggest that AIF functions as an electron transferase with a mechanism similar to that of the bacterial ferredoxin reductases, its closest evolutionary homologs.
Abstract: Mitochondria play a key role in apoptosis due to their capacity to release potentially lethal proteins. One of these latent death factors is cytochrome c, which can stimulate the proteolytic activation of caspase zymogens. Another important protein is apoptosis-inducing factor (AIF), a flavoprotein that can stimulate a caspase-independent cell-death pathway required for early embryonic morphogenesis. Here, we report the crystal structure of mouse AIF at 2.0 A. Its active site structure and redox properties suggest that AIF functions as an electron transferase with a mechanism similar to that of the bacterial ferredoxin reductases, its closest evolutionary homologs. However, AIF structurally differs from these proteins in some essential features, including a long insertion in a C-terminal β-hairpin loop, which may be related to its apoptogenic functions.
Journal Article•
TL;DR: In vivo, the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice.
Abstract: The mitochondrial peripheral benzodiazepine receptor (mPBR) is involved in a functional structure designated as the permeability transition pore, which controls apoptosis. Binding of Fas/APO-1/CD95 triggers a prototypic apoptosis-inducing pathway. Using four different human tumor cell lines (T-cell Jurkat, neuroblastoma SHEP, osteosarcoma 143N2, and glioblastoma SNB79 cell lines), all of which express CD95 and mPBR, we investigated the potential role of mPBR ligands in CD95-induced apoptosis. We show that, in vitro, the three mPBR ligands tested (RO5-4864, PK11195, and diazepam) enhanced apoptosis induced by anti-CD95 antibody in Jurkat cells, as demonstrated by mitochondrial transmembrane potential drop and DNA fragmentation. In contrast, RO5-4864, but not PK11195 or diazepam, enhanced anti-CD95 apoptosis in all other cell lines. These effects were obtained in Bcl-2-overexpressing SHEP cell lines, but not in Bcl-X(L) SHEP cell lines. Enhancement of anti-CD95 antibody-induced apoptosis by RO5-4864 was characterized by an increased mitochondrial release of cytochrome c and Smac/DIABLO proteins and an enhanced activation of caspases 9 and 3, suggesting a mitochondrion-dependent mechanism. Preincubation of cells with the different mPBR ligands or anti-CD95 did not affect the levels of expression of either mPBR or CD95. In vivo, we found that the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice. Peripheral benzodiazepine receptor ligands may therefore act as chemosensitizing agents for the treatment of human neoplasms.
TL;DR: Recent insights into the cellular death program as revealed by mice are discussed, including inactivation of genes involved in cell death using homologous recombination.
TL;DR: HIV‐1 elicits a pro‐apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53, which prevents karyogamy, m TOR activation, p53S15 phosphorylation and apoptosis.
Abstract: Syncytia arising from the fusion of cells expressing the HIV‐1‐encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR‐mediated phosphorylation of p53 on Ser15 (p53 S15 ), p53‐dependent upregulation of Bax and activation of the mitochondrial death pathway. p53 S15 phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase‐like dismantling of the nuclear envelope. Inhibition of cyclin‐dependent kinase‐1 (Cdk1) prevents karyogamy, mTOR activation, p53 S15 phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV‐1‐infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53 S15 phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV‐1 infection of primary CD4 lymphoblasts. Thus, HIV‐1 elicits a pro‐apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53.
TL;DR: The working hypothesis is that MMP may be a rate-limiting event of apoptosis induction even when cell death is initiated through a primary stimulus affecting other organelles than mitochondria such as nuclei, lysosomes or the endoplasmic reticulum.
Abstract: Accumulating evidence suggests that mitochondrial membrane permeabilization (MMP) is a rate-limiting step of programmed (developmental) cell death as well as stressinduced cell death, including in the context of anti-cancer chemotherapy or viral infection. This notion is re-enforced by the observation that the knock-out of proteins involved in the pathways leading to MMP or closely linked to MMP (Bax, Bak, Bim, AIF, cytochrome c, Apaf-1, caspase-9 etc.) results in a major phenotype. Recently, we have launched the working hypothesis that MMP may be a rate-limiting event of apoptosis induction even when cell death is initiated through a primary stimulus affecting other organelles than mitochondria such as nuclei (via p53 activation), lysosomes (via activation of cathepsins) or the endoplasmic reticulum (ER). The proapoptotic agents that specifically act on the ER include tunicamycin (TM, which inhibits N-linked glycosylation), brefeldin A (BFA, which inhibits ER-Golgi transport) and thapsigargin (TG, which inhibits the sarcoplasmic/endoplasmic Ca-ATPase SERCA). Whereas BFA and TG elicit a local unfolded protein response, TG depletes ER Ca and thus impinges on Ca signaling. Mouse embryonic fibroblasts lacking both Bax and Bak become resistant to apoptosis induction by TM and BFA, an observation that may be attributed to the obligatory participation of Bax and Bak in MMP induction, or alternatively, suggests an as yet poorly characterized function of Bax and Bak at the ER level. Indeed, Bax redistributes both to mitochondria and to ER upon apoptosis induction, and overexpression of Bax reportedly causes a loss of ER Ca content. To probe the importance of MMP for ER stress-induced cell death, we assessed the effects of two local MMP inhibitors, Bcl-XL and vMIA on cellular alterations provoked by TM, BFA, and TG (Figure 1). Bcl-XL is found inserted in intracellular membranes (in particular the outer mitochondrial membrane) and is known to stabilize the mitochondrial membrane barrier function by local interactions with pore forming proteins contained in the permeability transition pore complex, namely the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and pro-apoptotic Bcl-2 family members. In contrast to Bcl-2, ± 14 no local ER effects have been described for Bcl-XL. 15 Viral mitochondrial inhibitor of apoptosis (vMIA) is encoded by the Cytomegalovirus UL37 gene, has no obvious structural similarity with Bcl-2-like molecules, and is exclusively found in mitochondrial membranes, where it specifically interacts with ANT but not with VDAC, as shown by mass spectroscopic identification of vMIAinteracting proteins and confirmed by co-immunoprecipitation assays. ± 18 TM, BFA, and TG induced signs of MMP in several different cell lines including B cell lymphoma BJAB (Figure 1a) and cervical carcinoma HeLa cells (Figure 1b). Such signs consist in the loss of the mitochondrial transmembrane potential (DCm), as determined by means of the DCm-sensitive fluorochrome, DiOC6 3 (Figure 1a), and the release of cytochrome c from mitochondria, as determined by immunofluorescence analysis (Figure 1b,c). Thus, both the permeability of the inner membrane (on which the DCm builds up) and that of the outer membrane (which retains cytochrome c) were compromised by the three different ER-targeted toxins. ER stress-induced MMP was an early event since it was detectable in a fraction of cells that still lack signs of chromatin condensation (not shown). Stable transfection with vMIA and Bcl-XL prevented ER stressinduced MMP (Figure 1a,b,c), both in BJAB and in HeLa cells. It has been reported that, in determined circumstances, for instance in type I cells stimulated by CD95 ligation, Bcl-2-mediated MMP inhibition is not sufficient for the prevention of apoptosis. Therefore, we assessed whether MMP inhibition would suffice to suppress apoptosis induction by TM, BFA, or TG. Clearly, vMIA and Bcl-XL overexpression did reduce the frequency of cells which manifest caspase activation (not shown), nuclear chromatin condensation (Figure 1b), phosphatidylserine exposure on the outer leaflet of the plasma membrane (as determined with an Annexin V-FITC conjugate, Figure 1d), and loss of viability (as determined by staining with propidium iodide, PI, Figure 1e). In conclusion, it appears that MMP inhibition by Bxl-XL or vMIA can protect cells against apoptosis induction by ERspecific toxins. Using a monoclonal antibody specific for the apoptogenic conformation of Bax (6A7), we determined the putative link between vMIA-induced (presumably ANTmediated ± 18 apoptosis inhibition and the Bax/Bak mediated MMP induced by ER stress. ER stress did induce an apoptosis-associated change in Bax conformation, linked to its aggregation in cytoplasmic spots (Figure 2f), some of which coincide with mitochondria (as determined by confocal microscopy, not shown). vMIA did prevent the apoptosis associated conformational change of Bax, as well as its aggregation, a finding that may link our previous observation that Bax and ANT can interact to induce MMP. In conclusion, it appears that ER stress can induce apoptosis through a reaction that depends on pro-apoptotic members of the Bcl-2 family (Bax, Bak) and which involves MMP as a critical step towards cellular demise. Rather than Cell Death and Differentiation (2002) 9, 465 ± 467 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00
TL;DR: Recent advances in the cooperation between ANT and Bax/Bcl-2 family members, the multiplicity of agents affecting ANT pore function and the putative role of ANT isoforms in apoptosis control are discussed.
TL;DR: This corrects the article DOI: 10.1038/sj.onc.1205018 to include the word “onc” in the title of the paper rather than the text, saving it as a single word.
Abstract: Mitochondrial dysfunction is an essential step for killing of non-small cell lung carcinomas resistant to conventional treatment
TL;DR: In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH 3, which overcomes cytoprotection by B cl-2 and Bcl-XL.
Abstract: Peptides corresponding to the BH3 domains of Bax (BaxBH3) or Bcl-2 (Bcl2BH3) are potent inducers of apoptosis when fused to the Atennapedia plasma membrane translocation domain (Ant). BaxBH3Ant and Bcl2BH3Ant caused a mitochondrial membrane permeabilization (MMP) and apoptosis, via a mechanism that was not inhibited by overexpressed Bcl-2 or Bcl-XL, yet partially inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore. When added to isolated mitochondria, BaxBH3 and Bcl2BH3 induced MMP, which was inhibited by CsA. However, Bcl-2 or Bcl-XL failed to inhibit MMP induced by BaxBH3 and Bc2BH3 in vitro, while they efficiently suppressed the induction of MMP by the Vpr protein (from human immunodeficiency virus-1), a ligand of the adenine nucleotide translocator (ANT). BaxBH3 but not Bcl2BH3 was found to interact with ANT, and only BaxBH3 (not Bcl2BH3) permeabilized ANT proteoliposomes and induced ANT to form non-specific channels in electrophysiological experiments. In contrast, both BaxBH3 and Bcl2BH3 were able to stimulate channel formation by recombinant Bax protein. Thus, BaxBH3 might induce MMP via an action on at least two targets, ANT and Bax-like proteins. In contrast, Bcl2BH3 would elicit MMP in an ANT-independent fashion. In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH3. In conclusion, BaxBH3 and Bcl2BH3 induce MMP and apoptosis through a mechanism which overcomes cytoprotection by Bcl-2 and Bcl-XL.
TL;DR: It appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects.
Abstract: Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.
TL;DR: Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.
Abstract: As shown here, mitochondria purified from different organs (liver, brain, kidney, spleen and heart) contain both pro-caspase-9 and the processed, mature form of caspase-9. Purified liver mitochondria release mature caspase-9 upon induction of permeability transition in vitro. This is accompanied by a discrete increase in the enzymatic cleavage of pro-caspase-9 substrates. We found that SHEP neuroblastoma cells constitutively contain pre-processed caspase-9 in their mitochondria, using a combination of subcellular fractionation and immunofluorescence with an antibody specific for the processed caspase. This is a cell type-specific phenomenon since HeLa cells mitochondria mainly contain pro-caspase-9 and comparatively little processed caspase-9. Upon introduction of apoptosis, mitochondrial pro-caspase-9 translocates to the cytosol and to the nucleus. This phenomenon is inhibited by transfection with Bcl-2. In synthesis, we report the unexpected finding that mitochondria can contain a pre-processed caspase isoform in non-apoptotic cells. Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.
TL;DR: Data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo, and the cytotoxic effects of Atr are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP).
Abstract: Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.
TL;DR: It is shown that apoptosis of infected cells may be considered as a pristine defense against viruses, and suppression of the apoptotic program, thereby avoiding premature death of the host cell.
Abstract: Viruses mercilessly exploit their host cells to guarantee their own proliferation and propagation. To achieve this goal, many viruses suppress the apoptotic program, thereby avoiding premature death of the host cell. Indeed, apoptosis of infected cells may be considered as a pristine defense against
TL;DR: The data suggest that sulfasalazine‐induced apoptosis of T‐lymphocytes is mediated by mitochondrio‐nuclear translocation of AIF and occurs in a caspase‐independent fashion, and might provide the molecular basis for the beneficial therapeutic effects of sulf asalazine in the treatment of chronic inflammatory diseases.
Abstract: Impaired apoptosis of T-lymphocytes is involved in the development of chronic inflammatory disorders. Previously we have shown that the anti-inflammatory drug sulfasalazine induces apoptosis in a murine T-lymphocyte cell line. The aims of the present study were to expand these observations to human systems and to analyse the molecular basis for sulfasalazine-induced apoptosis. Sulfasalazine induces apoptosis both in Jurkat cells, a human T-leukaemia cell line (ED50 value approximately 1.0 mM), and in primary human peripheral blood T-lymphocytes (ED50 value approximately 0.5 mM). In contrast SW620 colon carcinoma cells or primary human synoviocytes are not affected at these concentrations suggesting a cell type-specific sensitivity to sulfasalazine. Sulfasalazine triggers the mitochondrial accumulation of Bax and induces a collapse of the mitochondrial transmembrane potential (deltapsi(m)). Sulfasalazine causes cytochrome c release from mitochondria and subsequent activation of caspase-3 and downstream substrates. However, the pan-caspase inhibitor Z-VAD.fmk fails to inhibit sulfasalazine-induced apoptosis. Sulfasalazine stimulates mitochondrio-nuclear translocation of the novel apoptogenic factor apoptosis-inducing factor (AIF) and triggers large-scale DNA fragmentation, a characteristic feature of AIF-mediated apoptosis. Sulfasalazine-induced DeltaPsi(m) loss, AIF redistribution, and cell death are fully prevented by overexpression of Bcl-2. In conclusion, our data suggest that sulfasalazine-induced apoptosis of T-lymphocytes is mediated by mitochondrio-nuclear translocation of AIF and occurs in a caspase-independent fashion. Sulfasalazine-induced apoptosis by AIF and subsequent clearance of T-lymphocytes might thus provide the molecular basis for the beneficial therapeutic effects of sulfasalazine in the treatment of chronic inflammatory diseases.
TL;DR: Recent data reveal unexpected functional links between apoptosis and autophagic cell death, in the sense that MMP can trigger autophagy of damaged mitochondria.
TL;DR: The data indicate that the resistance of p53-mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization, and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF to the cytotoxic action of this cytokine.
Abstract: In the present study, we have investigated the mechanisms by which the restoration of wild-type (wt) p53 functions in p53 mutant cells increases their susceptibility to the cytotoxic action of tumor necrosis factor (TNF). Our data indicate that the resistance of p53-mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization. Moreover, cl.1001 cells failed to translocate the mitochondrial AIF and cytochrome c to the nucleus and to the cytosol, respectively, in response to TNF. Sensitization of these cells, following infection with a recombinant adenovirus encoding wtp53, to TNF-induced cytotoxicity resulted in the restoration of caspase-8 cleavage and the reestablishment of mitochondrial signs of apoptosis. These findings suggest that the cross-talk between p53 and TNF-induced cell death depends on mitochondria and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.
TL;DR: The spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide, which could provide optimal conditions for triggering a specific immune response.
Abstract: REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.
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TL;DR: Dans la voie « intrinsèque » of l’apoptose, il semble que la perméabilisation mitochondriale est nécessaire and suffisante for le déclenchement du processus létal, dont le morphotype est ensuite partiellement déterminé par les caspases activées après le dysfonctionnement mitochondrial.
Abstract: des changements morphologiques caractéristiques, en particulier un rétrécissement de la taille cellulaire et une condensation de la chromatine qui sont en grande partie déterminés par l’activation de caspases, une classe spécifique de protéases. Y-a-t-il aussi des points de contrôle impliqués dans l’apoptose ? Chez les mammifères, dans la plupart des modèles d’apoptose, il apparaît que l’activation des caspases n’est pas le point de départ de la mort cellulaire mais participe plutôt à la destruction de la cellule dont la mort a déjà été programmée. Ce qui scelle le sort de la cellule serait la perméabilisation des membranes mitochondriales. Cette perméabilisation mitochondriale est induite par un grand nombre de molécules pro-apoptotiques endogènes et exogènes et résulte de l’activation de nombreuses voies de transduction de signaux létaux [1]. La perméabilisation des mitochondries entraîne le relargage d’activateurs de caspases et d’effecteurs létaux indépendants des caspases, ainsi qu’une perturbation métabolique majeure, avec un arrêt de la formation d’ATP et un déséquilibre du potentiel de réduction/oxydation. Les emplacements hiérarchiques des checkpoints impliquant la perméabilisation mitochondriale et l’activation des caspases varient en fonction du stimulus primaire menant à la mort. Dans la voie « intrinsèque » de l’apoptose, il semble que la perméabilisation mitochondriale est nécessaire et suffisante pour le déclenchement du processus létal, dont le morphotype est ensuite partiellement déterminé par les caspases activées après le dysfonctionnement mitochondrial. En revanche, dans la voie « extrinsèque », qui met en jeu les récepteurs dits de mort, dont le prototype est CD95/Fas, l’activation des caspases est primordiale et peut, dans certains types cellulaires, déclencher l’apoptose sans participation mitochondriale. Si ce scénario est amplement démontré pour les mammifères, que ce soit à l’échelle de la biologie cellulaire [2] ou de la génétique [3], il est très différent dans d’autres espèces considérées comme espèces-modèles d’apoptose. Chez Caenorhhabditis elegans et chez Drosophila melanogaster, l’apoptose est clairement contrôlée par les caspases et La mort de l’individu est une expérience que l’on ne peut transmettre. « Mourir » est donc un verbe qui, en toute logique, ne peut être employé que pour décrire le sort d’autrui et ne peut pas être employé à la première personne. La mort est perçue comme une menace de la rupture de la continuité de l’individu, menace qui se dissipe quand nous analysons le dynamisme de notre devenir subjectif et corporel. La mort fait partie de la vie : tant sur le plan de nos expériences tout adulte survit à un enfant mort qu’à l’échelle de la cellule, nous mourons en permanence. À l’équilibre, chaque division cellulaire doit être compensée par un événement de mort cellulaire, et c’est aussi bien l’excès que le défaut de mort cellulaire qui nous rend malades. Qu’une cellule qui devait mourir ne le fasse pas et nous risquons de développer un cancer, ce qui introduit la notion paradoxale de « mort vitale », la mort qui est nécessaire à notre survie. Si le lecteur doute de l’intérêt de la question, qu’il se rappelle que notre peau est nécessairement couverte de cellules mortes, ce qui n’est pas toujours si désagréable à toucher... Il n’est donc pas étonnant que la mort cellulaire programmée, fréquemment appelée apoptose, constitue l’une des thématiques les plus dynamiques de la recherche biomédicale actuelle. Plus de quarante-trois mille articles concernant l’apoptose ont été publiés dans des revues scientifiques entre 1991 et 2001 (voir http://esi-topics.com/apoptosis/index.html). Ce travail sans précédent a-t-il permis de percer le mystère de la mort cellulaire ? Et annonce-t-il des avancées dans le diagnostic et le traitement des grandes pathologies ? Le visage de l’apoptose, cette inconnue, compagne indispensable à notre survie, a-t-il été dévoilé ? Considérant ces interactions entre la vie et la mort, une réponse pourrait être obtenue en recherchant les analogies entre le cycle cellulaire et l’apoptose. Le déroulement du cycle cellulaire nécessite l’activation de molécules de signalisation, obéissant à une séquence stricte d’étapes cumulatives. Si la cellule détecte une imperfection quelconque dans l’exécution de ces étapes, elle active des points de contrôle (checkpoints) qui arrêtent le processus. L’apopGuido Kroemer