SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis

Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH](i), nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH](i) can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH](i) in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and alpha-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the execution phase of apoptosis.     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

Anaplasma phagocytophilum delays spontaneous human neutrophil apoptosis by modulation of multiple apoptotic pathways

Anaplasma phagocytophilum infects human neutrophils and inhibits the intrinsic pathway of spontaneous neutrophil apoptosis by protecting mitochondrial membrane integrity. In the present study, we investigated the molecular signalling of the extrinsic pathway and the interaction between the intrinsic and extrinsic pathways in the inhibition of spontaneous human neutrophil apoptosis by A. phagocytophilum. Cell surface Fas clustering during spontaneous neutrophil apoptosis was significantly blocked by A. phagocytophilum infection. The cleavage of pro-caspase 8, caspase 8 activation and the cleavage of Bid, which links the intrinsic and extrinsic pathways, in the extrinsic pathway of spontaneous neutrophil apoptosis were inhibited by A. phagocytophilum infection. Inhibition of this pathway was active as the cleavage of pro-caspase 8 and Bid in anti-Fas-induced neutrophil apoptosis was also inhibited by A. phagocytophilum infection. Likewise, A. phagocytophilum infection inhibited the pro-apoptotic Bax translocation to mitochondria, activation of caspase 9, the initiator caspase in the intrinsic pathway, and the degradation of a potent caspase inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP), during spontaneous neutrophil apoptosis. These data point to a novel mechanism induced by A. phagocytophilum involving both extrinsic and intrinsic pathways to ensure to delay the apoptosis of host neutrophils.     

Glutathione export during apoptosis requires functional multidrug resistance-associated proteins

GSH is released in cells undergoing apoptosis, and the present study indicates that the multidrug resistance-associated proteins (MRPs/ABCC) are responsible for this GSH release. Jurkat cells released approximately 75-80% of their total intracellular GSH during both Fas antibody- and staurosporine-induced apoptosis. In contrast, Raji cells, a lymphocyte cell line that is deficient in phosphatidylserine externalization, did not release GSH during apoptosis, and other apoptotic features appeared more slowly in these cells. Jurkat and Raji cell lines expressed comparable MRP and OATP/SLCO (organic anion-transporting polypeptide) mRNA levels, and MRP1 protein levels; however, differences existed in MRP1 localization and function. In Jurkat cells, MRP1 was largely localized to the plasma membrane, and these cells exported the MRP substrate calcein. Calcein release was enhanced during apoptosis. In contrast, Raji cells had little MRP1 at the plasma membrane and did not export calcein under basal or apoptotic conditions, indicating that these cells lack functional MRPs at the plasma membrane. GSH release in Jurkat cells undergoing apoptosis was inhibited by the organic anion transport inhibitors MK571, sulfinpyrazone, and probenecid, supporting a role for the MRP transporters in this process. Furthermore, when MRP1 expression was decreased with RNA interference, GSH release was lower under both basal and apoptotic conditions, providing direct evidence that MRP1 is involved in GSH export.     

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

Lipid raft connection between extrinsic and intrinsic apoptotic pathways

Apoptosis in mammalian cells is modulated by extrinsic and intrinsic signaling pathways through the formation of death receptor-mediated death-inducing signaling complex (DISC) and mitochondrial-derived apoptosome, respectively. We found by ultrastructural approaches that the antitumor drug edelfosine induced aggregates of lipid rafts containing Fas/CD95 receptor and Fas-associated death domain-containing protein in leukemic cells. Death receptors together with DISC and apoptosome constituents were recruited in rafts during edelfosine treatment in multiple myeloma cells. This apoptotic response involved caspases-8/-9/-10 that were translocated to rafts. Lipid raft disruption by cholesterol depletion inhibited loss of mitochondrial transmembrane potential, caspase activation and apoptosis, whereas cholesterol replenishment restored these responses. Our data indicate that rafts act as scaffolds where extrinsic and intrinsic apoptotic signaling pathways concentrate, forming clusters of apoptotic signaling molecule-enriched rafts (CASMER), which function as novel supramolecular entities in the triggering of apoptosis, and play an important role in edelfosine-induced apoptosis in blood cancer cells.     

Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.     

Increased expression of the multidrug resistance-associated protein 1 (MRP1) in kidney glomeruli of streptozotocin-induced diabetic rats

Oxidative stress has been linked to the podocytopathy, mesangial expansion and progression of diabetic nephropathy. The major cell defence mechanism against oxidative stress is reduced glutathione (GSH). Some ABC transporters have been shown to extrude GSH, oxidised glutathione or their conjugates out of the cell, thus implying a role for these transporters in GSH homeostasis. We found a remarkable expression of mRNA for multidrug resistance-associated proteins (MRP/ABCC) 1, 3, 4 and 5 in rat glomeruli. Three weeks after induction of diabetes in glomeruli of streptozotocin-treated rats, we observed a decline in reduced GSH levels and an increase in the expression and activity of MRP1 (ABCC1). These lower GSH levels were improved by ex vivo treatment with pharmacological inhibitors of MRP1 activity (MK571). We conclude that increased activity of MRP1 in diabetic glomeruli is correlated with an inadequate adaptive response to oxidative stress.     

Bid activation during induction of extrinsic and intrinsic apoptosis in eosinophils

Eosinophils readily undergo apoptosis when removed from a physiological environment via activation of the intrinsic cell death pathway. This can be further enhanced by certain chemicals (for example, glucocorticoid), or by extrinsic means (that is, Fas receptor engagement). In this investigation, we examined the relative importance of these pathways in cultured human peripheral blood eosinophils in the context of expression and activation of the BH3-only Bcl2 homologue Bid. Bid activation was examined under conditions where programmed cell death was either stimulated (via Fas engagement or glucocorticoid treatment) or inhibited (interleukin-5 (IL-5)) relative to control. Full-length Bid was found to be highly expressed in eosinophils, and processed to a similar extent during either agonist anti-Fas or glucocorticoid treatment. IL-5 blocked intrinsic Bid activation during factor withdrawal or glucocorticoid treatment, and partially attenuated that caused by Fas activation. Caspase 8 (but not caspase 9) antagonism partly but significantly affected receptor-mediated Bid activation and cell death; these processes were not altered by either caspase inhibitor during simple factor withdrawal or glucocorticoid treatment. Bid processing appears to be central to both intrinsic and extrinsic pathways of cell death in eosinophils. The role of IL-5 in blocking the intrinsic pathway of eosinophil apoptosis is underscored. Results of specific inhibition support the existence of Bid activation pathways in eosinophils other than those mediated by the classic initiator caspases.     

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.     

Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.     

Stress-induced apoptosis: Toward a symmetry with receptor-mediated cell death

Apoptosis is a form of programmed cell death executed by caspases activated along signalling pathways initiated by ligation of cell-surface death receptors ( extrinsic pathway ) or by perturbation of the mithocondrial membrane promoted by physical or chemical stress agents ( intrinsic pathway ). In metazoans, this evolutionary conserved, genetically controlled process has a role in a variety of physiological settings, as development, homeostasis of tissues and maintenance of the organism integrity. When deranged by impaired regulation or inappropriate activation apoptosis contributes to the pathogenesis of diseases as autoimmunity, cancer, restenosis, ischaemia, heart failure and neurodegenerative disorders. In this review we will present a survey of the stress-induced intrinsic, mithochondrial, pathway and, based on recent experimental data, we will propose a view compatible with an emergent conceptual symmetry between the two apoptogenic extrinsic and intrinsic pathways. Elements of symmetry present in both the apoptogenic signalling pathways include: early activation of initiator caspases (feed-forwarded by a direct or post-mitocondrial effector caspase-mediated amplification loop in some cell types) and mitochondrial membrane permeabilization with required release of antagonists of active caspase inhibitors (IAPs) in high-level IAPs-expressing cells and apoptosome-mediated amplification of the caspase cascade more or less needed in different cell types.     

Neurotoxic mechanisms caused by the Alzheimer's disease-linked Swedish amyloid precursor protein mutation: oxidative stress,caspases, and the JNK pathway

Autosomal dominant forms of familial Alzheimer's disease (FAD) are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2. Simultaneously, evidence is provided that increased oxidative stress might play a crucial role in the rapid progression of the Swedish FAD. Here we investigated the effect of the Swedish double mutation (K670M/N671L) in the beta-amyloid precursor protein on oxidative stress-induced cell death mechanisms in PC12 cells. Western blot analysis and cleavage studies of caspase substrates revealed an elevated activity of the executor caspase 3 after treatment with hydrogen peroxide in cells containing the Swedish APP mutation. This elevated activity is the result of the enhanced activation of both intrinsic and extrinsic apoptosis pathways, including activation of caspase 2 and caspase 8. Furthermore, we observed an enhanced activation of JNK pathway and an attenuation of apoptosis by SP600125, a JNK inhibitor, through protection of mitochondrial dysfunction and reduction of caspase 9 activity. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a result of an increased vulnerability of neurons through activation of different apoptotic pathways as a consequence of elevated levels of oxidative stress.     

Binding of a photoaffinity analogue of glutathione to MRP1 (ABCC1) within two cytoplasmic regions (L0 and L1) as well as transmembrane domains 10-11 and 16-17

MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural products as well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH is required for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating the influence of GSH on MRP1 is poorly understood, and the location of GSH binding site(s) within MRP1 have yet to be determined. To address these issues, we have synthesized a [(125)I] labeled azido-derivative of GSH (IAAGSH) to photoaffinity label MRP1. Our results revealed that IAAGSH labeled MRP1 with high specificity, and binding was inhibited by MRP1 substrates leukotriene C(4) and MK571. Interestingly, verapamil and vincristine enhanced IAAGSH photolabeling of MRP1, in agreement with observations that both drugs enhance GSH transport. We observed GSH to be the best inhibitor of photoaffinity labeling, as compared to oxidized glutathione (GSSG) and four different GSH alkyl derivatives. These observations indicate that IAAGSH interacted with MRP1 in a similar manner as unmodified GSH. Moreover, using eight MRP1-HA variants, each containing hemagglutinin A (HA) epitopes inserted at different sites in MRP1, we mapped the GSH binding sites in MRP1. Our GSH analogue photoaffinity labeled four MRP1 polypeptides that were located within two cytoplasmic domains in linker sequences (L0 and L1) as well as transmembrane domains 10-11 and 16-17. The photoaffinity labeling of polypeptides within L0 and L1 domains is further confirmed using two MRP1-specific monoclonal antibodies (MRPr1 and QCRL1) with epitopes within the linker domains. Taken together, this study provides the most precise information to date on the location of GSH binding sites in MRP1.     

Capsular polysaccharide induction of apoptosis by intrinsic and extrinsic mechanisms

A purified microbial capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), induces Fas ligand (FasL) upregulation on macrophages and, as a consequence, apoptosis of lymphocytes. The mechanisms that lead to lymphocyte apoptosis in both in vitro and in vivo systems were investigated by cytofluorimetric analysis and Western blotting experiments. Caspase 8 cleaves caspase 3 in two different pathways: directly as well as indirectly by activation of Bcl-2 interacting domain, which initiates caspase 9 cleavage. Therefore, the caspase 8 and caspase 9 pathways cooperate in an amplification loop for efficient cell death, and noteworthily we provide evidence that they are both activated in one single cell. Furthermore, both activation of GXM-mediated caspase 8 and apoptosis were also found in in vivo systems in an experimental model of murine candidiasis. Collectively, our data show that GXM-induced apoptosis involves, in a single cell, a cross-talk between extrinsic and intrinsic pathways. Such a finding offers opportunities for the therapeutic usage of this polysaccharide in appropriate clinical settings for taming T-cell responses.     

The phospholipid scramblase PLSCR1 increases UV induced apoptosis primarily through the augmentation of the intrinsic apoptotic pathway and independent of direct phosphorylation by protein kinase C delta

Cell death by apoptosis can be caused by the DNA mutagen UV light whose exposure causes the direct activation of both the caspase 9 regulated cell damage intrinsic pathway and the caspase 8 regulated plasma membrane extrinsic pathway. We determined that increased activity of the plasma membrane phospholipid scramblase, PLSCR1, amplified UV mediated apoptosis primarily through the activation of the intrinsic apoptotic pathway. The caspase 8 inhibitor z-IETD-fmk was not as effective an inhibitor of PLSCR1 augmented UV induced apoptosis compared to treatment with caspase 3, caspase 9, or pan-caspase inhibitors. The inability of the caspase 8 inhibitor to decrease UV induced apoptosis was dependent on PLSCR1, as UV induced apoptosis was decreased by a similar amount in the control cells in the presence of inhibitors of caspase 8, caspase 9, caspase 3, or the pan-caspase inhibitor. PKC-delta directly phosphorylates human PLSCR1 resulting in increased PLSCR1 scramblase activity. PKC-delta can also be activated by caspase mediated cleavage resulting in the release of a constitutively active kinase domain. We observed that replacing the PKC-delta phosphorylation site of PLSCR1 with an alanine did not affect the ability of PLSCR1 to enhance UV induced apoptosis implying that PKC-delta does not directly phosphorylate PLSCR1 to increase plasma membrane scramblase activity during apoptosis. Cells transfected with a PLSCR1 mutant that contained an alanine substitution at its known PKC-delta phosphorylation site underwent UV induced apoptosis at a level similar to those transfected with wild type PLSCR1. The combined results indicate that UV exposure in cells possessing PLSCR1 increases apoptosis primarily by enhancement of the intrinsic apoptotic pathway, and also imply that the increased apoptosis observed upon exposure to UV light is not through direct phosphorylation of PLSCR1 by PKC-delta.     

The MRP1-mediated effluxes of arsenic and antimony do not require arsenic-glutathione and antimony-glutathione complex formation

Arsenic trioxide is an effective treatment for acute promyelocytic leukemia, but resistance to metalloïd salts is found in humans. Using atomic absorption spectroscopy, we have measured the rate of uptake of arsenic trioxide and of antimony tartrate in GLC4 and GLC4/ADR cells overexpressing MRP1 and the rate of their MRP1-mediated effluxes as a function of the intracellular GSH concentration. In sensitive cells, after 1 h, a pseudosteady state is reached where intra- and extracellular concentrations of metalloid are the same. This precludes the formation, at short term, of complexes between arsenic or antimony with GSH. In resistant cells reduced intracellular accumulation of arsenic (or antimony), reflecting an increased rate of arsenic (or antimony) efflux from the cells, is observed. No efflux of the metalloid is observed in GSH depleted cells. The two metalloïds and GSH are pumped out by MRP1 with the same efficiency. Moreover for the three compounds 50% of the efflux is inhibited by 2 M MK571. This led us to suggest that As- and Sb-containing species could be cotransported with GSH.     

Flock house virus induces apoptosis by depletion of Drosophila inhibitor-of-apoptosis protein DIAP1

The molecular mechanisms by which RNA viruses induce apoptosis and apoptosis-associated pathology are not fully understood. Here we show that flock house virus (FHV), one of the simplest RNA viruses (family, Nodaviridae), induces robust apoptosis of permissive Drosophila Line-1 (DL-1) cells. To define the pathway by which FHV triggers apoptosis in this model invertebrate system, we investigated the potential role of Drosophila apoptotic effectors during infection. Suggesting the involvement of host caspases, the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone (z-VAD-fmk) prevented FHV-induced cytopathology and prolonged cell survival. RNA interference-mediated ablation of the principal Drosophila effector caspase DrICE or its upstream initiator caspase DRONC prevented FHV-induced apoptosis and demonstrated direct participation of this intrinsic caspase pathway. Prior to the FHV-induced activation of DrICE, the intracellular level of inhibitor-of-apoptosis (IAP) protein DIAP1, the principal caspase regulator in Drosophila melanogaster, was dramatically reduced. DIAP1 was depleted despite z-VAD-fmk-mediated caspase inhibition during infection, suggesting that the loss of DIAP1 was caused by an upstream FHV-induced signal. The RNA interference-mediated knockdown of DIAP1 caused rapid and uniform apoptosis of DL-1 cells and thus indicated that DIAP1 depletion is sufficient to trigger apoptosis. Confirming this conclusion, the elevation of intracellular DIAP1 levels in stable diap1-transfected cells blocked caspase activation and prevented FHV-induced apoptosis. Collectively, our findings suggest that DIAP1 is a critical sensor of virus infection, which upon virus-signaled depletion relieves caspase inhibition, which subsequently executes apoptotic death. Thus, our study supports the hypothesis that altering the level or the activity of cellular IAP proteins is a general mechanism by which RNA viruses trigger apoptosis.     

Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice

The signaling events leading to apoptosis can be divided into two major pathways, involving either mitochondria (intrinsic) or death receptors (extrinsic). In a recent study, we have shown the involvement of the mitochondria-dependent apoptotic pathway in heat-induced male germ cell apoptosis in the rat. In additional studies, using the gld (generalized lymphoproliferation disease) and lprcg (lymphoproliferation complementing gld) mice, which harbor loss-of-function mutations in Fas L and Fas, respectively, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system is not required for heat-induced germ cell apoptosis in the testis. In the present study, we have found that the initiation of apoptosis in wild-type mice was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. The relocation of Bax is accompanied by sequestration of ultracondensed mitochondria into paranuclear areas of apoptotic germ cells, cytosolic translocation of mitochondrial cytochrome c and DIABLO, and is associated with activation of the initiator caspase 9 and the executioner caspase 3. Similar events were also noted in both gld and lprcg mice. Taken together, these results indicate that the mitochondria-dependent pathway is the key apoptotic pathway for heat-induced male germ cell death in mice.