Autoimmune lymphoproliferative syndrome with defective Fas: genotype influences penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.     

Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds

Mutations in the death domain of the death receptor CD95 (APO-1/Fas) cause lymphoproliferation and autoimmune disease in both lpr(cg) mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr(cg) mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-kappaB (NF-kappaB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-kappaB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.     

Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk

Background

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and Principal Findings

The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and Significance

Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.     

SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane

Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.     

Mutations in apoptosis genes: a pathogenetic factor for human disease

Cell death by apoptosis is exerted by the coordinated action of many different gene products. Mutations in some of them, acting at different levels in the apoptosis process, have been identified as cause or contributing factor for human diseases. Defects in the transmembrane tumor necrosis factor receptor 1 (TNF-R1) lead to the development of familial periodic fever syndromes. Mutations in the homologous receptor Fas (also named CD95; Apo-1) are observed in malignant lymphomas, solid tumors and the autoimmune lymphoproliferative syndrome type I (ALPS I). A mutation in the ligand for Fas (Fas ligand; CD95 ligand, Apo-1 ligand), which induces apoptosis upon binding to Fas, was described in a patient with systemic lupus erythematodes and lymphadenopathy. Perforin, an other cytotoxic protein employed by T- and NK-cells for target cell killing, is mutated in chromosome 10 linked cases of familial hemophagocytic lymphohistiocytosis. Caspase 10, a representative of the caspase family of proteases, which plays a central role in the execution of apoptosis, is defect in autoimmune lymphoproliferative syndrome type II (ALPS II). The intracellular pro-apoptotic molecule bcl-10 is frequently mutated in mucosa-associated lymphoid tissue (MALT) lymphomas and various non-hematologic malignancies. The p53, an executioner of DNA damage triggered apoptosis, and Bax, a pro-apoptotic molecule with the ability to perturb mitochondrial membrane integrity, are frequently mutated in malignant neoplasms. Anti-apoptotic proteins like bcl-2, cellular-inhibitor of apoptosis protein 2 (c-IAP2) and neuronal apoptosis inhibitory protein 1 (NAIP1) are often altered in follicular lymphomas, MALT lymphomas and spinal muscular atrophy (SMA), respectively. This article reviews the current knowledge on mutations of apoptosis genes involved in the pathogenesis of human diseases and summarises the gradual transformation of discoveries in apoptosis research into benefits for the clinical management of diseases.     

Autoimmune lymphoproliferative syndromes: genetic defects of apoptosis pathways

Human and mouse natural mutants presenting with lymphoproliferative syndrome and autoimmunity (ALPS) have enlightened the role of the Fas and FasL in lymphocyte cell death and peripheral tolerance. Further study of the genetic basis of the human pathology led to the identification of apoptosis signaling defect, and pointed out to the crucial role of caspase-10 in the process of apoptosis induction. In contrast, the absence of lymproliferation in engineered mutants of 'death pathways' suggests that additional events are necessary to recapitulate the overt phenotype of ALPS patients or MRL/lpr mice. Moreover, these models highlight the roles of Fas and associated molecules, such as FADD and caspase-8, in lymphocyte development or activation. This review will discuss the main findings provided by the study of mouse models and human conditions.     

Progesterone is a suppressor of apoptosis in bovine luteal cells

Progesterone is suggested to be a suppressor of apoptosis in bovine luteal cells. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells. Furthermore, apoptosis is known to be controlled by the bcl-2 gene/protein family and caspases. This study was undertaken to determine whether intraluteal progesterone (P4) is involved in Fas L-mediated luteal cell death in the bovine corpus luteum (CL) in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 and the activity of caspase-3 in the bovine CL. Luteal cells obtained from the cows in the midluteal phase of the estrous cycle (Days 8-12 of the cycle) were exposed to a specific P4 antagonist (onapristone [OP], 10(-4) M) with or without 100 ng/ml Fas L. Although Fas L alone did not show a cytotoxic effect, treatment of the cells with OP alone or in combination with Fas L resulted in killing of 30% and 45% of the cells, respectively (P <0.05). DNA fragmentation was observed in the cells treated with Fas L in the presence of OP. The inhibition of P4 action by OP increased the expression of Fas mRNA (P <0.01); however, it did not affect bax or bcl-2 mRNA expression (P >0.05). Moreover, OP stimulated expression of caspase-3 mRNA (P <0.01). The overall results indirectly show that intraluteal P4 suppresses apoptosis in bovine luteal cells through the inhibition of Fas and caspase-3 mRNA expression and inhibition of caspase-3 activation.     

IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression

We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.     

Role of inherited defects decreasing Fas function in autoimmunity

Fas is a death receptor belonging to the TNFR superfamily and induces cell apoptosis by both activating a caspase cascade and altering mitochondria. In the immune system, Fas is involved in the switching-off of the immune responses and cell mediated cytotoxicity. In humans, genetic defects decreasing Fas function cause the Autoimmune Lymphoproliferative Syndrome (ALPS) where autoimmunities are associated with accumulation of polyclonal lymphocytes in the secondary lymphoid tissues and expansion of T cells lacking both CD4 and CD8 (DN cells). Expansion of DN cells is absent in an ALPS variant, named Dianzani's Autoimmune Lymphoproliferative Disease (DALD). The observation that DALD patients' families display increased frequency of autoimmune diseases different from ALPS suggests that defects of Fas function may also play a role in development of "common" autoimmune diseases. This possibility is supported by detection of defective Fas function in substantial proportions of patients with the multiple autoimmune syndrome or aggressive forms of type 1 diabetes or multiple sclerosis. This article reviews data suggesting that development of autoimmune/lymphoproliferative patterns may involve several alterations hitting the Fas system, but might also involve alterations in other systems contributing to the switching-off or proliferation of lymphocytes.     

Role of complement-binding CD21/CD19/CD81 in enhancing human B cell protection from Fas-mediated apoptosis

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIP(L). This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.     

Inhibition of TCR-induced CD8 T cell death by IL-12: regulation of Fas ligand and cellular FLIP expression and caspase activation by IL-12

In this study we demonstrate the anti-apoptotic effect of IL-12 and its underlying mechanism in CD8 T cells. The prolonged stimulation of CD8 T cells with anti-CD3 alone caused apoptosis mediated by Fas and the caspase signaling pathway. However, costimulation with IL-12 significantly prevented anti-CD3-induced apoptosis of CD8 T cells. IL-12 decreased the number of Fas ligand-positive CD8 T cells and inhibited the activation of caspase-8 and caspase-3. In addition, IL-12 up-regulated cellular FLIPs but not Bcl-2 family proteins or cellular inhibitor of apoptosis proteins. These data suggest that IL-12 provides survival signals to CD8 T cells by down-regulating Fas ligand and up-regulating cellular FLIPs, followed by inhibiting caspase activation, which implies a role for IL-12 in peripheral responses of CD8 T cells in vivo.     

Chemotherapy-induced apoptosis of S-type neuroblastoma cells requires caspase-9 and is augmented by CD95/Fas stimulation

Stromal or S-type tumor cells are a distinct lineage found in neuroblastoma tumors and have an important role in the biology of this disease. Anticancer agents induce apoptosis through death receptor- and mitochondria-initiated pathways. The object of this work was to determine the involvement of these pathways in the response to doxorubicin (Dox) and cisplatin (CDDP) in S-type neuroblastoma cells. Both drugs activated caspase-9 and caspase-3 but not caspase-8. Caspase-9-specific inhibition blocked S-type cell death induced by Dox. SH-EP1 cells transfected to express dominant negative mutant caspase-9, but not those expressing DN caspase-8, were resistant to Dox- and CDDP-induced apoptosis. The lack of caspase-8 involvement in chemotherapy-induced death was not the result of an intrinsic inability of these cells to activate this enzyme because when they were treated with tumor necrosis factor-related apoptosis-inducing ligand, caspase-8 was activated. We also found that both drugs up-regulated CD95/Fas expression but that CD95/Fas signaling was not necessary for cell killing. Experiments testing the response of chemotherapy-treated cells to agonists of the CD95/Fas receptor established that Dox and CDDP treatment sensitizes cells to CD95/Fas killing. Together, these results are consistent with a model in which caspase-9 is of central importance in the death mechanism utilized by these drugs in S-type cells. Although the death response is not dependent on CD95/Fas, concomitant stimulation of this receptor amplifies the death response in drug-treated cells.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.     

Synthetic glycosidated phospholipids induce apoptosis through activation of FADD,caspase-8 and the mitochondrial death pathway

Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD−/− and caspase-8−/− cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas−/− Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.     

Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein.

Apoptosis induction through CD95 (APO-1/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-x(L) in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-x(L) transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-x(L) protected cells, raising the question of how Bcl-x(L)-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-x(L) cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIP(L), caspase-8-dependent clustering, and internalization of CD95, as well as processing of pro-caspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-x(L)-expressing cells. Our data suggest that, in Bcl-x(L)-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-x(L).     

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N:-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.     

A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells

In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. Deficiencies in the apoptotic pathways may lead to chemoresistance. Here we report that glutathione (GSH) plays a critical role in activation of apoptosis pathways by CD95 (APO-1/Fas) or anticancer drugs. Upon treatment with anticancer drugs or CD95 triggering, CD95-resistant or Bcl-x(L) overexpressing CEM cells were deficient in activation of apoptosis pathways. CD95-resistant and Bcl-x(L) overexpressing CEM cells exhibited higher intracellular GSH levels in comparison to parental cells. Downregulation of GSH by L-buthionine-(S,R)-sulfoxime (BSO), a specific inhibitor of glutathione synthesis, reversed deficiencies in activation of apoptosis pathways by anticancer drugs or CD95. Interestingly, downregulation of GSH by BSO increased CD95 DISC formation in type I cells. In hybrids of CD95-resistant cells with sensitive cells and hybrids of overexpressing Bcl-x(L) cells with sensitive cells, the phenotype of apoptosis resistance was dominant. Also, in these hybrids, downregulation of GSH reversed CD95- and chemoresistance. We conclude that dominant apoptosis resistance depends, at least in part, on intracellular GSH levels, which may affect apoptosis signaling at different compartments, for example, the death receptor or mitochondria.