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Background: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL., Methods: Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson's correlation., Results: Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas., Conclusions: These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis., (Copyright: © 2020 Permanyer.)

We investigated the effect of apigenin, a dietary flavonoid, on isoproterenol hydrochloride (ISO)-induced apoptotic signaling in cardiomyoblast H9C2 cells. The results showed that apigenin treatment (10 µM) prevented ISO (31.25 μM)-induced lipid peroxidative levels and antioxidants status in H9C2 cells. Furthermore, apigenin inhibited expression of inflammatory markers in ISO-treated cells. In addition, apigenin prevented ISO-induced DNA damage and apoptotic signaling through modulating the expression of Bax, caspase-3, -8 and -9, cytochrome c, and Fas proteins in H9C2 cells. It is concluded that apigenin prevents ISO-induced antioxidants depletion, oxidative DNA damage, inflammatory, and apoptotic signaling in H9C2 cells. Thus, the present results demonstrated that apigenin has a cardioprotective effect on cardiomyoblasts cells., (© 2018 Wiley Periodicals, Inc.)

Based on the assumed oestrogenic and apoptotic properties of soya isoflavones (genistein, daidzein), and following the current OECD test-guidelines and principle of 3Rs, we have studied the potential toxicity of phytochemicals on the zebrafish embryos test (ZFET). For this purpose, zebrafish embryos at 2-3 h post-fertilisation (hpf) were exposed to both soya isoflavones (from 1.25 mg/L to 20 mg/L) and assayed until 96 hpf. Lethal and sub-lethal endpoints (mortality, hatching rates and malformations) were estimated in the ZFET, which was expanded to potential gene expression markers, determining the lowest observed effect (and transcriptional) concentrations (LOEC, LOTEC), and the no-observable effect (and transcriptional) concentrations (NOEC, NOTEC). The results revealed that genistein is more toxic (LC50-96 hpf: 4.41 mg/L) than daidzein (over 65.15 mg/L). Both isoflavones up-regulated the oestrogen (esrrb) and death receptors (fas) and cyp1a transcript levels. Most thyroid transcript signals were up-regulated by genistein (except for thyroid peroxidase/tpo), and the hatching enzyme (he1a1) was exclusively up-regulated by daidzein (from 1.25 mg/L onwards). The ZFET proved suitable for assessing toxicant effects of both isoflavones and potential disruptions (i.e. oestrogenic, apoptotic, thyroid, enzymatic) during the embryogenesis and the endotrophic larval period., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tissue factor (TF) is the primary activator of the blood coagulation cascade. Liver parenchymal cells (ie, hepatocytes) express TF in a molecular state that lacks procoagulant activity. Hepatocyte apoptosis is an important feature of acute and chronic liver diseases, and Fas-induced apoptosis increases hepatocyte TF procoagulant activity in vitro. We determined the impact of a pan-caspase inhibitor, IDN-7314, on hepatocyte TF activity in vitro and TF-mediated coagulation in vivo. Treatment of primary mouse hepatocytes with the Fas death receptor ligand (Jo2, 0.5 μg/ml) for 8 h increased hepatocyte TF procoagulant activity and caused release of TF-positive microvesicles. Pretreatment with 100 nM IDN-7314 abolished Jo2-induced caspase-3/7 activity and significantly reduced hepatocyte TF procoagulant activity and release of TF-positive microvesicles. Treatment of wild-type C57BL/6 mice with a sublethal dose of Jo2 (0.35 mg/kg) for 4.5 h increased coagulation, measured by a significant increase in plasma thrombin-antithrombin and TF-positive microvesicles. Total plasma microvesicle-associated TF activity was reduced in mice lacking hepatocyte TF; suggesting TF-positive microvesicles are released from the apoptotic liver. Fibrin(ogen) deposition increased in livers of Jo2-treated wild-type mice and colocalized primarily with cleaved caspase-3-positive hepatocytes. Pretreatment with IDN-7314 reduced caspase-3 activation, prevented the procoagulant changes in Jo2-treated mice, and reduced hepatocellular injury. Overall, the results indicate a central role for caspase activity in TF-mediated activation of coagulation following apoptotic liver injury. Moreover, the results suggest that liver-selective caspase inhibition may be a putative strategy to limit procoagulant and prothrombotic changes in patients with chronic liver disease.

Aims: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to β-cell failure in type 2 diabetes, cultured and transplanted islets. We previously showed that biosynthetic hIAPP aggregates induce β-cell Fas upregulation and activation of the Fas apoptotic pathway. We used cultured human and hIAPP-expressing mouse islets to investigate: (1) the role of interleukin-1β (IL-1β) in amyloid-induced Fas upregulation; and (2) the effects of IL-1β-induced β-cell dysfunction on pro-islet amyloid polypeptide (proIAPP) processing and amyloid formation., Research Design and Methods: Human and h IAPP -expressing mouse islets were cultured to form amyloid without or with the IL-1 receptor antagonist (IL-1Ra) anakinra, in the presence or absence of recombinant IL-1β. Human islets in which amyloid formation was prevented (amyloid inhibitor or Ad-prohIAPP-siRNA) were cultured similarly. β-cell function, apoptosis, Fas expression, caspase-8 activation, islet IL-1β, β-cell area, β-/α-cell ratio, amyloid formation, and (pro)IAPP forms were assessed., Results: hIAPP aggregates were found to increase IL-1β levels in cultured human islets that correlated with β-cell Fas upregulation, caspase-8 activation and apoptosis, all of which were reduced by IL-1Ra treatment or prevention of amyloid formation. Moreover, IL-1Ra improved culture-induced β-cell dysfunction and restored impaired proIAPP processing, leading to lower amyloid formation. IL-1β treatment potentiated impaired proIAPP processing and increased amyloid formation in cultured human and h IAPP -expressing mouse islets, which were prevented by IL-1Ra., Conclusions: IL-1β plays a dual role by: (1) mediating amyloid-induced Fas upregulation and β-cell apoptosis; (2) inducing impaired proIAPP processing thereby potentiating amyloid formation. Blocking IL-1β may provide a new strategy to preserve β cells in conditions associated with islet amyloid formation., (© 2017 John Wiley & Sons Ltd.)

The role of reactive metabolites and inflammatory stress has been largely evoked in idiosyncratic hepatotoxicity of diclofenac (DCF); however mechanisms remain poorly understood. We aimed to evaluate the influence of liver cell phenotype on the hepatotoxicity of DCF combined or not with TNF-α using differentiated and undifferentiated HepaRG cells, and for comparison, HepG2 cells. Our results demonstrate that after a 24h-treatment metabolizing HepaRG cells were less sensitive to DCF than their undifferentiated non-metabolizing counterparts as shown by lower oxidative and endoplasmic reticulum stress responses and lower activation of caspase 9. Differentiated HepaRG cells were also less sensitive than HepG2 cells. Their lower sensitivity to DCF was related to their high content in glutathione transferases. DCF-induced apoptotic effects were potentiated by TNF-α only in death receptor-expressing differentiated HepaRG and HepG2 cells and were associated with marked activation of caspase 8. TNF-α co-treatment did not aggravate DCF-induced cholestatic features. Altogether, our results demonstrate that (i) lower sensitivity to DCF of differentiated HepaRG cells compared to their non-metabolically active counterparts was related to their high detoxifying capacity, giving support to the higher sensitivity of nonhepatic tissues than liver to this drug; (ii) TNF-α-potentiation of DCF cytotoxicity occurred only in death receptor-expressing cells., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background/aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury., Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry., Results: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3., Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Advanced glycation end-products (AGEs) are extremely accumulated in the retinal vascular and epithelial cells of diabetes mellitus (DM) patients, particularly with diabetic retinopathy (DR). To elucidate the pathogenesis of the AGE-induced toxicity to retinal epithelial cells, we investigated the role of Fas-Fas ligand (FasL) signaling and mitochondrial dysfunction in the AGE-induced apoptosis. Results demonstrated that the AGE-BSA- induced apoptosis of retinal ARPE-19 cells. And the AGE-BSA treatment caused mitochondrial dysfunction, via deregulating the B-cell lymphoma 2 (Bcl-2) signaling. Moreover, the Fas/FasL and its downstreamer Caspase 8 were promoted by the AGE-BSA treatment, and the exogenous α-Fas exacerbated the activation of Caspase 3/8. On the other side, the siRNA-mediated knockdown of Fas/FasL inhibited the AGE-BSA-induced apoptosis. Taken together, we confirmed the activation of Fas-FasL signaling and of mitochondrial dysfunction in the AGE-BSA-promoted apoptosis in retinal ARPE-19 cells, implying the important role of Fas-FasL signaling in the DR in DM.

Extrinsic apoptosis is initiated by recognition and clustering of the single-pass transmembrane proteins Fas ligand and Fas expressed at the surface of closely apposed lymphocytes and target cells, respectively. Since Fas-mediated death response was mainly studied with soluble antibodies, the mobility constraints for receptor activation by a membrane embedded agonist is not well understood. We explored this influence by stimulating apoptosis on functionalized supported lipid bilayers, where we quantified agonist mobility by z-scan fluorescence correlation spectroscopy. Using different lipid compositions, we show that the apoptotic response correlates with increased lateral mobility of the agonist in the lipid bilayer., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Rheumatoid Arthritis (RA) is a chronic inflammatory disease affecting synovial joints. Tumor necrosis factor (TNF) α is a key component of RA pathogenesis and blocking this cytokine is the most common strategy to treat the disease. Though TNFα blockers are very efficient, one third of the RA patients are unresponsive or present side effects. Therefore, the development of novel therapeutic approaches is required. RA pathogenesis is characterized by the hyperplasia of the synovium, closely associated to the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLS), which invade and destroy the joint structure. Hence, depletion of RA FLS has been proposed as an alternative therapeutic strategy. The TNF family member Fas ligand (FasL) was reported to trigger apoptosis in FLS of arthritic joints by binding to its receptor Fas and therefore suggested as a promising candidate for targeting the hyperplastic synovial tissue. However, this cytokine is pleiotropic and recent data from the literature indicate that Fas activation might have a disease-promoting role in RA by promoting cell proliferation. Therefore, a FasL-based therapy for RA requires careful evaluation before being applied. In this review we aim to overview what is known about the apoptotic and non-apoptotic effects of Fas/FasL system and discuss its relevance in RA., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Arctigenin (ARG) has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC) was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose) polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.

TLRs play an important role in mediating intestinal inflammation and homeostasis. Fas is best studied in terms of its function in apoptosis, but recent studies demonstrate that Fas signaling may mediate additional functions such as inflammation. The role of Fas, and the Fas ligand (FasL), in the intestine is poorly understood. The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal epithelial cells (IECs). IECs were stimulated with TLR ligands, and expression of Fas and FasL was investigated. Treatment with TLR4 and TLR5 ligands, but not TLR2 and 9 ligands, increased expression of Fas and FasL in IECs in vitro. Consistent with this finding, expression of intestinal Fas and FasL was reduced in vivo in the epithelium of TLR4 knockout (KO), 5KO, and germ-free mice, but not in TLR2KO mice. Modulating Fas signaling using agonistic anti-Fas augmented TLR4- and TLR5-mediated TNF-α and IL-8 production by IECs. In addition, suppression of Fas in IECs reduced the ability of TLR4 and TLR5 ligands and the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8. In conclusion, this study demonstrates that extensive cross-talk in IECs occurs between the Fas and TLR signaling pathways, with the FasL/Fas system playing a role in TLR-mediated inflammatory responses in the intestine., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: Argininosuccinate synthase (ASS) is an enzyme shared by the urea cycle and the l-citrulline/nitric oxide (NO·) cycle. ASS is the rate-limiting enzyme in the urea cycle and along with nitric oxide synthase 2 (NOS2), it endows cells with the l-citrulline/NO· salvage pathway to continuously supply l-arginine from l-citrulline for sustained NO· generation. Thus, ASS conditions NO· synthesis by NOS2. Because of the relevance of NOS2 activation for liver injury, we examined the contribution of ASS to NO· generation and how it impacts liver injury., Methods: Wild-type (WT) mice and Ass(+/-) mice (Ass(-/-) mice are lethal) were intraperitoneally injected with ethanol (EtOH) at a dose of 2.5 g/kg of body weight twice a day for 3 days. Two hours after the last dose of EtOH, mice were administered the agonistic Jo2 anti-mouse Fas monoclonal antibody (Ab) at a dose of 0.2 μg/g of body weight. Mice were sacrificed 8 hours after the Jo2 Ab injection. Markers of nitrosative and oxidative stress as well as liver damage were analyzed., Results: EtOH plus Jo2 injection induced liver injury as shown by serum alanine aminotransferase and aspartate aminotransferase activity, liver pathology, TUNEL, and cleaved caspase-3 were lower in Ass(+/-) mice compared with WT mice, suggesting that ASS contributes to EtOH plus Jo2-mediated liver injury. CYP2E1 induction, glutathione depletion, and elevated thiobarbituric acid reactive substances were comparable in both groups of mice, suggesting that CYP2E1-mediated oxidative stress is not linked to ASS-induced liver injury. In contrast, NOS2 induction, 3-nitrotyrosine adducts formation and elevated nitrites, nitrates, and S-nitrosothiols were higher in livers from WT mice than from Ass(+/-) mice., Conclusion: Decreased nitrosative stress causes lower EtOH plus Jo2-induced liver injury in Ass(+/-) mice., (Copyright © 2013 by the Research Society on Alcoholism.)

The influence of UV-light (240-390 nm) in doses 151-3020 J/m2 on the nature of the death of lymphocytes cells of donor's blood (using markers of apoptotic and necrotic death of cells) and on the level of expression of the marker of apoptotic pre-preparation--CD95-receptor has been investigated. We have shown that UV-radiation increases expression of CD95-receptors which is caused mainly by de novo synthesis of the receptors. It has been revealed that during daily incubation of photo-modified lymphocytes (151 and 755 J/m2) without autological blood cell death occurs by receptor-involved apoptosis. Exposure to high doses of radiation (1510 and 3020 J/m2) causes massive necrotic death of immunocytes. The use of autologous blood plasma during incubation of photo-modified lymphocytes allows decreasing the number of both apoptotic and necrotic cells.

The lungs are the most common site for the metastatic spread of osteosarcoma. Success in using chemotherapy to improve overall survival has reached a plateau. Understanding the biologic properties that permit osteosarcoma cells to grow in the lungs may allow the identification of novel therapeutic approaches-the goal being to alter the tumor cells' expression of cell surface proteins so that there is no longer compatibility with the metastatic niche. We have demonstrated that the Fas Ligand positive (FasL(+)) lung microenvironment eliminates Fas(+) osteosarcoma cells that metastasize to the lungs. Indeed, osteosarcoma lung metastases from patients are Fas(-), similar to what we found in several different mouse models. The Fas(+) cells are cleared from the lungs through apoptosis induced by the Fas signaling pathway following interaction of Fas on the tumor cell surface with the lung FasL. Blocking the Fas signaling pathway interferes with this process, allowing the Fas(+) cells to grow in the lungs. Our investigations show that Fas expression in osteosarcoma cells is regulated epigenetically by the micro-RNA miR-20a, encoded by the miR-17-92 cluster. Our studies support the feasibility of finding agents that can re-induce Fas expression as a novel therapeutic approach to treat osteosarcoma patients with lung metastases. We have identified two such agents, the histone deacetylase inhibitor entinostat and the chemotherapeutic agent gemcitabine (GCB). Aerosol GCB and oral entinostat induce the upregulation of Fas and the regression of established osteosarcoma lung metastases. Aerosol GCB was not effective in the FasL-deficient gld mouse confirming that the lung microenvironment was central to the success of this therapy. Our studies establish the critical role of the lung microenvironment in the metastatic process of osteosarcoma to the lungs and suggest an alternative focus for therapy, that is, incorporating the lung microenvironment as part of the treatment strategy against established osteosarcoma disease in the lungs.

Although numerous studies have shown the cancer-preventive properties of butylidenephthalide (BP), there is little report of BP affecting human prostate cancer cells. In the present study, proteomic-based approaches were used to elucidate the anticancer mechanism of BP in LNCaP human prostate cancer cells. BP treatment decreased the viability of LNCaP human prostate cancer cells in a concentration- and time-dependent manner, which was correlated with G0/G1 phase cell cycle arrest. Increased cell cycle arrest was associated with a decrease in the level of CCND1, CDK2, and PCNA proteins and an increase in the level of CDKN2A, CDKN1A, and SFN proteins. Proteomic studies revealed that among 48 differentially expressed proteins, 25 proteins were down-regulated and 23 proteins were up-regulated and these proteins fall into one large protein protein interaction network. Among these proteins, FAS, AIFM1, BIK, CYCS, SFN, PPP2R1A, CALR, HSPA5, DDIT3, and ERN1 are apoptosis and endoplasmic reticulum (ER) stress associated proteins. Proteomic data suggested that multiple signaling pathways including FAS-dependent pathway, mitochondrial pathway, and ER stress pathway are involved in the apoptosis induced by BP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Fas/CD95 is the best-studied member of the death receptor (DR) superfamily in the central nervous system where it can trigger cellular responses other than apoptosis, including the promotion of neurogenesis and neuritogenesis, stimulation of the progression of gliomas, and regulation of the immune response of astrocytes., Methods: We have investigated the role of Fas/CD95 in the regulation of the proliferation of fetal astrocytes in vitro, as well as the signalling pathways involved., Results: Fas/CD95 ligation stimulated the proliferation of primary fetal astrocytes, through a mechanism that depends on the activation of caspase 8 and subsequent phosphorylation of extracellular signal regulated kinase (ERK). Interestingly this proliferative effect is only observed with a low dose of the Fas/CD95 agonist. In contrast, when primary astrocytes are challenged with a high dose of the Fas/CD95 agonist significant cell death occurs., Conclusions: Our findings support that, besides its effects on cell survival, Fas/CD95 may play a complex and prominent role in the regulation of astrocyte proliferation during development., (Copyright © 2013 S. Karger AG, Basel.)

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.

Excessive apoptosis and prolonged inflammation of alveolar cells are associated with the pathogenesis of pulmonary emphysema. We aimed to determine whether CD40 affects alveolar epithelial cells and endothelial cells, with regard to evoking apoptosis and inflammation. Mice were repeatedly treated with agonistic-anti CD40 antibody (Ab), with or without agonistic-anti Fas Ab, and evaluated for apoptosis and inflammation in lungs. Human pulmonary microvascular endothelial cells and alveolar epithelial cells were treated with agonistic anti-CD40 Ab and/or anti-Fas Ab to see their direct effect on apoptosis and secretion of proinflammatory molecules in vitro. Furthermore, plasma soluble CD40 ligand (sCD40L) level was evaluated in patients with chronic obstructive pulmonary disease (COPD). In mice, inhaling agonistic anti-CD40 Ab induced moderate alveolar enlargement. CD40 stimulation, in combination with anti-Fas Ab, induced significant emphysematous changes and increased alveolar cell apoptosis. CD40 stimulation also enhanced IFN-γ-mediated emphysematous changes, not via apoptosis induction, but via inflammation with lymphocyte accumulation. In vitro, Fas-mediated apoptosis was enhanced by CD40 stimulation and IFN-γ in endothelial cells and by CD40 stimulation in epithelial cells. CD40 stimulation induced secretion of CCR5 ligands in endothelial cells, enhanced with IFN-γ. Plasma sCD40L levels were significantly increased in patients with COPD, inversely correlating to the percentage of forced expiratory volume in 1 s and positively correlating to low attenuation area score by CT scan, regardless of smoking history. Collectively CD40 plays a contributing role in the development of pulmonary emphysema by sensitizing Fas-mediated apoptosis in alveolar cells and increasing the secretion of proinflammatory chemokines.

Background & Aims: Apoptosis is crucially involved in acute and chronic liver injury, including viral, cholestatic, toxic, and metabolic liver disease. Additionally, dysregulation of apoptosis signaling pathways has been implicated in hepatocarcinogenesis. The most prominent members of the apoptosis-mediating tumor necrosis factor receptor superfamily are the TNF-R1 (CD120a) and the CD95 (Apo-1/Fas) receptor. Although extensively studied, the intracellular signaling events in hepatocytes are only incompletely understood., Methods: To examine the role of the caspase-8 homolog cellular FLICE-inhibitory protein (c-FLIP) in liver injury, we generated mice with hepatocyte specific deletion of c-FLIP. Three models of acute liver injury were employed: the agonistic anti-CD95 antibody Jo2, d-galactosamine and LPS (GalN/LPS), and concanavalin A., Results: Conditional ablation of c-FLIP in hepatocytes augmented liver injury and cell death in all three models of liver injury. CD95- and GalN/LPS-induced liver injury was ameliorated by a pancaspase inhibitor, while ConA-induced injury was unaffected by caspase inhibition. Augmented activation of the MAPK JNK was observed in parallel to liver injury in c-FLIP knockout mice in all injury models; however, inhibition of JNK only affected TNF- and ConA-mediated injury., Conclusions: In summary, c-FLIP is a central regulator of cell death in hepatocytes, involving increased activation of caspases and the MAPK JNK., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4(+) T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation., (© 2011 Macmillan Publishers Limited)

Death receptor targeting has emerged as one of the promising novel approaches of cancer therapy. The activation of one such prototypic death receptor, CD95 (Fas/APO-1), has remained controversial because CD95 agonistic molecules have exhibited either too strong toxicity or too little activity. The natural CD95 ligand (CD95L) is a cytokine, which needs to trimerize to mediate a cell death signal. Mega-Fas-Ligand, now referred to as APO010, is a synthetic hexameric CD95 agonist that exhibits strong antitumor activity in various tumor models. Here, we studied the effects of APO010 in human glioma models in vitro and in vivo. Compared with a cross-linked soluble CD95L or a CD95-agonistic antibody, APO010 exhibited superior activity in glioma cell lines expressing CD95 and triggered caspase-dependent cell death. APO010 reduced glioma cell viability in synergy when combined with temozolomide. The locoregional administration of APO010 induced glioma cell death in vivo and prolonged the survival of tumor-bearing mice. A further exploration of APO010 as a novel antiglioma agent is warranted.

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ(0) cells. βLox5 ρ(0) cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0) cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

The size of the peripheral T-lymphocyte compartment is governed by complex homeostatic mechanisms that balance T-cell proliferation and death. Proliferation and survival signals are mediated in part by recurrent self-peptide/MHC-TCR interactions and signaling by the common γ chain-containing cytokine receptors, including those for IL-7 and IL-15. We have previously shown that the death receptor Fas (CD95/APO-1) regulates apoptosis in response to repeated TCR stimulation, whereas the Bcl-2 homology domain 3-only protein Bim mediates cytokine withdrawal-induced apoptosis. We therefore reasoned that these two molecules might cooperate in the regulation of homeostatic proliferation. In this study, we observe that the combined loss of Fas and Bim synergistically enhances the accumulation of T cells in lymphopenic host mice, and this is particularly pronounced for the unusual CD4(-) CD8(-) TCRαβ(+) T cells that are characteristic of Fas-deficient (Fas(lpr/lpr) ) mice. Our findings demonstrate that these CD4(-) CD8(-) TCRαβ(+) T cells arise from homeostatic proliferation of CD8(+) T cells. These studies also underscore the profound rate of baseline T-cell proliferation that likely occurs in wild-type mice even in the absence of foreign antigen, and the consequent need for its coordinated regulation by multiple death-signaling pathways., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Keratins 8 and 18 (K8 and K18) are heteropolymeric intermediate filament phosphoglycoproteins of simple-type epithelia. Mutations in K8 and K18 predispose the affected individual to liver disease as they protect hepatocytes from apoptosis. K18 undergoes dynamic O-linked N-acetylglucosamine glycosylation at Ser 30, 31 and 49. We investigated the function of K18 glycosylation by generating mice that overexpress human K18 S30/31/49A substitution mutants that cannot be glycosylated (K18-Gly(-)), and compared the susceptibility of these mice to injury with wild-type and other keratin-mutant mice. K18-Gly(-) mice are more susceptible to liver and pancreatic injury and apoptosis induced by streptozotocin or to liver injury by combined N-acetyl-D-glucosaminidase inhibition and Fas administration. The enhanced apoptosis in the livers of mice that express K18-Gly(-) involves the inactivation of Akt1 and protein kinase Ctheta as a result of their site-specific hypophosphorylation. Akt1 binds to K8, which probably contributes to the reciprocal hyperglycosylation and hypophosphorylation of Akt1 that occurs on K18 hypoglycosylation, and leads to decreased Akt1 kinase activity. Therefore, K18 glycosylation provides a unique protective role in epithelial injury by promoting the phosphorylation and activation of cell-survival kinases.

Activation of T-cells triggers store-operated Ca(2+) entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca(2+) entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis., (Copyright 2010 Elsevier Inc. All rights reserved.)

In recent years, 4-hydroxy-2-nonenal (4-HNE) has emerged as an important second messenger in cell cycle signaling. Here, we demonstrate that 4-HNE induces signaling for apoptosis via both the Fas-mediated extrinsic and the p53-mediated intrinsic pathways in HepG2 cells. 4-HNE induces a Fas-mediated DISC independent apoptosis pathway by activating ASK1, JNK, and caspase-3. Parallel treatment of 4-HNE to HepG2 cells also induces apoptosis by the p53 pathway through activation of Bax, p21, JNK, and caspase-3. Exposure of HepG2 cells to 4-HNE leads to the activation of both Fas and Daxx, promotes the export of Daxx from the nucleus to cytoplasm, and facilitates Fas-Daxx binding. Depletion of Daxx by siRNA results in the potentiation of apoptosis, indicating that Fas-Daxx binding in fact is inhibitory to Fas-mediated apoptosis in cells. 4-HNE-induced translocation of Daxx is also accompanied by the activation and nuclear accumulation of HSF1 and up-regulation of heat shock protein Hsp70. All these effects of 4-HNE in cells can be attenuated by ectopic expression of hGSTA4-4, the isozyme of glutathione S-transferase with high activity for 4-HNE. Through immunoprecipitation and liquid chromatography-tandem mass spectrometry, we have demonstrated the covalent binding of 4-HNE to Daxx. We also demonstrate that 4-HNE modification induces phosphorylation of Daxx at Ser668 and Ser671 to facilitate its cytoplasmic export. These results indicate that while 4-HNE exhibits toxicity through several mechanisms, in parallel it evokes signaling for defense mechanisms to self-regulate its toxicity and can simultaneously affect multiple signaling pathways through its interactions with membrane receptors and transcription factors/repressors.

As opioid receptors modulate proliferation and apoptosis of immune cells, we hypothesized that they could reduce malignant haematopoietic cells. After screening, we selected the human multiple myeloma LP-1 cells which express mu- (MOP-) and kappa-opioid receptors (KOP-R). U50 488 produces a modest but significant decrease in viability associated with an arrest in the G0/G1 phase, but not antagonized by NorBNI and not associated with modulation of p21(Cip1), p27(Kip1) or p53 expression. In contrast, no effect was observed with dynorphin, U69 593 and morphine. In conclusion, the anti-proliferative effects of U50 488 are not mediated by KOP-R in the LP-1 cells., (Copyright 2010 Elsevier B.V. All rights reserved.)

C(2)-ceramide (C(2)-cer) and binding of the CD95/APO-1/FAS (aCD95) receptor are acknowledged inducers of apoptosis. In spite of that, their effects on the endoplasmic reticulum (ER) and mitochondria during early phases of apoptotic onset are poorly characterized. Here, by employing various approaches, we followed structural and functional modifications of these organelles at the beginning of cellular demise. In detail, we observed that C(2)-cer, but not CD95 activation, markedly modifies the morphology of the ER and promotes Ca(2+) release. Accordingly, mitochondria of C(2)-cer-treated, but not of CD95-stimulated, cells are fragmented, show reduced Ca(2+) uptake, and collapsed membrane potential (DeltaPsi(m)). Most notably, C(2)-cer-mediated morphological aberrations of the ER are prevented neither by the pan-caspase inhibitor Z-VADfmk nor by the cell cytoskeleton dissembler cytochalasin-D, while on the contrary they are reduced by incubation in the presence of the intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). We conclude that initiation of apoptosis via the intrinsic (i.e. C(2)-cer mediated) pathway causes an early structural and functional alteration of both ER and mitochondria, thus underlying a final "non return" point in the apoptotic pathway., (Copyright 2009 Elsevier Inc. All rights reserved.)

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

The serine-threonine kinase RIP1 was originally identified through its ability to bind to the death domain of Fas (CD95). RIP1 has been shown to be recruited to the Fas death-inducing signaling complex (DISC) and is required for the induction of necrotic cell death. In this study, we show that in Jurkat T lymphocytes, RIP1 is also necessary for the most efficient activation of downstream caspases by Fas when treated with membrane-bound Fas ligand, but not with agonistic Abs or cross-linked soluble Fas ligand. RIP1 participates in the Fas-associated death domain protein-mediated recruitment of caspase-8 to the Fas receptor complex in a manner that promotes caspase-8 activation. Cross-linking Abs, such as CH11, bypass the requirement for RIP1 in caspase activation by initiating larger, though less efficient, DISC complexes, while membrane-bound Fas ligand initiates a smaller but more efficient DISC that functions, in part, by effectively incorporating more RIP1 into the complex. Consequently, RIP1 is likely a more integral part of physiological signaling through the Fas/CD95 receptor complex than previously recognized; at least when the signal is mediated by full-length membrane-bound FasL. Cross-linked soluble FasL, which also occurs physiologically, behaves similarly to the CH11 Ab, and may therefore be more likely to initiate nonapoptotic Fas signaling due to less RIP1 in the receptor complex. Thus, agonists that bind the same Fas receptor initiate mechanistically distinct pathways resulting in differential cytotoxicity.

We have previously shown that treatment of mice with pyrazole or acute ethanol potentiated Fas agonistic Jo2 antibody-induced liver injury by a mechanism involving induction of CYP2E1 and elevated oxidative stress. The current study evaluated whether chronic alcohol feeding potentiates Fas-induced liver injury and whether CYP2E1 plays a role in any enhanced hepatotoxicity. Wild-type and CYP2E1 knockout mice were fed ethanol or isocaloric dextrose for 4 weeks followed by a single treatment with either saline or Jo2. Mice were killed 8 h after the Jo2 challenge. There were three- to five fold increases in transaminases and more extensive eosinophilic necrosis, hemorrhage, and infiltration of inflammatory cells in the central zone of the hepatic lobule in the ethanol-fed mice treated with Jo2 compared to the dextrose/Jo2- or ethanol/saline-treated mice. Liver injury was blunted in ethanol-fed CYP2E1 knockout mice treated with Jo2. The chronic ethanol feeding produced steatosis, elevation of CYP2E1, and oxidative stress in wild-type but not CYP2E1 knockout mice. These changes in wild-type mice fed ethanol were similar after saline or Jo2 treatment. The Jo2 treatment produced activation of JNK and P38 MAP kinase, increased activity of caspase-8 and -3, and lowered hepatic GSH levels in both the dextrose- and the alcohol-fed mice. JNK was activated at early times after Jo2 treatment in the ethanol-fed mice. Serum TNF-alpha levels were strikingly elevated in the wild-type ethanol/Jo2 group, which showed liver injury, compared to all the other groups, which did not show liver injury. Inhibition of JNK or P38 MAPK partially, but not completely, prevented the elevated liver injury in the wild-type ethanol/Jo2 mice. These results show that chronic ethanol feeding enhances Fas-induced liver injury by a mechanism associated with induction of CYP2E1, elevated serum TNF-alpha levels, and activation of MAPK.

Within the lesions of inflammatory myopathies, muscle fibres and invading mononuclear cells express Fas and Fas ligand (FasL), respectively. However, the roles of the Fas/FasL interaction in the pathogenesis of inflammatory myopathies are not fully understood. In the present study, we investigated the roles of proinflammatory cytokines and the Fas/FasL system in the pathogenesis of inflammatory myopathies. In vitro culturing of muscle cells with the proinflammatory cytokines interferon-gamma, tumour necrosis factor-alpha, and interleukin (IL)-1beta synergistically increased Fas expression, susceptibility to Fas-mediated apoptosis, and the expression of cytoplasmic caspases 8 and 3. In addition, culturing of muscle cells with activated CD4(+) T cells induced muscle cell apoptosis, which was partially inhibited by anti-FasL antibody. We also tested the possibility that T helper (Th) 17, which is an IL-17-producing helper T-cell subset that plays crucial roles in autoimmune and inflammatory responses, participates in the pathogenesis of inflammatory myopathies. Interestingly, in vitro culturing of dendritic cells with anti-Fas immunoglobulin M (IgM) or activated CD4(+) T cells induced the expression of mRNA for IL-23p19, but not for IL-12p35, in addition to proinflammatory cytokines. Furthermore, IL-23p19 and IL-17 mRNAs were detected in the majority of biopsy samples from patients with inflammatory myopathies. Taken together, these results suggest that proinflammatory cytokines enhance Fas-mediated apoptosis of muscle cells, and that the Fas/FasL interaction between invading dendritic cells and CD4(+) T cells induces local production of IL-23 and proinflammatory cytokines, which can promote the proliferation of Th17 cells and enhance Fas-mediated apoptosis of muscle cells, respectively.

Glioblastoma is the most common primary brain tumor, characterized by its resistance to treatments. To define efficient therapy, the origin of tumor-forming cells needs to be elucidated in order to search for new therapeutic pathways. The objective of this study was to determine the different cell populations constituting a human glioblastoma cell line, U-87 MG and their sensitivity to apoptosis induced through the activation of Fas, a membranous death receptor. By a cell sorting method, the sedimentation field flow fractionation, two major cell subpopulations were identified, a most differentiated cell fraction, containing large and adherent cells, sensitive to Fas-induced apoptosis and another one, characterized by small cells forming aggregates, expressing CD133, a marker of stem cells and more resistant to Fas-activated apoptosis. By using a selective method of culture, adapted for neural stem cell cultures, we have verified that the U-87 MG cell line contained cancer stem cells similar to the immature ones obtained by the cell sorting method. Interestingly, while these tumor stem cells, expressing CD133, were resistant to Fas-induced apoptosis, monomeric form of Fas protein was detected predominantly in these cells. In contrast, the most mature cells, responsive to Fas-activated apoptosis, collected in another cell fraction, contained oligomeric aggregates of Fas protein, a pre-signalling form of the Fas receptor, essential for the initiation of apoptosis through its activation. These results suggest that these immature stem cells in glioma could be an important factor of resistance to chemotherapy requiring apoptosis through Fas signalling system. Indeed, future strategies of treatment, inducing differentiation of these stem cells need to be considered to enhance therapeutic efficiency.

Caspase-8 is a key initiator of death receptor-induced apoptosis. Here we report a novel short isoform of caspase-8 (caspase-8s), which encodes the first (Death Effector Domain) DED and part of the second DED, missing the C-terminal caspase domain. In vivo binding assays showed that transfected caspase-8s bound to (Fas-associated death domain protein) FADD, the adaptor protein in (death-induced signal complex) DISC. To investigate the potential effects of caspase-8s on cell apoptosis, Jurkat cells were stably transfected with caspase-8s. Overexpression of caspase-8s increased sensitivity to the apoptotic stimuli, Fas-agonistic antibody CH11. These results suggest that caspase-8s may act as a promoter of apoptosis through binding to FADD and is involved in the regulation of apoptosis. In addition, the results also indicate that the first DED was an important structure mediating combination between caspase-8 and FADD.

Fas has been reported to play an important role in apoptosis; however, Fas-mediated autophagy and its mechanism are still unclear. Here, we found that Fas agonistic antibody CH11-induced autophagy in HeLa cells, and inhibition of autophagy by 3-MA increased CH11-induced apoptosis. A Fas antagonistic antibody (UB2) suppressed both CH11-induced autophagy and apoptosis. In addition, the CH11-induced autophagy was blocked by JNK inhibitor (SP600125), but it was not affected by caspase 8 inhibitor (Z-IETD); whereas the CH11-induced apoptosis was increased by SP600125, and it was suppressed by Z-IETD. Further experiments confirmed that JNK was activated by CH11 dose-dependently, and the activation was suppressed when autophagy was blocked by 3-MA. Together, our results suggest that JNK, but not caspase 8, involves in Fas-mediated CH11-induced autophagy in HeLa cells, and this autophagy plays a protective role in CH11-induced cell death.

Stimulation of CD95 (APO-1/Fas) by its natural ligand CD95L (APO-1L/FasL) leads to the formation of the death-inducing signaling complex. Here we report that upon CD95 stimulation in several T and B cell lines, a novel signaling complex is formed, which we term complex II. Complex II is composed of the death effector domain proteins as follows: procaspase-8a/b, three isoforms of c-FLIP (c-FLIP(L), c-FLIP(S), c-FLIP(R)), and FADD. Notably, complex II does not contain CD95. Based on our findings we suggest that CD95 signaling includes two steps. The first step involves formation of the death-inducing signaling complex at the cell membrane. The second step involves formation of the cytosolic death effector domain protein-containing complex that may play an important role in amplification of caspase activation.

The current study was designed to investigate the effect and potential mechanism of exogenous administration of S-adenosyl-l-methionine (SAM) on the enhanced hepatotoxicity induced by the Fas agonistic Jo2 antibody plus acute ethanol pretreatment in C57BL/6 mice. Acute ethanol plus Jo2 treatment produces liver toxicity under conditions in which ethanol alone or Jo2 alone do not. SAM significantly attenuated this elevated hepatotoxicity in mice as manifested by a decrease of serum aminotransferases and morphological amelioration. Levels of SAM and activity of methionine adenosyltransferase were lowered by the ethanol plus Jo2 treatment but restored by administration of SAM. The ethanol plus Jo2 treatment increased activity and content of CYP2E1, iNOS content and TNF-alpha levels; these increases were blunted by SAM. SAM also protected against the elevated oxidative and nitrosative stress found after ethanol plus Jo2, likely due to the decreases in CYP2E1, iNOS and TNF-alpha. Calcium-induced swelling of mitochondria was enhanced by the ethanol plus Jo2 treatment and this was prevented by SAM. JNK and P38 MAPK were activated by the ethanol plus Jo2 treatment; JNK activation was partially prevented by SAM. It is suggested that SAM protects against the ethanol plus Jo2 toxicity by restoring hepatic SAM levels, preventing the increase in iNOS, CYP2E1 and TNF-alpha and there by lowering the elevated oxidative/nitrosative stress and activation of the JNK signal pathway, ultimately preventing mitochondrial damage.

Background and Objectives: Previous studies have presented evidence that human immunoglobulin G preparations for intravenous use contain antibodies directed against the death receptor Fas (CD95). The function of these antibodies was described as either antagonistic or agonistic; therefore, inhibiting or stimulating Fas-dependent apoptosis. Based on these reports, we asked whether the proportion of antagonistic and agonistic anti-Fas activities differs between different lots of intravenous immunoglobulin (IVIG). Variations between lots would open the possibility to preselect suitable lots of IVIG for different therapeutic purposes., Materials and Methods: Eleven lots of IVIG were tested for their ability to induce or inhibit Fas-dependent apoptosis. The biological significance of anti-Fas antibodies was confirmed by including anti-Fas antibodies purified from IVIG and IVIG depleted of anti-Fas antibodies in the study., Results: All 11 lots inhibited FasL-induced apoptosis. In addition, five lots stimulated apoptosis in the absence of FasL. Depletion of anti-Fas antibodies from IVIG abolished the capacity of IVIG to inhibit FasL-induced apoptosis, but reduced the ability to induce apoptosis only slightly., Conclusion: The inhibition of FasL-induced apoptosis by IVIG is because of the presence of antagonistic anti-Fas antibodies. The activity of these antibodies differs considerably between different lots. On the other hand, the induction of apoptosis by IVIG is probably because of the concerted action of a range of different antibodies. The variation in the proportion of stimulating and inhibiting anti-Fas activities between different lots of IVIG opens the possibility to preselect suitable lots for different therapeutic purposes.

In species with hemochorial placentation, such as the mouse and human, trophoblast cells of the implanting blastocyst induce apoptosis and displace endometrial epithelial cells (EEC) to cross the luminal epithelium of the endometrium. Since Fas and Fas ligand (FasL) are expressed in EEC and trophoblast cells respectively and mitogen-activated protein kinases (MAPKs) mediate Fas-induced apoptosis, the roles of Fas/FasL and MAPK signaling in trophoblast-EEC interactions were studied. By co-culturing BeWo trophoblast spheroids with RL95-2 EEC monolayers to mimic blastocyst-endometrial interactions, we found that trophoblast spheroid outgrowth on EEC was significantly enhanced by anti-Fas activating antibody. Since anti-Fas activating antibody had no effect on spheroid expansion on EEC-free culture surfaces, its enhancing effect on spheroid outgrowth on EEC may be mediated by acting on EEC to facilitate trophoblast-induced EEC apoptosis and displacement. Valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (VAD-FMK) staining showed that the percentage of apoptotic EEC at the spheroid-EEC interface was markedly increased by anti-Fas activating antibody. Moreover, the pancaspase inhibitor benzyloxycarbonyl-VAD-FMK was able to suppress the enhancing effect of anti-Fas activating antibody on spheroid expansion on EEC. Upon anti-Fas activating antibody stimulation, both p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) were activated. Furthermore, the anti-Fas activating antibody-enhanced EEC apoptosis and spheroid expansion on EEC were significantly inhibited by the p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125. Our results establish that anti-Fas activating antibody could activate p38 MAPK and JNK to induce EEC apoptosis, thereby promoting trophoblast outgrowth on EEC.

Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide- and protein kinase Czeta-dependent pathway, which leads to an activating phosphorylation of p47(phox). The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl(-)]. Bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47(phox) phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl(-)], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction.

Objective: Very limited information regarding the cardiac molecular mechanism in obesity is available. The purpose of this study was to evaluate the cardiac Fas receptor-dependent (type I) apoptotic pathway in obese Zucker rats., Research Methods and Procedures: Sixteen obese Zucker rats were studied at 5 to 6 months of age, and 16 age-matched lean Zucker rats served as controls. Heart weight index, myocardial architecture, key components of the Fas receptor-dependent apoptotic pathway, apoptotic activity, and fibrosis in the excised left ventricle of rats were measured by weight scales, hematoxylin and eosin staining, Western blotting, TUNEL assay, and Masson trichrome staining., Results: Body weight, whole heart weight, left ventricular weight, ratio of whole heart weight to tibia length, percentage of TUNEL-positive cardiac myocytes, and percentage of cardiac fibrosis were significantly increased in the obese group. Cardiomyocyte disarray and increased cardiac interstitial space were observed in obese rats. Protein levels of Fas ligand, Fas death receptors, and Fas-associated Death Domain were all significantly increased in the obese group. In addition, pro-caspase-8 and pro-caspase-3 were significantly decreased, whereas activated caspase-8 and activated caspase-3 were significantly increased in the obese group, which implies that pro-forms of caspase-8 and caspase-3 were cleaved into active-forms caspase-8 and caspase-3., Conclusions: Cardiac Fas receptor-dependent apoptotic pathways were more activated in obese rats' hearts, which may provide one of the possible apoptotic mechanisms for developing cardiac abnormality in obesity.

Recently we generated a mathematical model (Bentele, M., Lavrik, I., Ulrich, M., Stosser, S., Heermann, D. W., Kalthoff, H., Krammer, P. H., and Eils, R. (2004) J. Cell Biol. 166, 839-851) of signaling in CD95(Fas/APO-1)-mediated apoptosis. Mathematical modeling in combination with experimental data provided new insights into CD95-mediated apoptosis and allowed us to establish a threshold mechanism of life and death. Here, we further assessed the predictability of the model experimentally by a detailed analysis of the threshold behavior of CD95 signaling. Using the model predictions for the mechanism of the threshold behavior we found that the CD95 DISC (death-inducing signaling complex) is formed at the cell membrane upon stimulation with low concentrations of agonistic anti-APO-1 monoclonal antibodies; however, activation of procaspase-8 at the DISC is blocked due to high cellular FLICE-inhibitory protein recruitment into the DISC. Given that death signaling does not occur upon CD95 stimulation at low (threshold) anti-APO-1 concentrations, we also analyzed survival signaling, focusing on mitogen-activated protein kinase activation. Interestingly, we found that mitogen-activated protein kinase activation takes place under threshold conditions. These findings show that triggering of CD95 can signal both life or death, depending on the strength of the stimulus.

Fas triggering by agonistic antibodies or by its cognate ligand, FasL, induces apoptotic cell death, whereas mutation in the Fas death domain is associated with lymphoma progression. On prolonged culture in the presence of an agonistic anti-Fas antibody, we raised a Jurkat cell line resistant to agonistic antibodies but still sensitive to soluble FasL, which carried at the heterozygous state, a point mutation into the Fas death domain. Down-modulation of c-FLIP expression reversed the blockade of the Fas pathway. We show that the activation threshold for the Fas receptor is more easily overcome by multimeric FasL than by agonistic antibodies and that the increase of this threshold due to mutation in the Fas death domain can be overcome by acting on a downstream effector of the Fas signal, c-FLIP. These findings put forward a new approach to eradicate Fas-resistant tumor cells.

Targeting the mitotic motor kinesin kinesin spindle protein (KSP) is a new strategy for cancer therapy. We have examined the molecular events induced by KSP inhibition and explored possible mechanisms of resistance and sensitization of tumor cells to KSP inhibitors. We found that KSP inhibition induced cell death primarily via activation of the mitochondrial death pathway. In HeLa cells, inhibition of KSP by small-molecule inhibitor monastrol resulted in mitotic arrest and rapid caspase activation. BclXL phosphorylation and loss of mitochondrial membrane potential was detected before significant caspase activation, which was required to trigger the subsequent apoptotic pathway. In A549 cells, however, KSP inhibition did not induce mitochondrial damage, significant caspase activity, or cell death. A549 cells aberrantly exited mitosis, following a prolonged drug-induced arrest, and arrested in a G(1)-like state with 4N DNA content in a p53-dependent manner. Overexpression of BclXL provided a protective mechanism, and its depletion rescued the apoptotic response to monastrol. In addition, Fas receptor was up-regulated in A549 cells in response to monastrol. Treatment with Fas receptor agonists sensitized the cells to monastrol-induced cell death, following exit from mitosis. Thus, activation of the death receptor pathway offered another mechanism to enhance KSP inhibitor-induced apoptosis. This study has elucidated cellular responses induced by KSP inhibitors, and the results provide insights for a more effective cancer treatment with these agents.