Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 is associated with enhanced synthesis of bcl-2 and mediated by inhibition of caspase-3 activation

We have previously shown that generation of sublytic C5b-9, the membrane attack complex of complement, induces oligodendrocytes to enter cell cycle and reduces apoptotic cell death in vitro. In the present study, the cellular factors involved in apoptosis of oligodendrocyte progenitor cells and oligodendrocytes, and the inhibitory effect of C5b-9 on apoptotic process were investigated. Oligodendrocyte progenitor cells identified by mAb A2B5 that were isolated from neonatal rat brains were differentiated into oligodendrocytes in serum-free defined medium. The differentiation, which occurs simultaneously with apoptotic cell death, was associated with a rapid loss of bcl-2 mRNA and increased expression of caspase-3 mRNA. Activation of caspase-3 in differentiating cells was demonstrated by the generation of 17- and 12-kDa fragments of caspase-3 proenzyme and by cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 substrate. Cell death associated with differentiation was inhibited by the caspase-3 inhibitor DEVD-CHO in a dose-dependent manner. Assembly of sublytic C5b-9 resulted in inhibition of caspase-3 activation. In addition, synthesis of BCL-2 protein in oligodendrocytes was significantly increased by C5b-9. The TNF-alpha-induced apoptosis of oligodendrocytes was also inhibited by C5b-9. These results indicate that up-regulation of BCL-2 protein and inhibition of caspase-3 activation are potential mechanisms by which C5b-9 increases survival of oligodendrocyte in vitro and possibly in vivo during inflammation and immune-mediated demyelination affecting the CNS.     

Proliferative versus apoptotic functions of caspase-8 Hetero or homo: the caspase-8 dimer controls cell fate

Caspase-8, the initiator of extrinsically-triggered apoptosis, also has important functions in cellular activation and differentiation downstream of a variety of cell surface receptors. It has become increasingly clear that the heterodimer of caspase-8 with the long isoform of cellular FLIP (FLIP(L)) fulfills these pro-survival functions of caspase-8. FLIP(L), a catalytically defective caspase-8 paralog, can interact with caspase-8 to activate its catalytic function. The caspase-8/FLIP(L) heterodimer has a restricted substrate repertoire and does not induce apoptosis. In essence, caspase-8 heterodimerized with FLIP(L) prevents the receptor interacting kinases RIPK1 and -3 from executing the form of cell death known as necroptosis. This review discusses the latest insights in caspase-8 homo- versus heterodimerization and the implication this has for cellular death or survival. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

C5b-9 terminal complement complex protects oligodendrocytes from death by regulating Bad through phosphatidylinositol 3-kinase/Akt pathway

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a G(i)alpha-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.     

Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells

The caspase-8 homologue FLICE-inhibitory protein (FLIP) functions as a caspase-8 dominant negative, blocking apoptosis induced by the oligomerization of the adapter protein FADD/MORT-1. FLIP expression correlates with resistance to apoptosis induced by various members of the tumor necrosis factor family such as TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are largely unknown. In this report, we examined the roles of the MAP kinase and phosphatidylinositol (PI) 3-kinase signaling pathways in the regulation of FLIP expression in tumor cells. We observed that the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor cell lines tested. In contrast, disruption of the PI 3-kinase pathway with the specific inhibitor LY294002 reduced Akt (protein kinase B) phosphorylation and the levels of FLIP protein and mRNA in all cell lines evaluated. The introduction of a dominant negative Akt adenoviral construct also consistently reduced FLIP expression as well as the phosphorylation of the Akt target glycogen synthase kinase-3. In addition, infection of the same cell lines with a constitutively active Akt adenovirus increased FLIP expression and the phosphorylation of GSK-3. These data add FLIP to the growing list of apoptosis inhibitors in which expression or function is regulated by the PI 3-kinase-Akt pathway.     

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.     

S-nitrosylation of FLICE inhibitory protein determines its interaction with RIP1 and activation of NF-κB

Death receptor (DR) ligation can lead to divergent signaling pathways causing either caspase-mediated cell death or cell proliferation and inflammation. These variations in cellular fate are determined by adaptor proteins that are recruited to the DR signaling complex. FLICE inhibitory protein (FLIP) is an established inhibitor of caspase-8-mediated apoptosis, and it is also involved in NF-κB activation. However, the molecular mechanism that regulates FLIP within this complex is unknown. In this study, we provide new evidence for the regulation of NF-κB by FLIP through S-nitrosylation, which involves covalent modification of the protein’s cysteine thiol by nitric oxide to form S-nitrosothiol. Point mutations of FLIP at cysteine residues 254 and 259 prevent FLIP S-nitrosylation and its ability to activate NF-κB. The mechanism by which FLIP nitrosylation regulates NF-κB activity involves RIP1 binding and redistribution, whereas TRAF2 binding and distribution are unaffected. We further show that FLIP processing and cleavage is dependent on its nitrosylation status. Collectively, our study reveals a novel pathway for FLIP regulation of NF-κB through protein S-nitrosylation, which is a key posttranslational mechanism controlling DR-mediated cell death and survival. Since increased expression of FLIP and nitric oxide are frequently observed in chemotherapy-resistant tumors, S-nitrosylation of FLIP could be a key mechanism of chemoresistance and tumor growth.     

FLICE-like inhibitory protein blocks transforming growth factor beta 1-induced caspase activation and apoptosis in prostate epithelial cells

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.     

Sensitization of osteosarcoma cells to death receptor-mediated apoptosis by HDAC inhibitors through downregulation of cellular FLIP

Fas-mediated apoptosis plays an important role in elimination of tumor cells in vivo, but some tumor-derived cells are resistant to this mechanism. Here, we show that treatment with the histone deacetylase (HDAC) inhibitor FR901228 renders Fas-resistant osteosarcoma cell lines sensitive to Fas-mediated apoptosis by downregulating expression of cellular FLIP (cellular FLICE-inhibitory protein), an inhibitor of Fas-mediated activation of caspase-8. Moreover, sensitization to Fas-mediated apoptosis was also induced in Fas-resistant osteosarcoma cells by suppressing FLIP expression using FLIP-specific RNA interference. HDAC inhibitors including FR901228 were shown to induce downregulation of cellular FLIP through inhibiting generation of FLIP mRNA, rather than stimulating degradation at either protein or mRNA level, and the inhibition was independent of de novo protein synthesis. These results clearly indicate that some tumor cells exhibit a phenotype resistant to death receptor-mediated apoptosis by expressing cellular FLIP, and that HDAC inhibitors sensitize such resistant tumor cells by directly downregulating cellular FLIP mRNA.     

Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection

Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an anti-apoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.     

Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin-proteasome-mediated degradation of FLICE inhibitory protein

Stimulation of cell surface Fas (CD95) results in recruitment of cytoplasmic proteins and activation of caspase-8, which in turn activates downstream effector caspases leading to programmed cell death. Nitric oxide (NO) plays a key role in the regulation of apoptosis, but its role in Fas-induced cell death and the underlying mechanism are largely unknown. Here we show that stimulation of the Fas receptor by its ligand (FasL) results in rapid generation of NO and concomitant decrease in cellular FLICE inhibitory protein (FLIP) expression without significant effect on Fas and Fas-associated death domain (FADD) adapter protein levels. FLIP down-regulation as well as caspase-8 activation and apoptosis induced by FasL were all inhibited by the NO-liberating agent sodium nitroprusside and dipropylenetriamine NONOate, whereas the NO synthase inhibitor aminoguanidine and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had opposite effects, indicating an anti-apoptotic role of NO in the Fas signaling process. FasL-induced down-regulation of FLIP is mediated by a ubiquitin-proteasome pathway that is negatively regulated by NO. S-nitrosylation of FLIP is an important mechanism rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL. Deletion analysis shows that the caspase-like domain of FLIP is a key target for S-nitrosylation by NO, and mutations of its cysteine 254 and cysteine 259 residues completely inhibit S-nitrosylation, leading to increased ubiquitination and proteasomal degradation of FLIP. These findings indicate a novel pathway for NO regulation of FLIP that provides a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.     

Proliferative versus apoptotic functions of caspase-8 : Hetero or homo: The caspase-8 dimer controls cell fate

Caspase-8, the initiator of extrinsically-triggered apoptosis, also has important functions in cellular activation and differentiation downstream of a variety of cell surface receptors. It has become increasingly clear that the heterodimer of caspase-8 with the long isoform of cellular FLIP (FLIP_L) fulfills these pro-survival functions of caspase-8. FLIP_L, a catalytically defective caspase-8 paralog, can interact with caspase-8 to activate its catalytic function. The caspase-8/FLIP_L heterodimer has a restricted substrate repertoire and does not induce apoptosis. In essence, caspase-8 heterodimerized with FLIP_L prevents the receptor interacting kinases RIPK1 and -3 from executing the form of cell death known as necroptosis. This review discusses the latest insights in caspase-8 homo- versus heterodimerization and the implication this has for cellular death or survival. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

The c-FLIPL Cleavage Product p43FLIP Promotes Activation of Extracellular Signal-regulated Kinase (ERK), Nuclear Factor κB (NF-κB), and Caspase-8 and T Cell Survival

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIP_L can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIP_L. This triggers cleavage of c-FLIP_L at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.     

Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal α-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.     

Mitomycin C induces apoptosis and caspase-8 and -9 processing through a caspase-3 and Fas-independent pathway

Caspase-3 activity has been described to be essential for drug-induced apoptosis. Recent results suggest that in addition to its downstream executor function, caspase-3 is also involved in the processing of upstream caspase-8 and -9. To test the absolute requirement for caspase-3, we examined mitomycin C (MMC)-induced apoptosis in the caspase-3 deficient human breast cancer cell line MCF-7. MMC was used as anticancer drug since this agent was preferentially active compared to chemotherapeutic compounds with differing mechanisms of action such as cisplatin, docetaxel, or lovastatin. MMC treatment led to pronounced caspase-8, -9, and -7 processing and early morphological features of apoptosis within 48 h. This could be inhibited by the broad-spectrum caspase inhibitor z-VAD.fmk and to a lesser extent by z-IETD.fmk and z-LEHD.fmk, which have a certain preference for inhibiting caspase-8 and -9, respectively. MMC induced apoptosis in MCF-7 cells was not mediated by the death receptor pathway as demonstrated by experiments using the inhibiting anti-Fas antibody ZB4 and transfections with CrmA, a viral serpin inhibitor of caspase-8, and the dominant negative Fas-associated death domain (FADD-DN). Stable expression with Bcl-2 significantly prevented the processing of caspase-9 but also of caspase-8 and blocked the induction of apoptosis. Thus, we provide evidence that caspase-3 activity is dispensable for MMC-induced apoptosis and for caspase-8 and -9 processing in MCF-7 cells.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate,FLICE-like inhibitory protein (FLIP)

We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSSLLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p43) and a C-terminal one (p12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD(376)GPAMKNVEF, identified on N-terminal sequencing of the p12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblotting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-gamma-treated U937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U937 cells treated with vitamin D(3) or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U937 differentiation occur upstream of caspase-8 processing/activation.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.