CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis

We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.     

Resistance to Fas-mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP

The death receptor Fas transduces apoptotic death signaling mediated by caspases. In the present study, human hepatoma HepG2 cells showed the Fas-mediated apoptosis mediated by caspase, especially caspase 3, only in the presence of actinomycin D. Interestingly, cytosolic proteins extracted from intact HepG2 cells induced caspase 3 inactivation. Our results reveal that this inactivation was triggered by the direct inhibition of activated caspase 3 by IAP gene family ILP. In addition, a 53 kDa protein was co-immunoprecipitated with anti-human caspase 3 antibody from intact HepG2 cells. This protein was a complex-protein of procaspase 3 and the cell cycle regulator p21WAF1 (p21). P21 bound to only procaspase 3, but not to activated caspase 3. We also demonstrate that p21 protein-loaded HepG2 cells resist to Fas-mediated apoptosis even in the presence of actinomycin D. Here we report that caspase 3 inactivation for the resistance to Fas-mediated apoptosis is induced by a procaspase 3/p21 complex formation and direct inhibition of activated caspase 3 by ILP.     

Structure-function analysis of an evolutionary conserved protein, DAP3, which mediates TNF-alpha- and Fas-induced cell death

A novel approach to the isolation of positive mediators of programmed cell death, based on random inactivation of genes by expression of anti sense RNAs, was employed to identify mediators of interferon-gamma-induced apoptosis. One of the several genes identified is DAP3, which codes for a 46 kDa protein with a potential nucleotide-binding motif. Structure-function studies of the protein indicate that the intact full-length protein is required for its ability to induce apoptosis when overexpressed. The N-terminal 230 amino acids, on the other hand, act in a dominant-negative fashion. Both of these functions are dependent on the integrity of the nucleotide binding motif. Expression of anti-sense DAP3 RNA and of the dominant interfering form of DAP3 both protected cells from apoptosis induced by activation of Fas and tumor necrosis factor alpha (TNF-alpha) receptors. Thus, DAP3 is implicated as a positive mediator of these death-inducing stimuli. It functions downstream of the receptor signaling complex and its death promoting effects depend on caspase activity. In the nematode Caenorhabditis elegans, a potential homolog of DAP3 showing 35% identity and 64% similarity to the human protein was isolated. Overexpression of the nematode DAP3 cDNA in mammalian cells induced cell death, indicating that the protein is conserved at the functional level as well as the structural level.     

Inhibition of caspase activity prevents anti-IgM induced apoptosis but not ceramide generation in WEHI 231 B cells

In apoptosis induced by Reaper in Drosophila, as well as in a number of other systems, it has been suggested that the increased synthesis of ceramide might be a consequence of the activation of the caspase/ICE (Interleukin-1beta converting enzyme) protease pathway involved in cell death, implying that ceramide generation might often be the result rather than the cause of apoptosis. WEHI 231 B cells have previously been shown to undergo apoptosis following exposure to exogenous ceramide and to produce increased amounts of ceramide in response to anti-IgM crosslinking. We show here that in WEHI 231 cells a peptide inhibitor of caspase activity blocks cell death in response to both anti-IgM and exogenous ceramide. However, the induction of ceramide synthesis by WEHI 231 cells in response to anti-IgM crosslinking is not blocked by this peptide. These results indicate that antigen receptor induced ceramide generation in WEHI 231 cells does not require caspase activation, and support the view that ceramide generation in immature B cells may be the cause rather than the consequence of activation of the caspase dependent death pathway.     

Stress signals for apoptosis: ceramide and c-Jun kinase

Mammalian systems respond to environmental stress by either adapting or undergoing programmed cell death. While there is general agreement that the caspase family of proteases serve as the effectors of the apoptotic death response, the signaling apparatus involved in the decision to activate the caspase system is less clear. In the past few years, the sphingomyelin and c-Jun Kinase (JNK)/Stress-activated Protein Kinase (SAPK) pathways have been linked to the death response in many cellular systems. These signaling systems are found throughout the animal kingdom, and ceramide signaling is conserved through yeast. Since yeast do not undergo apoptosis, the sphingomyelin pathway appears evolutionarily older than the caspase-mediated death programs. While recent reviews by several groups have broadly surveyed ceramide signaling in apoptosis, this paper examines the role of sphingomyelinases and the JNK/SAPK pathway in coordinate signaling of apoptosis.     

JNK/SAPK activity is not sufficient for anticancer therapy-induced apoptosis involving CD95-L, TRAIL and TNF-alpha

We report here that stress stimuli such as gamma-irradiation or the anticancer drug doxorubicin activate expression of the death-inducing ligands (DILs) CD95-L, TNF-alpha and TRAIL. Apoptosis induced by gamma-irradiation or doxorubicin engages a FADD- and caspase-dependent apoptosis pathway which is inhibited by dominant negative FADD or the caspase inhibitor zVAD. zVAD did not prevent activity of JNK/SAPKs in response to doxorubicin suggesting that JNK/SAPK activity is independent of death receptor triggering during cellular stress-induced apoptosis. In addition, JNK/SAPKs remained activated by doxorubicin in resistant cell lines in which cleavage of caspases and apoptosis was not observed. These data uncouple JNK/SAPK activation and apoptosis signaling and indicate that cellular stress-induced apoptosis involves signaling via DILs which is paralleled by activation of JNK/SAPKs. Activation of these kinases may contribute e.g., to the expression of molecules involved in apoptosis but is not sufficient for induction of the apoptosis program following cellular stress.     

Facing death in the fly: genetic analysis of apoptosis in Drosophila

Apoptosis, a gene-directed form of cell death, occurs normally during development and plays a major role in many diseases, including cancer and neurodegenerative disorders. Molecular genetic studies in Drosophila have revealed the existence of three novel apoptotic activators, reaper, head involution defective and grim. Additionally, Drosophila homologs of evolutionarily conserved IAPs (inhibitor of apoptosis proteins) and CED-3/ICE-like proteases have been identified and characterized. Through the combined use of genetic, molecular, biochemical and cell biological techniques in Drosophila it should now be possible to elucidate the precise mechanism by which apoptosis occurs, and how the death program is activated in response to many distinct death-inducing signals.     

Apoptosis induced by Drosophila reaper and grim in a human system. Attenuation by inhibitor of apoptosis proteins (cIAPs)

Previous genetic studies have established Reaper and Grim as central regulators of apoptosis in Drosophila melanogaster. Reaper and Grim induce extensive apoptosis in Drosophila, yet share no homology to known vertebrate proteins. In this study, we show for the first time that ectopic expression of Reaper or Grim induced substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis was inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivo binding studies demonstrated that both Reaper and Grim physically interacted with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolished binding to cIAPs. In vitro binding experiments indicated that Reaper and Grim bound specifically to the BIR domain-containing region of cIAPs as deletion of this region resulted in loss of binding. The physical interaction was further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localized with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolished co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, we demonstrate that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain that is the only region having significant homology between these Drosophila death inducers.     

Mitochondria and apoptosis

A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.     

Drosophila grim induces apoptosis in mammalian cells

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.     

IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs

Survivin is a member of the inhibitor of apoptosis protein (IAP) family. We investigated the antiapoptotic mechanism of Survivin, as well as its expression in 60 human tumor cell lines used for the National Cancer Institute's anticancer drug screening program. In cotransfection experiments, cell death induced by Bax or Fas (CD 95) was partially inhibited (mean +/- SD, 65% +/- 8%) by Survivin, whereas XIAP, another IAP family member, almost completely blocked cell death (93% +/- 4%) under the same conditions. Survivin and XIAP also protected 293 cells from apoptosis induced by overexpression of procaspase-3 and -7 and inhibited the processing of these zymogens into active caspases. In vitro binding experiments indicated that, like other IAP-family proteins, Survivin binds specifically to the terminal effector cell death proteases, caspase-3 and -7, but not to the proximal initiator protease caspase-8. Using a cell-free system in which cytosolic extracts were derived from control- or Survivin-transfected cells and where caspases were activated either by addition of cytochrome c and dATP or by adding recombinant active caspase-8, Survivin was able to substantially reduce caspase activity, as measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in intact cells when Survivin was overexpressed by gene transfection and caspase activation was induced by the anticancer drug etoposide. Survivin was expressed in all 60 cancer cell lines analyzed, with highest levels in breast and lung cancers and lowest levels in renal cancers. These findings indicate that Survivin, which is commonly expressed in human tumor cell lines, can bind the effector cell death proteases caspase-3 and -7 in vitro and inhibits caspase activity and cell death in cells exposed to diverse apoptotic stimuli. Although quantitative differences may exist, these observations suggest commonality in the mechanisms used by IAP-family proteins to suppress apoptosis.     

Induction of apoptosis by the novel retinoid AHPN in human T-cell lymphoma cells involves caspase-dependent and independent pathways

Retinoids play an important role in the control of lymphocyte function and homeostasis in the thymus. In this study, we show that the induction of growth arrest and apoptosis in a variety of T-cell lymphoma cell lines, including Jurkat and Molt-4 cells, is highly specific for the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) since all-trans retinoic acid (RA), the RAR-selective retinoid TTAB, the RXR-selective retinoid SR11217 and the retinoid SR11302 exhibiting selective anti-AP1 activity, do not induce apoptosis or cause growth arrest. These findings support the concept that the effects of AHPN on proliferation and induction of apoptosis are mediated by a novel signaling pathway. AHPN-induced apoptosis is associated with an induction of internucleosomal DNA-fragmentation, increased annexin V binding and a 30-fold stimulation of caspase-3-like activity. Overexpression of Bcl-2 in Molt-4 cells greatly inhibits the induction of apoptosis by AHPN as indicated by the inhibition of DNA-fragmentation, annexin V binding and caspase-3-like activity. However, Bcl-2 overexpression does not interfere with the ability of AHPN to cause growth arrest or accumulation of cells in the early S-phase of the cell cycle, indicating that the effects of AHPN on growth arrest can be uncoupled from the effects on apoptosis. The caspase inhibitor Z-VAD-FMK, at concentrations that totally block caspase activity, delays but does not prevent cell death and does not affect the accumulation of cells in the S-phase of the cell cycle. Our results show that induction of caspase-3-like activity plays an important role in the execution of AHPN-induced apoptosis but cells can undergo cell death in the absence of this activity suggesting that AHPN-induced cell death involves caspase-dependent and -independent mechanisms.     

Differential use of immunoglobulin light chain genes and B lymphocyte expansion at sites of disease in rheumatoid arthritis (RA) compared with circulating B lymphocytes

Patients infected with HIV-1 often exhibit cognitive deficits that are related to progressive neuronal degeneration and cell death. The protein Tat, which is released from HIV-1-infected cells, was recently shown to be toxic toward cultured neurons. We now report that Tat induces apoptosis in cultured embryonic rat hippocampal neurons. Tat induced caspase activation, and the caspase inhibitor zVAD-fmk prevented Tat-induced neuronal death. Tat induced a progressive elevation of cytoplasmic-free calcium levels, which was followed by mitochondrial calcium uptake and generation of mitochondrial-reactive oxygen species (ROS). The intracellular calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red protected neurons against Tat-induced apoptosis. zVAD-fmk suppressed Tat-induced increases of cytoplasmic calcium levels and mitochondrial ROS accumulation, indicating roles for caspases in the perturbed calcium homeostasis and oxidative stress induced by Tat. An inhibitor of nitric oxide synthase, and the peroxynitrite scavenger uric acid, protected neurons against Tat-induced apoptosis, indicating requirements for nitric oxide production and peroxynitrite formation in the cell death process. Finally, Tat caused a delayed and progressive mitochondrial membrane depolarization, and cyclosporin A prevented Tat-induced apoptosis, suggesting an important role for mitochondrial membrane permeability transition in Tat-induced apoptosis. Collectively, our data demonstrate that Tat can induce neuronal apoptosis by a mechanism involving disruption of calcium homeostasis, caspase activation, and mitochondrial calcium uptake and ROS accumulation. Agents that interupt this apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in AIDS patients.     

Genetic analysis of protein kinase B (AKT) in Drosophila

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.     

Oxidative stress induces DNA fragmentation and caspase activation via the c-Jun NH2-terminal kinase pathway in H9c2 cardiac muscle cells

The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.     

TNF-alpha activates at least two apoptotic signaling cascades

Apoptosis, the process whereby cells activate an intrinsic death program, can be induced in HeLa cells by TNF-alpha treatment. The aims of the present study were (i) to examine the precise role and the origin of Reactive Oxygen Species (ROS) in the TNF-alpha-induced programmed cell death, (ii) to characterize and order the morphological and mitochondrial changes associated with this process and (iii) to link these events with the activation of caspases. Analyses were performed on TNF-alpha-treated cells in the presence of an anti-oxidant, or of a general caspase inhibitor. To assess the role of mitochondria in the cell death signal transduction, these studies were also realized on HeLa-variant cell lines lacking functional mitochondrial respiratory chain. We show that at least two separate signaling cascades, both mediated by Z-VAD-sensitive caspase(s), contribute to the TNF-alpha-induced apoptosis of HeLa cells. One signaling pathway involves an early mitochondria-dependent ROS production, the other being ROS-independent.     

Role of early and late replication events in induction of apoptosis by baculoviruses

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.     

Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the "protective" TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.     

CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor

DFF45 is a subunit of the DNA fragmentation factor (DFF) that is cleaved by caspase-3 during apoptosis. However, the mechanism by which DFF45 regulates apoptotic cell death remains poorly understood. Here we report the identification and characterization of two mammalian genes, CIDE-A and CIDE-B, encoding highly related proteins with homology to the N-terminal region of DFF45. CIDE-A and CIDE-B were found to activate apoptosis in mammalian cells, which was inhibited by DFF45 but not by caspase inhibitors. Expression of CIDE-A induced DNA fragmentation in 293T cells, which was inhibited by DFF45, further suggesting that DFF45 inhibits the apoptotic activities of CIDEs. In addition to mammalian CIDE-A and CIDE-B, we identified DREP-1, a Drosophila melanogaster homolog of DFF45 that could inhibit CIDE-A-mediated apoptosis. Mutant analysis revealed that the C-terminal region of CIDE-A was necessary and sufficient for killing whereas the region with homology to DFF45 located in the N-terminus was required for DFF45 to inhibit CIDE-A-induced apoptosis. CD95/Fas-mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. These studies suggest that DFF45 is evolutionarily conserved and implicate CIDEs as DFF45-inhibitable effectors that promote cell death and DNA fragmentation.