The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm

TNFR1 associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain along with nuclear import and export sequences that cause shuttling between the cytoplasm and nucleus. The death domain of TRADD contains the nuclear import sequence and expression of the core death domain (nuclear TRADD) results in exclusive nuclear localization and activation of a distinct apoptotic pathway. Cytoplasmic TRADD activates apoptosis through Fas-associated death domain protein (FADD) and caspase-8 activation that was blocked by caspase inhibitors or dominant-negative FADD. These inhibitors did not inhibit death induced by nuclear TRADD, which could only be inhibited by combining caspase inhibitors and a serine protease inhibitor. The pathway activated by nuclear TRADD requires caspase-9 catalytic activity. However, apoptosis activating factor deficiency confers only partial protection from death. This pathway represents an alternate means by which TRADD can regulate cell death independently of FADD and caspase-8 that occurs from the nucleus rather than the cytoplasm.     

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.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Fibrillogenic amylin evokes islet beta-cell apoptosis through linked activation of a caspase cascade and JNK1

Fibrillogenic human amylin elicits pancreatic beta-cell apoptosis that may contribute to development of type-2 diabetes. Here, we demonstrated that activation of a caspase cascade is necessary for induction of apoptosis by fibrillogenic amylin variants in two pancreatic beta-cell lines. Human amylin, as well as truncated 8-37human amylin, evoked sequential activation of caspases-8 and -3, and apoptosis, whereas non-beta-sheet forming and non-fibrillogenic homologs, such as [25,28,29triprolyl]human amylin, did not, implying that the beta-sheet conformer is required for human amylin-induced caspase activation. Significant inhibition of apoptosis was evoked by a selective caspase-1 inhibitor, indicating that caspase-1 is also essential for activation of the caspase cascade. Furthermore, we showed that specific jnk1 antisense oligonucleotides, which suppress phospho-JNK1 expression, effectively decreased human amylin-induced activation of c-Jun. Studies of the interplay between the caspase cascade and the JNK pathway showed that both apoptosis and caspase-3 activation were suppressed by treatment with a JNK inhibitor and by transfection of antisense jnk1 oligonucleotides or antisense-c-jun, whereas a selective inhibitor of caspases-1 and -3 prevented apoptosis but not c-Jun activation. Thus, the JNK1 activation preceded activation of caspases-1 and -3. However, selective JNK inhibition had no effect on caspase-8 activation, and selective caspase-8 inhibition only partially suppressed apoptosis and c-Jun activation, indicating that caspase-8 may partially act upstream of the JNK pathway. Our studies demonstrate a functional interaction of a caspase cascade and JNK1. Fibrillogenic amylin can evoke a JNK1-mediated apoptotic pathway, which is partially dependent and partially independent of caspase-8, and in which caspase-3 acts as a common downstream effector.     

Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis

Autophagy and apoptosis are two evolutionarily conserved processes that regulate cell fate in response to cytotoxic stress. However, the functional relationship between these two processes remains far from clear. Here, we demonstrate an autophagy-dependent mechanism of caspase-8 activation and initiation of the apoptotic cascade in response to SKI-I, a pan-sphingosine kinase inhibitor, and bortezomib, a proteasome inhibitor. Autophagy is induced concomitantly with caspase-8 activation, which is responsible for initiation of the caspase cascade and the mitochondrial amplification loop that is required for full execution of apoptosis. Inhibition of autophagosome formation by depletion of Atg5 or Atg3 results in a marked suppression of caspase-8 activation and apoptosis. Although caspase-8 self-association depends on p62/SQSTM1, its self-processing requires the autophagosomal membrane. Caspase-8 forms a complex with Atg5 and colocalizes with LC3 and p62. Moreover, FADD, an adaptor protein for caspase-8 activation, associates with Atg5 on Atg16L- and LC3-positive autophagosomal membranes and loss of FADD suppresses cell death. Taken together, these results indicate that the autophagosomal membrane serves as a platform for an intracellular death-inducing signaling complex (iDISC) that recruits self-associated caspase-8 to initiate the caspase-8/-3 cascade.     

The RIP-like kinase, RIP3, induces apoptosis and NF-kappaB nuclear translocation and localizes to mitochondria

A RIP-like protein, RIP3, has recently been reported that contains an N-terminal kinase domain and a novel C-terminal domain that promotes apoptosis. These experiments further characterize RIP3-mediated apoptosis and NF-kappaB activation. Northern blots indicate that rip3 mRNA displays a restricted pattern of expression including regions of the adult central nervous system. The rip3 gene was localized by fluorescent in situ hybridization to human chromosome 14q11.2, a region frequently altered in several types of neoplasia. RIP3-mediated apoptosis was inhibited by Bcl-2, Bcl-x(L), dominant-negative FADD, as well as the general caspase inhibitor Z-VAD. Further dissection of caspase involvement in RIP3-induced apoptosis indicated inhibition by the more specific inhibitors Z-DEVD (caspase-3, -6, -7, -8, and -10) and Z-VDVAD (caspase-2). However, caspase-1, -6, -8 and -9 inhibitors had little or no effect on RIP3-mediated apoptosis. Mutational analysis of RIP3 revealed that the C-terminus of RIP3 contributed to its apoptotic activity. This region is similar, but distinct, to the death domain found in many pro-apoptotic receptors and adapter proteins, including FAS, FADD, TNFR1, and RIP. Furthermore, point mutations of RIP3 at amino acids conserved among death domains, abrogated its apoptotic activity. RIP3 was localized by immunofluorescence to the mitochondrion and may play a key role in the mitochondrial disruptions often associated with apoptosis.     

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.     

Role of caspase-8 in thymus function

The thymus is the primary organ responsible for de novo generation of immunocompetent T cells that have a diverse repertoire of antigen recognition. During the developmental process, 98% of thymocytes die by apoptosis. Thus apoptosis is a dominant process in the thymus and occurs through either death by neglect or negative selection or through induction by stress/aging. Caspase activation is an essential part of the general apoptosis mechanism, and data suggest that caspases may have a role in negative selection; however, it seems more probable that caspase-8 activation is involved in death by neglect, particularly in glucocorticoid-induced thymocyte apoptosis. Caspase-8 is active in double-positive (DP) thymocytes in vivo and can be activated in vitro in DP thymocytes by T-cell receptor (TCR) crosslinking to induce apoptosis. Caspase-8 is a proapoptotic member of the caspase family and is considered an initiator caspase, which is activated upon stimulation of a death receptor (e.g., Fas), recruitment of the adaptor molecule FADD, and recruitment and subsequent processing of procaspase-8. The main role of caspase-8 seems to be pro-apoptotic and, in this review, we will discuss about the involvement of caspase-8 in (1) TCR-triggered thymic apoptosis; (2) death receptor-mediated thymic apoptosis; and (3) glucocorticoid-induced thymic apoptosis. Regarding TCR triggering, caspase-8 is active in medullary, semi-mature heat-stable antigen^hi (HAS^hi SP) thymocytes as a consequence of strong TCR stimulation. The death receptors Fas, FADD, and FLIP are involved upstream of caspase-8 activation in apoptosis; whereas, Bid and HDAC7 are involved downstream of caspase-8. Finally, caspase-8 is involved in glucocortocoid-induced thymocyte apoptosis through an activation loop with the protein GILZ. GILZ activates caspase-8, promoting GILZ sumoylation and its protection from proteasomal degradation.     

Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion

We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 +/- 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH(2)F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.     

Activation of NF-kappaB by FADD, Casper, and caspase-8

Fas-associated death domain protein (FADD), caspase-8-related protein (Casper), and caspase-8 are components of the tumor necrosis factor receptor type 1 (TNF-R1) and Fas signaling complexes that are involved in TNF-R1- and Fas-induced apoptosis. Here we show that overexpression of FADD and Casper potently activates NF-kappaB. In the presence of caspase inhibitors, overexpression of caspase-8 also activates NF-kappaB. A caspase-inactive point mutant, caspase-8(C360S), activates NF-kappaB as potently as wild-type caspase-8, suggesting that caspase-8-induced apoptosis and NF-kappaB activation are uncoupled. NF-kappaB activation by FADD and Casper is inhibited by the caspase-specific inhibitors crmA and BD-fmk, suggesting that FADD- and Casper-induced NF-kappaB activation is mediated by caspase-8. FADD, Casper, and caspase-8-induced NF-kappaB activation are inhibited by dominant negative mutants of TRAF2, NIK, IkappaB kinase alpha, and IkappaB kinase beta. A dominant negative mutant of RIP inhibits FADD- and caspase-8-induced but not Casper-induced NF-kappaB activation. A mutant of Casper and the caspase-specific inhibitors crmA and BD-fmk partially inhibit TNF-R1-, TRADD, and TNF-induced NF-kappaB activation, suggesting that FADD, Casper, and caspase-8 function downstream of TRADD and contribute to TNF-R1-induced NF-kappaB activation. Moreover, activation of caspase-8 results in proteolytic processing of NIK, which is inhibited by crmA. When overexpressed, the processed fragments of NIK do not activate NF-kappaB, and the processed C-terminal fragment inhibits TNF-R1-induced NF-kappaB activation. These data indicate that FADD, Casper, and pro-caspase-8 are parts of the TNF-R1-induced NF-kappaB activation pathways, whereas activated caspase-8 can negatively regulate TNF-R1-induced NF-kappaB activation by proteolytically inactivating NIK.     

Survival function of the FADD-CASPASE-8-cFLIP(L) complex

Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, its adapter molecule, FADD, required for caspase-8 activation, and cFLIPL, a caspase-8-like protein that lacks a catalytic site and blocks caspase-8-mediated apoptosis, are each essential for embryonic development. Animals deficient in any of these genes present with E10.5 embryonic lethality. Recent studies have shown that development in caspase-8-deficient mice is rescued by ablation of RIPK3, a kinase that promotes a form of programmed, necrotic cell death. Here, we show that FADD, RIPK3 double-knockout mice develop normally but that the lethal effects of cFLIP deletion are not rescued by RIPK3 deficiency. Remarkably, in mice lacking FADD, cFLIP, and RIPK3, embryonic development is normal. This can be explained by the convergence of two cell processes: the enzymatic activity of the FADD-caspase-8-cFLIPL complex blocks RIPK3-dependent signaling (including necrosis), whereas cFLIPL blocks RIPK3-independent apoptosis promoted by the FADD-caspase-8 complex.     

The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis.

Oncogenic Ras induces cells to undergo apoptosis after inhibition of protein kinase C (PKC) activity. The integration of differential signaling pathways is required for full execution of apoptosis. In this study, we used Jurkat as well as Fas/FADD-defective cell lines expressing v-ras to determine the upstream elements required for activation of the caspase cascade in PKC/Ras-mediated apoptosis. During this Ras-induced apoptotic process, caspase-8 was activated, possibly through its binding to Fas-associated death domain (FADD), in Jurkat/ras and Jurkat/Fas(m)/ras cells but not in Jurkat/FADD(m)/ras cells. c-Jun NH(2)-terminal kinase (JNK) was activated in all three cell lines expressing ras in response to apoptotic stimulation. Suppression of JNK by dn-JNK1 blocked the interaction of FADD and caspase-8 and partially protected Jurkat/ras and Jurkat/Fas(m)/ras cells from apoptosis. However, dn-JNK1 had no effect on PKC/Ras-induced apoptosis in Jurkat/FADD(m)/ras cells. The results indicate that FADD/caspase-8 signaling is involved in PKC/Ras-mediated apoptosis, and JNK may be an upstream effector of caspase activation.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis

In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

Expression of dominant-negative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard

DNA damaging agents up-regulate levels of the Fas receptor or its ligand, resulting in recruitment of Fas-associated death domain (FADD) and autocatalytic activation of caspase-8, consequently activating the executioner caspases-3, -6, and -7. We found that human epidermal keratinocytes exposed to a vesicating dose (300 microm) of sulfur mustard (SM) exhibit a dose-dependent increase in the levels of Fas receptor and Fas ligand. Immunoblot analysis revealed that the upstream caspases-8 and -9 are both activated in a time-dependent fashion, and caspase-8 is cleaved prior to caspase-9. These results are consistent with the activation of both death receptor (caspase-8) and mitochondrial (caspase-9) pathways by SM. Pretreatment of keratinocytes with a peptide inhibitor of caspase-3 (Ac-DEVD-CHO) suppressed SM-induced downstream markers of apoptosis. To further analyze the importance of the death receptor pathway in SM toxicity, we utilized Fas- or tumor necrosis factor receptor-neutralizing antibodies or constructs expressing a dominant-negative FADD (FADD-DN) to inhibit the recruitment of FADD to the death receptor complex and block the Fas/tumor necrosis factor receptor pathway following SM exposure. Keratinocytes pretreated with Fas-blocking antibody or stably expressing FADD-DN and exhibiting reduced levels of FADD signaling demonstrated markedly decreased caspase-3 activity when treated with SM. In addition, the processing of procaspases-3, -7, and -8 into their active forms was observed in SM-treated control keratinocytes, but not in FADD-DN cells. Blocking the death receptor complex by expression of FADD-DN additionally inhibited SM-induced internucleosomal DNA cleavage and caspase-6-mediated nuclear lamin cleavage. Significantly, we further found that altering the death receptor pathway by expressing FADD-DN in human skin grafted onto nude mice reduces vesication and tissue injury in response to SM. These results indicate that the death receptor pathway plays a pivotal role in SM-induced apoptosis and is therefore a target for therapeutic intervention to reduce SM injury.     

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-alpha-induced apoptosis. STS activated I kappa B kinase and nuclear factor-kappa B (NF-kappa B) nuclear translocation and DNA binding but inhibited transactivation of I kappa B-alpha, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-kappa B reporter in transfected Huh-7 cells.     

Caspase activation by adenovirus e4orf4 protein is cell line specific and Is mediated by the death receptor pathway

Adenovirus E4orf4 protein has been shown to induce transformed cell-specific, protein phosphatase 2A-dependent, and p53-independent apoptosis. It has been further reported that the E4orf4 apoptotic pathway is caspase-independent in CHO cells. Here, we show that E4orf4 induces caspase activation in the human cell lines H1299 and 293T. Caspase activation is required for apoptosis in 293T cells, but not in H1299 cells. Dominant negative mutants of caspase-8 and the death receptor adapter protein FADD/MORT1 inhibit E4orf4-induced apoptosis in 293T cells, suggesting that E4orf4 activates the death receptor pathway. Cytochrome c is released into the cytosol in E4orf4-expressing cells, but caspase-9 is not required for induction of apoptosis. Furthermore, E4orf4 induces accumulation of reactive oxygen species (ROS) in a caspase-8- and FADD/MORT1-dependent manner, and inhibition of ROS generation by 4,5-dihydroxy-1, 3-benzene-disulfonic acid (Tiron) inhibits E4orf4-induced apoptosis. Thus, our results demonstrate that E4orf4 engages the death receptor pathway to generate at least part of the molecular events required for E4orf4-induced apoptosis.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Curcumin Induces Apoptosis in Human Melanoma Cells through a Fas Receptor/Caspase-8 Pathway Independent of p53

In this study, we investigated the molecular pathways targeted by curcumin during apoptosis of human melanoma cell lines. We found that curcumin caused cell death in eight melanoma cell lines, four with wild-type and four with mutant p53. We demonstrate that curcumin-induced apoptosis is both dose- and time-dependent. We found that curcumin did not induce p53, suggesting that curcumin activates other apoptosis pathways. Our data show that curcumin activates caspases-3 and -8 but not caspase-9, supporting the rationale that apoptosis occurs via a membrane-mediated mechanism. Both a caspase-8 and broad-based caspase inhibitor, but not a caspase-9 specific inhibitor, suppressed curcumin-induced cell death. To further support our hypothesis that curcumin induces activation of a death receptor pathway, we show that curcumin induces Fas receptor aggregation in a FasL-independent manner and that low-temperature incubation, previously shown to inhibit receptor aggregation, prevented curcumin-induced cell death. Moreover, we demonstrate that expression of dominant negative FADD significantly inhibited curcumin-induced cell death. In addition, our results indicate that curcumin also blocks the NF-kappaB cell survival pathway and suppresses the apoptotic inhibitor, XIAP. Since melanoma cells with mutant p53 are strongly resistant to conventional chemotherapy, curcumin may overcome the chemoresistance of these cells and provide potential new avenues for treatment.