Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Polycystin-1 promotes PKCalpha-mediated NF-kappaB activation in kidney cells

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-kappaB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293(CTT)), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-kappaB nuclear levels and NF-kappaB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-kappaB promoter activation was mediated by PKCalpha because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293(CTT) cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-kappaB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCalpha-mediated NF-kappaB signalling and cell survival.     

Mechanism of apoptosis induced by IFN-alpha in human myeloma cells: role of Jak1 and Bim and potentiation by rapamycin

Interferon-alpha (IFN-alpha) has been used for the last 20 years in the maintenance therapy of multiple myeloma (MM), though it is only effective in some patients. Congruent with this, IFN-alpha induces apoptosis in some MM cell lines. Understanding the mechanism of IFN-alpha-induced apoptosis could be useful in establishing criteria of eligibility for therapy. Here we show that IFN-alpha-induced apoptosis in the MM cell lines U266 and H929 was completely blocked by a specific inhibitor of Jak1. The mTOR inhibitor rapamycin mitigated apoptosis in U266 but potentiated it in H929 cells. IFN-alpha induced PS exposure, DeltaPsi(m) loss and pro-apoptotic conformational changes of Bak, but not of Bax, and was fully prevented by Mcl-1 overexpression in U266 cells. IFN-alpha treatment caused the release of cytochrome c from mitochondria to cytosol and consequently, a limited proteolytic processing of caspases. Apoptosis induced by IFN-alpha was only slightly prevented by caspase inhibitors. Levels of the BH3-only proteins PUMA and Bim increased during IFN-alpha treatment. Bim increase and apoptosis was prevented by transfection with the siRNA for Bim. PUMA-siRNA transfection reduced electroporation-induced apoptosis but had no effect on apoptosis triggered by IFN-alpha. The potentiating effect of rapamycin on apoptosis in H929 cells was associated to an increase in basal and IFN-alpha-induced Bim levels. Our results indicate that IFN-alpha causes apoptosis in myeloma cells through a moderate triggering of the mitochondrial route initiated by Bim and that mTOR inhibitors may be useful in IFN-alpha maintenance therapy of certain MM patients.     

Curcumin inhibits interferon-alpha induced NF-kappaB and COX-2 in human A549 non-small cell lung cancer cells

The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-alpha treatment. The IFN-alpha-treated A549 cells showed increase in protein expression levels of NF-kappaB and COX-2. IFN-alpha induced NF-kappaB binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-alpha-induced COX-2 expression in A549 cells. Within 10 min, IFN-alpha rapidly induced the binding activity of a gamma-(32)P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-alpha-induced activations of NF-kappaB and COX-2 were inhibited by the addition of curcumin in A549 cells.     

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.     

Relationship between constitutive nuclear factor-kappaB (NF-kappaB) and inhibitor kappaB-alpha (IkappaB-alpha) in an interferon-alpha-sensitive human Burkitt lymphoma cell line

The human Burkitt lymphoma Daudi cell line expresses constitutively active nuclear factor kappaB (NF-kappaB) in the nucleus in spite of high levels of inhibitor kappaB-alpha (IkappaB-alpha) in the cytoplasm. The antiproliferative response of these cells to interferon-alpha (IFN-alpha) correlated with the inhibition of the constitutive NF-kappaB activity by the cytokine. The present study shows that IFN-alpha caused an increase in p53 level, inhibited cell proliferation by [(3)H]thymidine incorporation, and stimulated cytotoxicity and apoptosis by PARP-cleavage in the Daudi cells. In order to study the relationship between the constitutively active NF-kappaB and IkappaB-alpha, a dominant negative mutant IkappaB-alpha (IkappaB-alphaDN), lacking the N-terminal 36 amino acids required for the activation of NF-kappaB by tumor necrosis factor-alpha (TNF-alpha), was expressed in the Daudi cells. The expression of IkappaB-alphaDN protein did not inhibit the constitutive NF-kappaB activity, but it inhibited cell proliferation, antiproliferative response to IFN-alpha, and phosphorylated mitogen activated protein kinase (p-MAPK) level. Thus, our results suggest that constitutive NF-kappaB activity in the human Burkitt lymphoma Daudi cells is maintained by a mechanism independent of IkappaB-alpha degradation, and that the IkappaB-alpha is involved in the proliferation of these cells, possibly through the MAP kinase pathway. Therefore, in addition to IFN-alpha treatment, both NF-kappaB and IkappaB-alpha may be used as drug targets for inhibiting cell proliferation in the lymphomas.     

The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover

The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).     

Interferon-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.     

NF-kappaB activation but not PI3K/Akt is required for dexamethasone dependent protection against TNF-alpha cytotoxicity in L929 cells

Tumor necrosis factor alpha (TNF-alpha) is one of the best-described cell death promoters. In murine L929 fibroblasts, dexamethasone inhibits TNF-alpha-induced cytotoxicity. Since phosphatidyl inositol 3 kinase (PI3K) and nuclear factor kappa B (NF-kappaB) proteins regulate several survival pathways, we evaluated their participation in dexamethasone protection against TNF-alpha cell death. We interfered with these pathways by overexpressing a negative dominant mutant of PI3K or a non-degradable mutant of inhibitor of NF-kappaB alpha (IkappaBalpha) (the cytoplasmic inhibitor of NF-kappaB) in L929 cells. The mutant IkappaB, but not the mutant PI3K, abrogated dexamethasone-mediated protection. The loss of dexamethasone protection was associated with a diminished accumulation in XIAP and c-IAP proteins.     

Heat shock protein 70 inhibits the nuclear import of apoptosis-inducing factor to avoid DNA fragmentation in TF-1 cells during erythropoiesis

Loss of mitochondrial membrane potential (DeltaPsi(m)) and release of AIF (apoptosis-inducing factor) from mitochondria are key steps in apoptosis. In TF-1 model, DeltaPsi(m) was depolarized with AIF release during erythroid development. Yet, no DNA fragmentation was observed. When DeltaPsi(m) depolarization had been blocked, erythropoiesis was suppressed. Interestingly, heat shock protein 70 (Hsp70) was found transiently upregulated during depolarization and it retained AIF in the cytosol to avoid DNA damages. When Hsp inhibitor was added, DNA fragmentation occurred. We show this mechanism for the first time in erythropoiesis how cells with DeltaPsi(m) depolarization and AIF release escape apoptosis.     

Time-dependent onset of Interferon-alpha2b-induced apoptosis in isolated hepatocytes from preneoplastic rat livers

We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.     

Apo2L/TRAIL is an indirect mediator of apoptosis induced by interferon-alpha in human myeloma cells

Interferon-alpha (IFN-alpha) is currently used for the therapy of multiple myeloma (MM) though it is only effective in some patients. IFN-alpha induces apoptosis in some MM cell lines and it has been proposed to occur through an autocrine loop involving Apo2L/TRAIL. We have analysed the sensitivity to IFN-alpha and Apo2L/TRAIL of five MM cell lines and found no correlation between the apoptosis inducing ability of both cytokines. IFN-alpha-induced apoptosis in MM cells was not prevented by a caspase-8 selective inhibitor (Z-IETD-fmk) or blocking Apo2L/TRAIL. However, human monocytes treated with IFN-alpha release bioactive Apo2L/TRAIL to culture media which was cytotoxic for MM cells resistant to IFN-alpha. We propose that Apo2L/TRAIL released from IFN-alpha-stimulated blood monocytes would be a major mediator of the anti-myeloma effect of IFN-alpha in vivo.     

Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma

We investigated whether Epstein--Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)-alpha. EBV-negative subclones isolated from EBV-positive Burkitt's lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN-alpha treatment. On the other hand, EBV-positive counterparts exhibited striking resistance against IFN-alpha-induced apoptosis. Transfection of an individual EBV latent gene into EBV-negative BL cells revealed that EBV-encoded poly(A)(-) RNAs (EBERs) were responsible for IFN resistance. EBERs bound double-stranded (ds) RNA-activated protein kinase (PKR), a key mediator of the antiviral effect of IFN-alpha, and inhibited its phosphorylation. Transfection of dominant-negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV-negative BL cells resistant to IFN-alpha-induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA-binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN-alpha-induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN-induced apoptosis in virus-associated tumors.     

A role for c-FLIPL in the regulation of apoptosis,autophagy, and necroptosis in T lymphocytes

Caspase 8 plays a dual role in the survival of T lymphocytes. Although active caspase 8 mediates apoptosis upon death receptor signaling, the loss of caspase 8 activity leads to receptor-interacting protein (RIP)-1/RIP-3-dependent necrotic cell death (necroptosis) upon TCR activation. The anti-apoptotic protein c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) suppresses death receptor-induced caspase 8 activation. Moreover, recent findings suggest that c-FLIP is also involved in inhibiting necroptosis and autophagy. It remains unclear whether c-FLIP protects primary T lymphocytes from necroptosis or regulates the threshold at which autophagy occurs. Here, we used a c-FLIP isoform-specific conditional deletion model to show that c-FLIP_L-deficient T cells underwent RIP-1-dependent necroptosis upon TCR stimulation. Interestingly, although previous studies have only described necroptosis in the absence of caspase 8 activity, we found that pro-apoptotic caspase 8 activity and apoptosis were also enhanced in c-FLIP_L-deficient T lymphocytes. Furthermore, c-FLIP_L-deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. Together, these findings indicate that c-FLIP_L plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes.     

The histone deacetylase inhibitor sodium butyrate interacts synergistically with phorbol myristate acetate (PMA) to induce mitochondrial damage and apoptosis in human myeloid leukemia cells through a tumor necrosis factor-alpha-mediated process

Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release). Neutralization of endogenous TNFalpha by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G(2)M populations, and dephosphorylation of p34(cdc2). These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFalpha-dependent pathway in which procaspase 8 and Bid activation play key roles.     

Selective inhibition of IkappaB kinase sensitizes mantle cell lymphoma B cells to TRAIL by decreasing cellular FLIP level

In an attempt to circumvent the intrinsic resistance of mantle cell lymphoma (MCL) cells to apoptosis, we have analyzed their sensitivity to the extrinsic apoptotic signal triggered by TRAIL. We show here that TRAIL can trigger apoptosis in a majority of MCL cell lines and primary cultures, irrespective of receptor levels, Bcl-2 family members, or caspase regulator expression. MCL sensitivity to TRAIL was closely linked to the activity of the NF-kappaB p50 factor and to the consequent expression of cellular FLIP (c-FLIP), which accumulated into the TRAIL-dependent complex in resistant cells. c-FLIP transient knockdown overcame MCL resistance to TRAIL, while NF-kappaB inhibitors differentially modulated TRAIL cytotoxicity. Indeed, bortezomib increased TRAIL cytotoxic effects in sensitive cells, but led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction. In contrast, the IkappaB kinase inhibitor BMS-345541 led to decreased c-FLIP expression and allowed all MCL samples to undergo TRAIL-mediated apoptosis. These results present the combination of TRAIL stimulation and IkappaB kinase inhibition as a new approach to MCL therapy.     

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-induced CXCL8 production is mediated through PKCalpha-dependent activation of the IKKalphabeta signaling pathway in epithelial cells

Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of NF-kappaB-dependent chemokine gene expression. However, downstream signaling pathways, especially that of Ca(2+)-dependent protein kinase C alpha (PKCalpha), and in particular, the identity of the IKKalphabeta signal pathway for CXCL8 secretion in Mycobacterium bovis BCG-induced epithelial cells are still unknown. In this study, we demonstrated that the phosphoinositide-phospholipase C (PI-PLC) downstream signaling pathway is involved in M. bovis BCG-induced CXCL8 release, since A549 cells pretreated with U73122, a PI-PLC inhibitor, inhibited CXCL8 release, whereas U73343 the inactive analog had no effect. In addition, our results demonstrated that M. bovis BCG-induced CXCL8 production by A549 cells was significantly blocked by using neomycin (another well-described inhibitor of PI-PLC with a different mechanism of action), Ro-32-0432 and Ro-31-8220 (two PKCalpha inhibitors), PP1 and PP2 (two potent and selective inhibitors of the Src-family tyrosine kinases), and Bay 11-7082 (an IkappaB phosphorylation inhibitor). We also demonstrated that M. bovis BCG can rapidly induce translocation of PKCalpha from the cytosol to the membrane, and that treatment of cells with M. bovis BCG caused time-dependent increases in phosphorylation of c-Src at tyrosine 416. Finally, our studies revealed that M. bovis BCG induced the association of c-Src and IKKalphabeta during the interaction of PKCalpha and IKKalphabeta. Altogether, these results represent the first evidence to date suggesting that M. bovis BCG activates the PI-PLC/PKCalpha/c-Src/IKKalphabeta signaling pathway to induce CXCL8 release in human epithelial cells.     

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.