AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX^−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.     

Activation of calcium-independent phospholipase A (iPLA) in brain mitochondria and release of apoptogenic factors by BAX and truncated BID

Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C-terminal fragment (BAXDeltaC) were shown to induce modest cytochrome c (Cyt c) release from rat brain mitochondria when activated by tBID. We now show that tBID plus monomeric full-length BAX induce extensive release of Cyt c, Smac/DIABLO, and Omi/HtrA2 (but not endonuclease G and the apoptosis inducing factor) comparable to the release induced by alamethicin. This occurs independently of the permeability transition without overt changes in mitochondrial morphology. The mechanism of the release may involve formation of reactive oxygen species (ROS) and activation of calcium-independent phospholipase A(2) (iPLA(2)). Indeed, increased ROS production and activated iPLA(2) were observed prior to massive Cyt c release. Furthermore, the extent of inhibition of Cyt c release correlated with the degree of suppression of iPLA(2) by the inhibitors propranolol, dibucaine, 4-bromophenacyl bromide, and bromenol lactone. Consistent with a requirement for iPLA(2) in Cyt c release from brain mitochondria, synthetic liposomes composed of lipids mimicking the outer mitochondrial membrane (OMM) but lacing iPLA(2) failed to release 10 kDa fluorescent dextran (FD-10) in response to tBID plus BAX. We propose that tBID plus BAX activate ROS generation, which subsequently augments iPLA(2) activity leading to changes in the OMM that allow translocation of certain mitochondrial proteins from the intermembrane space.     

Dissimilar mechanisms of cytochrome c release induced by octyl glucoside-activated BAX and by BAX activated with truncated BID

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAX_oligo) and by a combination of monomeric BAX (BAX_mono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, t^cBID). We also examined whether the effects induced by BAX_oligo or by BAX_mono activated with t^cBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that t^cBID plus BAX_mono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAX_oligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAX_oligo could be at least partially suppressed by mitochondrial depolarization. The effects of t^cBID plus BAX_mono were insensitive to depolarization. BAX_oligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K⁺ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAX_oligo, but failed to influence the effects produced by t^cBID plus BAX_mono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAX_oligo and by BAX activated with t^cBID.     

UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation

Ultraviolet irradiation resistance-associated gene (UVRAG) is a well-known regulator of autophagy by promoting autophagosome formation and maturation. However, little is known about the non-autophagic functions of UVRAG. Here, we present evidence that UVRAG functions as an unusual BCL2-associated X protein (Bax) suppressor to regulate apoptosis. Chemotherapy and radiation induces UVRAG expression and subsequently upregulates autophagy and apoptosis in tumour cells. Depletion of UVRAG expression by RNA interference renders tumour cells more sensitive to chemotherapy- and radiation-induced apoptosis in vitro and in vivo. Moreover, UVRAG interacts with Bax, which inhibits apoptotic stimuli-induced mitochondrial translocation of Bax, reduction of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. Our findings show that UVRAG has an essential role in the intrinsic mitochondrial pathway of apoptosis by regulating the localization of Bax. This pathway represents a target for clinical intervention against tumours.     

Targeting BAX ubiquitin-binding sites reveals that BAX activation is essential for its ubiquitin-dependent degradation

BAX is an important proapoptotic protein of the BCL-2 family, and its stability is essential for the regulation of the mitochondrial apoptotic pathway. A previous study revealed that BAX could undergo degradation through the ubiquitin-proteasome pathway. In this study, we identified two lysine sites, K21 and K123, that were critical ubiquitin-binding sites in BAX. Mutation of these two sites prolonged the half-life of BAX and also affected its proapoptotic ability. Intriguingly, we found that ABT-737, a BCL-2 inhibitor, significantly enhanced TRAIL-induced BAX degradation in HCT116 cells and increased TRAIL-induced apoptosis in the HCT116 only with the BAX K21R/K123R mutant, not other BAX mutants. In addition, overexpression of PARKIN, an E3 ubiquitin ligase targeting BAX, dramatically decreased BAX protein level when only treated with ABT-737 in HCT116 cells. Therefore, we speculated that BAX activation is essential for its ubiquitin-dependent degradation.     

Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X(L), MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.     

Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro-Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline-treated rats. Immunohistochemistry and western blot analysis for the calpain-derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus .     

Maternal behavior regulates long-term hippocampal expression of BAX and apoptosis in the offspring

Naturally occurring variations in maternal care influence hippocampal development in the rat. In the present study we found that variations in maternal licking/grooming (LG) during the first week of life are associated with altered hippocampal expression of BAX (group-1 tumor necrosis factor family mediated cell death effector) in 90-day-old male offspring. BAX-like immunoreactivity on western blots is significantly increased in the adult offspring of low-level LG mothers. There is no effect of maternal care on levels of either B-cell lymphoma-2 (BCL-2) (group-II mitochondria mediated cell death suppressor) or BAD (group-III endoplasmic reticulum mediated cell death effector). The most striking biochemical event in apoptosis is DNA fragmentation. Terminal deoxynucleotidyl transerferase (Tdt)-mediated dUTP-biotin nick-end labeling (TUNEL) and 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the number of TUNEL-positive cells in both the dentate gyrus and CA1 region of the hippocampus is significantly increased in the adult offspring of low-level LG mothers. In conclusion, we propose that hippocampal neurons in the offspring of low-level LG mothers may be more vulnerable to loss through apoptosis.     

The role of cardiolipin in promoting the membrane pore-forming activity of BAX oligomers

BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5–α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.     

A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX

The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.     

Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes

Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.     

BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane

Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this study, we have identified a native complex containing caspase-8 and BID on the mitochondrial membrane, and showed that death receptor activation by Fas or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced the cleavage of BID (tBID formation) within this complex. tBID then shifted to separate mitochondria-associated complexes that contained other BCL-2 family members, such as BAK and BCL-X(L). We report that cells stabilize active caspase-8 on the mitochondria in order to specifically target mitochondria-associated BID, and that BID cleavage on the mitochondria is essential for caspase-8-induced cytochrome c release. Our findings indicate that during extrinsic apoptosis, caspase-8 can specifically target BID where it is mostly needed, on the surface of mitochondria.     

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.     

TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation

Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.     

Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis

Nucleic acids are potent triggers for innate immunity. Double‐stranded DNA and RNA adopt different helical conformations, including the unusual Z‐conformation. Z‐DNA/RNA is recognised by Z‐binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM‐1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock‐in models, we report that mutation of key amino acids involved in Z‐DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1‐ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross‐linked to endogenous RNA. These observations show that Z‐RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.     

Gossypol induces apoptosis in human PC-3 prostate cancer cells by modulating caspase-dependent and caspase-independent cell death pathways

The rate of gossypol-induced apoptosis does not correlate very well with the same dose of gossypol-induced cell growth inhibition, indicating an anti-proliferative effect of gossypol. Using a co-immunoprecipitation assay, it was observed that the level of Bcl-X(L) protein bound to Bax was clearly lower than that of Bcl-2 protein at 5 micro M of gossypol treatment, and the level of Bim protein bound to Bcl-X(L) was lowered at 20 micro M of gossypol treatment for 24 h, implicating that gossypol inhibits the heterodimerization of Bcl-X(L) with Bax and Bim. Gossypol-induced apoptosis is partly suppressed by as low as 0.5 micro M, but not abolished by as high as 50 micro M of a broad range caspase inhibitor, Z-VAD-FMK, suggesting that gossypol-induced apoptosis is both caspase-dependent and -independent. Furthermore, the release of apoptosis inducing factor (AIF), which triggers caspase-independent apoptosis, from mitochondria to cytosol was observed in PC-3 cells exposed to gossypol treatment. In conclusion, gossypol inhibits the proliferation and induces apoptosis in PC-3 cells. Gossypol-induced apoptosis is, at least, through inhibiting the heterodimerization of Bcl-X(L)/Bcl-2 with pro-apoptosis molecules, followed by a caspase-dependent and -independent process which involves the release of AIF from the mitochondria to cytosol.     

Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts

Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.     

TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members

TNF receptor 1 signaling induces NF-κB activation and necroptosis in L929 cells. We previously reported that cellular inhibitor of apoptosis protein-mediated receptor-interacting protein 1 (RIP1) ubiquitination acts as a cytoprotective mechanism, whereas knockdown of cylindromatosis, a RIP1-deubiquitinating enzyme, protects against tumor necrosis factor (TNF)-induced necroptosis. We report here that RIP1 is a crucial mediator of canonical NF-κB activation in L929 cells, therefore questioning the relative cytoprotective contribution of RIP1 ubiquitination versus canonical NF-κB activation. We found that attenuated NF-κB activation has no impact on TNF-induced necroptosis. However, we identified A20 and linear ubiquitin chain assembly complex as negative regulators of necroptosis. Unexpectedly, and in contrast to RIP3, we also found that knockdown of RIP1 did not block TNF cytotoxicity. Cell death typing revealed that RIP1-depleted cells switch from necroptotic to apoptotic death, indicating that RIP1 can also suppress apoptosis in L929 cells. Inversely, we observed that Fas-associated protein via a death domain, cellular FLICE inhibitory protein and caspase-8, which are all involved in the initiation of apoptosis, counteract necroptosis induction. Finally, we also report RIP1-independent but RIP3-mediated necroptosis in the context of TNF signaling in particular conditions.