MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly

When T cells are activated, the expression of the CD95 ligand is elevated, with the purpose of inducing apoptosis in target cells and to later eliminate the activated T cells. We have shown previously that mitogen-activated protein kinase (MAPK or ERK) signaling suppresses CD95-mediated apoptosis in different cellular systems. In this study we examined whether MAPK signaling controls the persistence and CD95-mediated termination of an immune response in activated T cells. Our results show that activation of Jurkat T cells through the T cell receptor immediately suppresses CD95-mediated apoptosis, and that this suppression is mediated by MAPK activation. During the phase of elevated MAPK activity, the activation of caspase-8 and Bid is inhibited, whereas the assembly of a functional death-inducing signaling complex (DISC) is not affected. These results explain the resistance to CD95 responses observed during the early phase of T cell activation and suggest that MAPK-activation deflects DISC signaling from activating caspase-8 and Bid. The physiological relevance of the results was confirmed in activated primary peripheral T cells, in which inhibition of MAPK signaling markedly sensitized the cells to CD95-mediated apoptosis.     

TCR-mediated up-regulation of c-FLIPshort correlates with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity

To investigate apoptosis resistance upon restimulation in human peripheral blood T lymphocytes, we used the following in vitro model. This model represents the main features of T cell reactivity: freshly isolated PHA-activated T cells cultured in IL-2 for a prolonged period of time develop a CD95 (APO-1/Fas) apoptosis-sensitive phenotype. These T cells represent activation-induced cell death-sensitive T cells during the down phase of an immune response. A fraction of apoptosis-sensitive activated T cells becomes apoptosis resistant upon TCR/CD3 restimulation. CD95 apoptosis sensitivity requires formation of a functional receptor associated death-inducing signaling complex (DISC), i.e., a protein complex of CD95 receptors, the adaptor Fas-associated death domain protein (FADD)/MORT1 and caspase-8 (FADD-like IL-1ss-converting enzyme (FLICE), MACH, Mch5). We identified activation of procaspase-8 at the DISC as the main target for the protective activity of TCR/CD3 restimulation. We found that procaspase-8 cleavage is reduced in T cells after TCR/CD3 restimulation. In addition, we detected up-regulation of c-FLIP(S) (the short splice variant of the cellular FLICE inhibitory protein) and strongly enhanced recruitment of c-FLIP(S) into the DISC. These data suggest that the recruitment of c-FLIP(S) into the DISC results in reduced DISC and caspase-8 activity.     

Adhesion-mediated intracellular redistribution of c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long confers resistance to CD95-induced apoptosis in hematopoietic cancer cell lines

Evasion of immune surveillance is a key step in malignant progression. Interactions between transformed hematopoietic cells and their environment may initiate events that confer resistance to apoptosis and facilitate immune evasion. In this report, we demonstrate that beta(1) integrin-mediated adhesion to fibronectin inhibits CD95-induced caspase-8 activation and apoptosis in hematologic tumor cell lines. This adhesion-dependent inhibition of CD95-mediated apoptosis correlated with enhanced c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long (c-FLIP(L)) cytosolic solubility compared with nonadhered cells. Cytosolic c-FLIP(L) protein preferentially associated with cytosolic Fas-associated death domain protein (FADD) and localized to the death-inducing signal complex after CD95 ligation in adherent cells. The incorporation of c-FLIP(L) in the death-inducing signal complex prevented procaspase-8 processing and activation of the effector phase of apoptosis. Adhesion to fibronectin increased c-FLIP(L) cytosolic solubility and availability for FADD binding by redistributing c-FLIP(L) from a preexisting membrane-associated fraction. Increased cytosolic availability of c-FLIP(L) for FADD binding was not related to increased levels of RNA or protein synthesis. These data show that adhesion of anchorage-independent cells to fibronectin provides a novel mechanism of resistance to CD95-mediated programmed cell death by regulating the cellular localization and availability of c-FLIP(L).     

Constitutive caspase activation and impaired death-inducing signaling complex formation in CD95-resistant,long-term activated,antigen-specific T cells

Elimination of T cells during an immune response is mediated by activation-induced cell death (AICD) and CD95-mediated apoptosis. Chronic graft-vs-host disease and T cell-mediated autoimmune diseases are caused by the persistence of activated T cells that escaped tolerance induction by deletion or silencing. To mimic the in vivo situation of long-term activated T cells, we generated an in vitro system using HLA-A1-specific T cells, weekly restimulated by Ag. While short-term activated T cells (two to five rounds of stimulation) were CD95 sensitive and susceptible to AICD, T cells stimulated more than eight times acquired constitutive CD95 resistance and exhibited reduced AICD. Phenotypically, these long-term activated T cells could be identified as effector/memory T cells. The expression of the proforms of the CD95 receptor initiator caspases, caspase-8 and -10, and the effector caspase-3 was strongly decreased in these cells, and only active caspase fragments were detected. In contrast to short-term activated T cells, constitutive CD95 receptor clustering was observed on the cell surface, and caspase-8 was bound to the CD95 receptor in the absence of receptor triggering. After further cross-linking of CD95, additional formation of the death-inducing signaling complex (DISC) was strongly impaired. Reduced DISC formation in long-term activated T cells was associated with the loss of PTEN expression and the increased phosphorylation of protein kinase B. Inhibitors of phosphoinositol 3-kinase restored CD95 sensitivity and DISC formation in long-term activated T cells. These data suggest that defective CD95 signaling in effector/memory T cells may contribute to the apoptosis resistance toward physiological stimuli in T cells mediating tissue destruction in vivo.     

Differential modulation of apoptosis sensitivity in CD95 type I and type II cells.

We have recently identified two different pathways of CD95-mediated apoptosis (Scaffidi, C., Fulda, S., Srinivasan, A., Feng, L., Friesen, C., Tomaselli, K. J., Debatin, K.-M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675-1687). CD95-mediated apoptosis in type I cells is initiated by large amounts of active caspase-8 formed at the death-inducing signaling complex (DISC) followed by direct cleavage of caspase-3. In contrast, in type II cells very little DISC and small amounts of active caspase-8 sufficient to induce the apoptogenic activity of mitochondria are formed causing a profound activation of both caspase-8 and caspase-3. Only in type II cells can apoptosis be blocked by overexpressed Bcl-2 or Bcl-x(L). We now show that a number of apoptosis-inhibiting or -inducing stimuli only affect apoptosis in type II cells, indicating that they act on the mitochondrial branch of the CD95 pathway. These stimuli include the activation of protein kinase C, which inhibits CD95-mediated apoptosis resulting in a delayed cleavage of BID, and the induction of apoptosis by the ceramide analog C(2)-ceramide. In addition, we have identified the CD95 high expressing cell line Boe(R) as a CD95 apoptosis-resistant type II cell that can be sensitized by treatment with cycloheximide without affecting formation of the DISC. This also places the effects of cycloheximide in the mitochondrial branch of the type II CD95 pathway. In contrast, c-FLIP was found to block CD95-mediated apoptosis in both type I and type II cells, because it acts directly at the DISC of both types of cells.     

Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at?the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the?stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.     

Inhibition of RIP and c-FLIP enhances TRAIL-induced apoptosis in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it is capable of preferentially inducing apoptosis in human cancer over normal cells. The majority of human pancreatic cancers, unfortunately, are resistant to TRAIL treatment. Here, we show that the inhibition of caspase-8 cleavage is the most upstream event in TRAIL resistance in pancreatic cancers. TRAIL treatment led to the cleavage of caspase-8 and downstream caspase-9, caspase-3, and DNA fragmentation factor 45 (DFF45) in TRAIL-sensitive pancreatic cancer cell lines (BXPC-3, PACA-2). This caspase-8-initiated caspase cascade, however, was inhibited in TRAIL-resistant pancreatic cancer cell lines (PANC-1, ASPC-1, CAPAN-1, CAPAN-2). The long and short forms of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP(L), c-FLIP(S)) were highly expressed in the TRAIL-resistant as compared to the sensitive cells; knockdown of c-FLIP(L) and c-FLIP(S) by a short hairpin RNA (shRNA) rendered the resistant cells sensitive to TRAIL-induced apoptosis through the cleavage of caspase-8 and activation of the mitochondrial pathway. Receptor-interacting protein (RIP) has been reported in TRAIL-induced activation of NF-kappaB and we show here that knockdown of RIP sensitized the resistant cells to TRAIL-induced apoptosis. These results indicate the role of c-FLIP and RIP in caspase-8 inhibition and thus TRAIL resistance. Treatment of the resistant cells with camptothecin, celecoxib and cisplatin resulted in the downregulation of c-FLIP and caused a synergistic apoptotic effect with TRAIL. These studies therefore suggest that combination treatment with chemotherapy can overcome TRAIL resistance and enhance TRAIL therapeutic efficacy in treating pancreatic cancers.     

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP_L, and the short c-FLIP isoform, c-FLIP_R, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.     

Cellular FLIP (long form) regulates CD8+ T cell activation through caspase-8-dependent NF-kappa B activation

Cellular FLIP long form (c-FLIP(L)) was originally identified as an inhibitor of Fas (CD95/Apo-1). Subsequently, additional functions of c-FLIP(L) were identified through its association with receptor-interacting protein (RIP)1 and TNFR-associated factor 2 to activate NF-kappaB, as well as by its association with and activation of caspase-8. T cells from c-FLIP(L)-transgenic (Tg) mice manifest hyperproliferation upon activation, although it was not clear which of the various functions of c-FLIP(L) was involved. We have further explored the effect of c-FLIP(L) on CD8(+) effector T cell function and its mechanism of action. c-FLIP(L)-Tg CD8(+) T cells have increased proliferation and IL-2 responsiveness to cognate Ags as well as to low-affinity Ag variants, due to increased CD25 expression. They also have a T cytotoxic 2 cytokine phenotype. c-FLIP(L)-Tg CD8(+) T cells manifest greater caspase activity and NF-kappaB activity upon activation. Both augmented proliferation and CD25 expression are blocked by caspase inhibition. c-FLIP(L) itself is a substrate of the caspase activity in effector T cells, being cleaved to a p43(FLIP) form. p43(FLIP) more efficiently recruits RIP1 than full-length c-FLIP(L) to activate NF-kappaB. c-FLIP(L) and RIP1 also coimmunoprecipitate with active caspase-8 in effector CD8(+) T cells. Thus, one mechanism by which c-FLIP(L) influences effector T cell function is through its activation of caspase-8, which in turn cleaves c-FLIP(L) to allow RIP1 recruitment and NF-kappaB activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.     

Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8

Caspase activity is required not only for the death of T cells, but also for their activation. A delicate balance of caspase activity is thus required during T cell activation at a level that will not drive cell death. How caspase activity is initiated and regulated during T cell activation is not known. One logical candidate for this process is cellular FLIP long form (c-FLIP(L)), because it can block caspase-8 recruitment after Fas (CD95) ligation as well as directly heterodimerize with and activate caspase-8. The current findings demonstrate that after T cell activation, caspase-8 and c-FLIP(L) associate in a complex enriched for active caspases. This occurs coincidently with the cleavage of two known caspase-8 substrates, c-FLIP(L) and receptor interacting protein 1. Caspase activity is higher in wild-type CD8(+) than CD4(+) effector T cells. Increased expression of c-FLIP(L) results in augmented caspase activity in resting and effector T cells to levels that provoke cell death, especially of the CD8 subset. c-FLIP(L) is thus not only an inhibitor of cell death by Fas, it can also act as a principal activator of caspases independently of Fas.     

Fever-like hyperthermia controls T Lymphocyte persistence by inducing degradation of cellular FLIPshort

Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIP(S)) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIP(S), the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIP(S). Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.     

Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

Cellular FADD-like interleukin-1β–converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP_L], c-FLIP short [c-FLIP_S], and c-FLIP Raji [c-FLIP_R]) regulate caspase-8 activation and death receptor (DR)–induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP_L has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP_S or c-FLIP_R. Our findings resolve the present controversial discussion on the function of c-FLIP_L as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Effector CD4+ T cells generate intermediate caspase activity and cleavage of caspase-8 substrates

Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4(+) T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIP(L), receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4(+) T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

CD21/CD19 coreceptor signaling promotes B cell survival during primary immune responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2-/- MD4 B cells do not. In contrast, Cr2-/- MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.     

Glutathione dependence of caspase-8 activation at the death-inducing signaling complex.

Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.     

Expression level of c-FLIP versus Fas determines susceptibility to Fas ligand-induced cell death in murine thymoma EL-4 cells

The caspase-8 inhibitor c-FLIP blocks death receptor-mediated cell death and plays an essential role in the regulation of lymphocyte homeostasis and the immune escape of tumors. The murine thymoma cell line EL-4 was resistant to Fas ligand (FasL)-induced apoptosis by constitutive expression of FLIP (L). Cycloheximide downregulated the expression of FLIP (L) and markedly sensitized EL-4 cells to FasL-induced apoptosis. In contrast, DNA-damaging agents sensitized EL-4 cells to FasL-induced cell death via an increase of cell-surface Fas without any influence on FLIP (L) expression. Enforced expression of transfected Fas rendered EL-4 cells highly susceptible to FasL-induced cell death. These findings demonstrate that susceptibility to FasL-induced cell death mainly depends on the expression level of c-FLIP versus cell-surface Fas.     

Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis

Death receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP(S)) and a long, 55-kDa form (c-FLIP(L)). The role of c-FLIP(S) as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIP(L) remains controversial. Although overexpression of transfected c-FLIP(L) inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIP(L) on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIP(L) in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIP(L) augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIP(L) functions primarily as an inhibitor of death receptor-mediated apoptosis.