cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment

A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist–induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA–mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIP_L and not cFLIP_S interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor–mediated cell death.     

Interaction between cFLIPL and Itch,a ubiquitin ligase,is obstructed in Trypanosoma cruzi‐infected human cells

Death receptor‐mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite‐infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP_L, a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP_L, leading to proteasomal degradation, is inhibited in parasite‐infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP_L, was found to be equivalent in T. cruzi‐infected and in uninfected cells. However, co‐immunoprecipitation analysis showed that the interaction between cFLIP_L and Itch is strongly inhibited in T. cruzi‐infected cells. This unique parasite strategy, which has not been reported in any other pathogen‐infected cells, may allow the host cell to accumulate cFLIP_L, eventually resulting in the inhibition of apoptosis of T. cruzi‐infected cells.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIP_L, a protein that blocks caspase 8 activation. cFLIP_L levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIP_L in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.     

Bisindolylmaleimide IX facilitates extrinsic and initiates intrinsic apoptosis in TNF-α-resistant human colon adenocarcinoma COLO 205 cells

Human COLO 205 colon adenocarcinoma cells are immune to extrinsic apoptosis induced by immunomodulatory cytokines. Among the antiapoptotic mechanisms responsible for the immune escape, the overexpression of the cFLIP protein seems to be critical. cFLIP appears to inhibit the TNF-α-induced death receptor signal. The application of the metabolic inhibitor bisindolylmaleimide IX (Bis-IX), known as a potent PKC repressor, sensitized COLO 205 cells to TNF-α-mediated apoptosis. The Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP_L and TRADD protein levels. At the same time, the level of FADD protein was up-regulated. Additionally, the combined TNF-α and Bis-IX treatment caused cleavages of Bid and procaspase-9, as well as cytochrome c release. Thus, the evidence of this study indicates that Bis-IX facilitates the death receptor signal mediated by TNF-R1. Moreover, Bis-IX alone initiated intrinsic apoptosis, which could be abolished by Bcl-2 delivery. It heralds the involvement of mitochondria in caspase-8-independent intrinsic apoptosis. In turn, the treatment with bisindolylmaleimide III (Bis-III) did not assist TNF-α-dependent apoptosis.     

Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a mammalian dNTP hydrolase (dNTPase) that regulates intracellular dNTP balance. We have previously reported that SAMHD1 mRNA and protein levels are significantly downregulated in CD4⁺ T-cells of patients with cutaneous T-cell lymphoma (CTCL), a disease characterized by infiltration of neoplastic CD4⁺ T-lymphocytes into the skin. However, functional significance of SAMHD1 in CTCL development and progression remains unknown. Here we investigate the mechanism by which SAMHD1 induces apoptosis in CTCL-derived CD4⁺ T-cells. We stably expressed exogenous SAMHD1 in the CTCL-derived HuT78 T-cell line containing a very low level of endogenous SAMHD1 protein. We found that low-level exogenous expression of SAMHD1 led to a significant reduction in HuT78 cell growth, proliferation, and colony formation. Exogenous SAMHD1 expression in HuT78 cells also resulted in increased spontaneous and Fas ligand (Fas-L)-induced apoptosis levels via activation of the extrinsic pathway, including caspase-8, ?3 and ?7. Additionally, increased SAMHD1 significantly reduced the protein and mRNA expression of the short isoform of cFLIP (cFLIP_S), an important negative regulator of Fas-L-mediated apoptotic signaling. Our results indicate that exogenous SAMHD1 expression inhibits HuT78 cell growth and proliferation in part by increasing apoptosis. These findings implicate that SAMHD1 acts as an inhibitor in CTCL cell growth, suggesting that downregulation of SAMHD1 expression in neoplastic T-cells can facilitate uncontrolled cell proliferation.     

Sodium butyrate sensitizes human colon adenocarcinoma COLO 205 cells to both intrinsic and TNF-α-dependent extrinsic apoptosis

Overexpression of cFLIP protein seems to be critical in the antiapoptotic mechanism of immune escape of human COLO 205 colon adenocarcinoma cells. Actually, cFLIP appears to inhibit the death receptor ligand-mediated cell death. Application of the metabolic inhibitor sodium butyrate (NaBt), short-chain volatile fatty acid, sensitized COLO 205 cells to TNF-α-mediated apoptosis. Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP and simultaneous up-regulation of TNF-R1 protein levels. Additionally, the combined TNF-α and NaBt treatment caused cleavage of Bid and caspase-9 activation, as well as cytochrome c release from mitochondria. Thus, the evidence of this study indicates that NaBt facilitates the death receptor signal evoked by TNF-α. Moreover, NaBt alone initiated intrinsic apoptosis, that in turn was abolished by intracellular BCL-2 delivery. It confirms the involvement of mitochondria in the proapoptotic activity of NaBt. The activation of mitochondrial pathway was substantiated by up-regulated expression of BAK with concomitant reduction of antiapoptotic BCL-x_L, XIAP and survivin proteins. These findings suggest that NaBt could represent a good candidate for the new therapeutic strategy aimed to improve chemo- and immunotherapy of colon cancer.     

DED or alive: assembly and regulation of the death effector domain complexes

Death effector domains (DEDs) are protein–protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.     

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.     

Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G₁ cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-x_L, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.     

c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIP_L interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIP_L interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIP_L positively regulates autophagy, by enhancing Beclin-1 protein stability.Subject terms: Biochemistry, Autophagy     

Acute organ failure following the loss of anti-apoptotic cellular FLICE-inhibitory protein involves activation of innate immune receptors

Apoptosis signaling is involved in both physiological tissue homeostasis and acute and chronic diseases. The role of regulatory apoptosis signaling molecules and their organ-specific functions are less defined. Therefore, we investigated the loss of the anti-apoptotic cellular FLICE-inhibitory protein (cFLIP) and the mechanisms of the resulting lethal organ failure in vivo using inducible knockout mice. These were generated by crossing floxed cFLIP mice to a tamoxifen inducible Rosa26-creERT2 mouse strain. Death following global loss of cFLIP resulted from liver failure, accumulation of M1-polarized macrophages and accompanying hepatic cell death and inflammation. Apoptosis was also prominent in immune cells, the kidney and intestinal epithelial cells (IECs) but not in cardiomyocytes. Cellular injury led to the release of damage-associated molecular patterns (DAMPs) and the induction of innate immune receptors including toll-like receptors (TLRs) 4 and 9, and stimulator of interferon genes (STING). Transplantation of bone marrow with intact cFLIP or depletion of macrophages prevented the phenotype of acute liver failure. Interestingly, compound deletion of cFLIP in bone marrow-derived cells and hepatocytes did not promote organ failure. Thus, cFLIP exerts a critical role in tissue homeostasis by preventing the activation of monocytic cells and innate immunity, which causes cell death and inflammation in susceptible tissues. These results encourage the development of organ-specific anti-apoptotic and anti-inflammatory therapies in acute organ failure.The mortality of acute organ failure is high and the underlying pathophysiological mechanisms are poorly understood. Cellular injury from controlled (apoptosis and necroptosis) or uncontrolled (necrosis) cell death and metabolic, regulatory modes of tissue turnover (autophagy) contribute to the regulation of tissue homeostasis. Even minor alterations in the finely tuned balance of proliferation and cell death can lead to severe organ dysfunction or cancer.¹ In hepatocytes, apoptosis can be initiated through an extrinsic or intrinsic signaling pathway. Activation of the extrinsic signaling cascade involves cell surface bound receptors among which the tumor necrosis factor (TNF)-receptor superfamily is the most prominent.² Receptor-mediated apoptosis involves formation of an intracellular death-inducing signaling complex (DISC), which includes procaspase 8 and cellular FLICE-inhibitory protein (cFLIP) among others.² cFLIP is a caspase 8 homolog and exerts anti-apoptotic function by blocking caspase 8 activation. Loss of cFLIP has been shown to result in embryonic lethality from increased apoptosis of cardiomyocytes.³ cFLIP is critically involved in apoptosis- and stress-signaling pathways in IECs,⁴ hepatocytes,^{5, 6, 7} lymphocytes⁸ and myeloid lineage-derived cells.⁹ Recently, Piao et al.¹⁰ showed that deletion of cFLIP using different transgenic mouse strains impaired hepatocyte and IEC survival by inducing cell death dependent on TNF, Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL). Additionally, a Mx1-Cre line was used to assess the role of cFLIP in global cellular homeostasis and in these mice a phenotype with fatal hepatitis was observed. However, these studies did not exclude that interferon (IFN)-dependent signals triggered by poly I:C could promote cellular injury through, for example, TLR3 and melanoma differentiation-associated protein 5 (MDA-5). Also, the underlying pathomechanisms of this phenotype remained unresolved.To investigate the mechanisms of cFLIP-induced organ failure, we generated mice with conditional, ubiquitous deletion of cFLIP by crossing floxed cFLIP^f/f mice to a tamoxifen-inducible Rosa26-creERT2 mouse strain. Loss of cFLIP resulted in acute liver failure characterized hypoglycemia and hyperbilirubinemia, and was accompanied by depletion of intrahepatic leukocytes and the activation of inflammatory macrophages. All mice died within 96 h. Interestingly, this phenotype was not explained by the loss of cFLIP in hepatocytes or hematopoietic cells alone, indicating an organ-spanning crosstalk or the involvement of further compartments. Furthermore, we show that DAMPs, including cell-free double-stranded (ds)DNA, released during cell death induce upregulation and activation of dsDNA-sensing endosomal and cytosolic signaling pathways, namely TLR9 and STING, which contribute to an overwhelming inflammatory immune response and cell death. This phenotype was prevented by replenishment of cFLIP in bone marrow-derived cells (BMC) or depletion of macrophages.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Mapping protein interactions by combining antibody affinity maturation and mass spectrometry

Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (V_H) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 10⁸ to 10⁹ new variable light (V_L) genes. Improved binders were isolated by stringent selections from these new “chain-shuffled” libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein–protein interaction networks.     

The α1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells

Background

We have previously shown that c-FLIP_L is a more potent inhibitor than c-FLIP_S of Fas ligand-induced apoptosis and that c-FLIP_L physically binds to Daxx, an alternative Fas-signaling adaptor. Here we examined whether c-FLIP_S effectively inhibits TNFR1-mediated apoptosis and triggers JNK activation through its interaction with TRAF2.

Results

Some cancer cell lines, such as DU145, AGS, and PC3, have higher levels of c-FLIP_S than other cell lines, such as SNU-719 and T24. The expression of c-FLIP_S correlated with the susceptibility to TNFR1-mediated apoptosis. In contrast to DU145 and PC3, which are resistant to TNFR1-mediated apoptosis, T24 and SNU719 were sensitive to TNF-α treatment. To address the role of c-FLIP_S in TNFR1-mediated apoptosis, we examined the molecular interaction between c-FLIP_S and TRAF2. As expected, western blot analysis revealed that TRAF2 antibody immunoprecipitated a greater amount of c-FLIP_S than c-FLIP_L. Also, we measured the involvement of c-FLIP_S in TNF-α-induced JNK activation and apoptosis by comparing these in TNF-α-resistant and TNF-α-sensitive cell lines. Treatment with TNF-α increased the phosphorylated JNK level in SNU719 and T24 cells, whereas DU145 and AGS cells were resistant to TNF-α-mediated apoptosis.

Conclusion

We now report that the short form of c-FLIP_S is a more efficient inhibitor of TNF-receptor 1-mediated apoptosis signaling than the long form of the protein.     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.