Death Receptor Stimulation Research Articles

Palmitoylation Controls Death Receptor Function in Chronic Lymphocytic Leukemia

Abstract 2829 Introduction:Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Palmitoylation is a common post-translational modification and defined as the addition of palmitic acid to internal cysteins. Interestingly, in contrast to other lipid modifications, it is reversible. Control over the palmitoylation cycle therefore provides indirect control over protein localization and function. While a number of proteins with palmitoyl transferase activity are known, LYPLA1 (lysophospholipase 1) is the only enzyme known to be responsible for the process of depalmitoylation. CLL cells are known to be resistant to TRAIL-mediated apoptosis. While TRAIL-R1 is reported to be palmitoylated, TRAIL-R2 seems to contain a region with basic amino acids in its membrane-proximal cytoplasmatic domain. Some studies showed that palmitoylation is crucial for several steps of death receptor signaling. Therefore, regulation of depalmitoylation by LYPLA1 seems to be an important tool for the regulation of death receptor function.Methods and Results: Global palmitoylation in CLL cells was investigated by screening for all palmitoylated proteins via a click chemistry assay. There, cells were metabolically labeled, coupled to a specific reporter group and then analyzed by in-gel fluorescence. Comparison of healthy B cells, healthy PBMCs and CLL cells revealed a significant difference in global palmitoylation (+38.5 % in B cells, n=6, p<0.001; +57.8 % in healthy PBMCs, n=6, p<0.001 compared to CLL cells, n=10). We identified LYPLA1 as overexpressed in CLL compared to healthy controls on both protein and mRNA level. We generated a potent LYPLA1 inhibitor. We could show, that inhibition of LYPLA1 led to a significant increase of the overall protein palmitoylation level in CLL cells (+24.7 % n=6, p=0.0118). Ours and other groups have shown, that treatment of cancer cells with TRAIL and X-linked inhibitor of apoptosis protein (XIAP)-inhibition lead to apoptosis in otherwise TRAIL resistant CLL cells. Since death receptors might be palmitoylated, we extended these studies. Treatment of CLL cells with TRAIL, XIAP- and LYPLA1-inhibition led to significantly increased apoptosis compared to TRAIL treatment and XIAP-inhibition alone (+43.2 %, n=12, p=0.0089). Palmitoylation of death receptors was investigated with the help of acyl-biotin exchange chemistry. We could show that palmitoylation of TRAIL-R1 was significantly increased after LYPLA1-inhibiton (+58.7 %, n=3, p=0.0169). It could be demonstrated, that inhibition of LYPLA1 in combination with death receptor stimulation increased the amount of activated caspase-8 in comparison to solely TRAIL and DMSO treated cells (+41.8 %, n=3, p=0.0199), indicating that palmitoylation plays a crucial role in apoptotic signaling far from XIAP. In addition to that, we could show that inhibition of depalmitoylation of TRAIL-R1 led to more death receptor located to lipid rafts. To understand how LYPLA1 is regulated, we investigated two highly conserved miRNAs which were predicted as key regulators of LYPLA1 and which are significantly downregulated in CLL. Indeed, luciferase assays revealed that both miRNAs were able to downregulate LYPLA1 expression. Conclusion:We show for the first time, that LYPLA1 is a central enzyme which regulates the apoptotic signaling of TRAIL. Furthermore, we identified LYPLA1 to be regulated by miRNAs, which are deregulated in CLL. These novel findings allow speculation, that LYPLA1 inhibitors could be used for the treatment of CLL. Future experiments should therefore aim at investigating the LYPLA1 signaling pathway as a potential target for CLL/ cancer therapy.L.P.F. and V.F. contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

The Prevalence of TNFα-Induced Necrosis over Apoptosis Is Determined by TAK1-RIP1 Interplay

Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIPL. The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.

Harnessing Tumor Necrosis Factor Receptors to Enhance Antitumor Activities of Drugs

Cancer is the second-leading cause of death in the U.S. behind heart disease and over stroke. The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The inhibition of cell death pathways is one of these tumor characteristics which also include sustained proliferative signaling, evading growth suppressor signaling, replicative immortality, angiogenesis, and promotion of invasion and metastasis. Cell death is mediated through death receptor (DR) stimulation initiated by specific ligands that transmit signaling to the cell death machinery or through the participation of mitochondria. Cell death involving DR is mediated by the superfamily of tumor necrosis factor receptor (TNF-R) which includes TNF-R type I, CD95, DR3, TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 (TRAIL-R1) and -2 (TRAIL-R2), DR6, ectodysplasin A (EDA) receptor (EDAR), and the nerve growth factor (NGF) receptor (NGFR). The expression of these receptors in healthy and tumor cells induces treatment side effects that limit the systemic administration of cell death-inducing therapies. The present review is focused on the different therapeutic strategies such as targeted antibodies or small molecules addressed to selective stimulated DR-mediated apoptosis or reduce cell proliferation in cancer cells.

Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α and death receptor stimulation in resistant variants of the human U937 cell line

Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

Dysregulation of TNFα-Induced Necroptotic Signaling In Chronic Lymphocytic Leukemia: Suppression of CYLD Gene by LEF1

AbstractAbstract 377Impaired cell death program has been noted as one of the hallmarks of Chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through apoptosis or necrosis pathway. While apoptosis is known as a regulated cellular program, necrosis is known as an accidental event caused by overwhelming stress. However, accumulating evidence suggests that necrosis can also be executed by regulated mechanisms, especially in apoptotic-deficient conditions. Recently, the term necroptosis has been used to designate one particular form of programmed necrosis induced by stimulating death receptors with agonists such as TNFα, FasL, and TRAIL. Apoptosis suppression by caspase inhibitors such as zVAD may switch apoptotic response to necroptosis or enhance necroptosis. In contrast to well-characterized apoptotic pathway, the detailed molecular mechanisms underlying necroptosis are still not fully understood. A genome wide siRNA screen revealed two members of the receptor interacting protein (RIP) kinase family, RIP1 and RIP3P, to be essential for necroptosis. Upon stimulation of death receptors, RIP3 is recruited to RIP1 to form a necroptosis-inducing complex which is essential for cell death execution. The deubiquitinase cylindromatosis (CYLD) is recruited to TNFα receptor upon its activation and directly regulates RIP1 ubiquitination. In addition, by activating key enzymes of metabolic pathways, RIP3 regulates TNFα-inducing mitochondrial reactive oxygen species (ROS) production, which partly accounts for its ability to potentiate necroptosis. Until now, much less is known about the significance of necroptosis in malignant disease.Here we demonstrate that primary CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor zVAD. Upon TNFα+zVAD stimulation, normal CD19+ B cells increased ROS production > 8 fold, while same treatment only resulted in ∼ 2 fold induction in ROS generation in CLL samples. Two core components of necroptotic machine, RIP3 and CYLD, are markedly down-regulated in CLL compared with normal B cells, at both protein and transcription levels.Moreover, we identified LEF1, a downstream effector of Wnt/β-catenin pathway, as a transcription repressor of CYLD in CLL. LEF1 is highly expressed in CLL cells, whereas normal B cells have very low levels of LEF1 expression. Attenuation of LEF1 expression through RNAi technology resulted in a dramatic increase in CYLD levels in CLL cells, as determined by western blot and real time RT-PCR analysis. Dual-luciferase assays showed that forced expression of LEF1 markedly decreased CYLD promoter activity compared with controls. Mutation of LEF1 responsive elements (LERs) on CYLD promoter significantly abolished transcriptional repression of CYLD by LEF1. Chromatin immunoprecipitation assays showed that LEF1 is recruited to LER region within the CYLD promoter in CLL cells. Additionally, Knocking down LEF1 sensitizes CLL cells to TNFα-induced necroptosis.The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine such as LEF1 to restore this pathway may represent a novel approach for CLL treatment. Disclosures:No relevant conflicts of interest to declare.

ChemInform Abstract: Albanol A from the Root Bark of Morus alba L. Induces Apoptotic Cell Death in HL60 Human Leukemia Cell Line.

Albanol A (1), isolated from the root bark extract of Morus alba (mulberry), was evaluated for the cytotoxic and apoptosis-inducing activities in human leukemia (HL60) cells, and for the inhibitory activity in human DNA topoisomerases (Topo) I and II. This compound showed potent cytotoxic activity (IC(50) 1.7 microM) on the cells, and potent inhibitory activity on topoisomerase II (IC(50) 22.8 microM). In addition, albanol A induced early apoptosis which was detected by observing the membrane phospholipid exposure in flow cytometry. Western blot analysis showed that albanol A markedly reduced the levels of procaspases-3, 8, and 9, while being increased the levels of cleaved caspases-3, 8, and 9. The Bax/Bcl-2 ratio was significantly increased by albanol A treatment. Furthermore, albanol A induced caspase-2 activation. These results suggested that albanol A induces apoptotic cell death in HL60 via both the cell death receptor pathway by stimulation of death receptor, and the mitochondrial pathway by Topo II inhibition through caspase-2 activation. Therefore, albanol A may be a promising lead compound for developing an effective drug for treatment of leukemia.

Cryptocaryone, a Natural Dihydrochalcone, Induces Apoptosis in Human Androgen Independent Prostate Cancer Cells by Death Receptor Clustering in Lipid Raft and Nonraft Compartments

Androgen refractory prostate cancer is a major clinical challenge. Treatment approaches to prostate cancer are based on various mechanisms that cause malignant cell apoptosis. Of these strategies the anticancer effect of triggering death receptors is well substantiated. Several pharmacological and biochemical assays were used to characterize the apoptotic signaling pathways of the natural dihydrochalcone cryptocaryone in prostate cancer cells. Cryptocaryone induced antiproliferative and apoptotic effects in human androgen independent prostate cancer cells. It induced caspase-8 and 3 activation but did not change total protein levels of death receptors and their ligands. DR5 surface expression was moderately increased by cryptocaryone. Confocal immunofluorescence examination showed that cryptocaryone induced Fas clustering and the association of downstream signaling molecules, including FADD and procaspase-8. DR4 and DR5 aggregation was also induced by cryptocaryone. Data were confirmed by protein profile analysis of detergent resistant membranes showing that Fas, DR4, DR5, FADD and procaspase-8 levels were increased 1.3, 3.5, 4.1, 13.1 and 4.1-fold, respectively, in the lipid raft compartment. Cryptocaryone mediated clustering of death receptors and associated molecules was also detected in nonraft compartments. The distribution between lipid raft and nonraft compartments was validated by the cholesterol depleting agent methyl-beta-cyclodextrin. Cryptocaryone significantly potentiated FasL induced apoptosis in PC-3 cells. We suggest that cryptocaryone has anticancer activity via the stimulation of death receptor and associated molecule clustering, leading to caspase-8 and 3 activation, and apoptosis in prostate cancer cells.

Albanol A from the Root Bark of Morus alba L. Induces Apoptotic Cell Death in HL60 Human Leukemia Cell Line

Albanol A (1), isolated from the root bark extract of Morus alba (mulberry), was evaluated for the cytotoxic and apoptosis-inducing activities in human leukemia (HL60) cells, and for the inhibitory activity in human DNA topoisomerases (Topo) I and II. This compound showed potent cytotoxic activity (IC(50) 1.7 microM) on the cells, and potent inhibitory activity on topoisomerase II (IC(50) 22.8 microM). In addition, albanol A induced early apoptosis which was detected by observing the membrane phospholipid exposure in flow cytometry. Western blot analysis showed that albanol A markedly reduced the levels of procaspases-3, 8, and 9, while being increased the levels of cleaved caspases-3, 8, and 9. The Bax/Bcl-2 ratio was significantly increased by albanol A treatment. Furthermore, albanol A induced caspase-2 activation. These results suggested that albanol A induces apoptotic cell death in HL60 via both the cell death receptor pathway by stimulation of death receptor, and the mitochondrial pathway by Topo II inhibition through caspase-2 activation. Therefore, albanol A may be a promising lead compound for developing an effective drug for treatment of leukemia.

A primate-specific POTE-actin fusion protein plays a role in apoptosis

The primate-specific gene family, POTE, is expressed in many cancers but only in a limited number of normal tissues (testis, ovary, prostate). The 13 POTE paralogs are dispersed among 8 human chromosomes. They evolved by gene duplication and remodeling from an ancestral gene, Ankrd26, recently implicated in controlling body size and obesity. In addition, several POTE paralogs are fused to an actin retrogene producing POTE-actin fusion proteins. The biological function of the POTE genes is unknown, but their high expression in primary spermatocytes, some of which are undergoing apoptosis, suggests a role in inducing programmed cell death. We have chosen Hela cells as a model to study POTE function in human cancer, and have identified POTE-2alpha-actin as the major transcript and the protein it encodes in Hela cells. Transfection experiments show that both POTE-2alpha-actin and POTE-2gammaC are localized to actin filaments close to the inner plasma membrane. Transient expression of POTE-2alpha-actin or POTE-2gammaC induces apoptosis in Hela cells. Using wild-type and mutant mouse embryo cells, we find apoptosis induced by over-expression of POTE-2gammaC is decreased in Bak ( -/- ) or Bak ( -/- ) Bax ( -/- ) cells indicating POTE is acting through a mitochondrial pathway. Endogenous POTE-actin protein levels but not RNA levels increased in a time dependent manner by stimulation of death receptors with their cognate ligands. Our data indicates that the POTE gene family encodes a new family of proapoptotic proteins.

A New C-Terminal Cleavage Product of Procaspase-8, p30, Defines an Alternative Pathway of Procaspase-8 Activation

Caspase-8 is the main initiator caspase in death receptor-induced apoptosis. Procaspase-8 is activated at the death-inducing signaling complex (DISC). Previous studies suggested a two-step model of procaspase-8 activation. The first cleavage step occurs between the protease domains p18 and p10. The second cleavage step takes place between the prodomain and the large protease subunit (p18). Subsequently, the active caspase-8 heterotetramer p18(2)-p10(2) is released into the cytosol, starting the apoptotic signaling cascade. In this report, we have further analyzed procaspase-8 processing upon death receptor stimulation directly at the DISC and in the cytosol. We have found an alternative sequence of cleavage events for procaspase-8. We have demonstrated that the first cleavage can also occur between the prodomain and the large protease subunit (p18). The resulting cleavage product, p30, contains both the large protease subunit (p18) and the small protease subunit (p10). p30 is further processed to p10 and p18 by active caspases. Furthermore, we show that p30 can sensitize cells toward death receptor-induced apoptosis. Taken together, our data suggest an alternative mechanism of procaspase-8 activation at the DISC.

Uncovering operational interactions in genetic networks using asynchronous Boolean dynamics

Biological networks of large dimensions, with their diagram of interactions, are often well represented by a Boolean model with a family of logical rules. The state space of a Boolean model is finite, and its asynchronous dynamics are fully described by a transition graph in the state space. In this context, a model reduction method will be developed for identifying the active or operational interactions responsible for a given dynamic behaviour. The first step in this procedure is the decomposition of the asynchronous transition graph into its strongly connected components, to obtain a “reduced” and hierarchically organized graph of transitions. The second step consists of the identification of a partial graph of interactions and a sub-family of logical rules that remain operational in a given region of the state space. This model reduction method and its usefulness are illustrated by an application to a model of programmed cell death. The method identifies two mechanisms used by the cell to respond to death-receptor stimulation and decide between the survival and apoptotic pathways.

PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability

Cellular FLICE-inhibitory protein (c-FLIP) proteins are crucial regulators of the death-inducing signaling complex (DISC) and caspase-8 activation. To date, three c-FLIP isoforms with distinct functions and regulation have been identified. Our previous studies have shown that the stability of c-FLIP proteins is subject to isoform-specific regulation, but the underlying molecular mechanisms have not been known. Here, we identify serine 193 as a novel in vivo phosphorylation site of all c-FLIP proteins and demonstrate that S193 phosphorylation selectively influences the stability of the short c-FLIP isoforms, as S193D mutation inhibits the ubiquitylation and selectively prolongs the half-lives of c-FLIP short (c-FLIP(S)) and c-FLIP Raji (c-FLIP(R)). S193 phosphorylation also decreases the ubiquitylation of c-FLIP long (c-FLIP(L)) but, surprisingly, does not affect its stability, indicating that S193 phosphorylation has a different function in c-FLIP(L). The phosphorylation of this residue is operated by the protein kinase C (PKC), as S193 phosphorylation is markedly increased by treatment with 12-O-tetradecanoylphorbol-13-acetate and decreased by inhibition of PKCalpha and PKCbeta. S193 mutations do not affect the ability of c-FLIP to bind to the DISC, although S193 phosphorylation is increased by death receptor stimulation. Instead, S193 phosphorylation affects the intracellular level of c-FLIP(S), which then determines the sensitivity to death-receptor-mediated apoptosis. These results reveal that the differential stability of c-FLIP proteins is regulated in an isoform-specific manner by PKC-mediated phosphorylation.

Identification of a Molecular Signaling Network that Regulates a Cellular Necrotic Cell Death Pathway

Stimulation of death receptors by agonists such as FasL and TNFalpha activates apoptotic cell death in apoptotic-competent conditions or a type of necrotic cell death dependent on RIP1 kinase, termed necroptosis, in apoptotic-deficient conditions. In a genome-wide siRNA screen for regulators of necroptosis, we identify a set of 432 genes that regulate necroptosis, a subset of 32 genes that act downstream and/or as regulators of RIP1 kinase, 32 genes required for death-receptor-mediated apoptosis, and 7 genes involved in both necroptosis and apoptosis. We show that the expression of subsets of the 432 genes is enriched in the immune and nervous systems, and cellular sensitivity to necroptosis is regulated by an extensive signaling network mediating innate immunity. Interestingly, Bmf, a BH3-only Bcl-2 family member, is required for death-receptor-induced necroptosis. Our study defines a cellular signaling network that regulates necroptosis and the molecular bifurcation that controls apoptosis and necroptosis.

NDR Kinase Is Activated by RASSF1A/MST1 in Response to Fas Receptor Stimulation and Promotes Apoptosis

Human NDR1 and 2 (NDR1/2) are serine-threonine protein kinases in a subgroup of the AGC kinase family. The mechanisms of physiological NDR1/2 activation and their function remain largely unknown. Here we report that Fas and TNF-alpha receptor stimulation activates human NDR1/2 by promoting phosphorylation at the hydrophobic motif (Thr444/442). Moreover, NDR1/2 are essential for Fas receptor-induced apoptosis as shown by the fact that NDR knockdown significantly reduced cell death whereas overexpression of the NDR1 kinase further potentiated apoptosis. Activation of NDR1/2 by death receptor stimulation is mediated by the tumor suppressor RASSF1A. Furthermore, RASSF1A-induced apoptosis largely depends on the presence of NDR1/2. Fas receptor stimulation promoted direct phosphorylation and activation of NDR1/2 by the mammalian STE20-like kinase 1 (MST1), a downstream effector of RASSF1A. Concurrently, the NDR1/2 coactivator MOB1 induced MST1-NDR-MOB1 complex formation, which is crucial for MST1-induced NDR1/2 phosphorylation upon induction of apoptosis. Our findings identify NDR1/2 as novel proapoptotic kinases and key members of the RASSF1A/MST1 signaling cascade.

Identification of an antiapoptotic protein complex at death receptors

Stimulation of death receptors activates the extrinsic apoptotic signaling pathway that leads to cell death. Although many steps of this apoptotic signaling cascade are known, few mechanisms that counterbalance the death signal have been described. We identified an antiapoptotic protein complex associated with death receptors that contains glycogen synthase kinase-3 (GSK3), DDX3 and cellular inhibitor of apoptosis protein-1 (cIAP-1). GSK3, DDX3 and cIAP-1 are associated in cells with each other and with death receptors. Blocking the actions of GSK3 or DDX3 potentiated caspase-3 activation induced by stimulation of four different death receptors in several types of cells. GSK3 restrained apoptotic signaling by inhibiting formation of the death-inducing signaling complex and caspase-8 activation. Stimulated death receptors surmount the antiapoptotic complex by causing GSK3 inactivation and cleavage of DDX3 and cIAP-1 to enable progression of the apoptotic signaling cascade, but the antiapoptotic complex remains functional in cancer cells resistant to death receptor stimulation, a resistance that is overcome by GSK3 inhibitors. Thus, an antiapoptotic complex of GSK3, DDX3 and cIAP-1 caps death receptors, providing a checkpoint to counterbalance apoptotic signaling.

A Life or Death Decision

Stimulation of lymphocyte death receptors leads to activation of the cysteine protease caspase-8, which undergoes autoproteolysis to produce a mature form that cleaves effector caspases (such as caspase-3) as part of a proteolytic cascade that ends in apoptosis. In contrast, caspase-8 activation during antigenic stimulation, which involves its association with a complex that contains Bcl10 and MALT1, is required for lymphocyte proliferation. Kawadler et al . found that interleukin-2 (IL-2) production was reduced in activated human T cells (stimulated with antibodies directed against CD3 and a chimeric form of CD28) in which caspase-8 was knocked down with siRNA. Caspase-8 activated by antibody stimulation underwent limited autoproteolysis and failed to cleave substrate effector caspases appreciably. Activation of the transcription factor NF-κB in response to coexpression of MALT1 and Bcl10 was attenuated in Jurkat lymphoma cells lacking caspase-8; moreover, MALT1 knockdown prevented caspase-8 activation in primary human T cells. Noting that MALT1 contains a paracaspase domain (distantly related to the caspase proteolytic domain), the authors used a controlled heterodimerization system to show that heterodimerization of MALT1 and caspase-8 led to cleavage of the latter. The MALT1 paracaspase domain bound to the caspase-8 protease domain; however, mutational analysis established that caspase-8 cleavage was autoproteolytic and did not depend on MALT1 protease activity. MALT1 failed to stimulate caspase-8–mediated proteolysis of caspase-3, although it did stimulate proteolysis of the caspase homolog c-FLIP L . c-FLIP L was cleaved after antibody stimulation of lymphocytes, and its knockdown inhibited IL-2 production. Moreover, mutational analysis revealed that MALT1-dependent activation of caspase-8 and proteolysis of c-FLIP L were critical to NF-κB activation and IL-2 production. Thus, MALT1 appears to activate caspase-8 in a manner that affects its processing and substrate specificity, which leads to proliferative—rather than apoptotic—signaling. H. Kawadler, M. A. Gantz, J. L. Riley, X. Yang, The paracaspase MALT1 controls caspase-8 activation during lymphocyte proliferation. Mol. Cell 31 , 415-421 (2008). [PubMed]

MUC1 Oncoprotein Blocks Death Receptor–Mediated Apoptosis by Inhibiting Recruitment of Caspase-8

Stimulation of the death receptor superfamily induces the activation of caspase-8 and thereby the apoptotic response. The MUC1 oncoprotein is aberrantly overexpressed by diverse human malignancies and inhibits stress-induced apoptosis. The present results show that MUC1 blocks activation of caspase-8 and apoptosis in the response of malignant cells to tumor necrosis factor alpha, tumor necrosis factor-related apoptosis-inducing ligand, and Fas ligand. The results show that MUC1 associates constitutively with caspase-8. The MUC1 cytoplasmic domain (MUC1-CD) binds directly to the caspase-8 p18 fragment upstream to the catalytic Cys(360) site. The results also show that MUC1-CD binds to Fas-associated death domain (FADD) at the death effector domain. In nonmalignant epithelial cells, MUC1 interacts with caspase-8 and FADD as an induced response to death receptor stimulation. The functional significance of these interactions is supported by the demonstration that MUC1 competes with caspase-8 for binding to FADD and blocks recruitment of caspase-8 to the death-inducing signaling complex. These findings indicate that MUC1 is of importance to the physiologic regulation of caspase-8 activity and that overexpression of MUC1, as found in human malignancies, could contribute to constitutive inhibition of death receptor signaling pathways.

Cytotoxicity of contact lens multipurpose solutions: Role of oxidative stress, mitochondrial activity and P2X7 cell death receptor activation

Objective To investigate the cytotoxicity of multipurpose solutions used for contact lens disinfection. Methods Four multipurpose solutions (Optifree ® containing quaternary ammonium as preservative, Renu ®, Solocare ® and Complete ® containing polyhexamethylene biguanide as preservative) were incubated on a conjunctival cell line to evaluate their capacity to induce necrosis (neutral red test), intracellular redox status alteration (Alamar Blue test), reactive oxygen species overproduction (DCFH-DA and dihydroethidium tests), mitochondrial alterations (NonylAcridine Orange and JC-1 tests) and to activate P2X7 cell death receptor (YO-PRO-1 test). Tests were performed using cytofluorometry and inverted fluorescence microscopy. Results Our results showed that multipurpose solutions induced necrosis, in addition to oxidative stress. Optifree ® induced oxidative stress with increased mitochondrial mass, Renu ®, Solocare ® and Complete ® induced whether a decrease in reactive oxygen species production with mitochondrial alterations, or an increase in reactive oxygen species production, but each solution stimulated P2X7 cell death receptor activation. Early stimulation of P2X7 cell death receptor, prior to any superoxide production, demonstrated the possible involvement of this receptor in the oxidative stress process. Conclusion These new findings suggest that most of multipurpose solutions are toxic with oxidative stress and apoptosis induction.

Quercetin enhances TRAIL-mediated apoptosis in colon cancer cells by inducing the accumulation of death receptors in lipid rafts

Cytokines such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in colon cancer cells through engagement of death receptors. Nevertheless, evading apoptosis induced by anticancer drugs characterizes many types of cancers. This results in the need for combination therapy. In this study, we have investigated whether the flavonoid quercetin could sensitize human colon adenocarcinoma cell lines to TRAIL-induced apoptosis. We report that quercetin enhanced TRAIL-induced apoptosis by causing the redistribution of DR4 and DR5 into lipid rafts. Nystatin, a cholesterol-sequestering agent, prevented quercetin-induced clustering of death receptors and sensitization to TRAIL-induced apoptosis in colon adenocarcinoma cells. In addition, our experiments show that quercetin, in combination with TRAIL, triggered the mitochondrial-dependent death pathway, as shown by Bid cleavage and the release of cytochrome c to the cytosol. Together, our findings propose that quercetin, through its ability to redistribute death receptors at the cell surface, facilitates death-inducing signaling complex formation and activation of caspases in response to death receptor stimulation. Based on these results, this study provides a challenging approach to enhance the efficiency of TRAIL-based therapies.

RIP1, a kinase on the crossroads of a cell's decision to live or die

Binding of inflammatory cytokines to their receptors, stimulation of pathogen recognition receptors by pathogen-associated molecular patterns, and DNA damage induce specific signalling events. A cell that is exposed to these signals can respond by activation of NF-kappaB, mitogen-activated protein kinases and interferon regulatory factors, resulting in the upregulation of antiapoptotic proteins and of several cytokines. The consequent survival may or may not be accompanied by an inflammatory response. Alternatively, a cell can also activate death-signalling pathways, resulting in apoptosis or alternative cell death such as necrosis or autophagic cell death. Interplay between survival and death-promoting complexes continues as they compete with each other until one eventually dominates and determines the cell's fate. RIP1 is a crucial adaptor kinase on the crossroad of these stress-induced signalling pathways and a cell's decision to live or die. Following different upstream signals, particular RIP1-containing complexes are formed; these initiate only a limited number of cellular responses. In this review, we describe how RIP1 acts as a key integrator of signalling pathways initiated by stimulation of death receptors, bacterial or viral infection, genotoxic stress and T-cell homeostasis.