Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells
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Current models for Fas (CD95)-mediated apoptosis suggest that FLICE/caspase-8 is recruited and activated, which results in cell death. However, the role of additional molecules in Fas signaling and FLICE activation is not clear. A chimeric Fas/FLICE (F/F) receptor, containing the extracellular/transmembrane portion of Fas and the caspase region of FLICE, mediated anti-Fas apoptosis. FLICE protease subunits were generated from the F/F precursor. Killing induced by Fas, but not F/F, was blocked by a dominant negative FADD. Apoptosis triggered through Fas and F/F was inhibited by coexpression of CrmA and p35, but not Bcl-xL. F/F bypassed Fas resistance in COS-7 cells and blocking by the death effector domain (DED)-containing viral protein MC159. These results show that: 1) F/F induces cell death, indicating that FLICE activation is sufficient for apoptosis and does not require additional Fas- or FADD-binding proteins; and 2) F/F bypasses proximal defects in Fas signaling that prevent FLICE recruitment or activation.
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Arsenite, cadmium, and mercury are among the most abundant toxic metals (TM) in the environment. Although the most common renal manifestation of TM toxicity is proximal tubular dysfunction, significant glomerular injury can also occur. We hypothesized that glomerular injury following TM exposure results from TM-induced apoptosis of podocytes. To test this hypothesis we examined the extent of apoptosis and the apoptotic pathways induced in cultured murine podocytes incubated for three days with arsenite, cadmium, or mercury, and with equimolar combinations of all three metals. Apoptosis was detected by DNA laddering, and the number of apoptotic nuclei determined by Tunel assay. Treatment for three days with each TM resulted in DNA laddering and induced a dose-dependent increase in apoptotic nuclei. In contrast, treatment with equimolar combinations of TM induced significantly fewer apoptotic nuclei than individual TM treatments. Apoptosis induced by each TM was associated with a significant (∼400%) increase in caspase 8 activity, but no change in caspase 9 activity, and Western analyses revealed a marked up-regulation of Fas (∼500%) and FADD (∼300%) with no change in expression of Bax, Bcl-2, or Bcl-xL. Similar to the apoptotic response, combinations of TM induced less caspase 8 activity and Fas/FADD expression than individual TM treatments. Collectively, these results demonstrate that (1) TM induced apoptosis in cultured murine podocytes via the extrinsic Fas-FADD caspase 8 pathway, rather than the mitochondrial apoptotic pathway, and (2) combination TM exposure induced less apoptosis than individual TM, indicating an antagonistic rather than an additive or synergistic toxicity.
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Oncogenic Ras induces cells to undergo apoptosis after inhibition of protein kinase C (PKC) activity. The integration of differential signaling pathways is required for full execution of apoptosis. In this study, we used Jurkat as well as Fas/FADD-defective cell lines expressing v-ras to determine the upstream elements required for activation of the caspase cascade in PKC/Ras-mediated apoptosis. During this Ras-induced apoptotic process, caspase-8 was activated, possibly through its binding to Fas-associated death domain (FADD), in Jurkat/ras and Jurkat/Fas(m)/ras cells but not in Jurkat/FADD(m)/ras cells. c-Jun NH(2)-terminal kinase (JNK) was activated in all three cell lines expressing ras in response to apoptotic stimulation. Suppression of JNK by dn-JNK1 blocked the interaction of FADD and caspase-8 and partially protected Jurkat/ras and Jurkat/Fas(m)/ras cells from apoptosis. However, dn-JNK1 had no effect on PKC/Ras-induced apoptosis in Jurkat/FADD(m)/ras cells. The results indicate that FADD/caspase-8 signaling is involved in PKC/Ras-mediated apoptosis, and JNK may be an upstream effector of caspase activation.
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Objective: To study the expression of FADD and caspase-3 and its relation to apoptosis in cutaneous squamous cell carcinoma(SCC) and basal cell carcinoma(BCC).Methods: Apoptosis and expression of FADD and caspase-3 in 48 SCCs and 41 BCCs were determined by TUNEL and SABC immunohistochemical technique,respectively.Results: Apoptosis index(AI) and FADD and caspase-3 expression in SCC were superior to those in BCC(P0.01).There were positive correlations between AI and FADD or caspase-3 expression,and between FADD and caspase-3 expression in SCC and BCC(P0.05).AI and caspase-3 expression were downregulated in poorly differentiated SCC(P0.05),and AI increased in primary lesion(P0.01).Conclusion: Fas-mediated pathway may be the primary apoptotic pathway in SCC and BCC;FADD and caspase-3 could play an important role in the apoptosis of SCC and BCC.
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The Fas-associated death domain (FADD) has long been regarded as a crucial adaptor protein in the extrinsic apoptotic pathway.Despite the non-apoptotic function of FADD is gradually being discovered and confirmed, its corresponding physiological and pathological significance is still unclear.Based on the database of GWAS catalog and GTEx Portal, 17 SNPs associated with leukemia susceptibility were found to be linked to FADD expression.We then investigated a regulatory role of FADD in T-acute lymphoblastic leukemia (T-ALL) using Jurkat cells as a model.Jurkat cells stably depleted of FADD (FADD -/-Jurkat) expression exhibited dampened proliferation, hypersensitivity to Etoposide-induced intrinsic apoptosis whereas near total resistance to TRAIL-induced extrinsic apoptosis.Comparison between wild type and FADD -/-Jurkat cells using iTRAQ-based proteomics revealed considerably altered expression spectrum of genes, and led us to focus on metabolic pathways.Investigation of glycolytic and mitochondrial pathways and relevant enzymes revealed that FADD knockout triggered a metabolic shift from glycolysis to mitochondrial respiration in Jurkat cells.Re-expression of FADD in FADD -/-Jurkat cells partially rescued glycolytic capacity.FADD loss triggers global metabolic reprogramming in Jurkat cells and therefore remains as a potential druggable target for ALL treatment.
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Death receptors, a subset of the tumor necrosis factor (TNF) receptor (TNFR) superfamily that includes TNFR1, CD95 (Fas, Apo-1), and the TRAIL (TNF-related apoptosis-inducing ligand) receptors, transduce signals capable of engaging apoptosis or necrosis,1 depending on the status of signaling molecules in the cells. Ligation of one such receptor, cluster of differentiation 95 (CD95), has catastrophic consequences in vivo, because this results in lethal, fulminant liver destruction.2 In this issue of HEPATOLOGY, Hikita et al.3 employ a conditional gene deletion model to explore the molecular mechanisms of this liver failure. CD95, cluster of differentiation 95; FADD, Fas-associated protein with death domain; TNF, tumor necrosis factor; XIAP, X-linked inhibitor of apoptosis protein. Upon ligation, CD95 rapidly recruits an intracellular adapter molecule, Fas-associated protein with death domain (FADD), which in turn binds to and activates the initiator caspase, caspase-8.1 The activation of caspase-8 requires two steps: dimerization of the inactive "pro-form" of the molecule, followed by autocleavage, which generates a stable, active protease.4 (Note that cleavage alone does not activate caspase-8, despite claims of "feedback activation" via cleavage by other enzymes5; there is no biochemical support for such feedback.) Upon activation of caspase-8, apoptosis can proceed in two ways (Fig. 1). The first route is via caspase-8–mediated cleavage and activation of the executioner caspases, caspase-3 and caspase-7 (unlike caspase-8, executioner caspases exist as preformed dimers, and cleavage alone is sufficient for their activation). This pathway of caspase-8–mediated cleavage and activation of executioner caspases occurs in so-called "Type I" cells.6 However, an endogenous caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), if present in sufficient amounts, can bind to and block the activation of the executioner caspases, preventing apoptosis. This occurs in "Type II" cells.7 In this case, apoptosis proceeds when caspase-8 cleaves a BCL-2 family protein, BID, which in turn activates the proapoptotic effectors of the BCL-2 family, BAX and BAK. The latter permeabilize the mitochondrial outer membrane, releasing proteins (e.g., Smac and Omi) that antagonize XIAP, allowing executioner caspase activation, and thus apoptosis, to proceed. Although mitochondrial outer membrane permeabilization also releases cytochrome c, which triggers the activation of caspase-9, this is not required for apoptosis to proceed in this scenario.7 Type I versus Type II death receptor signaling. Ligation of CD95 induces the recruitment of an adaptor protein FADD via homotypic death domain (DD) interactions between FADD and the cytoplasmic portion of the CD95 receptor. FADD then recruits caspase-8 via death effector domain (DED) interactions. Depending on the levels of the executioner-inhibitor protein XIAP, apoptosis will then proceed via one of two ways. In Type I cells, XIAP levels are low, and caspase-8–mediated cleavage and activation of the executioner caspases, caspase-3 and caspase-7, is sufficient to drive apoptosis. In Type II cells, XIAP is present in sufficient amounts to bind to and block the activation of the executioner caspases, preventing apoptosis. Thus, in Type II cells, caspase-8–mediated cleavage of BID, subsequent activation of BAX and BAK, and release of the XIAP inhibitors Smac and Omi from mitochondria during mitochondrial outer membrane permeabilization (MOMP) are required for efficient executioner caspase activation and apoptosis to proceed. Mice lacking BID are completely resistant to ligation of CD95 in vivo,7, 8 leading to the idea that hepatocytes (and indeed, any cell contributing to CD95-mediated lethality) are Type II cells. This is further supported by the finding that mice lacking both BID and XIAP are highly sensitive to CD95 ligation, and display liver destruction.7 Now, Hikita et al.3 have examined the effects of removing BAX and BAK from hepatocytes by conditional deletion of BAX in a BAK−/− background. BAX and BAK are generally redundant proteins; the presence of one or the other is sufficient for BID-induced cell death in many cells, but in the absence of both, active BID is harmless.9 Liver mainly expresses BAK, and indeed, the authors observed less CD95-induced hepatocyte apoptosis in mice lacking BAK alone than in wild-type mice. In the absence of both BAX and BAK, they observed even less cell death. Nevertheless, the mice underwent catastrophic liver destruction. Therefore, we have a paradox. BID is required for CD95-induced liver damage, and BAX/BAK are required for BID function. Yet, removal of BAX and BAK did not protect the animals from CD95-induced lethality. How can this be reconciled? One possibility is that, in the mixed strain background employed in this study, the levels of XIAP were insufficient to protect the liver, and therefore the death proceeded via a "Type I" pathway as opposed to the Type II pathway typically observed in hepatocytes. The authors note that XIAP levels fell several hours after CD95 ligation in these animals. Previous studies have shown that hepatocytes, cultured ex vivo, convert from Type II to Type I cells,10 lending some credence to this idea. However, BID-deficient mice are fully resistant to CD95 ligation, without showing even a delayed liver failure,7 but in those studies, a pure C57Bl/6 background was employed. Strain differences may therefore account for the divergent results. There is, however, another explanation. Hikita et al.3 deleted BAX only in hepatocytes of the BAK-deficient mice, using a CRE (cyclization recombination) transgene driven by the albumin promoter. Hepatocytes not expressing this CRE would die in response to CD95-induced BID cleavage, as would any cell that does not drive this promoter. Might other cells, dying in this BID-dependent (Type II) manner, cause hepatic injury? In an earlier study, hepatocyte expression of a BCL-2 transgene driven by the albumin promoter (BCL-2 efficiently blocks BID-induced cell death) reportedly blocked hepatocyte apoptosis, but not liver destruction.11 This is completely consistent with the findings of Hikita et al.3 Previously, it was noted that CD95 ligation in vivo induces destruction of vascular endothelium in the liver.12-14 This produces the sinusoidal hemorrhage characteristic of this treatment. As a result, CD95 ligation would be lethal even if hepatocytes were protected. Therefore, although deletion of BID throughout the animal protects hepatocytes, endothelial cells, and the animal as a whole, deletion of BAX and BAK (or expression of BCL-2) specifically in hepatocytes does not. It is an attractive resolution to the apparent paradox. Hikita et al.,3 however, noted some apoptosis in hepatocytes in their engineered animals upon ligation of CD95. These might be cells that had failed to flox BAX, as mentioned above, or perhaps more intriguingly, may be dying independently of BAX and BAK. The latter possibility is supported by studies showing that metabolic stress (e.g., glucose deprivation) can sensitize cells for CD95-induced death.15 Certainly, a failure of the blood supply, as discussed above, would cause such stress, and it will be of interest to ascertain if this can convert Type II cells to Type I cells. Finally, one might be enticed to consider the possibility that liver destruction via CD95 ligation may proceed not only by apoptosis but also by necrosis. Several molecular mechanisms whereby necrosis can be "programmed" are known.1 However, Hikita et al.3 showed that cyclophilin D, which is required for some forms of necrosis,16 does not play a role in CD95-induced liver damage in the absence of BAX and BAK. Furthermore, because it has long been known that caspase inhibitors (which preferentially go to the liver in vivo) block CD95 ligation-induced lethality,17 the authors also confirmed that the lethality in their mice was similarly blocked by caspase inhibitors. Tellingly, a recently uncovered pathway of necrosis is antagonized by caspase-8,18, 19 but based on these results, it does not appear to play a role in CD95-mediated liver destruction. The liver is more than hepatocytes. Understanding how the ligation of death receptors, either in experimental models or disease states, causes liver injury and how this can be abated remains a priority.
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Activation‐induced cell death (AICD) in activated T lymphocytes is largely mediated by Fas/Fas ligand (FasL) interaction. The cytoplasmic adaptor molecule Fas‐associated death domain protein (FADD) plays an essential role in the apoptotic signalling of the Fas death pathway. In the present study, we observed that FADD deficient (FADD −/− ) Jurkat T cells undergo AICD to a similar extent as wild‐type cells. AICD in wild‐type Jurkat T cells is via apoptosis, whereas it is non‐apoptotic in FADD −/− cells. The latter took up propidium iodide, exhibit a loss in mitochondrial membrane potential and have no detectable cleavage products of caspase‐8 or ‐3 activation, suggesting that these cells die by necrosis. Wild‐type Jurkat T cells undergo apoptosis when incubated with recombinant FasL and Trail but not with TNF‐α. In contrast, FADD −/− Jurkat T cells are resistant to FasL and Trail but die of necrosis when incubated with TNF‐α. We showed that neutralising anti‐TNF‐α blocked AICD as well as TNF‐α‐induced necrosis in FADD −/− Jurkat T cells. Furthermore, downregulating the receptor interacting protein, RIP, with geldanamycin treatment, which is essential for TNF‐α signalling, markedly inhibited AICD in FADD −/− Jurkat T cells. In addition, caspase‐8‐deficient Jurkat T cells are resistant to Fas‐ and TNF‐α‐induced cell death. Taken together, our results suggest that a deficiency in FADD and not caspase‐8 or the inhibition of the Fas signalling pathway sensitises Jurkat T cells to TNF‐α‐dependent necrosis during AICD.
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