Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon ...stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere.
The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either ...apoptosis or necroptosis. This 2 MDa intracellular complex that we designate “Ripoptosome” is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIPL prevents Ripoptosome formation, whereas, intriguingly, cFLIPS promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the “rheostat” that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.
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► Formation of the Ripoptosome, an intracellular death platform, is inhibited by cIAPs ► This high MW platform can be recruited to TLR3, leading to apoptosis or necroptosis ► cFLIPS blocks caspase-8 activity in the complex required for disassembly/stability ► cFLIPS promotes necroptosis in the absence of cIAPs
Abstract Background Anti-programmed cell death receptor-1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma as well as for other cancer entities. They act via blockade ...of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects affect skin, gastrointestinal tract, liver, endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. Methods and findings In total, 496 patients with metastatic melanoma from 15 skin cancer centers were treated with pembrolizumab or nivolumab; 242 side-effects were described in 138 patients. In 116 of the 138 patients, side-effects affected the skin, gastrointestinal tract, liver, endocrine, and renal system. Rare side-effects included diabetes mellitus, lichen planus, and pancreas insufficiency due to pancreatitis. Conclusion Anti-PD1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.
The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) ...or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.
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•c-FLIP isoforms (L/S) do not directly compete with caspase-8 for binding to FADD•c-FLIP binds to the DISC via a co-operative hierarchical caspase-8-dependent process•Co-operative and hierarchical binding crucially explains the dual function of c-FLIPL•Our unified model defines how c-FLIP isoforms differentially direct cell fate
It is unclear how c-FLIP isoforms can differentially regulate caspase-8 activation to direct cell fate. Hughes et al. show that c-FLIPL/S recruitment to FADD is indirect and requires caspase-8. This co-operative hierarchical binding process explains the conundrum of the dual role of c-FLIPL and defines how c-FLIPL/S differentially control cell fate.
Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat ...melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor β-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.
Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune ...system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as “find-me” signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.
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► Fas-induced apoptosis is associated with secretion of cytokines and chemokines ► Fas-driven MCP-1 and IL-8 were chemotactic for monocytes and neutrophils, respectively ► IL-8 and MCP-1 can act as “find-me” signals for apoptotic cells ► Apoptotic cells can actively communicate with cells of the immune system
The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we ...demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in Hoip
and Hoil-1
mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.
Formation of the death-inducing signaling complex (DISC) initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By ...elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.
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•Caspase-10 negatively regulates DISC-mediated caspase-8 activation and cell death•DISC formation and caspase-10 recruitment depend on caspase-8 scaffold function•Caspase-10 rewires DISC signaling to NF-κB-induced gene induction/cell survival•The catalytic activity of caspase-10 and caspase-8 are redundant in gene induction
It has been assumed that caspase-10, and its homolog caspase-8, have redundant functions in cell death signaling. Horn et al. now reveal a role for caspase-10 in switching CD95 signaling from caspase-8-induced cell death to NF-κB activation/cell survival. DISC recruitment of caspase-10 and NF-κB activation critically depend upon caspase-8 scaffold function.
Programmed necrosis and necroptosis signalling Feoktistova, Maria; Leverkus, Martin
The FEBS journal,
January 2015, 2015-Jan, 2015-01-00, 20150101, Letnik:
282, Številka:
1
Journal Article
Recenzirano
Odprti dostop
In recent years, the paradigm of cell death regulation has changed. Nowadays, not only apoptosis but also several forms of necrosis (e.g. necroptosis) are considered to be regulated. The central ...roles of receptor‐interacting serine/threonine protein kinase1 (RIPK1), RIPK3, and mixed‐lineage kinase domain‐like protein, and the molecular signalling platforms in which these molecules participate, are being intensively studied. In particular, the role of RIPK1, being both a kinase and a scaffold molecule, in different cell death regulatory complexes is of great relevance for the field. This minireview aims to introduce the emerging and dynamic field of necroptosis to the reader, with a specific focus on intracellular signalling pathways involved in this process.
The understanding of cell death has dramatically changed. Necrosis (e.g. RIPK‐dependent necroptosis) is also executed in a genetically controlled manner. This minireview aims to introduce the process of necroptosis with a specific focus on intracellular signalling pathways. The central role of RIPK1/RIPK3/MLKL, the molecular signalling platforms where these molecules are activated, and recent in vivo evidence are described.
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 cFLIPL and not cFLIPS 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.
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