Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Summary Receptor interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pro-necrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound; whereas, D161G, D143N, and K51A mutants only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3K51A/K51A) are viable and fertile, in stark contrast to the perinatal lethality of Rip3D161N/D161N mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.

Summary Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Although there are many assays for detection of apoptosis, relatively few assays are available for measuring necrosis. A key signature for necrotic cells is the permeabilization of plasma membrane. This event can be quantified in tissue culture settings by measuring the release of the enzyme lactate dehydrogenase (LDH). When combined with other methods, measuring LDH release is a useful method for detection of necrosis. In this chapter, we describe the step-by-step procedure for detection of LDH release from necrotic cells using a microtiter plate based colorimetric absorbance assay.

No abstract

Caspases are cysteine proteases that mediate programmed cell death in phylogenetically diverse multicellular organisms. We report here two kindreds with autoimmune lymphoproliferative syndrome (ALPS) type II, characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects, that harbor independent missense mutations in Caspase 10. These encode amino acid substitutions that decrease caspase activity and interfere with death receptor-induced apoptosis, particularly that stimulated by Fas ligand and TRAIL. These results provide evidence that inherited nonlethal caspase abnormalities cause pleiotropic apoptosis defects underlying autoimmunity in ALPS type II.

The serine/threonine kinase RIPK1 is recruited to the TNF receptor 1 to mediate pro-inflammatory signalling and to regulate TNF-induced cell death. A RIPK1 deficiency results in perinatal lethality, impaired NFκB and MAPK signalling and sensitivity to TNF-induced apoptosis. Chemical inhibitor and in vitro reconstitution studies suggested RIPK1 displays distinct kinase activity dependent and independent functions. To determine the contribution of RIPK1 kinase to inflammation in vivo, we generated knock-in mice endogenously expressing catalytically inactive RIPK1 D138N. Unlike Ripk1−/− mice, which die shortly after birth, RIPK1D138N/D138N mice are viable. Cells expressing RIPK1 D138N are resistant to TNF- and poly (I:C)-induced necroptosis in vitro and RIPK1D138N/D138N mice are protected from TNF-induced shock in vivo. Moreover, RIPK1D138N/D138N mice fail to control Vaccinia virus replication in vivo. This study provides genetic evidence that the kinase activity of RIPK1 is not required for survival but is essential for TNF-, TRIF- and viral-initiated necroptosis.

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G 1 /S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G 1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G 1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G 1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.Apoptosis in immature T cells and T-cell hybridomas, which may relate to negative selection during T-cell development, can be initiated by signals through the T-cell receptor-CD3 complex (18,32,33). This process of activation-induced apoptosis (anti-CD3 apoptosis) consists of two distinct phases. The first phase is the cell cycle block at the G 1 /S transition; it is followed by the second phase, with the generation of apoptotic DNA ladders (18). The second phase requires extracellular calcium and can be inhibited by the immunosuppressive drug cyclosporin A (18). We and others have shown that Nur77 (NGFI-B) orphan steroid receptor is induced during anti-CD3 apoptosis through the calcium signals and it plays an essential role in the cell death process (17, 35). Dominant negative Nur77 can block apoptosis but not the interleukin 2 production of anti-CD3-treated T-cell hybridomas (35). Thus, Nur77 is involved in the second phase of anti-CD3 T-cell apoptosis.As alluded to above, anti-CD3 death in T-cell hybridomas is also accompanied by a G 1 cell cycle block. In all organisms studied so far, cell cycle progression is mediated by cyclindependent kinases (CDKs) that consist of a catalytic subunit CDK and a regulatory subunit cyclin. In mammalian cells, cyclin E-CDK2 together with cyclin D-CDK4 and/or cyclin D-CDK6, which are active in the G 1 phase, control the G 1 -to-S transition (23, 24, 28, 31 and references therein). There are at least three different D-type cyclins, with T cells expressing cyclins D2 and D3 and two cyclin D-associating kinases, CDK4 and CDK6. One of their substrates is the retinobl...