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.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for~60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (ΔΨ m ). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-x L overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-x L blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the deathinducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of ΔΨ m . Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-x L , converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-x L . In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

APO‐1 (Fas/CD95), a member of the tumor necrosis factor receptor superfamily, induces apoptosis upon receptor oligomerization. In a search to identify intracellular signaling molecules coupling to oligomerized APO‐1, several cytotoxicity‐dependent APO‐1‐associated proteins (CAP) were immunoprecipitated from the apoptosis‐sensitive human leukemic T cell line HUT78 and the lymphoblastoid B cell line SKW6.4. CAP1–3 (27–29 kDa) and CAP4 (55 kDa), instantly detectable after the crosslinking of APO‐1, were associated only with aggregated (the signaling form of APO‐1) and not with monomeric APO‐1. CAP1 and CAP2 were identified as serine phosphorylated MORT1/FADD. The association of CAP1–4 with APO‐1 was not observed with C‐terminally truncated non‐signaling APO‐1. In addition, CAP1 and CAP2 did not associate with an APO‐1 cytoplasmic tail carrying the lprcg amino acid replacement. Moreover, no APO‐1‐CAP association was found in the APO‐1+, anti‐APO‐1‐resistant pre‐B cell line Boe. Our data suggest that in vivo CAP1–4 are the APO‐1 apoptosis‐transducing molecules.