Key Points
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In response to cytotoxic insults, cells die by different subroutines that are mainly based on apoptosis and necrosis and often preceded by a global cytoprotective response centred on autophagy. Thus, cell-based tests that measure cell death-related phenomena must answer two critical questions: do the cells die and, if so, through which lethal pathway do they die?
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Cells should be considered as dead when they fulfil at least one of the following criteria: the plasma membrane has lost its integrity, the cell has fragmented into apoptotic bodies, or the corpse or its fragments have been taken up by neighbouring cells.
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Cell death assays fall into two major groups: assays that measure bona fide cell death and tests that quantify biochemical processes that are viewed as surrogate viability markers.
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Apoptosis assays can be grossly subdivided into two categories: methods that detect events occurring in most (if not all) instances of apoptotic cell death and tests that measure pathway-specific processes, which depend on the apoptosis-initiating stimulus and the precise cellular context of lethal signalling.
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Autophagy can be quantified by steady-state assessment of the subcellular morphology, long-lived molecule content and the lipidation status of the autophagosomal membrane protein LC3. In addition, flux measurements allow discrimination between efficient and stalled autophagic degradation. Efficient autophagic degradation generally exerts cytoprotective functions, whereas stalled autophagic degradation frequently represents a pathological event.
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Necrotic cell death can be differentiated from other cell death subroutines by monitoring the kinetics of appearance of common markers, such as disintegration of the plasma membrane.
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Sophisticated mitotic catastrophe assays are based on fluorescent biosensor cell lines that are assessed by robotized video microscopy and automated image analysis, both of which allow us to monitor several indicators of the nuclear status and establish cell fate profiles.
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It can be anticipated that the combination of ever more sophisticated high-content and high-throughput microscopy methods with microfluidic devices or on-chip technologies will accelerate the cell-based identification of cell death-modulatory drugs.
Abstract
Cell death has an important role in many human diseases, and strategies aimed at modulating the associated pathways have been successfully applied to treat various disorders. Indeed, several clinically promising cytotoxic and cytoprotective agents with potential applications in cancer, ischaemic and neurodegenerative diseases have recently been identified by high-throughput screening (HTS), based on appropriate cell death assays. Given that different cell death modalities may be dysregulated in different diseases, it is becoming increasingly clear that such assays need to not only quantify the extent of cell death, but they must also be able to distinguish between the various pathways. Here, we systematically describe approaches to accurately quantify distinct cell death pathways, discuss their advantages and pitfalls, and focus on those techniques that are amenable to HTS.
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Acknowledgements
G.K. is supported by the Ligue Nationale contre le Cancer (Equipes labellisée), the Agence Nationale pour la Recherche (ANR), the European Commission (Active p53, Apo-Sys, ChemoRes, ApopTrain and ArtForce), the Fondation pour la Recherche Médicale (FRM), the Institut National du Cancer (INCa), Cancéropôle Ile-de-France and the AXA Chair for Longevity Research. J.Y. is supported by a US National Institutes of Health Director's Pioneer Award, grants from the National Institute of Aging (USA) and the National Cancer Institute (USA). We are indebted to S. Shen for providing fluorescence microscopy images.
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Supplementary information Table S1
Recent successful high-throughput cell-based assays for the detection of cell death/survival-related variables (PDF 435 kb)
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Glossary
- Apoptotic bodies
-
Membrane-surrounded vesicles that are shed from dying cells during the late stages of apoptosis, and that may include portions of the nucleus and/or seemingly normal organelles.
- Autophagic cell death
-
For a long time, this term has erroneously been used to indicate cell death that manifests with autophagic vacuolization; thus implying that cell death is mediated by rather than accompanied by autophagy.
- Caspases
-
Cysteine proteases that cleave their substrate after an aspartic acid residue. Caspases have a critical role in both the initiation (caspase 2, caspase 8, caspase 9 and caspase10) and execution (caspase 3, caspase 6 and caspase 7) of apoptosis.
- Necroptosis
-
A regulated form of necrosis that requires the catalytic activity of receptor-interacting protein kinase 1 (RIPK1) and RIPK3.
- Anoikis
-
A particular form of apoptosis that is triggered by the detachment of cells from the extracellular matrix.
- Entosis
-
A recently discovered and debated cell death mode in which one cell engulfs one of its live neighbours, which may then die within the phagosome.
- Pyroptosis
-
A pyrogenic cell death modality that is accompanied by the activation of caspase 1 and the secretion of the pro-inflammatory cytokine interleukin-1β. Pyroptosis can manifest with features of apoptosis or necrosis.
- Parthanatos
-
Caspase-independent cell death induced by poly(ADP-ribose) polymerase 1 and mediated by apoptosis-inducing factor.
- Autoschizis
-
A cell death modality that is initiated in certain cancer cells by combinatorial treatment with vitamins C and K3, and leads to the self-excision of cytoplasmic portions.
- Intrinsic apoptosis
-
Intracellular stress is sensed by mitochondria, which undergo mitochondrial membrane permeabilization and hence activate caspase-dependent and caspase-independent cell death executioner mechanisms.
- Extrinsic apoptosis
-
Apoptosis can be initiated by extracellular molecules that bind to 'death' receptors (for example, FAS), in turn activating the caspase 8–caspase 3 cascade.
- Necrostatin 1
-
The tryptophan-based molecule 5-(1H-indol-3-ylmethyl)-3-methyl-2-thioxo-4-imidazolidi-none that was first identified as a specific and potent inhibitor of necroptosis.
- Micronucleation and multinucleation
-
The presence of multiple nuclei often derives from mitotic problems — for instance, from chromosomes or chromosomal fragments that have not been evenly distributed at anaphase — or from deficient cytokinesis.
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Kepp, O., Galluzzi, L., Lipinski, M. et al. Cell death assays for drug discovery. Nat Rev Drug Discov 10, 221–237 (2011). https://doi.org/10.1038/nrd3373
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DOI: https://doi.org/10.1038/nrd3373
