Different types of cell death have classically been identified by discrete morphological changes. The hallmarks of apoptosis include cell shrinkage, nuclear fragmentation and membrane blebbing whereas necroptosis is characterized by cell swelling and plasma membrane breakdown. While these two forms of cell death are clearly distinct, substantial crosstalk occurs between them. Accordingly, it is becoming increasingly important to understand how these processes differ and to understand ways to differentiate them in cellular populations.
Apoptosis is a genetically programmed mechanism of cell death that is activated in response to cell stress, infection or developmental cues. Apoptosis is split into two main pathways, the intrinsic and extrinsic pathways, and can be triggered by granzymes from natural killer (NK) cells and cytotoxic T lymphocytes. The intrinsic pathway is regulated by the Bcl-2 family members and results in the loss of the mitochondrial integrity, whereas the extrinsic pathway is regulated by TNF ligands and death receptors. In most cases, the characteristic morphological and biochemical changes associated with apoptosis are governed by caspases. Executioner caspases are responsible for cleaving proteins involved in DNA fragmentation, cytoskeletal architecture, cell membrane integrity and more.
In contrast, necroptosis is a form of programmed cell death that acts independently of caspase activity. While necroptosis is much less characterized than apoptosis, it is established that serine/threonine kinases RIP1, RIP3, and the mixed lineage kinase domain-like protein, MLKL, are critical for this form of cell death. Another indicator of necroptosis is the formation of what’s known as the “Ripoptosome” which is characterized by recruitment of RIP1, FADD and Caspase 8. Interestingly, the Ripoptosome can lead to either caspase-independent necrosis or caspase-mediated apoptosis.
While chemical inhibitors targeting caspases, RIPK1, or MLKL provide evidence of the specific signaling pathways involved, these tools can affect both apoptosis and necroptosis. For instance, Necrostatin-1, a RIPK1 inhibitor, prevents necroptosis but can also induce RIPK1-mediated apoptosis. zVAD-fmk (a broad-spectrum caspase inhibitor) blocks apoptosis and in some cases, as with L929 cells, can stimulate necroptosis. Due to the considerable overlap between these two forms of cell death, it can be challenging to analyze the regulatory components involved. Multiple approaches to monitor and modulate apoptosis and necroptosis such as combining gene silencing or knockout studies with experiments using pharmacological inhibitors are essential to validate the contribution of specific pathways.
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