How to differentiate apoptotic from necroptotic cell death? Part I.

Figure 1. Example of a necroptotic cell (left) versus an
apoptotic cell (right). Image courtesy of the Vadenabeele lab.

Since the discovery that at least two forms of programmed cell death exist, apoptosis and necroptosis, a need has arisen to accurately discriminate between these two forms of cell death. Determining whether a cell is death or alive seems straightforward enough but all commonly used cell death assays have certain caveats and exceptions that one should be aware of before accurate conclusions can be drawn. In the following essays, I’ll go over the different cell death assays that I’m familiar with and discuss their applicability in discriminating between apoptotic and necroptotic cell death as well as common pitfalls and limitations using examples from my own experiments.

What discriminates apoptotic from necroptotic cell death?

Simply put: apoptosis is caspase-dependent cell death, whereas necroptosis is RIP-kinase-dependent cell death (for a brief review see Walsh, 2014) . Apoptosis occurs in an orderly manner: the cell’s DNA is digested into chunks of roughly 300 base pairs and the cell contents are packaged in small vesicles, the apoptotic bodies, that are phagocytosed by neighbouring cells and tissue macrophages for recycling. During necroptosis, on the other hand, the plasma membrane ruptures and the cell contents are spilled into the environment, stimulating a local inflammatory response. Apoptosis is the dominant form of cell death, since caspases have the ability to inactivate the RIPK signalling pathway. Thus, if your cells are dying while caspases are active, they’re most likely dying by apoptosis. However, caspase activity also occurs outside apoptosis and although vendors might claim that their product accurately detects the activity of one caspase or another, no simple method exists to make this distinction.

In general, caspase activity can be measured by the rate of cleavage of a tetra peptide linked to a fluorophore. For example, products to determine caspase-3/7 activity are usually based on the tetra peptide ‘DEVD’ linked to, for example, an AMC or AFC fluorescent moiety. Active caspase-3 will cleave the peptide after the last ‘D’, releasing the fluorescent moiety. An increase in fluorescence can then be said to correlate with an increase of caspase-3 activity. However, no single tetra peptide is exclusively cleaved by one caspase or another. DEVD is indeed a preferred substrate of both caspase-3 and -7 but can also be processed readily by caspase-8 and even the proteasome. Thus, an increase in DEVDase activity in your sample doesn’t necessarily indicate an increase in caspase-3 activity. The same holds true for every other tetra-peptide-based assay.

In addition, as I mentioned earlier, there are many scenarios wherein moderate caspase activity is not followed by cell death. Caspase-3 activity, for example, normally associated with end-stage apoptosis, also plays a role in memory formation in the brain in the absence of cell death. Caspase-7 may be involved in inflammation and initial activation of caspase-8 signals cell survival, rather than death. Thus, although apoptosis is invariably associated with caspase activity, caspase activity does not necessarily lead to apoptosis.

Unfortunately, no simple methods exists to determine the activity of the RIP kinases or their downstream effectors. However, there are several potent inhibitors of RIPK1 on the market: the necrostatins. The first of these, necrostatin-1, has now been shown to inhibit at least one additional enzyme, indoleamine-2,3-dioxygenase (IDO; Vandenabeele et al), and should therefore be used with caution. An alternative is now available in the form of necrostatin-1s (Nec-1s) which still prevents necroptotic cell death, in the absence of IDO inhibition. However, we can’t know what we haven’t looked for and even this inhibitor may have off-target effects.

Notwithstanding the fact that all chemical inhibitors may, to a greater or lesser extent, have off-target effects, utilization of these inhibitors still provides us with relatively simple means of discriminating the two forms of cell death. Thus, if you want to investigate whether a death-inducing compound kills your cells by apoptosis or necroptosis you could do a control experiment in the presence of a broad spectrum caspase inhibitor, such as z-VAD-fmk or boc-D-fmk, and/or necrostatin. If the caspase inhibitor rescues the death phenotype, the cells were most likely killed by apoptosis, if the necrostatin rescues the phenotype, the cells were most likely dying by necroptosis. However, there is a caveat. Certain substances have the ability to induce necroptotic cell death, but only in the absence of caspase activity as a consequence of autocrine TNFa signalling. To determine whether your compound induces such ‘secondary necroptosis’ I advice using a combination of z-VAD-fmk and necrostatin as a control besides z-VAD-fmk and necrostatin alone. If your compound appears to induce secondary necroptosis, perform a control in the presence of an anti-TNFa antibody to make sure that the observed cell death isn't a consequence of autocrine TNFa signalling. 

Figure 2. Secondary necroptosis in U937 cells. For this experiment, U937 cells where stimulated with the DNA-damaging agent etoposide in the presence of various inhibitors, as indicated. As you can see in panel A, the cells produce large amounts of TNFa upon etoposide stimulation in the presence of zVAD. Addition of necrostatin-1 (Nec1) inhibits this TNFa production. In panel B, you can see that the TNFa production is so high that the cells undergo secondary necroptosis. Vability is restored by addition of necrostatin-1 or an anti-TNFa antibody (aTNF). See van Raam et al., 2012 for a detailed description.