Apoptosis Assays: Caspases and Beyond

Researchers examine cell death for a variety of reasons, from the obvious, like find out whether a particular environment is toxic, to determining the mechanism of action of a pharmacologically active substance. Often, how the cell is killing itself is perhaps more important than whether it dies. Many assays can determine whether suicidal cells have undergone apoptosis (programmed cell death), necrosis (cell death associated with inflammation), necroptosis (programmed necrosis) or have experienced some other self-destructive behavior. 

Diseases such as myocardial infarction and neurodegeneration are associated with excessive apoptosis, and nearly every known cancer is due in part to the inhibition of apoptosis.

But apoptosis itself is not just one thing. There are at least two major pathways—dubbed “intrinsic” and “extrinsic”—that lead to programmed cell death, generally culminating in a cascade of caspase proteases that converge on caspase 3 and ultimately destroy vital cellular proteins. The need to identify which of these is triggered, at which step the process is blocked and whether an inhibitor can even be inhibited has resulted in a proliferation of apoptosis assays.

Is it apoptosis?

Different markers of apoptosis are used as the basis for the most common apoptotic assays, either separately or in concert with each other or more generic assays, such as membrane permeability assays. Morphological changes, such as cell shrinkage and membrane blebbing, can be seen under the microscope. The flipping of phosphatidylserine from the inner to the outer leaflet of the cell membrane can be queried with Annexin V (calcium-dependent phospholipid-binding proteins that bind to phosphatidylserines as a measure of apoptosis). Chromosomal degradation is seen by labeling nicked DNA in a TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. And activation of the caspase cascade is typically probed by looking either for activated caspases (typically caspase 3) or their cleaved products. Which to choose depends on a host of factors, including cell type and amount of sample; available equipment and expertise; and whether it’s important to know which stage of the apoptotic process is being detected.

Physicians typically don’t care which pathways and mechanisms and molecules are being triggered or blocked, because it’s “assumed that work has already been done by the time the chemo agent is in your hands, and you’re using it,” says Bill Jacqmein, chief operations and technology officer at Vergent Bioscience. But these details may be exactly what the researcher is trying to determine.

Finding caspase

Caspases exist constitutively in cells as zymogens. Some (like “executioner” caspase 3 and caspase 7) are constitutive dimers but must be cleaved and rearranged to create the active site. Others (like the “initiator” caspases 8, 9 and 10) are constitutive monomers that become activated only after they have dimerized and been recruited to oligomeric multiprotein complexes [1].

Antibodies have been generated to most, if not all, caspases of interest. Yet because caspases are constitutive, to query whether they’re involved in apoptosis it’s important to use a reagent that can discern the active or cleaved form from the proenzyme. Antibodies are a key reagent used in a variety of assays, including flow cytometry, immunohistochemistry, ELISA, Western blotting and immunofluorescence.

Many assays look for activity instead of the caspase itself. “There are substrates that are fairly ubiquitously cleaved by caspases, like PARP [poly(ADP-ribose) polymerase 1] and lamin,” and we have antibodies specific for those cleavage products as well, says Gary Kasof, fellow in product development at Cell Signaling Technology (CST). Other substrates are cleaved by only a single or a select few caspases.

Non-antibody-based assays are also available. For example, many vendors market both pan- and specific caspase kits based on the FLICA (fluorescent labeled inhibitor of caspases) assay, in which a small fluorescent molecule passes through the live cell membrane and irreversibly binds to the caspase’s active site, with unbound reagent being washed away. The same concept has been used to create PET probes, with an F-18 (fludeoxyglucose) PET label in place of fluorescence, points out Jacqmein.

Another option is to use a cell line that’s missing the gene for the particular caspase you’re interested in. “If a researcher thinks they’ve developed a new drug that triggers apoptosis in cancer cell lines, for example, that would be a quick and easy way of assessing whether their new drug works through that pathway,” says Nicola McCarthy, oncology program manager at Horizon Discovery, which offers several haploid caspase knockouts.

Beyond caspases

“We also have other haploid cell lines that are missing various components of the apoptosis pathway,” McCarthy adds.

“If you look at just the downstream caspase family members”—there are at least 12 unique caspase proteins in the family—“you will see the convergence of basically every apoptotic pathway,” says Daniel Braunschweig, Bio-Rad Laboratories’ global product manager for Bio-Plex assays. But if you just look at caspases, you’ll miss out on finding the cause of intrinsic pathway-regulated apoptosis: “Lack of apoptosis in cancer, or excessive apoptosis in the case of neurodegeneration or heart disease or something like that, typically runs through the Bcl-2 family members.” For this reason, he says, Bio-Rad focuses its Bio-Plex (Luminex-based) apoptosis panels on the Bcl-2 family. A single marker is often insufficient to get a clear understanding of the mechanism underlying the process, Braunschweig adds.

“Any time you’re trying to establish if a therapeutic regimen is working, or to stage a disease, you always go for multiple markers,” concurs Sunetra Ray, technical support specialist at BioVision. This helps explain the popularity of flow cytometry, for example, in apoptosis research.

There is also a lot of interest lately in the host of other molecules and pathways that interact with the intrinsic and extrinsic apoptotic pathways. These include the inhibitor of apoptosis (IAP) family and SMAC (an inhibitor of IAPs), small-molecule mimetics of which have been developed to promote apoptosis in cancer.

There are many ways to interrogate apoptosis—far more than have been surveyed here—from the generic (did it happen?) to the very specific (were both caspase 9 and Bax active following treatment?). Choose carefully. It may be a matter of matter of life and (cell) death.

Reference

[1] Ashkenazi, A, Salvesen, G, “Regulated cell death: signaling and mechanisms,” Annu Rev Cell Dev Biol, 30:337-56, 2014. [PMID: 25150011]