In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

Apoptosis assays: caspase activation, membrane alterations, DNA fragmentation, and mitochondrial changes A broad range of chemotherapies work by interfering with the mechanisms of cell death, and inducing apoptosis remains a promising strategy for cancer drug discovery. Using apoptosis as an end point underlies several novel approaches of cell death assays. Apoptosis involves caspase activation, cleavage of cellular protein substrates, cleavage of DNA into size fragments, and formation of apoptotic bodies. Therefore, all these characteristics represent potential targets for novel specific drugs. Despite the fact that caspase 8 and caspase 3 might be also involved in alternative nonlethal functions, as suggested by Yi et al, the detection of apoptosis using caspase enzymes remains reliable and widely used. 30 The occurrence of caspase activity in a cell during apoptosis leads to the cleavage of specific substrates that can later be easily detected as a marker of caspase activation. Indeed, several antibodies are available and can be used against a myriad of caspase substrates, including PARP-1 (poly-ADP-ribose-polymerase-1), for instance, through immunoblot analysis, immunofluorescence, and flow cytometry. Furthermore, the detection of early apoptosis may be possible, thanks to antibodies that only detect the caspase-cleaved form of a substrate protein with site-specific cleavage. 31, 32 Alternatively, caspase activation can be assessed by other reagents that are compatible with HTS applications, such as exogenous proluminescent or fluorogenic caspase substrates. A real-time assessment of caspase activity may then be allowed, thanks to substrates that are bifunctional compounds, and fluorescent biosensors can be introduced into cells. 33 Thereby, studying caspase activity remains a reliable and specific approach for assessing apoptosis. 34 Among the characteristics of apoptosis, the exposure of phospholipid phosphatidylserine (PS) on the cell membrane in response to proapoptotic stimuli represents a major feature of the caspase-dependent process in healthy cells. Therefore, a PS-binding protein such as annexin-V is experimentally used to detect PS exposure. Several annexin-V derivatives associated with different fluorochromes can be used for apoptosis measurement using multicolor flow cytometry or fluorescence microscopy. To distinguish apoptotic cells from nonapoptotic cells, annexin-V is usually combined with cell-impermeable dyes, such as propidium iodide, as apoptotic cells are annexin-V positive but with intact plasma membranes. It remains a sensitive and rapid method to assess apoptosis, but we must keep in mind that PS exposure can also occur in cell death-unrelated conditions, especially when T lymphocytes are activated. [35] [36] [37] DNA fragmentation during apoptosis is a caspase-mediated mechanism and leads to specific internucleosomal DNA double-strand breaks with fragments of multiple of 180 base pairs in size. Historically, a gel electrophoresis of genomic DNA was used for demonstrating internucleosomal DNA fragmentation as apoptotic cells display a characteristic DNA ladder, whereas necrotic cells show a smear of randomly degraded DNA, but it has been superseded by faster and easier methods. As a matter of fact, determination of DNA content and number of apoptotic cells is now preferentially investigated using flow cytometer with DNA-binding fluorochromes, such as propidium iodide, through the detection of hypodiploid nuc