The Death of a Cell

Another characteristic of apoptotic cells that can be exploited in this death quest is the appearance of phosphatidylserine (PS) on the outer surface of the membrane. Annexin V binds with high affinity to PS and can be used to spotlight dying cells. Fourteen companies offer Annexin V with a variety of tags and detectors attached that can be used immunohistochemically or with the flow cytometer (Alexis, BioWhittaker, Boehringer Mannheim, Chemicon, CLONTECH, Immunotech, Genzyme Diagnostics, Kamiya Biomedical, Oncogene Research Products, Oncor, Pharmingen, R & D Systems, Trevigen, Zymed). Some kits come supplied with reagents to differentiate necrotic cells from apoptotic cells, relying on the fact that the plasma membrane of apoptotic cells, flipping notwithstanding, is still intact. Thus certain dyes taken up by necrotic cells are excluded from apoptotic cells. These reagents have the advantage of being usable on intact, even live, cells; they can be used quantitatively; and they generally can be used within the first few hours after apoptosis is initiated. However, similar to the kits for DNA fragmentation, they do not provide insight into the mechanism of apoptosis. The events surrounding the initiation of programmed cell death captivate the interest of researchers, and delineating the activities of the caspases is currently the rage in research in this area. Called by various names in the literature-ICE protease, for interleukin converting enzyme (caspase 1, the first of this family to be described, has this activity), FLICE, MACH, apopain, CPP32-the family's proper name, cysteinyl aspartate-specific proteinases, references the absolute requirement for an aspartic acid residue immediately adjacent to the point of cleavage. An intriguing feature of this family of proteases is that the proteases themselves must undergo an activating cleavage in order for apoptosis to be initiated. Since the caspases contain their own cleavage site, the notion that the activation process is both autocatalytic and cascading is taking hold. The caspase family stands currently at 10 members that are divided into three groups based on differences in the amino acids downstream of the aspartic acid residue in the cleavage site. Functionally, each group appears to be responsible for different biochemical events: group 1, which includes caspases 1, 4, and 5, has both interleukins and caspases as substrates; group 2, with caspases 2, 3, and 7 as members, cleaves PARP and other caspases; and group 3, containing caspases 6, 8, 9, and 10, does damage to the cytoskeleton. The amino acid sequences of the catalytic sites of the three groups have been used in the design of substrates and inhibitors that are being used to dissect the early events of apoptosis. These reagents come in kits (Chemicon, Bio-Rad, CLONTECH, Oncogene Research Products, Oncoimmunin, Upstate Biotechnology) or alone, (Alexis, BIOMOL, Calbiochem, Enzyme Research Products, Kamiya Biochemicals, Oncogene Research Products, Pharmingen, Quality Controlled Biochemicals, Upstate Biotechnology) come with tags or not, and are specific for individual caspases or groups. While most substrates do not penetrate the cell membrane and hence must be used with cell lysates, newly available is a novel class of protease substrates introduced by Oncoimmuin that cross the membrane and can be used with intact cells. As a class, these reagents provide the earliest peek into apoptosis, some capable of detecting events within minutes of the induction of apoptosis. Given that a wide variety of events initiate apoptosis, it is not surprising that numerous inducers and inhibitors of apoptosis have been described. Almost as numerous is the number of companies that offer these reagents, either the biomolecules themselves or antibodies/ELISAs directed against them. They are far too numerous to name, but a listing of the companies that supply them is located on The Scientist web site (www.the-scientist.com). New research tools for studying apoptosis and new companies devoted to this research topic are springing up almost daily. A recent listing of companies that supply apoptosis reagents lists over 80 companies. Recognizing the importance of apoptosis in disease states, diagnostic and therapeutic reagents are under development. Apoptosis On The Web Research in this area is definitely a growth industry (see R. Lewis, The Scientist, Feb. 6, 1995, page 16), and growing along with it are the resources available to students of apoptosis. A number of apoptosis-related web sites are now running on the internet, among them: www.apopnet.com -apoptosis on line, a searchable site designed to address the most frequently asked questions www.cellsalive.com -where you can see a cell undergoing apoptosis by time-lapse video microscopy. www.nih.gov/sigs/aig -the web site of the NIH based Apoptosis Interest Group The author can be reached at ldefrancesco@the-scientist.com. Chart 1 Chart 2 Chart 3 Chart 4 Chart 5

The Death of a Cell

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