Dead Again: Adventures in Apoptosis

Date: March 1, 1999Caspase Substrates and Inhibitors, Apoptosis ProductsCast of Characters Apoptosis is no less a hot topic than it was a year ago when LabConsumer first looked at the available tools for studying this complex phenomenon.1 In that intervening time, roughly 20 papers per day have been published on apoptosis, an issue of Science devoted to apoptosis has been published,2 and countless new pieces have been added to the puzzle. To get an idea of the complexity of apoptosis in higher species, take a look at the cast of characters (Table on page 19), which is doubtless incomplete. The challenge to those studying this process is to understand how the individual members of this cast interact and function to facilitate cell death or protect cells from it. What makes understanding this process so intriguing is the fact that there are multiple pathways to apoptosis, even within a single cell type, and different cell types can respond to inducers of apoptosis differently, depending at least in part on their surface architecture. Detection of apoptosis with M30 CytoDeath. HeLa cells, treated with TNF and actinomycin D, were stained with M30 antibody, which detects cytokeratin 18 cleaved by caspases. Image provided by Roche Molecular Biochemicals. Along with gains in understanding this process have come some innovations in the methods for studying it. This article will examine some of the new tools and refinements of the old tools that have come to light (or to our attention) since last year. The first problem one faces when encountering a dying cell is deciding whether it is apoptosis--programmed cell death--that is driving the events or whether it is simply necrosis. A variety of tools that exploit some of the features that set apoptosis apart from necrosis are available to assist in this dilemma. The properties of the membranes of apoptotic cells are distinctly different from those of necrotic cells: necrotic cells are notoriously leaky and will take up dyes/molecules from the surrounding medium, while apoptotic cells will not. Thus, one way to differentiate necrosis from apoptosis is to observe the uptake of vital dyes, fluorescent (propidium iodide or PI) or colorimetric (trypan blue). However, as apoptosis progresses, cell membranes become more permeable, making this test useful for only the early stages of the process. Furthermore, apoptosis often takes place in only a fraction of the cells in a population, so simply ruling out necrosis doesn't tell you which cells, if any, might be apoptotic. For this, positive identification of apoptotic cells is needed. Among the simplest methods is the old saw Hoechst versus PI bivariate histogram, so familiar to students of flow cytometry. In this context, Hoechst 33342, which stains live cells poorly, fluoresces brightly when taken up into the condensed chromatin found in (late) apoptotic cells, giving the cells a characteristic morphology. Also in the simple-but-effective category (not to mention inexpensive) is Molecular Probe's YO-PRO™-1 dye, a viable fluorescent dye that is taken up selectively by apoptotic cells and not normal cells. Like Hoechst, when YO-PRO is used in conjunction with PI, necrotic versus apoptotic cells can be distinguished, but unlike Hoechst, YO-PRO is not toxic to cells. Another rather simple approach to identifying apoptotic cells takes advantage of the fact that during apoptosis, DNA content will slowly change as proteins are stripped off the chromosomes and the DNA becomes vulnerable to nicking by endonucleases. In permeabilized cells, low molecular weight DNA will leak out of the nucleus, leaving cells with a lowered DNA content, which can be documented with DNA stains. As simple as this may sound, data of this kind can nonetheless be difficult to interpret, as apoptosis does not result in the creation a single cell type but rather a continuum of cell types with respect to some of these properties, which can blur the distinction between apoptosis and necrosis. A fairly early event in apoptosis is the flipping of some components of the plasma membrane from the inside surface to the outside surface, principally phosphatidylserine (PS). "Flipping" is the transition from a normally asymmetric distribution of the charged head groups of PS to a random orientation, thus exposing the PS head group on the external surface of the plasma membrane. This feature provides an identification tag for apoptotic cells through Annexin V, a phospholipid binding protein with the convenient habit of binding to PS. Annexin V conjugated with a variety of labels and in kits of various kinds is available for marking apoptotic cells. In addition to the standard fluorocein isothiocyanate (FITC), biotin, and phycoerythrin (PE), Annexin is also available with allophycocyanin (APC) (ALEXIS), Alexa™ 488 (Molecular Probes, NeXins Research), Oregon Green™ (Molecular Probes, NeXins Research, R & D Systems), Alexa 568 (Molecular Probes, NeXins Research, Roche Molecular Biochemicals), and enhanced green fluorescent protein (CLONTECH). The virtue of some of these new dyes (Alexa 488 and 568, and APC) is that they allow for triple label experiments, going beyond the simple red-green double-label fluorescence. Holder of world-wide patents for the application of Annexin V to the identification of apoptotic cells, the Dutch company NeXins Research has recently described in vivo labeling of apoptotic cells using a biotin-conjugated Annexin V.4 This property of the apopotic membrane is also exploited by Miltenyi Biotec's microbeads complexed with Annexin V, which facilitate the isolation of apoptotic cells from a mixed population. This kit, in conjunction with a magnetic separation unit supplied by Miltenyi and others, can be used either to enrich for apoptotic cells, (MACS Apoptotic Cell Isolation Kit) or to deplete a culture of dead cells (MACS Dead Cell Removal Kit). All these assays must, of necessity, be done on whole, viable cells and therefore can not be used with archived material; upon fixation, cells becomes permeable to Annexin V, which, once in the cell, will bind to a number of intracellular membranes. Annexin V labeled cells can be assayed either by flow cytometry, when a fluorochrome is attached to Annexin, or on slides. Both techniques give information on the percentage of cells in a population undergoing apoptosis and, in the case of cytometry, can be used to isolate such cells. Particularly handy in this regard is CompuCyte's Laser Scanning Cytometer (LSC), a slide-based laser scanning instrument that scans a microscope slide and identifies, counts, and records the positions of fluorescently labeled cells. Since apoptosis is accompanied by morphological changes in cells, and cells can sometimes be misclassified by the standard apoptotic probe assays, the LSC offers the ability to return to a cell and confirm that it is in fact apoptotic. The fragmentation of the nuclear DNA and concomitant generation of free 3'-ends has been exploited by numerous TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) assays, which enzymatically incorporate modified nucleotides containing a variety of tags on the ends, marking apoptotic cells for flow cytometry or microscopy. Since last year, more companies have weighed in with TUNEL kits of their own. In addition, the labeling of apoptotic cells with a monoclonal antibody directed against single-stranded DNA, offered by APOSTAIN and ALEXIS, is being touted as a superior method for assaying apoptosis-induced DNA fragmentation, with respect to specificity.5 This stain is based on the increased sensitivity of apoptotic cells to heat; gentle heating unravels the DNA in apoptotic cells due to the loss of chromosomal protein. The antibody binds to this unraveled single-stranded DNA. An unusual entry into the collection of products is the PCDstik Apoptosis Detection Assay Kit from Stratagene, a dip stick that company literature claims can detect fragmented DNA-histone core complexes in lysates prepared from as few as 2,500 cells in as little as 10 minutes. It has long been known that mitochondria have a connection to apoptosis. One of the first mammalian apoptosis proteins identified by homology to CED-3, a nematode cell death protein, bcl-2, is a mitochondrial protein. More recent observations have strengthened that link. For example, the release of cytochrome c from the mitochondria has been found to be an initiating event in the cascade of proteases that accompanies and orchestrates apoptosis. While a variety of potential-sensitive mitochondrial probes have been available (rhodamines and cyanines, for example), one (offered by CLONTECH in its ApoAlert™ Mitochondrial Membrane Sensor Kit and by Molecular Probes, in its JC-1) has a direct application to the study of apoptosis. This fluorescent potential-sensitive dye partitions differently in normal cells than apoptotic cells: in normal, healthy cells, the dye accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. In apoptotic cells with altered mitochondrial membrane potential, the dye stays in the cytoplasm, fluorescing green, providing a ready discrimination between apoptotic and nonapoptotic cells. (However, be warned: other conditions besides apoptosis can lead to a loss in mitochondrial membrane potential and differential staining by potential-sensitive dyes). Another mitochondrial dye, MitoTracker® Red CMXRos (Molecular Probes) provides the ability to fix the cells after incubation with this mitochondrial membrane potential probe without loss of signal, allowing researchers to study other elements of apoptosis concurrently with changes in the mitochondria. The above-mentioned probe does not necessarily allow for this. In the works at CLONTECH is an antibody against another mitochondrial membrane protein involved with apoptosis. Co-localization of two different caspase activities. HL-60 cells, treated with staurosporine, were stained with DEVDGIase, substrate for caspase 3 and 3-like, and VEIDNase, substrate for caspase 6 and 6-like. Red represents caspase 3, green caspase 6, and yellow both caspases. Figure provided by Oncoimmunin. At the heart of the apoptosis pathway are the caspases (cysteinyl-aspartic acid proteases), which continue to attract the attention of many Dead Heads. Among the many tools for studying caspases are purified enzymes themselves, antibodies directed against them, substrates and inhibitors of the enzymes, and kits for assaying caspases, all of which have seen new entries into the marketplace in the last year. In addition, reagents that will detect the cleaved protein products of various caspases have started to make their way into use. A new entry into the pile of antibodies against apoptosis-related proteins is a kit offered by Roche Molecular Biochemicals: the M30 CytoDeath Assay for use with epithelial cells and tissues. This kit uses a monoclonal antibody directed against the cleavage site of cytokeratin 18 that detects the cleaved version of the protein. Given this specificity, the M30 antibody can be used in whole cells or in situ to quantitate apoptosis. A variety of polyclonal antibodies directed against apoptosis targets (for example PARP, DFF, or the caspases themselves, which undergo a protease cleavage to become activated) are available from a number of companies (Pharmingen, BIOMOL, Enzyme Systems Product, Affinity BioReagents, and others). Most, but not all, of these recognize both cleaved and native species and therefore must be used with separated proteins, on Westerns, for example. However, antibodies that recognize only the cleaved and hence activated caspase 3 are available from Pharmingen and Upstate Biotechnology. Promega will soon offer an Anti-PARP p85 Fragment polyclonal antibody (patent pending) that will specifically recognize the caspase cleavage fragment of PARP without recognizing the 116k Da intact molecule in formalin fixed samples for immunocytochemistry. Substrates and inhibitors are legion for the 14 caspases that have been isolated. The substrates can be either fluorogenic or chromogenic, producing a signal upon cleavage. Particularly handy are cell permeable substrates developed at OncoImmunin. These molecules allow for assays of intact, viable cells that are sensitive enough to be used in the early stage of the process, in contrast to antibodies or cell impermeable substrates that require permeabilization of cells. Another twist to assaying of caspases in lysates is the caspase 3 Fluorescence Immunoabsorbent Enzyme Assay (FIENA) Kit offered by Roche Molecular Biochemicals. This kit incorporates an adsorption step on microplates coated with anticaspase 3 antibody, and while it does require more steps, it adds a level of specificity not obtained by using the substrates alone, many of which can show some (albeit reduced) activity with more than a single caspase. Given the importance of apoptosis to the development and survival of organisms, it perhaps is not surprising that multiple pathways as well as multiple layers of controls exist. And particularly striking in the schemes of apoptosis that have been developed is the reliance on protein:protein interactions among this large cast of characters. From the outer surface of the cell to the innermost regions of the nucleus, proteins are hooking up with other proteins in the cascade of events that lead ultimately to death. This fact is reflected in what might otherwise appear to be rather strange names given to the various proteins--FADD and TRADD (Fas-associated death domain and TNF-1 R1-associated death domain) proteins, for example. And then there's my personal favorite, TWEAK, which interacts with TRAMP (tyrosine-rich acidic matrix protein aka DR3 (death receptor 3). Research that has worked out the details of these interactions has relied heavily on antibodies, and accordingly a large number of reagents are currently available. Enumerating the vast store of antibodies that are available and the companies that produce them is beyond the scope of this article. However, several groups of proteins seem to capture particular attention: the bcl family because of the critical role it plays in regulating apoptosis, being an inhibitor of the process; cytochrome c, as the role of this mitochondrial protein is becoming appreciated; and of course Fas and Fas ligand, because of the central role they play in cytotoxic T lymphocyte killing. Among the products listed in the Table of Apoptosis Products are some antibodies that are either new or among the products the company has chosen to highlight from its offerings. A notable feature of apoptosis is that many of the critical proteins exist in preformed inactive forms, the caspases being the quintessential example. Accordingly, greater attention has been paid to the proteins themselves rather than the genes or messages, since much of the control appears to be exerted at the level of protein activation rather than gene expression. Nonetheless, as researchers move through the process, interest in measuring gene expression appears to be on the rise, judging from the number of new nucleic acid-based probes currently available. Companies are now offering cDNA probes for a number of apoptosis-related genes (ALEXIS, Ambion, Upstate Biotechnology), primer pairs for PCR and RT-PCR (Maxim, whose primer pairs are optimized for multiplexed reaction, Stratagene), and ribonuclease protection assay, which provides the possibility of measuring groups of messages in one experiment (RiboQuant Assay, Pharmingen). Resources for those wishing to know more about apoptosis are plentiful. Several Web sites devoted to this topic are available; several companies have produced excellent manuals to take you through in detail much of what has been touched on in this article. Pharmingen has produced a manual (Apoptosis: Applied Reagents and Technologies) as has Roche Molecular Biochemicals (Apoptosis and Cell Proliferation). While each contains information on the respective company's products, they both are filled with background information and references useful to anyone with an interest in apoptosis. L. DeFrancesco, "The death of a cell: a profile of apoptosis kits and reagents," The Scientist, 11(24): 22, Dec. 8, 1997. Science, 281:1301-25, August 28,1998. USA patent number 5834196: "Method for detecting and/or quantifying and/or separating apoptotic cells in or from a sample." Eijnde et al., "In situ detection of apoptosis during embryogenesis with Annexin-V: From whole mount to ultrastructure," Cytometry, 29:313-20, 1997. O.S. Frankfurt et al., "Monoclonal Antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis," Experimental Cell Research, 226: 387-97, 1996. The author can be reached at ldefrancesco@the-scientist.com. Caspase Substrates and Inhibitors, Apoptosis ProductsCast of Characters

Dead Again: Adventures in Apoptosis

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