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Correspondence to: Mark C. Willingham, Dept. of Pathology, Medical Center Boulevard, WinstonSalem, NC 271571072.
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Summary |
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Detection of apoptotic cell death in cells and tissues has become of paramount importance in many fields of modern biology, including studies of embryonic development, degenerative disease, and cancer biology. In addition to methods that employ biochemical analysis of large populations of cells, cytochemical methods have recently been extensively used both in individual cells and in tissues. Most of these methods exploit properties of dying cells that are more or less specific for the apoptotic process. However, considerable confusion exists over the interpretation of some of these methods and their usefulness in all settings. This review attempts to summarize the more recent advances in cytochemical detection of apoptosis and emphasizes some of the pitfalls that confuse the interpretation of results of these methods. (J Histochem Cytochem 47:11011109, 1999)
Key Words: apoptosis, TUNEL, ISNT, ISEL, cell death, caspases, blebbing, annexin V, in situ end-labeling, mitochondria, time-lapse microscopy, DNA cytochemistry, DNA fragmentation
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Introduction |
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The field of cell death research has undergone an explosion of new knowledge over the past decade. The realization that programmed cell death operates by highly conserved ubiquitous mechanisms in cells, and that these events are pivotal in most important pathologic processes, has focused interest on cell death research. The need for histochemical and cytochemical methods to evaluate death of cells, especially in intact tissues, has led to the development of several techniques. These methods are now used extensively in the study of a wide variety of diseases and in the study of physiology and development. However, the interpretation and accuracy of these methods are not always clear. This topic has been comprehensively reviewed previously and the difficulties in interpretation of apoptosis have been highlighted (
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Cell Death |
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Cells can die by either of two major mechanisms: necrosis or apoptosis. Necrosis is the death of cells through external damage, usually mediated via destruction of the plasma membrane or the biochemical supports of its integrity. Such a death is analogous to "cell murder." The necrotic cell exhibits a swollen morphology and the plasma membrane lyses, releasing cytoplasmic components into the surrounding tissue spaces. This release of necrotic debris attracts inflammatory cells, leading to the tissue destruction characteristic of inflammation. The death of single cells by this mechanism might be resolvable in some tissues, but a large number of cells dying by necrosis usually results in inflammation and subsequent repair and scarring, leading to compromise and permanent alteration of tissue architecture. Necrosis can occur in a matter of seconds (
The other major form of cell death is based on the concept of programmed cell death. Programmed cell death is a paradigm in which cells contain a genetically coded program of elements that lead to the death of cells. The biochemical and morphological events that effect this death usually lead to a unique and highly controlled series of events. The terminal events of this process are termed apoptosis (
The terminal events of apoptosis involve the activation of a specific series of cytoplasmic proteases, termed caspases. The activation of these self-catalytic caspases in the cytoplasm is tightly regulated. The initiators of apoptosis that set off this cascade of events leading to caspase activation are multiple, and two major pathways of initiation of this terminal pathway have been identified. One pathway involves so-called death receptors at the cell surface, a pathway that can directly activate upstream caspases in the cytoplasm. Another pathway involves the participation of mitochondria through the induction of leakiness of the external mitochondrial membrane, leading to the release of cytochrome c into the cytosol. These two pathways also intersect, in that the death receptor pathway can be amplified through mitochondrial damage. Downstream from these initiator mechanisms are terminal caspases that lead to the morphological and biochemical consequences of apoptosis.
The historical recognition that apoptotic cell death was a unique series of events was initially based on morphological evidence of changes in cell structures, especially the segmentation of nuclei. Other studies have shown that cells in tissue culture usually go through a unique series of surface morphological changes (e.g.,
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Detection Methods in Individual Cells and in Tissues |
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Much of the knowledge about apoptosis mechanisms has derived from observations of isolated cells, such as tissue culture systems. However, the properties revealed in these assays are not always applicable to the study of intact tissues. For example, assays such as the binding of annexin V or the impermeability of propidium iodide depend on the ability to incubate intact impermeable cells with the reagent, a process not possible in fixed tissue sections. Assays based on the size of isolated cell fragments are not applicable to fixed tissue. The kinetic context related to the synchrony of apoptosis, the length of time necessary for the initiating event to lead to the changes that occur, and the removal of apoptotic cells in vivo through phagocytosis often make the assays of apoptosis that are useful in tissue culture less applicable to intact tissues. Furthermore, events that occur in individual cells can sometimes be implied from assays that are applicable only to the examination of large numbers of cells at one time. An analogy might be the ability to detect the low-level expression of products of specific transfected genes in mass culture through the use of sensitive enzyme assays (e.g., CAT assay) as opposed to the cytochemical identification of high levels of the specific protein product in a small number of individual cells (e.g., a GFP vector). These two approaches can sometimes yield conflicting results, often based on the heterogeneity or lack of synchrony of events in single cells vs the entire population. This has been a major point of confusion in the field of apoptosis.
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Apoptosis Is Not Always Synchronous |
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A major and often unappreciated aspect of apoptotic cell death is that cells within a population may begin apoptosis at very different times after the addition of an initiator, and the length of the various stages of apoptotic morphological change can vary from cell to cell (
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Surface Morphological and Biochemical Changes |
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For tissue culture systems, a good point at which to start the analysis of apoptosis is the use of video time-lapse microscopy, a simple and clearly interpretable approach to defining the kinetics of apoptosis in cell culture (
Externalization of phosphatidylserine (PS) and phosphatidylethanolamine is a hallmark of the changes in the cell surface during apoptosis (
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The permeability of the plasma membrane is also a central difference between necrosis and apoptosis. Large molecular weight DNA binding dyes, such as propidium iodide (PI), cannot enter intact cells because of their large size and, without permeabilization treatments, do not label apoptotic cells until the final lysis stage. On the other hand, smaller dyes, especially those that can attach to DNA, can label both apoptotic cells and normal cells. Using flow cytometry, one can distinguish apoptotic from necrotic cells as those that show internal DNA labeling with a small dye (such as DAPI, Hoechst 33342 or 33258, or calcein-AM), while not labeling with PI (
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Nuclear Morphology Changes and DNA Changes May Not Be the Same |
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An early observation concerning apoptosis was that cells that entered apoptosis from nonmitotic parts of the cell cycle showed dramatic and characteristic changes in nuclear shape and organization. It is perhaps correct to say that the characteristic change in nuclear morphology is the most accurate indicator of the involvement of apoptosis in the death of a cell. This is true even in the face of the ironic observation that nuclear segmentation is not actually required for other aspects of apoptosis. That is, cells that have been enucleated still undergo the other changes associated with apoptosis (
In addition to changes in nuclear morphology, loss of DNA integrity also characterizes apoptosis. It was assumed that these two events were in some way related, as they may be. However, there is no compelling reason necessarily to assume that they are. When DNA extracted from apoptotic cells was analyzed using gel electrophoresis, a characteristic internucleosomal "ladder" of DNA fragments was found. Larger DNA fragments have also been seen at earlier times in apoptotic cell cultures. These gel results have been used as a hallmark of apoptotic detection. However, such analysis requires the extraction of DNA from large numbers of cells. In addition, the apoptosis must be relatively synchronous for this analysis, a synchrony that is not always present. It has not been directly demonstrated that DNA fragments in this way in intact cells. That is, it is possible that the internucleosomal breaks in DNA occur after or during DNA extraction procedures because of the potential fragility of caspase-treated DNA in removing other components of chromatin structure. Furthermore, the detection of strand breaks may be so sensitive that only a small number of apoptotic cells may be needed in a population to produce a detectable signal. That is, the entry of the majority of cells into apoptosis might occur at a much later time point than the first detection of "ladders." Even so, these observations led to the development of in situ assays for the presence of single-strand or double-strand breaks in DNA. The interpretation and application of these methods have been somewhat controversial. Regardless of the specific methods used, the change in nuclear morphology often does not coincide with the appearance of detectable strand breaks in every cell.
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The Terminology of DNA and Nuclear Changes in Apoptosis |
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Although many cytochemical methods are based on the detection of strand breaks or on the appearance of DNA fragments in gel electrophoresis, there is no reason to assume that nuclear morphological changes and detectable DNA strand breaks occur at the same time. As a point of terminology, there has been considerable confusion generated by the term "nuclear fragmentation." This term mixes the concepts of the generation of DNA fragments (DNA fragmentation) with the changes in nuclear shape. A better alternative, which I prefer, is the use of the term "nuclear segmentation." This describes the change in nuclear shape in which the normally round or oval nucleus segments into smaller, compact, homogeneous, variably sized chromatin masses, the characteristic change seen in apoptosis. This term does not imply any generation of DNA fragments or strand breaks but refers only to the shape of the nucleus. Because there is reason to believe that the shape and the DNA breaks may not necessarily be related in each cell, it appears better to use this more precise term to describe shape changes.
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When Do DNA Strand Breaks Appear in Situ? |
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The major methods developed for detection of DNA strand breaks involve the detection of 3'-OH ends of single-stranded DNA (in situ end-labeling; ISEL). Addition of labeled nucleotides to these ends, either using E. coli polymerase (or its Klenow fragment) by in situ nick-translation (ISNT) (Figure 3) or using terminal transferase (TUNEL) (
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Apoptotic Cells May Not "Live" Long In Vivo |
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Dying cells in vivo are removed from tissues by phagocytic cells, a process that emphasizes the usefulness of the apoptotic mechanism for normal tissue remodeling. However, the removal of cells can occur with different efficiencies in different tissues. Massive apoptosis can overwhelm the phagocytic potential of a tissue, and virtually all apoptotic cells will remain for extended periods. In the case of limited apoptosis, however, phagocytic or other adjacent cells may remove the apoptotic remnants as rapidly as they are generated. Unlike cell culture, in which the cell remnants remain undisturbed, in vivo apoptosis may yield only a hint of an occasional cell remnant in a phagocytically active tissue. This problem makes in vivo measurements and interpretation of apoptosis by in situ cytochemical methods extremely difficult.
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Other Biochemical Changes |
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Recent studies have revealed many other steps in the apoptotic machinery inside cells. Among these are the activation of caspases and the cleavage of specific caspase substrates. Incorporation of specific caspase substrates into living cells and the detection of cleavage products (e.g., using fluorescence resonance energy transfer) have been presented as new assays of apoptosis (
Methods for detection of activity of endogenous caspase substrates (such as PARP) have also been presented recently (
Another example of antibodies specific for caspase-generated cleavage products was a study describing an antibody specific for a caspase-generated fragment of actin, termed "fractin" (
Transglutaminase activation is another biochemical change that has been proposed as an indicator of apoptosis, but whether or not this would be applicable to all systems is not yet clear (
Mitochondria have been implicated in the apoptotic pathway due to many death inducers, even those that act primarily through surface death receptors but are then amplified through mitochondrial damage. The primary event proposed as important for apoptotic induction is the leakiness of the external membrane of the mitochondrion, resulting in leakage of cytochrome c into the cytosol. This is different from the overall loss of membrane potential of the entire mitochondrion (the permeability transition) as measured by vital dyes such as rhodamine 123 or Mitotracker, but both events may occur in dying cells (
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Conclusions |
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This brief review emphasizes some of the recently employed cytochemical methods for the detection of apoptosis in cells. Unfortunately, there is no single assay that can be used blindly with perfect specificity and sensitivity. Ironically, the most specific assay is perhaps the oldest, the detection of nuclear shape changes in the early stages of apoptosis. In combination with other methods, this morphological interpretation usually allows a relatively accurate interpretation of apoptosis. For tissue sections, many investigators recommend labeling of DNA strand breaks (ISNT, TUNEL, anti-SS DNA) together with analysis of nuclear morphology. Although detection of DNA strand breaks is a cornerstone method for use in tissue sections, it requires care in interpretation, especially in the details of cell fixation, permeabilization, and processing. For cultured cells, a direct and easily interpretable assay for apoptosis is the observation of surface morphological features with time-lapse microscopy. For flow cytometry using DNA binding dyes, detection of apoptotic bodies as a pre-G1 peak is a simple and rapid assay for large numbers of cells. Labeling of surface-exposed phosphatidylserine is a useful although imperfect method for both microscopy and flow cytometry. Perhaps the only comforting conclusion from these observations is that the mechanism of apoptotic death appears to be extremely highly conserved in eukaryotes. As a consequence, the development of new techniques will have to deal with the many facets of only a single, albeit complex, biological mechanism.
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Acknowledgments |
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The author thanks Katherine Barrett for expert technical assistance, and Kristy K. Young and Jae A. Collins for help with the ISNT experiments shown in Figure 3.
Received for publication April 7, 1999; accepted April 23, 1999.
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