Cardiovascular Research

Cardiovascular Research 2000 45(3):528-537; doi:10.1016/S0008-6363(99)00384-3
© 2000 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Saraste, A. Articles by Pulkki, K. Search for Related Content PubMed PubMed Citation Articles by Saraste, A. Articles by Pulkki, K. Social Bookmarking          What's this?

Copyright © 2000, European Society of Cardiology

Morphologic and biochemical hallmarks of apoptosis

Antti Saraste^a,b,* and Kari Pulkki^c

^aDepartment of Anatomy, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
^bDepartment of Medicine, Turku University Central Hospital, Turku, Finland
^cDepartment of Clinical Chemistry, Turku University Central Hospital, Turku, Finland

^* Corresponding author. Tel.: +358-2-333-71; fax: +358-2-333-7352 antti.saraste{at}utu.fi

Received 18 August 1999; accepted 21 October 1999

	Abstract

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
Apoptosis is characterised by a series of typical morphological features, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells. This paper reviews the current knowledge on the molecular mechanisms of apoptosis as they relate to the morphologic hallmarks and their implications for the detection of apoptosis in cardiac tissue. Activation of cysteine proteases called caspases plays a major role in the execution of apoptosis. These proteases selectively cleave vital cellular substrates, which results in apoptotic morphology and internucleosomal fragmentation of DNA by selectively activated DNases. In response to several pro-apoptotic signals, mitochondria release caspase activating factors, that initiate an escalating caspase cascade and commit the cell to die. Members of the Bcl-2 oncoprotein family control mitochondrial events and are able to prevent, or induce, both apoptotic and non-apoptotic types of cell death. This suggests that different types of cell death share common mechanisms in the early phases, whereas activation of caspases determines the phenotype of cell death. Detection of apoptotic cells in tissue samples currently relies on the TUNEL assay. TUNEL-positive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in DNA electrophoresis. Thus, provided that the staining protocol is carefully standardised, this quantitative methodology provides reproducible results of the occurrence of cardiomyocyte apoptosis in cardiac samples. Recently, potentially more specific assays based on analysis of DNA fragmentation or demonstration of caspase activation have been developed. Applicability of these assays to demonstrate cardiomyocyte apoptosis should be tested.

KEYWORDS Apoptosis; Myocytes; Mitochondria; Histo(patho)logy

	1 Introduction

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
Apoptosis is characterised by a series of typical morphological events, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells [1]. Internucleosomal fragmentation of genomic DNA has been the biochemical hallmark of apoptosis for many years [2]. The cell death process is executed in an organised fashion reflecting the presence of well preserved molecular pathways. Recently, selective proteolysis of vital cellular substrates has been implicated as the key molecular mechanism of apoptotic changes.

This paper reviews the current knowledge on the molecular mechanisms of apoptosis as they relate to the morphological hallmarks, their implications in the detection of apoptosis and the differentiation between apoptosis and other forms of cell death. We discuss the implications of these issues in the assessment of cardiomyocyte apoptosis.

	2 Phases and time course of apoptosis

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
Under physiological conditions, the occurrence of apoptosis in tissues is typically a rare event. Thus, only a small number of apoptotic cells can be seen at any time point. Studies with cytosolic extracts of cells, which have been induced to undergo apoptosis in a synchronous manner, have shown that apoptosis can be divided into biochemically and morphologically distinct phases [3,4]. In the first, pro-apoptotic stimuli trigger activation of the central molecular machinery of apoptosis (initiation phase). In the second, committed or effector phase, the molecular executioner machinery becomes fully activated as shown by the ability of the cytosolic extracts of committed cells to induce apoptotic changes in nuclei [3,4]. Only after this, in the third, or degradation phase, do the hallmarks of apoptosis become evident. These include morphologic changes and DNA fragmentation.

The asynchronous nature of apoptotic death in cell populations is mainly due to the highly variable duration of the initiaton phase. In cell culture videomicroscopy studies the dynamic morphologic changes at the light microscopic level always take place in less than 2 h [5,6]. The point of no return occurs several hours before the appearance of morphologic features [6,7]. In vivo, the duration of an apoptotic cell death has been estimated to be between 6 and 24 h, although it may vary depending on the cell type [1,8]. As a result of the short time scale of apoptosis, only few cells undergoing apoptosis are present at a single time point and the quantitative significance of apoptosis may easily be underestimated.

	3 Morphologic hallmarks of apoptosis

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
The definition of apoptosis was first based on a distinct sequence of morphologic features by electron microscopy, described in 1972 by Kerr et al. [1,9]. The onset of apoptosis is characterised by shrinkage of the cell and the nucleus as well as condensation of nuclear chromatin into sharply delineated masses that become marginated against the nuclear membranes. Later on, the nucleus progressively condenses and breaks up (karyorrhexis). The cell detaches from the surrounding tissue and its outlines become convoluted and form extensions. The term budding has been coined for a process whereby the extensions separate and the plasma membrane seals to form a separate membrane around the detached solid cellular material. These apoptotic bodies are crowded with closely packed cellular organelles and fragments of nucleus. The fine structures, including membranes and mitochondria, are well preserved inside the bodies. The apoptotic bodies are rapidly phagocytosed into neighbouring cells, including macrophages and parenchymal cells. Apoptotic bodies can be recognised inside these cells, but eventually they become degraded. If the fragmented cell is not phagocytosed it will undergo degradation which resembles necrosis in a process called secondary necrosis [1,9]. Apoptotic shrinkage, disassembly into apoptotic bodies and engulfment of individual cells characteristically occur without associated inflammation, which would be the consequence of releasing intracellular contents into tissues.

Recent studies indicate that proteolytic cleavages of a set of key proteins by activated caspase proteases play a role in the accomplishment of apoptotic morphology (Table 1) [10]. Although the exact mechanism of how the degradation of these proteins results in apoptotic morphology remains unknown, many target proteins of caspases participate in the formation and regulation of the membrane-associated cortical microfilament cytoskeleton, which is an important determinant of the cell shape (Table 1) [11–20]. Overexpression of the caspase cleaved forms of Gas2 or gelsolin result in dramatic changes in cell shape, resembling apoptosis [18,19]. Also other proteases, such as calpains [11,14], have been implicated in the signalling of the apoptotic changes in the cytoskeleton.

View this table: [in this window] [in a new window]   Table 1 Structural proteins processed by caspases associated with apoptotic morphology

 
Two protein kinases at the cell-to-cell and cell-to-matrix attachment sites are also targets of caspases [21,22]. Their cleavage by caspases results in further enhancement of the pro-apoptotic signalling [21] and possibly detachment of the cell from the surrounding tissue [22]. Disassembly of the nuclear lamina, the supporting structure of the nuclear envelope, is also an essential feature of nuclear breakdown in apoptosis [3]. This process depends on caspase-mediated degradation of nuclear lamins A and B [23,24]. Proteins involved in the regulation of chromatin structure [25] or interactions between chromatin and nuclear matrix proteins, such as nuclear mitosis associated protein (NuMa) [26], are also cleaved. Caspase proteases have even been implicated in the externalisation of phosphatidyl serine, which mediates the recognition of apoptotic bodies by neighbouring cells [15,27,28].

In cardiomyocytes, several apoptotic features have been reported at the light microscopic level (Fig. 1) (review in [29]). These include condensation of nuclei, shrinkage of the cytoplasm, convolution of outlines, and detachment from the surrounding tissue. Using electron microscopy, morphologic features of apoptosis have been described in cardiomyocytes in the degenerating conduction system [30], in experimental heart failure [31] and in human hibernating myocardium [32]. Apoptotic bodies phagocytosed by macrophages and cardiomyocytes have also been observed, but at this stage the cell type from which the apoptotic body was derived from could not be recognized [30,31].

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (a) Ladder pattern of internucleosomal fragmentation in DNA electrophoresis. The DNA is isolated from human myocardium bordering the acutely infarcted tissue and from the remote non-infarcted myocardium of the same heart (left and right lanes, respectively). (b–e) Apoptotic cardiomyocytes detected with TUNEL assay and anti-myosin staining in a cardiomyopathic explanted failing human heart. Positive TUNEL reaction is in the nuclei (b and d, arrows) of the same cells that have been identified as cardiomyocytes by anti-myosin staining (c and e). The nuclei of both cells are condensed, which is typical of apoptosis. Moreover, the cell in panel d is shrunken, its outline is convoluted and detached from the surrounding tissue suggesting that it is in a late stage of apoptosis. Arrow in (a) shows a fragment size of 400 bp, bar in (b–e)=10 µm.

 

	4 Biochemical hallmarks of apoptosis

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
4.1 Internucleosomal DNA fragmentation
The biochemical hallmark of apoptosis is degradation of DNA by endogenous DNases, which cut the internucleosomal regions into double-stranded DNA fragments of 180–200 base pairs (bp) [2]. The DNA fragments contain blunt ends [33] as well as single base 3' overhangs [34]. Internucleosomal fragmentation has been demonstrated with well-characterised apoptotic morphology in a wide variety of situations and cell types (review in [35]).

A variety of caspase substrates are involved in the regulation of DNA structure, repair and replication [25]. The DNase enzymes responsible for the fragmentation during apoptosis include DNA fragmentation factor (DFF40) [36], caspase activated DNase (CAD) [37,38] and in hematopoietic cells NUC70 [39]. DFF40 and CAD are present in normal cells as inactive heterodimers with the inhibitor proteins DFF45 [40] and ICAD (inhibitor of CAD) [38]. These enzymes are selectively activated upon cleavage by caspase 3 [38,40] or by other members of the caspase family [41]. Exposure of nuclei to activated CAD or DFF40 is sufficient to induce the nuclear morphologic changes typical of apoptosis [36,37]. Formation of large size (50–300 kbp) DNA fragments precedes internucleosomal fragmentation, but the role of the above mentioned DNases in this process has not been studied [35].

Demonstration of the internucleosomal DNA fragmentation has been a major advance in the detection of apoptosis. The DNA fragments are detectable as a ladder pattern in the electrophoresis of isolated DNA [2]. With the terminal transferase mediated DNA nick end labelling (TUNEL) assay, cells containing DNA strand breaks become visible in light microscopic analysis [8]. The quantification of apoptosis in tissue samples relies on this or related methods.

TUNEL positive cardiomyocytes can be found in cardiac tissue samples in several physiological and pathological conditions (review in [29,42]). However, there has been a substantial variation in the quantitative results obtained in consecutive studies in the same situation. The validity of the TUNEL assay as a method to detect apopotosis has even been questioned [43]. These issues point to methodological complexities associated with the TUNEL assay. DNA damage is not a unique feature of apoptosis, but can occur in necrosis, during repair of reversibly damaged DNA and postmortem autolysis [34,44]. The terminal transferase enzyme adds labelled nucleotides into various kinds of DNA ends [45]. The staining kinetics of the TUNEL assay depends on the accessibility of the DNA strand breaks to the staining reagents. This varies between tissue types and tissue preparation procedures, e.g. fixation and proteolytic pre-treatments [29]. Thus, to avoid false positive or negative results, careful standardisation of the staining conditions is important [29].

Our experience [46,47] is that the optimal positive control of apoptosis is an adjacent myocardial tissue section treated with DNase I enzyme to induce the formation of DNA strand breaks. The tissue sections are heated in sodium citrate (80°C, 30 min) and digested with proteinase K (10 µg/ml, 37°C, 30 min) to expose DNA. To confirm optimal sensitivity and specificity of the assay, the development of the alkaline phosphatase staining reaction should be monitored using the light microscope and terminated upon appearance of intense positivity in the DNase treated control section. These procedures also normalise the staining results for differences in the tissue permeability of the reagents.

Using this methodology, the quantitative results are reproducible (r=0.88 for results of repeated procedures) [47] and the TUNEL-positive cardiomyocytes show some features of apoptotic morphology, such as condensed nuclei [46,47] (Fig. 1). In contrast, we have not observed TUNEL staining in cells showing features of coagulative necrosis [46]. In our hands, DNA ladders become demonstrable in myocardial samples when the proportion of TUNEL-positive cardiomyocytes exceeds approximately 0.04% [46] (Fig. 1). Others have shown that postmortem autolysis of 48 h in neural tissue [48] and 24 h in cardiac tissue [49] does not affect the reliability of the TUNEL assay.

4.2 Caspase activation
The fact that interleukin-1β -converting enzyme (ICE) is structurally related to the Caenorhabditis elegans cell death protein CED-3, which is necessary for the occurrence of apoptosis in this animal, first suggested that ICE and related enzymes could be implicated in apoptosis also in mammals [50,51]. ICE became the first member of a protease family named as caspases [52], which play a major role in the execution of apoptosis [53–58]. A number of genetic and biochemical studies suggest that caspase activation is essential for the occurrence of the apoptotic phenotype of cell death [59–61]. However, cell death per se is not necessarily dependent on caspases, because cells die through a non-apoptotic morphology even when caspase activity is inhibited [5,7,59,62].

Caspases are cysteine proteases that cleave their substrate proteins specifically behind an aspartate residue (review in [63]). They are formed constitutively and are normally present as inactive proenzymes. For the induction of full enzymatic activity, they require cleavage at specific internal aspartate residues, which separate large and small subunits from each other [63]. Studies on substrate specificity, prodomain structure and biological function [10,63–67] have revealed that caspases are activated during apoptosis in a self-amplifying cascade. Activation of the upstream caspases, such as caspases 2, 8, 9 and 10, by pro-apoptotic signals leads to proteolytic activation of the downstream or effector caspases 3, 6 and 7. The effector caspases actually cleave a set of vital proteins and thus, initiate and execute the apoptotic degradation phase including DNA degradation and the typical morphologic features.

Two major pathways of caspase activation have been characterised (Fig. 2). One is initiated by ligation of the death receptors (rev. in [68]), which include e.g. Fas and TNF receptors. Caspase 8 is the most apical caspase in this pathway [65,66], which is activated by a signalling complex at the receptors [68]. The other, mitochondrial pathway (review in [69,70]) integrates apoptotic signals caused by various cytotoxic agents, aberrant oncogene expression [71] and p53 [72]. It is a target of some apoptosis regulating proteins of the Bcl-2 family [73,74]. It also amplifies receptor mediated apoptosis [75]. The key checkpoint of this pathway is the release of cytochrome C and other caspase activating factors, such as the apoptosis inducing factor AIF, into the cytosol from the mitochondrial transmembrane space [69,70,76,77]. Caspase 9 is the most upstream caspase in this mitochondrial apoptotic pathway [67]. Activation of pro-caspase 9 requires a cytosolic complex that includes cytochrome C, the C. elegans death gene CED-4 homologue called apoptosis protease-activating factor (Apaf-1) and dATP [78,79]. Since the mitochondrial morphology remains intact [1], mechanisms of cytochrome C release during apoptosis have been a matter of discussion. Mitochondrial membrane permeability transition and subsequent opening of the permeability transition associated pores [70], and opening of selective release channels before swelling of the mitochondrial matrix, rupture of outer mitochondrial membrane, and permeability transition [69,80] have been suggested.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Major molecular pathways of apoptosis and the relationships between apoptotic and non-apoptotic cell death. Ligation of the death receptors and release of caspase activating factors, such as cytochrome C, from the mitochondria initiate caspase cascades via initiator caspases 8 and 9, respectively. Mitochondria also amplify receptor mediated apoptosis. The activation of caspases determines the phenotype of cell death. However, in some circumstances, apoptosis can progress into secondary necrosis. Apaf-1=apoptosis protease-activating factor, ROS=reactive oxygen species.

 
A novel approach to study the presence of apoptosis is to demonstrate the activation of downstream caspases. This can be detected by western blotting of target proteins that have been cleaved by caspases or by demonstration of caspase activity by enzyme assay. Cleavage site specific antibodies that detect only the cleaved forms of caspase substrates [81–83] or activated forms of caspases have also been generated [84–86]. They can be applied to detect apoptotic cells in tissue sections by light microscopy. In addition, caspase inhibitors have been used to study whether cell death is dependent on caspases.

Several studies have provided evidence of caspase activation in cardiomyocyte death. Active forms of caspases 2, 3 and 7 are generated in the ischemic–reperfused myocardium [87]. Caspase 3 substrate cleavage has been observed under oxidative stress, in ischemic–reperfused myocardium, during acute cardiac allograft rejection and in explanted failing human hearts [87–90]. Moreover, activated form of caspase 3 has been shown to colocalise with the TUNEL-positive cardiomyocytes in ischemic–reperfused myocardium [84]. Recently, translocation of cytochrome C, the major intermediate activator of caspases, from mitochondria into cytoplasm has been observed in explanted failing human hearts [90] and in response to oxygen radical treatment [88]. Caspase inhibitors have been shown to prevent cardiomyocyte death in response to simulated ischemia in vitro [91] and in ischemic–reperfused myocardium together with reduction of myocardial infarct size [87,92].

	5 Apoptosis and necrosis: the spectrum of cell death

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
The differences between apoptosis and other types of cell death have recently been a matter of discussion [93]. Apoptosis is usually contrasted to necrosis, which is considered as the general appearance of cell death following rapid loss of cellular homeostasis (accidental cell death) [1,9,93]. The term oncosis has been coined for cell death caused specifically by ischemia [93]. These types of cell death are characterised by swelling due to accumulation of water and electrolytes, early plasma membrane rupture and disruption of cellular organelles, including mitochondria. Budding and formation of apoptotic bodies are absent and the nuclear chromatin is irregularly clumped. Leakage of intracellular contents induces an inflammatory response. Phagocytosis of the remnants of dead cells is delayed until accumulation of inflammatory cells. The resulting lesions contain groups of contiguous necrotic cells and at later time points inflammatory leukocytes.

The exact morphology of necrotic cells is highly variable, depending on the features of the causative agent. Detection of non-apoptotic cell death relies mainly on the morphologic appearance of the lesions and on the use of assays that demonstrate cell membrane damage, such as uptake of a tracer dye or leakage of intracellular enzymes. However, the term necrosis is actually imprecise [93], as it not only refers to changes occurring during cell death but also to degradation after cells have died. Even apoptotic cells undergo secondary necrosis after the distinct apoptotic morphology has already been accomplished [1].

Recent biochemical studies have weakened the strong contrast between apoptosis and necrosis (Fig. 2) [69,70]. An emerging view is that the mitochondrial changes would be the critical step in commitment to both apoptotic and non-apoptotic types of cell death (Fig. 2). First, the mitochondrial permeability transition is involved in apoptosis and in many examples of well characterised necrosis [70]. Second, the anti-apoptotic proteins of the Bcl-2 family, which act on the mitochondrial level by blocking the release of cytochrome C, prevent both apoptotic and non-apoptotic cell death [5,7,62]. In contrast, the pro-apoptotic family members such as Bax, induce both types of cell death [94]. Thus, it appears that apoptosis and necrosis may share common mechanisms in the early stages of cell death. The activation of caspases would finally determine whether the cell death is phenotypically apoptotic or necrotic [59,69]. Still, the lack of ATP as well as increasing intensity of the stimulus may result in abortion of apoptosis and consequently, in a mixture of apoptotic and necrotic features [69,95,96]. Similarly, caspase mediated cell death can serve as a precursor of secondary necrosis [69].

The lack of uniform criteria to differentiate between apoptosis and other types of cell death has caused confusion in determining the relative contributions of each form of cell death, particularly in situations when apoptosis and necrosis are thought to co-exist. A typical example is acute myocardial ischemia–reperfusion injury [46,49,97–100]. The kinetics of cell death is very rapid as an area of myocardial necrosis evolves in the central ischemic area during the first hours–days after the onset of ischemia [97,98]. Apoptotic DNA fragmentation has been found in the very early phases of the process in the central ischemic areas and at later times in the border zones [46,98]. Moreover, there is a population of cells showing both internucleosomal DNA fragmentation and membrane damage typical of apoptosis and necrosis, respectively [98]. The proportion of apoptotic cells in the ischemic areas has been highly variable. Moreover, the classical apoptotic morphology has been absent in two consecutive studies [99,100]. Several possible explanations to this have been proposed. First, if the same stimulus induced both apoptosis and necrosis in a dose-dependent fashion [96], higher intensity of ischemia in the central areas could favour necrosis. Second, since the completion of the apoptotic program requires energy, progression of the process could be aborted by the loss of high energy ATP in severely ischemic tissue [95]. Even a specific association of apoptosis with reperfusion has been suggested [99,101]. Third, it has been suggested that biochemical pathways mediating both types of cell death could be activated in the same cell [100].

	6 Implications for studying apoptosis in cardiomyocytes

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
Increasing evidence shows that cardiomyocyte apoptosis occurs in various disease conditions. However, the exact significance of apoptosis and other types of cell death remains to be studied. Table 2 summarises some features of cardiomyocytes that must be taken into account when the results of in vitro apoptosis experiments are to be interpreted. Moreover, to address these questions a consensus on the definition of apoptosis and methodological issues associated with quantification of apoptosis would be needed.

View this table: [in this window] [in a new window]   Table 2 Special features of cardiomyocytes with possible influence on apoptosis

 
To quantify apoptotic cardiomyocytes in tissue samples, a method with high signal to noise ration is required. Quantification requires analysis of a large number of high power microscopic fields because the number of apoptotic cells may be very small [47] and highly variable in different parts of the sample [45]. The cardiomyocyte origin of the cells undergoing apoptosis should be confirmed by the presence of surrounding myofibers (see Fig. 1). This can be done using either non-specific counterstaining or specific antibodies, which are likely to be more sensitive in case of very small apoptotic bodies. The current method of choice for quantification of apoptotic cardiomyocytes is the TUNEL assay, provided that is carefully standardised (see above).

Confirmation of apoptosis is then based on other criteria, such as demonstration of the internucleosomal DNA fragmentation in electrophoresis or analysis of the morphological features. Electrophoresis does not specify the cell types undergoing apoptosis and it is not sensitive enough to detect small quantitative differences. Morphologic criteria are considered as the most reliable evidence of apoptosis. However, demonstration of complete apoptotic morphology by a single method is difficult. Nuclear condensation, shrinkage of the cell and fragmentation into apoptotic bodies can be visualised using light microscopy, which is suitable for screening of large numbers of cells. Electron microscopy is required to demonstrate the loss of intact intracellular structures. The major limitation of electron microscopy is that studying large numbers of cells is not feasible. Since the proportion of cells in the degradation phase of apoptosis at a single time point is small, finding even a single apoptotic cell by electron microscopy may be difficult. On the other hand, the use of morphologic criteria has some inherent limitations. The exact sequence of morphological changes may vary in different cell types. For example, there is less formation of apoptotic bodies in cells with a stiff intracellular structure [9].

Novel detection methods of apoptosis include the in situ ligation assay and assays based on the demonstration of caspase activation. By the in situ ligation assay using Taq polymerase it is possible to detect the single base DNA overhangs in apoptotic cells [34]. These DNA fragments are not found in necrotic tumour tissue and after non-apoptotic events, such as treatment of cells with hydrogen peroxide or exposing them to prolonged post-mortem autolysis [34]. Thus, assays using Taq polymerase are suggested to be very specific for the detection of apoptosis in tissue samples. In the myocardium, equal results have been obtained using both TUNEL- and Taq polymerase assays [102,103]. Activation of caspases is a potentially specific feature of apoptosis, as it reflects the underlying molecular changes. Recent studies have shown that methods based on demonstration of caspase activation can be applied to detect apoptotic cells in tissue sections by light microscopy. However, sufficient quantitative accuracy and reproducibility of these assays remains to be studied. In addition to detection of apoptosis, caspase inhibitors could be used to probe caspase-dependence of cardiomyocyte death in response to various apoptotic stimuli.

Time for primary review 26 days.

	Acknowledgments

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 
This study has been financially supported by the Finnish Heart Association, the Aarne Koskelo Foundation, the Finnish Cultural Foundation and the EVO-funds of Turku University Central Hospital. Liisa-Maria Voipio-Pulkki is acknowledged for reading the manuscript and for stimulating discussions.

	References

Top Abstract 1 Introduction 2 Phases and time... 3 Morphologic hallmarks of... 4 Biochemical hallmarks of... 5 Apoptosis and necrosis:... 6 Implications for studying... Acknowledgments References

 

1. Kerr J.F.R., Wyllie A.H., Currie A.R. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer (1972) 26:239–257.[Web of Science][Medline]
2. Wyllie A.H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (1980) 284:555–556.[CrossRef][Medline]
3. Lazebnik Y.A., Cole S., Cooke C.A., Nelson W.G., Earnshaw W.C. Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. (1993) 123:7–22.[Abstract/Free Full Text]
4. Solary E., Bertrand R., Kohn K.W., Pommier Y. Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase I and II inhibitors. Blood (1993) 81:1359–1368.[Abstract/Free Full Text]
5. McCarthy N.J., Whyte M.K.B., Gilbert C.S., Evan G.I. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. (1997) 136:215–227.[Abstract/Free Full Text]
6. Messam C.A., Pittman R.N. Asynchrony and commitment to die during apoptosis. Exp. Cell Res. (1998) 238:389–398.[CrossRef][Web of Science][Medline]
7. Brunet C.L., Gunby R.H., Benson R.S., Hickman J.A., Watson A.J., Brady G. Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers. Cell Death Different (1998) 5:107–115.[CrossRef][Web of Science][Medline]
8. Gavrieli Y., Sherman Y., Ben-Sasson S.A. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J. Cell. Biol. (1992) 119:493–501.[Abstract/Free Full Text]
9. Kerr J.F.R., Winterford C.M., Harmon B.V. Apoptosis: its significance in cancer and cancer therapy. Cancer (1994) 73:2013–2026.[CrossRef][Web of Science][Medline]
10. Martin S.J., Green D.R. Protease activation during apoptosis: Death by a thousand cuts? Cell (1995) 82:349–352.[CrossRef][Web of Science][Medline]
11. Brown S.B., Bailey K., Savill J. Actin is cleaved during constitutive apoptosis. Biochem. J. (1997) 323:233–237.[Web of Science][Medline]
12. Kayalar C., Ord T., Testa M.P., Zhong L.T., Bredesen D.E. Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition. Proc. Natl. Acad. Sci. (1996) 93:2234–2238.[Abstract/Free Full Text]
13. Mashima T., Naito M., Noguchi K., Miller D.K., Nicholson D.W., Tsuruo T. Actin cleavage by CPP-32/apopain during the development of apoptosis. Oncogene (1997) 14:1007–1012.[CrossRef][Web of Science][Medline]
14. Wang K.K.W., Posmantur R., Nath R., et al. Simultaneous degradation of II- and βII-spectrin by caspase 3 (CPP32) in apoptotic cells. J. Biol. Chem. (1998) 273:22490–22497.[Abstract/Free Full Text]
15. Vanags D.M., Porn-Ares M.I., Coppola S., Burgess D.H., Orrenius S. Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J. Biol. Chem. (1996) 271:31075–31085.[Abstract/Free Full Text]
16. Martin S.J., O’Brien G.A., Nishioka W.K., McGahon A.J., Saido T.C., Green D.R. Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem (1995) 270:6425–6428.[Abstract/Free Full Text]
17. Brancolini C., Lazarevic D., Rodriquez J., Schneider C. Dismantling cell–cell contacts during apoptosis is coupled to caspase-dependent proteolytic cleavage of beta-catenin. J. Cell Biol. (1997) 139:759–771.[Abstract/Free Full Text]
18. Kothakota S., Azuma T., Reinhard C., et al. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science (1997) 278:294–298.[Abstract/Free Full Text]
19. Brancolini C., Benedetti M., Schneider C. Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J. (1995) 14:5179–5190.[Web of Science][Medline]
20. Rudel T., Bokoch G.M. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science (1997) 276:1571–1574.[Abstract/Free Full Text]
21. Cardone M.H., Salvesen G.S., Widmann C., Johnson G., Frisch S.M. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell (1997) 90:315–323.[CrossRef][Web of Science][Medline]
22. Wen L.P., Fahrni J.A., Troie S., Guan J.L., Orth K., Rosen G.D. Cleavage of focal adhesion kinase by caspases during apoptosis. J. Biol. Chem. (1997) 272:26056–26061.[Abstract/Free Full Text]
23. Orth K., Chinnayan A.M., Garg M., Froelich C.J., Dixit V.M. The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A. J. Biol. Chem. (1996) 271:16443–16446.[Abstract/Free Full Text]
24. Takashi A., Alnemri E.S., Lazebnik Y.A., et al. Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin-1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc. Natl. Acad. Sci. (1996) 93:8395–8400.[Abstract/Free Full Text]
25. Nicholson D.W., Thornberry N.A. Caspases: killer proteases. Trends Biochem. Sci. (1997) 22:299–306.[CrossRef][Web of Science][Medline]
26. Casiano C.A., Martin S.J., Green D.R., Tan E.M. Selective cleavage of nuclear autoantigens during CD95 (Fas/APO-1) -mediated T cell apoptosis. J. Exp. Med. (1996) 184:765–770.[Abstract/Free Full Text]
27. Fadok V.A., Voelker D.R., Priscilla A.C., Cohen J.J., Bratton D.L., Henson P.M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. (1992) 148:2209–2216.
28. Martin S.J., Finucane D.M., Amarante-Mendes G.P., O’Brien G.A., Green D.R. Phosphatidylserine externalisation during CD95-induced apoptosis of cells and cytoplasts requires ICE/CED-3 protease activity. J. Biol. Chem. (1996) 271:28753–28756.[Abstract/Free Full Text]
29. Saraste A. Morphologic criteria and detection of apoptosis. Herz (1999) 24:189–195.[Web of Science][Medline]
30. James T.N., Terasaki F., Pavlovich E.R., Vikhert A.M. Apoptosis and pleomorphic micromitochondriosis in the sinus nodes surgically excised from five patients with the long QT syndrome. J. Lab Clin. Med. (1993) 122:309–323.[Web of Science][Medline]
31. Sharov V.G., Sabbah H.N., Shimoyama H., Goussev A.V., Lesch M., Goldstein S. Evidence of cardiocyte apoptosis in myocardium of dogs with chronic heart failure. Am. J. Pathol. (1996) 148:141–149.[Abstract]
32. Elsässer A., Schlepper M., Klöverkorn W.-P., et al. Hibernating myocardium: An incomplete adaptation to ischemia. Circulation (1997) 96:2920–2931.[Abstract/Free Full Text]
33. Alnemri E.S., Litwack G. Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non Ca²⁺-requiring mechanism(s). J. Biol. Chem. (1990) 265:17323–17333.[Abstract/Free Full Text]
34. Didenko V.V., Hornsby P.J. Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis. J. Cell Biol. (1996) 135:1369–1376.[Abstract/Free Full Text]
35. Bortner C.D., Oldenburg N.B.E., Cidlowski J.A. The role of DNA fragmentation in apoptosis. Trends Cell Biol. (1995) 5:21–26.[CrossRef][Web of Science][Medline]
36. Liu X., Li P., Widlak P., Zou H., Luo X., Garrard W.T., Wang X. The 40-kDa subunit of DNA fragmentation factor induce DNA fragmentation and chromatin condensation during apoptosis. Proc. Natl. Acad. Sci. (1998) 95:8461–8466.[Abstract/Free Full Text]
37. Enari M., Sakahira H., Yokoyama H., Okawa K., Iwamatsu A., Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature (1998) 391:43–50.[CrossRef][Medline]
38. Sakahira H., Enari M., Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature (1998) 391:96–99.[CrossRef][Medline]
39. Urbano A., McCaffrey R., Foss F. Isolation and characterization of NUC70, a cytoplasmic, hematopoietic apoptotic endonuclease. J. Biol. Chem. (1998) 273:34820–34927.[Abstract/Free Full Text]
40. Liu X., Zou H., Slaughter C., Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell (1997) 89:175–184.[CrossRef][Web of Science][Medline]
41. Tang D., Kidd V.J. Cleavage of DFF-45/ICAD by multiple caspases is essential for its function during apoptosis. J. Biol. Chem. (1998) 273:28549–28552.[Abstract/Free Full Text]
42. Haunstetter A., Izumo S. Apoptosis: Basic mechanisms and implications for cardiovascular disease. Circ. Res. (1998) 82:1111–1129.[Free Full Text]
43. Kanoh M., Takemura G., Misao J., Hayakawa Y., Aoyama T., Nishikagi K., Noda T., Fujiwara T., Fukuda K., Minatogutchi S., Fujiwara H. Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy. Not apoptosis but DNA repair. Circulation (1999) 99:2757–2764.[Abstract/Free Full Text]
44. Gold R., Schmied M., Giegerich G., Breitschopf H., Hartung H.P., Toyka K.V., Lassmann H. Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest (1994) 71:219–225.[Web of Science][Medline]
45. Grosse F., Manns A. Methods in molecular biology. Burrel M.M., ed. (1993) vol. 16. Enzymes of molecular biology. Totowa, N.J: Humana Press. 95–105.
46. Saraste A., Pulkki K., Kallajoki M., Henriksen K., Parvinen M., Voipio-Pulkki L.-M. Apoptosis in human acute myocardial infarction. Circulation (1997) 95:320–323.[Abstract/Free Full Text]
47. Saraste A., Pulkki K., Kallajoki M., et al. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur. J. Clin. Invest. (1999) 29:380–386.[CrossRef][Web of Science][Medline]
48. Petito C.K., Roberts B. Effect of postmortem interval on in situ end-labeling of DNA oligonucleosomes. J. Neuropath. Exp. Neur. (1995) 54:761–765.[Web of Science][Medline]
49. Veinot J.P., Gattinger D.A., Fliss H. Early apoptosis in human myocardial infarcts. Human Path (1997) 28:485–492.[CrossRef]
50. Yuan J., Shaham S., Ledoux S., Ellis H.M., Horvitz H.R. The C elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell (1993) 75:641–652.[CrossRef][Web of Science][Medline]
51. Thornberry N.A., Bull H.G., Calaycay J.R., et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature (1992) 356:768–774.[CrossRef][Medline]
52. Alnemri E.S., Livingston D.J., Nicholson D.W., et al. Human ICE/CED-3 protease nomenclature. Cell (1996) 87:171.[CrossRef][Web of Science][Medline]
53. Kumar S., Kinoshita M., Noda M., Copeland N.G., Jenkins N.A. Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme. Genes Dev. (1994) 8:1613–1626.[Abstract/Free Full Text]
54. Wang L., Miura M., Bergeron L., Zhu H., Yuan J. Ich-1, an ICE/Ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell (1994) 78:739–750.[CrossRef][Web of Science][Medline]
55. Fernandes T.A., Litwack G., Alnemri E.S. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J. Biol. Chem. (1994) 9:30761–30764.
56. Nicholson D.W., Ali A., Thornberry N.A., et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature (1995) 376:37–43.[CrossRef][Medline]
57. Tewari M., Quan L.T., O’Rourke K., et al. Yama/CPP32β, a mammalian homolog of CED-3, is a crmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell (1995) 81:801–809.[CrossRef][Web of Science][Medline]
58. Kuida K., Zheng T.S., Na S., et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Cell (1996) 384:368–372.
59. Hirsch T., Marchetti P., Susin S.A., et al. The apoptosis–necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene (1997) 15:1573–1581.[CrossRef][Web of Science][Medline]
60. Woo M., Hakem R., Soengas M.S., et al. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev. (1998) 12:806–819.[Abstract/Free Full Text]
61. Jänicke R.U., Sprengart M.L., Wati M.R., Porter A.G. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. (1998) 273:9357–9360.[Abstract/Free Full Text]
62. Amarente-Mendes G.P., Finucance D.M., Martin S.J., Cotter T.G., Salvesen G.S., Green D.R. Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death. Cell Death Different (1998) 5:298–306.[CrossRef][Web of Science][Medline]
63. Thornberry N.A., Lazebnik Y. Caspases: enemies within. Science (1998) 281:1312–1316.[Abstract/Free Full Text]
64. Thornberry N.A., Rano T.A., Paterson E.P., et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. (1997) 272:17907–17911.[Abstract/Free Full Text]
65. Srinivasula S.M., Ahmad M., Fernandes-Alnemri T., Litwack G., Alnemri E.S. Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. (1996) 93:14486–14491.[Abstract/Free Full Text]
66. Hirata H., Takahashi A., Kobayashi S., et al. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. (1998) 187:587–600.[Abstract/Free Full Text]
67. Slee E.A., Harte M.T., Kluck R.M., et al. Ordering the cytochrome C-initiated caspase cascade: hierarchical activation of caspases -2, -3, -6, -7, -8 and -10 in a caspase-9-dependent manner. J. Cell. Biol. (1999) 144:281–292.[Abstract/Free Full Text]
68. Ashkenazi A., Dixit V.M. Death receptors: signaling and modulation. Science (1998) 281:1305–1308.[Abstract/Free Full Text]
69. Green D.R., Reed J.C. Mitochondria and apoptosis. Science (1998) 281:1309–1312.[Abstract/Free Full Text]
70. Kroemer G., Zamzani N., Susin S.A. Mitochondrial control of apoptosis. Immunol. Today (1997) 18:44–51.[CrossRef][Web of Science][Medline]
71. Fearnhead H.O., Rodriguez J., Govek E.E., et al. Oncogene-dependent apoptosis is mediated by caspase-9. Proc. Natl. Acad. Sci. (1998) 95:13664–13669.[Abstract/Free Full Text]
72. Soengas M.S., Alarcon R.M., Yoshida H., et al. Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science (1999) 284:156–159.[Abstract/Free Full Text]
73. Kluck R.M., Bossy-Wetzel E., Green D.R., Newmeyer D.D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science (1997) 275:1132–1136.[Abstract/Free Full Text]
74. Yang J., Liu X., Bhalla K., et al. Prevention of apoptosis by Bcl-2; release of cytochrome C from mitochondria blocked. Science (1997) 275:1129–1132.[Abstract/Free Full Text]
75. Li H., Zhu H., Xu C.J., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell (1998) 94:491–501.[CrossRef][Web of Science][Medline]
76. Liu X., Kim C.N., Jemmerson R., Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome C. Cell (1996) 86:147–157.[CrossRef][Web of Science][Medline]
77. Kluck R.M., Martin S.J., Hoffman B.M., Zhou J.S., Green D.R., Newmeyer D.D. Cytochrome C activation of CPP32-like proteolysis plays a critical role in a xenopus cell-free apoptosis system. EMBO J. (1997) 16:4639–4649.[CrossRef][Web of Science][Medline]
78. Li P., Nijhawan D., Budihardjo I., et al. Cytochrome C and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell (1997) 91:479–489.[CrossRef][Web of Science][Medline]
79. Srinivasula S.M., Ahmad M., Fernandes-Alnemri T., Alnemri E.S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell (1998) 1:949–957.[CrossRef][Web of Science][Medline]
80. Dinsdale D., Zhuang J., Cohen G.M. Redistribution of cytochrome c precedes the caspase-dependent formation of ultracondensed mitochondria, with a reduced inner membrane potential, in apoptotic monocytes. Am. J. Pathol. (1999) 155:607–618.[Abstract/Free Full Text]
81. Caulin C., Salvesen G.S., Oshima R.G. Caspase cleavage of keratin 18 and reorganisation of intermediate filaments during epithelial cell apoptosis. J. Cell. Biol. (1997) 138:1379–1394.[Abstract/Free Full Text]
82. Yang F., Sun X., Beech W., et al. Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease. Am. J. Pathol. (1998) 152:379–389.[Abstract]
83. Suurmeijer A.J.H., Wijk J., Veldhuisen D.J., Yang F., Cole G.M. Fractin immunostaining for the detection of apoptotic cells and apoptotic bodies in formalin-fixed and paraffin-embedded tissue. Lab. Invest. (1999) 79:619–620.[Web of Science][Medline]
84. Black S.C., Huang J.Q., Rezaiefar P., et al. Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat. J. Mol. Cell Cardiol. (1998) 30:733–742.[CrossRef][Web of Science][Medline]
85. Kouroku Y., Urase K., Fujita E., et al. Detection of activated caspase-3 by a cleavage site-directed antiserum during naturally occurring DRG neuron apoptosis. Biochem Biophys Res Commun (1998) 29:780–784.
86. Srinivasan A., Roth K.A., Sayers R.O., et al. In situ detection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Different. (1998) 5:1004–1016.[CrossRef][Web of Science][Medline]
87. Holly T.A., Drincic A., Byun Y., et al. Caspase inhibition reduces myocyte cell death induced by myocardial ischemia and reperfusion in vivo. J. Mol. Cell Cardiol. (1999) 31:1709–1715.[CrossRef][Web of Science][Medline]
88. Harsdorf R., Li P-F., Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation (1999) 99:2934–2941.[Abstract/Free Full Text]
89. Koglin J., Granville D.J., Glysing-Jensen T., et al. Attenuated acute cardiac rejection in NOS2 –/– recipients correlates with reduced apoptosis. Circulation (1999) 99:836–842.[Abstract/Free Full Text]
90. Narula J., Pandey P., Abrustini E., et al. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc. Natl. Acad. Sci. (1999) 96:8144–8149.[Abstract/Free Full Text]
91. Gottlieb R.A., Gruol D.L., Zhu J.Y., Engler R.L. Preconditioning in rabbit cardiomyocytes. Role of pH, vacuolar proton ATPase, and apoptosis. J. Clin. Invest. (1996) 97:2391–2398.[Web of Science][Medline]
92. Yaoita H., Oqawa K., Maehara K., Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation (1998) 97:276–281.[Abstract/Free Full Text]
93. Majno G., Joris I. Apoptosis, oncosis and necrosis. An overview of cell death. Am. J. Pathol. (1995) 146:3–15.[Abstract]
94. Xiang J., Chao D.T., Korsmeyer S.J. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. (1996) 93:14559–14563.[Abstract/Free Full Text]
95. Leist M., Single B., Castoldi A.F., Kühnle S., Nicotera P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J. Exp. Med. (1997) 185:1481–1486.[Abstract/Free Full Text]
96. Leist M., Nicotera P. The shape of cell death. Biochem. Biophys. Res. Comm. (1997) 236:1–9.[CrossRef][Web of Science][Medline]
97. Buja L.M. Modulation of myocardial response to ischemia. Lab Invest. (1998) 78:1345–1373.[Web of Science][Medline]
98. Kajstura J., Cheng W., Reiss K., et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. (1996) 74:86–107.[Web of Science][Medline]
99. Gottlieb R.A., Burleson K.A., Kloner R.A., Babior B.M., Engler R.L. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J. Clin. Invest. (1994) 94:1621–1628.[Web of Science][Medline]
100. Ohno M., Takemura G., Ohno A., et al. Apoptotic myocytes in infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: Analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation (1998) 98:1422–1430.[Abstract/Free Full Text]
101. Webster K.A., Discher D.J., Kaiser S., Hernandez O., Sato B., Bishopric N.H. Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53. J. Clin. Invest. (1999) 104:239–252.[Web of Science][Medline]
102. Leri A., Claludio P.P., Li Q., et al. Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin–angiotensin system and decreases the Bcl-2 to Bax protein ratio in the cell. J. Clin. Invest. (1998) 101:1326–1342.[Web of Science][Medline]
103. Shizukuda Y., Buttrick P.M., Geenen D.L., Borczuk A.C., Kitsis R.N. Sonnenblick E.H. β-adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy. Am J Physiol (1998) 275:H961–H968.[Web of Science][Medline]
104. Cosulich S.C., Horiuchi H., Zerial M., Clarke P.R., Woodman P.G. Cleavage of rabaptin-5 blocks endosome fusion during apoptosis. EMBO J. (1997) 16:6182–6192.[CrossRef][Web of Science][Medline]
105. Koseki T., Inohara N., Chen S., Núñez G. ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart interacts selectively with caspases. Proc. Natl. Acad. Sci. (1998) 95:5156–5160.[Abstract/Free Full Text]
106. Cheng W., Li B., Kajstura J., et al. Stretch-induced programmed myocyte cell death. J. Clin. Invest. (1995) 96:2247–2259.[Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				X. Zhang, M. G. Paule, G. D. Newport, X. Zou, N. Sadovova, M. S. Berridge, S. M. Apana, J. P. Hanig, W. Slikker Jr, and C. Wang A Minimally Invasive, Translational Biomarker of Ketamine-Induced Neuronal Death in Rats: microPET Imaging Using 18F-Annexin V Toxicol. Sci., October 1, 2009; 111(2): 355 - 361. [Abstract] [Full Text] [PDF]

				C. E. Bell, N. M.K. Lariviere, P. H. Watson, and A. J. Watson Mitogen-activated protein kinase (MAPK) pathways mediate embryonic responses to culture medium osmolarity by regulating Aquaporin 3 and 9 expression and localization, as well as embryonic apoptosis Hum. Reprod., June 1, 2009; 24(6): 1373 - 1386. [Abstract] [Full Text] [PDF]

				Z. J. Gartner and C. R. Bertozzi Programmed assembly of 3-dimensional microtissues with defined cellular connectivity PNAS, March 24, 2009; 106(12): 4606 - 4610. [Abstract] [Full Text] [PDF]

				J.-H. Han, S. A. Kushner, A. P. Yiu, H.-L. Hsiang, T. Buch, A. Waisman, B. Bontempi, R. L. Neve, P. W. Frankland, and S. A. Josselyn Selective Erasure of a Fear Memory Science, March 13, 2009; 323(5920): 1492 - 1496. [Abstract] [Full Text] [PDF]

				D.-S. Yang, A. Kumar, P. Stavrides, J. Peterson, C. M. Peterhoff, M. Pawlik, E. Levy, A. M. Cataldo, and R. A. Nixon Neuronal Apoptosis and Autophagy Cross Talk in Aging PS/APP Mice, a Model of Alzheimer's Disease Am. J. Pathol., September 1, 2008; 173(3): 665 - 681. [Abstract] [Full Text] [PDF]

				A. Shalaby, T. Rinne, O. Jarvinen, A. Saraste, J. Laurikka, H. Porkkala, P. Saukko, and M. Tarkka Initial results of a clinical study: adenosine enhanced cardioprotection and its effect on cardiomyocytes apoptosis during coronary artery bypass grafting Eur. J. Cardiothorac. Surg., April 1, 2008; 33(4): 639 - 644. [Abstract] [Full Text] [PDF]

				S. Kawahara, Y. Hata, M. Miura, T. Kita, A. Sengoku, S. Nakao, Y. Mochizuki, H. Enaida, A. Ueno, A. Hafezi-Moghadam, et al. Intracellular Events in Retinal Glial Cells Exposed to ICG and BBG Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4426 - 4432. [Abstract] [Full Text] [PDF]

				Z.-g. Sheng, S. Peng, C.-y. Wang, H.-b. Li, R. K. Hajela, Y.-e. Wang, Q.-q. Li, M.-f. Liu, Y.-s. Dong, and G. Han Apoptosis in Microencapsulated Juvenile Rabbit Chondrocytes Induced by Ofloxacin: Role Played by beta1-Integrin Receptor J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 155 - 165. [Abstract] [Full Text] [PDF]

				S. Ghosh, S. De, and S. K. Dutta Altered Protein Expressions in Chronic PCB-153-Induced Human Liver (HepG2) Cells International Journal of Toxicology, May 1, 2007; 26(3): 203 - 212. [Abstract] [Full Text] [PDF]

				M. Scannell, M. B. Flanagan, A. deStefani, K. J. Wynne, G. Cagney, C. Godson, and P. Maderna Annexin-1 and Peptide Derivatives Are Released by Apoptotic Cells and Stimulate Phagocytosis of Apoptotic Neutrophils by Macrophages J. Immunol., April 1, 2007; 178(7): 4595 - 4605. [Abstract] [Full Text] [PDF]

				W. Ibe, A. Saraste, S. Lindemann, S. Bruder, M. Buerke, H. Darius, K. Pulkki, and L.-M. Voipio-Pulkki Cardiomyocyte apoptosis is related to left ventricular dysfunction and remodelling in dilated cardiomyopathy, but is not affected by growth hormone treatment Eur J Heart Fail, February 1, 2007; 9(2): 160 - 167. [Abstract] [Full Text] [PDF]

				S. Sarfaraz, F. Afaq, V. M. Adhami, A. Malik, and H. Mukhtar Cannabinoid Receptor Agonist-induced Apoptosis of Human Prostate Cancer Cells LNCaP Proceeds through Sustained Activation of ERK1/2 Leading to G1 Cell Cycle Arrest J. Biol. Chem., December 22, 2006; 281(51): 39480 - 39491. [Abstract] [Full Text] [PDF]

				M. Malmberg, T. Vahasilta, A. Saraste, V. Kyto, J. Kiss, E. Kentala, M. Kallajoki, and T. Savunen Cardiomyocyte apoptosis and duration of aortic clamping in pig model of open heart surgery. Eur. J. Cardiothorac. Surg., September 1, 2006; 30(3): 480 - 484. [Abstract] [Full Text] [PDF]

				Y. Yang, X. Pan, W. Lei, J. Wang, J. Shi, F. Li, and J. Song Regulation of Transforming Growth Factor-{beta}1-Induced Apoptosis and Epithelial-to-Mesenchymal Transition by Protein Kinase A and Signal Transducers and Activators of Transcription 3. Cancer Res., September 1, 2006; 66(17): 8617 - 8624. [Abstract] [Full Text] [PDF]

				E. Karaskov, C. Scott, L. Zhang, T. Teodoro, M. Ravazzola, and A. Volchuk Chronic Palmitate But Not Oleate Exposure Induces Endoplasmic Reticulum Stress, Which May Contribute to INS-1 Pancreatic {beta}-Cell Apoptosis Endocrinology, July 1, 2006; 147(7): 3398 - 3407. [Abstract] [Full Text] [PDF]

				J.-M. Yun, M.-H. Kweon, H. Kwon, J.-K. Hwang, and H. Mukhtar Induction of apoptosis and cell cycle arrest by a chalcone panduratin A isolated from Kaempferia pandurata in androgen-independent human prostate cancer cells PC3 and DU145 Carcinogenesis, July 1, 2006; 27(7): 1454 - 1464. [Abstract] [Full Text] [PDF]

				K. Aure, G. Fayet, J. P. Leroy, E. Lacene, N. B. Romero, and A. Lombes Apoptosis in mitochondrial myopathies is linked to mitochondrial proliferation Brain, May 1, 2006; 129(5): 1249 - 1259. [Abstract] [Full Text] [PDF]

				S. Miret, E. M. De Groene, and W. Klaffke Comparison of In Vitro Assays of Cellular Toxicity in the Human Hepatic Cell Line HepG2 J Biomol Screen, March 1, 2006; 11(2): 184 - 193. [Abstract] [PDF]

				T. Vahasilta, A. Saraste, V. Kyto, M. Malmberg, J. Kiss, E. Kentala, M. Kallajoki, and T. Savunen Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia Ann. Thorac. Surg., December 1, 2005; 80(6): 2229 - 2234. [Abstract] [Full Text] [PDF]

				J. P. Lamb, A. James, N. Carroll, L. Siena, J. Elliot, and A. M. Vignola{dagger} Reduced apoptosis of memory T-cells in the inner airway wall of mild and severe asthma Eur. Respir. J., August 1, 2005; 26(2): 265 - 270. [Abstract] [Full Text] [PDF]

				L. Deszcz, E. Gaudernak, E. Kuechler, and J. Seipelt Apoptotic events induced by human rhinovirus infection J. Gen. Virol., May 1, 2005; 86(5): 1379 - 1389. [Abstract] [Full Text] [PDF]

				D. M. Ray, F. Akbiyik, S. H. Bernstein, and R. P. Phipps CD40 Engagement Prevents Peroxisome Proliferator-Activated Receptor {gamma} Agonist-Induced Apoptosis of B Lymphocytes and B Lymphoma Cells by an NF-{kappa}B-Dependent Mechanism J. Immunol., April 1, 2005; 174(7): 4060 - 4069. [Abstract] [Full Text] [PDF]

				C. S. H. Ng, S. Wan, and A. P. C. Yim Pulmonary ischaemia-reperfusion injury: role of apoptosis Eur. Respir. J., February 1, 2005; 25(2): 356 - 363. [Abstract] [Full Text] [PDF]

				T. Schachner, A. Oberhuber, Y. Zou, A. Tzankov, H. Ott, G. Laufer, and J. Bonatti Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts Eur. J. Cardiothorac. Surg., February 1, 2005; 27(2): 302 - 306. [Abstract] [Full Text] [PDF]

				S. Philipp, I. Pagel, K. Hohnel, J. Lutz, J. Buttgereit, T. Langenickel, P. Hamet, R. Dietz, and R. Willenbrock Regulation of caspase 3 and Fas in pressure overload-induced left ventricular dysfunction Eur J Heart Fail, December 1, 2004; 6(7): 845 - 851. [Abstract] [Full Text] [PDF]

				B. S. Cummings, G. R. Kinsey, L. J. C. Bolchoz, and R. G. Schnellmann Identification of Caspase-Independent Apoptosis in Epithelial and Cancer Cells J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 126 - 134. [Abstract] [Full Text] [PDF]

				D. F. Goldspink, J. G. Burniston, G. M. Ellison, W. A. Clark, and L.-B. Tan Catecholamine-induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: the same or separate death pathways? Exp Physiol, July 1, 2004; 89(4): 407 - 416. [Abstract] [Full Text] [PDF]

				U. Ziegler and P. Groscurth Morphological Features of Cell Death Physiology, June 1, 2004; 19(3): 124 - 128. [Abstract] [Full Text] [PDF]

				F. Sayk, S. Kruger, J.F. M. Bechtel, A. C. Feller, H. H. Sievers, and C. Bartels Significant damage of the conduction system during cardioplegic arrest is due to necrosis not apoptosis Eur. J. Cardiothorac. Surg., May 1, 2004; 25(5): 801 - 806. [Abstract] [Full Text] [PDF]

				J. D. McCully, H. Wakiyama, Y.-J. Hsieh, M. Jones, and S. Levitsky Differential contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1923 - H1935. [Abstract] [Full Text] [PDF]

				A. M. A. Nasirudeen, Y. E. Hian, M. Singh, and K. S. W. Tan Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis Microbiology, January 1, 2004; 150(1): 33 - 43. [Abstract] [Full Text] [PDF]

				F. Sayk and C. Bartels Oncosis rather than apoptosis? Ann. Thorac. Surg., January 1, 2004; 77(1): 382 - 382. [Full Text] [PDF]

				T. M. Payne, R. E. Molestina, and A. P. Sinai Inhibition of caspase activation and a requirement for NF-{kappa}B function in the Toxoplasma gondii-mediated blockade of host apoptosis J. Cell Sci., November 1, 2003; 116(21): 4345 - 4358. [Abstract] [Full Text] [PDF]

				A. S. Galvez, J. A. Ulloa, M. Chiong, A. Criollo, V. Eisner, L. F. Barros, and S. Lavandero Aldose Reductase Induced by Hyperosmotic Stress Mediates Cardiomyocyte Apoptosis: DIFFERENTIAL EFFECTS OF SORBITOL AND MANNITOL J. Biol. Chem., October 3, 2003; 278(40): 38484 - 38494. [Abstract] [Full Text] [PDF]

				C. D. Bortner and J. A. Cidlowski Uncoupling Cell Shrinkage from Apoptosis Reveals That Na+ Influx Is Required for Volume Loss during Programmed Cell Death J. Biol. Chem., October 3, 2003; 278(40): 39176 - 39184. [Abstract] [Full Text] [PDF]

				C. J. Zeiss The Apoptosis-Necrosis Continuum: Insights from Genetically Altered Mice Vet. Pathol., September 1, 2003; 40(5): 481 - 495. [Abstract] [Full Text] [PDF]

				A. Gonzalez, M. A Fortuno, R. Querejeta, S. Ravassa, B. Lopez, N. Lopez, and J. Diez Cardiomyocyte apoptosis in hypertensive cardiomyopathy Cardiovasc Res, September 1, 2003; 59(3): 549 - 562. [Abstract] [Full Text] [PDF]

				Z.-K. Wu, J. Laurikka, A. Saraste, V. Kyto, E. J. Pehkonen, T. Savunen, and M. R. Tarkka Cardiomyocyte apoptosis and ischemic preconditioning in open heart operations Ann. Thorac. Surg., August 1, 2003; 76(2): 528 - 534. [Abstract] [Full Text] [PDF]

				P. B. Bushdid, H. Osinska, R. R. Waclaw, J. D. Molkentin, and K. E. Yutzey NFATc3 and NFATc4 Are Required for Cardiac Development and Mitochondrial Function Circ. Res., June 27, 2003; 92(12): 1305 - 1313. [Abstract] [Full Text] [PDF]

				D. J. Lips, L. J. deWindt, D. J.W. van Kraaij, and P. A. Doevendans Molecular determinants of myocardial hypertrophy and failure: alternative pathways for beneficial and maladaptive hypertrophy Eur. Heart J., May 2, 2003; 24(10): 883 - 896. [Abstract] [Full Text] [PDF]

				M. A. Fortuno, A. Gonzalez, S. Ravassa, B. Lopez, and J. Diez Clinical implications of apoptosis in hypertensive heart disease Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1495 - H1506. [Full Text] [PDF]

				L. Hinck, J.P. Thissen, and R. De Hertogh Identification of Caspase-6 in Rat Blastocysts and Its Implication in the Induction of Apoptosis by High Glucose Biol Reprod, May 1, 2003; 68(5): 1808 - 1812. [Abstract] [Full Text] [PDF]

				H. Hui, A. Nourparvar, X. Zhao, and R. Perfetti Glucagon-Like Peptide-1 Inhibits Apoptosis of Insulin-Secreting Cells via a Cyclic 5'-Adenosine Monophosphate-Dependent Protein Kinase A- and a Phosphatidylinositol 3-Kinase-Dependent Pathway Endocrinology, April 1, 2003; 144(4): 1444 - 1455. [Abstract] [Full Text] [PDF]

				G. Valen The basic biology of apoptosis and its implications for cardiac function and viability Ann. Thorac. Surg., February 1, 2003; 75(2): S656 - 660. [Abstract] [Full Text] [PDF]

				T. M. Hazzard, F. Xu, and R. L. Stouffer Injection of Soluble Vascular Endothelial Growth Factor Receptor 1 into the Preovulatory Follicle Disrupts Ovulation and Subsequent Luteal Function in Rhesus Monkeys Biol Reprod, October 1, 2002; 67(4): 1305 - 1312. [Abstract] [Full Text] [PDF]

				W. Chen, J. Sulcove, I. Frank, S. Jaffer, H. Ozdener, and D. L. Kolson Development of a Human Neuronal Cell Model for Human Immunodeficiency Virus (HIV)-Infected Macrophage-Induced Neurotoxicity: Apoptosis Induced by HIV Type 1 Primary Isolates and Evidence for Involvement of the Bcl-2/Bcl-xL-Sensitive Intrinsic Apoptosis Pathway J. Virol., August 12, 2002; 76(18): 9407 - 9419. [Abstract] [Full Text] [PDF]

				L Mazur, A Czyzewska, and M Bochenek Flow cytometric detection of apoptotic bone marrow cells with fractional DNA content after application of WR-2721, cyclophosphamide, cisplatin, and exposure of mice to gamma rays Human and Experimental Toxicology, June 1, 2002; 21(6): 335 - 341. [Abstract] [PDF]

				C. GILL, R. MESTRIL, and A. SAMALI Losing heart: the role of apoptosis in heart disease--a novel therapeutic target? FASEB J, February 1, 2002; 16(2): 135 - 146. [Abstract] [Full Text] [PDF]

				S. Segerer, F. Eitner, Y. Cui, K. L. Hudkins, and C. E. Alpers Cellular Injury Associated with Renal Thrombotic Microangiopathy in Human Immunodeficiency Virus-Infected Macaques J. Am. Soc. Nephrol., February 1, 2002; 13(2): 370 - 378. [Abstract] [Full Text] [PDF]

				M. N. Sharikabad, K. M. Ostbye, and O. Brors Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2113 - H2123. [Abstract] [Full Text] [PDF]

				S. M. Hawes, Y. Gie Chung, and K. E. Latham Genetic and Epigenetic Factors Affecting Blastomere Fragmentation in Two-Cell Stage Mouse Embryos Biol Reprod, October 1, 2001; 65(4): 1050 - 1056. [Abstract] [Full Text] [PDF]

				S. Meintieres, A. Biola, M. Pallardy, and D. Marzin Apoptosis can be a confusing factor in in vitro clastogenic assays Mutagenesis, May 1, 2001; 16(3): 243 - 250. [Abstract] [Full Text] [PDF]

				M. Borgers, L.-M. Voipio-Pulkki, and S. Izumo Apoptosis Cardiovasc Res, February 1, 2000; 45(3): 525 - 527. [Full Text] [PDF]

				M. Alvarez-Tejado, S. Naranjo-Suarez, C. Jimenez, A. C. Carrera, M. O. Landazuri, and L. del Peso Hypoxia Induces the Activation of the Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway in PC12 Cells. PROTECTIVE ROLE IN APOPTOSIS J. Biol. Chem., June 15, 2001; 276(25): 22368 - 22374. [Abstract] [Full Text] [PDF]

				B. S. Sayan, G. Ince, A. E. Sayan, and M. Ozturk NAPO as a novel marker for apoptosis J. Cell Biol., November 26, 2001; 155(5): 719 - 724. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Saraste, A. Articles by Pulkki, K. Search for Related Content PubMed PubMed Citation Articles by Saraste, A. Articles by Pulkki, K. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics