General introduction and overview of contents

When cells receive mixed signals for growth they usually die. For instance, when the developmental programme requires cell division but external growth signals are lacking, or when a growth-related gene such as c-myc is highly expressed but the cellular environment has insufficient nutrient content, or a toxic xenobiotic is present, the cell dies by a process termed apoptosis. Although there are differences in the phenomena observed during the apoptotic sequence of events, depending on the cell type, and agent or circumstance which initiates the cell's demise, there are morphological and biochemical similarities which suggest that these are variants of the same biological process, designed to control the size of cell populations.

It is important to distinguish apoptosis from the other major form of cell death, necrosis. First, at the tissue level, apoptosis produces little or no inflammation, since shrunken portions of the cell are engulfed by the neighbouring cells, especially macrophages, rather than being released into the extracellular fluid. In contrast, in necrosis, cellular contents are released into the extracellular fluid, and thus have an irritant effect on the nearby cells, causing inflammation. Secondly, there is the expectation that elucidation of the steps of the cellular mechanisms that lead to apoptosis may allow this form of cell death to be induced more effectively by cancer therapeutic agents. Thirdly, the apoptotic mechanism of cell death is fundamental to the normal development of tissues and organisms. In contrast, cell death by necrosis is usually accidental and therefore does not have such significance.

The role of apoptosis in cell population control during development has suggested that there are inherent cellular programmes that lead the cell to self-destruct. This has been confirmed in a number of instances; e.g. in a small nematode, Caenorhabditis elegans (C. elegans), where each individual cell can be recognized, it has been found that in the hermaphrodite form of the worm the same set of 113 cells is destined for programmed cell death during embryogenesis, and another set of 18 cells later in life, for a total of 131 cells (1). Also, inhibition of RNA or protein synthesis can, in many cases, abrogate cell death by apoptosis (2), although it usually accelerates necrosis. Thus, it appears that gene expression is necessary for cell death. Yet. there: is another level of complexity, as, in some instances, inhibition of protein or RNA synthesis, or even cxplusion of nuclei, does not prevent what otherwise appears to be programmed cell death (3). Such cells are thought to be primed for apoptosis.

The original use of the term apoptosis was primarily descriptive of the cellular morphology of dying cells (4). Although in the current literature most authors blur the precision of this term (3), it is still tenable to define apoptosis as cell death that differs from necrosis on a morphological basis, observable by light or by electron microscopy. The key features originally described included shrinkage and blobbing of the cytoplasm; preservation of the structure of cellular organelles, including the mitochondria; and condensation and margination of chromatin, although not all of these are seen in all cell types (Figure /), It is generally assumed that these morphological changes result from a developmental programme for cell death that can be triggered by deprivation of a growth factor, or by addition of a xenobiotic compound such as a cancer therapeutic drug. The morphological criteria are still the most important when complex cell populations, such as tissues, are examined.

Figure 1. An illustration of the light microscopic appearance of apoptotic cells and their modification by a differentiation-inducing agent, (a) HL60 cells were exposed to calcium ionophore A23187 (10 ^M for 8 h), embedded in epon, and 10 ^m sections were stained with toluidine blue. Note the densely stained fragments of chromatin in the nuclei and cytoplasm of most celts, (b) HL60 cells treated as in (a), but first exposed to 1,25-dihydroxyvitamin Ds (10"" M for 48 h), which protects HL60 cells against apoptosis (10). Note the smaller (differentiated) cells, only a few of which show apoptotic nuclei.

Figure 1. An illustration of the light microscopic appearance of apoptotic cells and their modification by a differentiation-inducing agent, (a) HL60 cells were exposed to calcium ionophore A23187 (10 ^M for 8 h), embedded in epon, and 10 ^m sections were stained with toluidine blue. Note the densely stained fragments of chromatin in the nuclei and cytoplasm of most celts, (b) HL60 cells treated as in (a), but first exposed to 1,25-dihydroxyvitamin Ds (10"" M for 48 h), which protects HL60 cells against apoptosis (10). Note the smaller (differentiated) cells, only a few of which show apoptotic nuclei.

and overall cell shrinkage and nuclear condensation are the easiest to recognize. These are presented in detail in Chapter 2, with special emphasis on the detection and quantitation of apoptosis in vivo, since this is a much more challenging task than recognition of apoptosis in tissue culture.

In pure cell populations, biochemical changes in chromatin and DNA degradation provide useful and often quantifiable means of detecting apoptosis. It is often forgotten, however, that random DNA degradation is not a specific test for apoptosis but simply demonstrates cell death. Although detection of DNA degradation may be useful as an adjunct method of quantitation, occurrence of apoptosis has be shown by morphological or by more specific biochemical methods.

The classical biochemical method for demonstrating apoptosis is the presence of oligonuclcosome-si/ed fragments of DNA, which, when run on agarose gels, produce 'ladders1, as discussed in Chapter 3 and illustrated in Figure 2 (5,6). It has been shown also that an earlier endonucleolytic cleavage of chromatin produces DNA fragments from 300 kb down to 50 kb in size (7, 8). Also, the observation that mitochondrial DNA is intact in early stages of apoptosis

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Figure2. DNA ladder formation in HL60-G1 cells (a subclone of human leukaemia HL6G cells), but not in K562 human leukaemia cells, following exposure to doxorubicin (5 ^.M for HL60 cells and 10 ^M for K562 cells, both for 24 h). DNA was extracted and run on 2% agarose gels and stained with ethidium bromide. DNA ladders indicative of nucleosomal DNA fragmentation became apparent after 8 h, concident with morphological appearances due to apoptosis (not shown). Microscopic examination of doxorubicin-treated K562 cells showed that these cells became necrotic [not shown}.

Figure2. DNA ladder formation in HL60-G1 cells (a subclone of human leukaemia HL6G cells), but not in K562 human leukaemia cells, following exposure to doxorubicin (5 ^.M for HL60 cells and 10 ^M for K562 cells, both for 24 h). DNA was extracted and run on 2% agarose gels and stained with ethidium bromide. DNA ladders indicative of nucleosomal DNA fragmentation became apparent after 8 h, concident with morphological appearances due to apoptosis (not shown). Microscopic examination of doxorubicin-treated K562 cells showed that these cells became necrotic [not shown}.

pro vides a basis for a method which can detect and quantify apoptosis (9), as illustrated in Fig 7 and discussed further in Section 6. These methods prove that apoptosis has occurred, although sometimes a lag period of several hours is necessary for the signs of apoptosis to become detectable, as will be discussed later in this chapter. It is possible that this lag can explain some situations in which morphological apoptosis is not accompanied by DNA fragmentation.

Analysis of all aspects of cell death has been greatly aided by application of flow cytometry (see Chapter 4). The instrumentation is now widely available in most academic or industrial centres, and can provide quantifiable data for practically every method of detection of apoptosis described in this volume. In the author's laboratory, determination of the sub-Gl/GO cellular DNA content of propidium iodide-stained cells (see Chapter 4, Protocol 4) has been found to be excellent for routine use in studies of the effects of chemo-therapeutic drugs on cultured cancer cells (e.g. ref. 10). A more specialized and less accessible instrument, the laser-scanning cytometer, combines the advantages of flow cytometry with image analysis, providing information on cell morphology, and, if necessary, tissue architecture. The recent modifications of flow cytometric procedures for laser-scanning cytometry presented in Chapter 4 may prove particularly valuable for groups with access to this instrument.

The cascades which signal and execute cell death by apoptosis can be initiated by internal cues or by agents present in the extracellular environment. The cell death receptors described in Chapter 5 participate in the T cellmediated cytotoxicity of target cells, and serve to illustrate the procedures used to study receptor-mediated pathways to apoptosis. Chapter 5 also presents an overview of pathways to cell-mediated cytotoxicity and methods which can distinguish cytoplasmic from nuclear manifestations of apoptosis.

Induction of apoptotic pathways by extracellular agents can also occur by membrane events that include liberation of sphingolipids, which act as messengers of cell death. Chapter 6 describes the sphingomyelin cycle in which a stress signal activates a cell membrane-associated enzyme, sphingomyelinase, resulting in the formation of ceramide from sphingomyelin. In this way various cytokines, such as TNF-a, interleukin-1, and 7-interferon, chemo-therapeutic agents, and serum starvation can induce ceramide formation, which activates cellular protein kinases and protein phosphatases involved in cellular life and death decisions.

A well-known event in cell death is exteriorization of phosphatidylserine on plasma membrane. This change allows binding of the anticoagulant protein annexin V to this negatively charged phospholipid with great affinity, but is not entirely specific for apoptosis. Approaches that allow the distinction of apoptosis from necrosis based on annexin V techniques are the principal focus of Chapter 7, which also discusses other cytoskeletal and nucleoskeletal alterations associated with apoptosis.

Signals for apoptosis generated within the cell do not appear to be well understood, but are known to include metabolic alterations in the mitochondria that result in the disruption of mitochondrial transmembrane potential and changes in the cellular redox state. Chapter 8 addresses the mitochondrial permeability transitions and the role of various aspects of oxidative stress in the process of apoptosis. In particular, determination of peroxide and superoxide levels and cellular glutathione content are presented, to illustrate how surrogate end-points can be used to investigate and quantitate apoptosis-related phenomena.

An intermediate level of co-ordination of cell death versus survival signals is largely controlled by members of the Bcl-2 family of protein. These are discussed in Chapter 9, with a wealth of techniques optimized in the Reed laboratory, while investigation of the functioning of the caspase cascade is considered in Chapter 10. Extensive tabulation of the properties of caspases and of polypeptides cleaved during apoptosis concludes Chapter 10 and this volume.

2. Historical perspective

It is not always realized that apoptosis affects cells one at a time. Indeed, this form of cell death had been recognized earlier, as 'single cell necrosis'. The original descriptions were reported over a hundred years ago by pathologists studying liver diseases, but referred to as 'hyaline' or 'acidophilic bodies'. For instance, Councilman described hyaline bodies in the livers of patients dying of yellow fever (11), and subsequent electron microscopic studies concluded that these structures, also often called 'Councilman bodies', represent the remains of single dead hepatocytes (12, 13). Until recently, they were stated to result from coagulative necrosis of single cells (14), but, interestingly, they were noted to be eventually phagocytosed by macrophages. When the description 'apoptosis' was introduced by Kerr et al. in 1972 (4), this form of cell death fitted right into the definition, and, indeed, this was experimentally confirmed (12).

In the strictest sense, apoptosis refers to manifestations of a process, a programme leading to cell death, recognizable by morphological or a variety of biochemical criteria, which are discussed in subsequent chapters. The process itself, cell death, cannot, of course, be seen, and is detected by a combination of its manifestations. Thus, the terms ' apoptosis' and 'programmed cell death' are not exactly equivalent, nor is the phrase 'physiological cell death', which generally implies a developmentally determined programme, as opposed to 'chemoaptosis', in which the programme is triggered by xenobiotics such as cancer chemotherapeutic drugs.

3. Distinction of apoptosis from other forms of cell death

The current literature contains many examples of loose use of the term 'apoptosis'. There are several forms of cell death, and in all of them nuclear

DNA becomes degraded, at some point. As mentioned above, demonstration of DNA damage or release of products of DNA degradation is by itself insufficient to justify description of the phenomenon as apoptosis. The distinction is more than a semantic debate, since the concept behind the term 'apoptosis' is the existence of an inherent cellular programme, somewhat similar to the programmes which drive cell differentiation, whereas 'necrosis' results entirely from circumstances outside the cell. Other forms of cell death, e.g. mitotic cell death, are insufficiently characterized to be considered at this time as biologically distinct entities.

The distinction between apoptosis and necrosis can be made biochemically (discussed on pp. 12-15) but is also very clear on purely morphological grounds. As sketched in Figure 3, the apoptotic cell shrinks, nuclear chromatin undergoes marked condensation, and it is expelled from the cells as apoptotic bodies that are phagocytosed by neighbouring cells. In contrast, necrotic cells first increase their cellular water content and thus their volume, the nuclei lose the typical chromatin structure which is often seen as irregular clumping and/or dissolution, and the cell membrane ruptures, discharging the cellular contents into the environment. Many of the manifestations of necrosis appear to be due to the depletion of cellular ATP by the agents or conditions precipitating necrosis, and it is known that there are steps in the apoptotic cascade that require ATP or dATP (15). This may be an incompletely explored area for the study of how to distinguish controversial cases of cell death; for further discussion of this interesting topic the report by Eguchi et al. should be consulted (16).

The criteria currently most useful for distinguishing apoptosis from necrosis are listed in Tables 1 and 2. Importantly, there are also similarities between

Table 1. Morphological differences and similarities between apoptosis and necrosis

Differences

Similarities or confounding variables

Apoptosis

Necrosis

1. Nuclei Pyknosis

Karyolysis preceded by irregular chromatin

Damage occurs in both and karyorrhexis (dense condensation of chromatin)

clumping Disrupted

2. Cytoplasmic Morphologically organelles intact

3. Cell membrane Apoptotic bodies, blebbing

4. Cell volume Cells shrink

Blebbing and loss of integrity Cells swell

Secondary damage in apoptosis Changes seen in both

There may be no detectable changes In epithelia superficial cells are apoptotic and in groups

5. In tissues

Single cells affected Groups of cells affected

6. Tissue response None

Inflammation

Apoptosis

Necrosis

Figure 3. A sketch of the key morphological differences between apoptosis and necrosis. When a normal cell, depicted in (a), receives overriding signals to undergo apoptosis, it first exhibits an irregular contour and appears smaller (b). The chromatin then shows dense condensation, especially at the nuclear periphery, and small pieces of the cell, usually containing condensed chromatin, break off (c). The pieces, called apoptotic bodies, are taken up by phagocytosis by neighbouring cells, particularly macrophages if these happen to be present (d). The apoptotic bodies are then gradually digested by the phagocytic cells. In necrosis, the cells swell and chromatin often is alternatively diffuse or finely clumped (e). The cytoplasmic organelles, such as the mitochondria, may be swollen but remain intact. The necrotic cell eventually lyses, releasing all of its contents into the extracellular space, and thus eliciting an inflammatory response by the tissue.

apoptosis and necrosis, and, in view of these and a frequent overlap of characteristic features, conclusive evidence of the occurrence of apoptosis should demonstrate more than one morphological or biochemical criterion of apoptosis.

Table 2. Biochemical differences and similarities between apoptosis and necrosis

Differences

1. Nuclear DNA damage

2. Nuclear gene expression

3. Mitochondrial DNA damage

(a) DNases

(b) Proteases

(c) Transglutaminase

5. Membrane function

6. Cell Internal milieu

Apoptosis

Nucleosomal and/or 50-300 kb fragments —»ladders on gels Usually needed

Spared

Necessary Necessary Frequent Inact

Slightly acidic (pH 6.4) Often increases May be intact Required

Necrosis

Random -»smears on gels Not needed

Occurs early

Not necessary

Loss of function

Similarities or confounding variables

Takes place in both, easier to detect in apoptosis Not needed in cells primed for apoptosis

Lysosomal DNase and proteases are activated in necrosis

Acidic Both acidic

Always increases Seen in both Defective —

4. Apoptotic cascades

There appears to be an intricate but precisely ordered cellular machinery for self-destruction. This machinery is a subject of intense current investigations (discussed in several chapters in this volume) but some general principles have already emerged. First, the initiating signals can be either extracellular or intracellular. Secondly, many proteolytic enzymes are involved in apoptosis, which can be subdivided into classes which either initiate the process, propagate and amplify the signal, and those which attack the cellular structures to cause their collapse (Figure 4). Third, the mitochondria play an important role in this process, and many serve to integrate the various signals for apoptosis. Fourth, the pro-apoptotic machinery interacts with cellular survival mechanisms at several levels, including the mitochondria and the execution caspases {Figure 5). The suggested role for mitochondria in activation of the executioner caspase-3 is further illustrated in Figure 6.

5. Time course of apoptotic cascades

A wide range of times has been reported for the duration of apoptosis. The discrepancy is particularly marked when in vivo and tissue culture experiments are compared. For instance, Figure 2 shows that DNA ladders, which signify a late stage of apoptosis, are evident at 8 h after addition of 5 |xM doxorubicin to HL60 cells, and disruption of the inner mitochondria trans-

Figure 4. A conceptual outline of the major steps in the proteolytic cascade characteristic of apoptosis. Note that signals for apoptosis can be generated from the outside or the inside of a cell. The sequential activation of successive proteases (caspases) provides a fail-safe mechanism that makes it possible to abort a premature apoptotic signal, and also serves to amplify the signal to allow rapid finalization of an irrevocable decision to self-destruct.

Figure 4. A conceptual outline of the major steps in the proteolytic cascade characteristic of apoptosis. Note that signals for apoptosis can be generated from the outside or the inside of a cell. The sequential activation of successive proteases (caspases) provides a fail-safe mechanism that makes it possible to abort a premature apoptotic signal, and also serves to amplify the signal to allow rapid finalization of an irrevocable decision to self-destruct.

membrane potential, an early event in apoptosis, can be detected in a similar in vitro system (human myeloma cells treated with a retinoid) within 1 h (17). In contrast, in an in vivo model of apoptosis that occurs in rat ventral prostate after castration, the process was reported to take 44 h, and more detailed analysis of this model suggested that the time gap between the apoptotic trigger and the appearance of minimally abnormal morphology was 12-16 h. The disassembly of apoptotic ceils into apoptotic bodies was estimated to take

_ Extracellular Stimuli /

Drugs Radiation Viruses

_ Extracellular Stimuli /

Drugs Radiation Viruses

IAP proteins eg Survivin

Actin

Cytosketetal changes Cell Condensation

Lamins, PARP, DFF, etc

IAP proteins eg Survivin

Actin

Cytosketetal changes Cell Condensation

Lamins, PARP, DFF, etc

Chromatin Condensation

Figure 5. A more detailed outline of apoptosis-signalling pathways. The mitochondria are shown to act as the principal integrators of signals for apoptosis that do not have dedicated membrane receptors for that purpose. The survival signals which counter the pro-death signals are not shown, but components of the intracellular machinery that promotes survival are indicated (Bcl-2, survivin). A few examples of cellular targets for executioner caspases are also shown.

4-5 h, but the digestion of apoptotic bodies by the neighbouring cells, which phagocytose these remnants, appeared to be a slow process, requiring approximately 24 h (18).

Although these observations suggest that finding evidence of apoptosis in animal tissues allows a reasonable window of time, this situation is not always the case. Apoptotic bodies are frequently cleared from the tissues more rapidly than in the central prostate, and there is an additional uncertainty as to how rapidly the in vivo stimuli for apoptosis can reach the cell and be summated before they exceed the threshold for initiation of cell death pro-

Mitochondria

Cytochrome C(Apaf-2)

Mitochondria

Bcl-2

Cytochrome C(Apaf-2)

Bcl-2

Caspase-3 Precursor

Activated Caspase-3

Figure 6. A detail of the role of mitochondria in triggering the caspase cascade. Note the release of cytochrome c and the requirement for dATP. (Modified from Li et at.. Cell, 91, 479,1997).

grammes. Thus, when looking for evidence of apoptosis in tissues, the old adage, 'the absence of proof is not necessarily the proof of absence', becomes particularly appropriate. The apoptotic cells and bodies may have been missed.

The importance of careful selection of the time parameters for detection of apoptotic is further discussed in Chapter 5.

Caspase-3 Precursor

Activated Caspase-3

Figure 6. A detail of the role of mitochondria in triggering the caspase cascade. Note the release of cytochrome c and the requirement for dATP. (Modified from Li et at.. Cell, 91, 479,1997).

grammes. Thus, when looking for evidence of apoptosis in tissues, the old adage, 'the absence of proof is not necessarily the proof of absence', becomes particularly appropriate. The apoptotic cells and bodies may have been missed.

The importance of careful selection of the time parameters for detection of apoptotic is further discussed in Chapter 5.

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