Apoptosis: Suicide,execution or murder?

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Apoptosis 1, an alternative mode of cell death to necrosis, has been the focus of considerable attention in recent years. Apoptosis is c o m m o n l y observed when ceil death is a programmed event during embryogenesis, metamorphosis or normal cell turnover, for example. For this reason, apoptosis is often referred to as 'programmed cell death', but the two terms are not synonymous since there are m a n y instances of apoptosis that are clearly not programmed but are the cell's response to changes in its local environment. Distinctive morphological and biochemical changes take place during apoptosis (see Refs 2 and 3 for recent reviews) and these lead to the controlled dismantling of the cell into small vesicles with intact cell membranes, termed apoptotic bodies, which are quickly recognized and engulfed by neighbouring cells or marauding macrophages. Morphologically, apoptotic cells are typified by the highly condensed and fragmented state of their nuclei, which can be visualized under light microscopy in m a n y cell types (Fig. 1). These early morphological changes usually correlate with fragmentation of the nuclear chromatin at internucleosomal sites as a result of activation of an endogenous endonuclease; when DNA is examined by electrophoresis this produces a highly characteristic 200 bp multiple 'ladder-pattern'. Necrosis, on the other hand, is a relatively uncontrolled process that usually follows a gross perturbation to the cellular environment and loss of plasma membrane integrity. This leads to ions and water flowing down their chemical and osmotic gradients, thus provoking rapid cell and organelle swelling and leading to rupture of the cell and spillage of its contents into the extracellular environment. A cell undergoing necrosis can thus cause further injury and even death to neighbouring cells and can provoke an inflammatory response.

Apoptosis: suicide, execution or murder? Apoptosis, a controlled form of cell death, appears to be regulated in several ways. Early studies indicated that de novo protein synthesis was required for apoptosis of thymocytes, but more recent studies have found that other cells can undergo apoptosis when protein synthesis is blocked or that inhibition of protein or RNA synthesis can induce apoptosis. Whether these findings reflect distinct forms of apoptosis or variations on a single pathway is not yet known. In this article the case for a single pathway to apoptosis, accessible at multiple points, is discussed.

several lines of evidence arguing against a universal requirement for de novo expression of such 'death genes' during apoptosis. First, m u c h of the evidence for a protein or RNA synthesis requirement for apoptosis comes from studies of rodent thymocytes or T-cell hybridomas. Since thymocytes are unique in that they are delicately poised on the brink of life (positive selection, resulting in maturation

Apoptosis as a f o r m of cell suicide

The biochemistry of the apoptotic process is still ill-defined but is currently under very active investigation. Apoptotic cell death is perceived as a type of cell suicide since it appears to be genetically controlled. This view stems from studies that suggest transcriptional and/or translational control is maintained by the dying cell over the apoptotic process. For example, apoptosis of rodent thymocytes induced by X/7-irradiation4, S, treatment with the Ca 2+ ionophore A23187 (Refs 6,7), cytotoxic drugs or nucleosides8, 9, apoptosis of T-cell lines induced by IL-2 withdrawal 10, or apoptosis of Tcell hybridomas induced by anti-CD3 antibody treatment 11, is delayed or abrogated in every case by inhibitors of RNA and/or protein synthesis. Such studies have led to the notion that macromolecular synthesis is an essential c o m p o n e n t of the apoptotic response in general, and that this represents synthesis of components of a cell suicide programme that are encoded by putative 'death genes'. However, it is becoming apparent that the situation is more complex than was first thought, with TRENDS IN CELL BIOLOGYVOL. 3 MAY 1993

FIGURE 1 Morphology of apoptosis in HL-60 cells. Apoptotic cells are readily distinguishable by the condensed and fragmented state of their nuclei. These cells then undergo further collapse into smaller membrane-bound vesicles (arrows) for phagocytosis by neighbouring cells or tissue macrophages. Magnification, 1000 x. © 1993 ElsevierSciencePublishersLtd (UK) 0962-8924/93/$06.00

SeamusMartin is at the Department of Immunology, UniversityCollege London Medical School,Arthur StanleyHouse, 40-50 Tottenham Street, London, UKW1P 9PG. ] 41

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TABLE I - APOPTOSIS IN THE PRESENCE OF PROTEIN AND/OR RNA SYNTHESIS INHIBITORS a Cell type

Apoptotic stimulus

Inhibitor

consequence of interruption macromolecular synthesis.

of

Refs

Inhibition of protein or RNA synthesis does not necessarily block CTL targets CTL attack CHX 12 apoptosis T-CLL Dexamethasone CHX 13 The protein and/or RNA synthesis Human medullary Dexamethasone CHX 14 requirements for apoptosis among thymocytes cell types other than thymocytes were not well documented until Apoptosis potentiated by protein/RNA synthesis inhibitors recent years, but these experiments T-ALL Spontaneous CHX 13 give a very different picture to the cALL Spontaneous CHX 13 generally accepted one that protein AML Spontaneous CHX 13 or RNA synthesis is a necessary reB-CLL Spontaneous CHX 15 quirement for this type of cell death Neutmphils Spontaneous CHX, Act D, EM. 16 (Table 1). One of the earliest demonBurkitt's lymphoma Hyperthermia CHX 17 strations that apoptosis could proHuman mammary TNF-c~ CHX 18 ceed in the presence of protein adenocarcinoma synthesis inhibitors was by Duke et Mouse lymphoma Photodynamic therapy CHX, Act D 19 al. 12 who reported that apoptosis of cytotoxic T lymphocyte (CTL) tarApoptosis triggered by protein/RNA synthesis inhibitors gets was not blocked in the presHL-60 A23187, colchicine CHX, Act D 20 ence of such drugs. This has been T-cell hybridoma Aphidicolin, camptothecin Act D 23 followed by other instances of cells Rodent macrophages Gliotoxin CHX 26 undergoing apoptosis while protein Rodent T-cell b l a s t s Gliotoxin CHX 26 synthesis is blocked13,14 (Table 1). Rodent thymocytes Gliotoxin CHX 26 There are also m a n y cases where Human medullary CHX 14 apoptosis is potentiated by protein thymocytes or RNA synthesis inhibition13,15q9 17 Burkitt's lymphoma CHX (Table 1). For example, the spontan20,21 MRC-5 Act D, CHX eous apoptosis of B-chronic lympho20,21 U937 Act D, CHX cytic human leukaemia cells (B-CLL)is 20,21 Molt-4 Act D, CHX and peripheral blood neutrophils 16 Daudi Act D, CHX 20,21 is enhanced rather than inhibited by 20,21 Raji Act D, CHX CHX, in a dose-dependent manner. Human T-cell blasts Act D, CHX 22 Even more surprisingly, there are T-cell hybridoma Act D 23 now numerous reports that protein 24,25 Rodent intestinal Act D, CHX or RNA synthesis inhibition alone crypt cells can directly trigger apoptosis in a Rodent macrophages CHX 26 variety of cell types 2°-26 (Table 1). For instance, the h u m a n promyeloaMacromolecular synthesis inhibitors used in the above reports were actinomycin D (Act D), cytic leukaemia cell line HL-60, emetine (EM) and cycloheximide (CHX). along with many other tumour cell types, is extremely sensitive to inhibitors of protein or RNA syntheinto functional CD4 + or CD8 + T cells) or death sis, with these cells undergoing apoptosis within se(negative selection by induction of apoptosis) veral hours of actinomycin D (Act D, an inhibitor of RNA synthesis) or CHX treatment 20. This effect these cells may be exceptions to the norm. Second, although in m a n y instances protein and RNA synis dose dependent and occurs at concentrations of inhibitor within the range used to demonstrate thesis inhibitors have been shown to delay/block apoptosis, this could be entirely due to side effects inhibition of apoptosis in other cell systems. of these agents on other cellular processes and not Several other leukaemic cell lines such as U937, Molt-4, Daudi and Raji cells also undergo extensive necessarily due to inhibition of macromolecular apoptosis when exposed to these agents20, zl. Prosynthesis per se. Furthermore, experiments demontein or RNA synthesis inhibition also triggers actistrating inhibition of apoptosis by cycloheximide (CHX) or other protein synthesis inhibitors merely vated h u m a n peripheral blood T cells to undergo apoptosis 22. suggest a requirement for continuing macromolecInterestingly, anti-CD3 mAb-induced apoptosis ular synthesis, so the concept of de novo synthesis of 'death gene' products need never be invoked. of cells from the h u m a n T-cell hybridoma A1.1 can be inhibited by Act D in a dose-dependent manner, Most significant of all, however, are the evergrowing number of studies that have either failed but this drug has no effect when apoptosis is to demonstrate inhibition of apoptosis in the presinduced in these cells by the chemotherapeutic agents aphidicolin (a DNA polymerase inhibitor) ence of inhibitors of protein or RNA synthesis, or or camptothecin (a topoisomerase I inhibitor) 23. have shown apoptotic cell death to be a direct Apoptosis not blocked by protein/RNA synthesis inhibitors

m

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Moreover, Act D on its own was found to induce massive apoptosis of these cells at higher concentrations. So here we have conflicting results in the same cell type, one result indicating that these cells have an RNA synthesis requirement for apoptosis to proceed, and the other indicating that inhibition of RNA synthesis is itself a trigger for apoptotic cell death in these cells.

Cell surface

Protein synthesis dependent

Is t h e r e a single c o m m o n p a t h w a y to apoptosis?

How can the variable effects of protein synthesis inhibitors on apoptosis be explained? The conflicting results with the A1.1 T-cell hybridoma indicate that there m a y be different pathways to apoptosis (one dependent on protein synthesis and the other independent of protein synthesis) in the same cell, or that there m a y be a single c o m m o n pathway (in which only early events require protein synthesis) that can be accessed at m a n y points along the line. In the latter case, certain stimuli m a y interact with the pathway at a fairly advanced stage, thereby circumventing the early events that would normally be required for apoptosis induced by other means (Fig. 2). After all, the difference between an apoptotic signal delivered by means of inappropriately engaging the T-cell receptor complex (as with antiCD3 antibodies on thymocytes) and a signal delivered as a result of a drug binding to DNA and inhibiting RNA synthesis (as with Act D) is likely to be considerable. However, it is also the case that the protein synthesis requirement of apoptosis induced by the same stimulus varies with the cell type, suggesting that different cell types may have distinct apoptotic machinery, or regulate it differently. Cells that do not require new protein synthesis to undergo apoptosis'in response to a particular stimulus m a y repress apoptosis effector molecules that are already present in the cell (e.g. the nonspecific endonuclease) by means of specific inhibitors (such as the apoptosis-suppressing protein Bcl-2; Ref. 27); such cell types would undergo apoptosis if protein synthesis were hindered, and can thus be thought of as being 'primed' for apoptosis by having already undergone the early steps involving protein synthesis in the pathway at s6me stage of their differentiation. The contradictory data with regard to a protein or RNA synthesis requirement for apoptosis m a y therefore represent opposite sides of the same coin, with some cells requiring de n o v o synthesis of apoptosis-related proteins upon receipt of a death-inducing stimulus, while others constitutively express these molecules but maintain control over t h e m with specific inhibitory proteins. A further possibility also exists in the form of cells that would normally require de novo protein synthesis to undergo apoptosis but can be relieved of this requirement if the missing component(s) are supplied by another cell. This situation appears to apply to cytotoxic T-cell killing, where the CTL delivers a lethal package to the target cell by means of secretory granules that contain perforin, a pore-forming protein, as well as serine proteases, several of which have recently been found TRENDS IN CELLBIOLOGYVOL. 3 MAY 1993

C Endonuclease activation

DNA fragmentation and chromatin condensation

I Protein synthesis independent

CELL DEATH

Recognition and engulfment by phagocytes FIGURE 2

Schematic representation of the apoptosis pathway. Apoptosis can be triggered at multiple points along the pathway ( ~ - ) , depending upon the initiating stimulus. Upon receipt of a stimulus that acts early in the apoptosis pathway (such as anti-CD3 antibody), the cell must transduce the signal by putative proteins, A, B and C, as far as the later (irreversible) events in the pathway such as endonucleaseactivation. Since stimuli that act early in the pathway depend on the synthesis of A-C for their transduction, these apoptosis-inducing signals can be blocked by inhibiting protein or RNA synthesis. Later-acting signals (such as cytotoxic drugs) may bypass this requirement if they act upon a long-lived or abundant component of the pathway (which does not require continuous synthesis). Specific inhibitory proteins (e.g. Bcl-2) appear to be present in many cells to control the tendency of such cells to undergo spontaneous apoptosis. Such proteins may act by suppressing the activity of the stable components of the apoptosis pathway. Should the levels of such suppressorsfall (e.g. due to protein synthesis inhibition), then apoptosis wouid ensue.

to be capable of triggering apoptosis in target cells 28. Similar ideas have also been mentioned by Cohen and colleagues 29 who divide apoptotic cell death into three distinct mechanisms: the induction mechanism (protein synthesis is required), the transduction mechanism (protein synthesis not required), and the release mechanism (where interruption of protein synthesis induces apoptosis). Thus, when a cell dies by apoptosis it may not -necessarily be committing suicide. Deaths that do not require de novo protein synthesis m a y be more appropriately thought of as either execution, where the victim is primed for death by an external influence, as in CTL killing for example, or outright murder, where a primed cell is prevented 1 43

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from synthesizing life-saving apoptosis-inhibitory protein(s) by a cytotoxic drug such as CHX or Act D, or possibly as a result of viral infection. References 1 KERR,J. F. R., WYLLIE, A. H. and CURRIE,A. R. (1972) Brit. J. Cancer 26, 239-257 2 FESUS,L., DAVIES, P. J. A. and PIACENTINI, M. (1991) Eur. J. Cell BioL 56, 170-177 3 WILLIAMS, G. T., SMITH, C. A., MCCARTHY,N. J. and GRIMES, E. A. (1992) Trends CellBioL 2, 263-267 4 YAMADA, T. and OHYAMA, H. (1980) Int. J. Radiat. BioL 37, 695-702 5 SELLINS,K. S. and COHEN, J. J. (1987) J. ImmunoL 139, 3199-3206 6 KIZAKI, H., TADAKUMA, T., ODAKA, C., MURAMATSU,J. and ISHIMURA, Y. (1989) J. ImmunoL 143, 1790-1794 7 WYLLIE,A. H., MORRIS, R. G., SMITH, A. L. and DUNLOP, D. (1984) J. PathoL 142, 67-77 8 MCCONKEY, D. J., HARTZELL,P., DUDDY, S. K., HAKANSSON, H. and ORRENIUS,S. (1988) Science242, 256-259 9 KIZAKI, H., SHIMADA, H., OHSAKA, F. and SAKURADA,T. (1988) J. Immunol. 141, 1652-1657 10 COHEN, J. J., DUKE, R. C., CHERVENAK,R., SELLINS,K. S. and OLSON, L. K. (1985) Adv. Exp. Med. BioL 184, 493-506 11 SHI,Y., SZALAY,M. G., PASKAR,L., BOYER,M., SINGH, B. and GREEN, D. R. (1990)J. Immunol. 144, 3326-3333 12 DUKE, R. C., CHERVENAK,R. and COHEN, J. J. (1983) Proc. Natl Acad. Sci. USA 80, 6361-6365

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