Proc. Natl. Acad. Sci. USAVol. 93, pp. 2239-2244, March 1996

Review

The molecular biology of apoptosisD. L. Vaux* and A. StrasserThe Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melboure Hospital, Wctoria 3050, Australia

ABSTRACT All multicellular organ-isms have mechanisms for killing theirown cells, and use physiological cell deathfor defence, development, homeostasis,and aging. Apoptosis is a morphologicallyrecognizable form of cell death that isimplemented by a mechanism that hasbeen conserved throughout evolutionfrom nematode to man. Thus homologs ofthe genes that implement cell death innematodes also do so in mammals, but inmammals the process is considerablymore complex, involving multiple iso-forms of the components of the cell deathmachinery. In some circumstances thisallows independent regulation of path-ways that converge upon a common endpoint. A molecular understanding of thismechanism may allow design of therapiesthat either enhance or block cell death atwill.

Physiological cell death occurs when a cellwithin an organism dies by a mechanismorchestrated by proteins encoded by thehost's genome. The purpose of this pro-cess is to kill unwanted host cells and is putto use in three situations: (i) for develop-ment and homeostasis; (ii) as a defencemechanism; and (iii) in aging.Where did physiological cell death

evolve, and what was its original purpose?No single-celled organism can have a celldeath program whose activation is oblig-atory, as this would lead to its extinction.Single-celled organisms are also notthought to age the way that plants andanimals with predetermined life-spans do,yet asymmetrically dividing yeast havebeen observed to die after giving rise to acertain number of daughters (1). Conven-tional aging and cellular senescence prob-ably required the evolution of multicellu-lar organisms with separate germ andsomatic cells.There are an increasing number of re-

ports of single-celled organisms that killthemselves by a mechanism whose activa-tion is not obligatory, but can be used inthreatening situations (2, 3). Altruistic, ordefensive, cell death might be used toprevent the death by infection or starva-

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tion of neighboring, related individuals.Thus certain bacteria will kill themselveswhen they detect a bacteriophage andthereby prevent spreading the infection tothe entire colony (2). The genes for thisdefensive cell death mechanism would notdie with the individual but could be per-petuated by its relatives.The fact that defensive cell death and

developmental/homeostatic cell death inmetazoans is achieved by similar processes(see below), whereas life-span of thewhole organism is not (4), makes it attrac-tive to speculate that cell death firstevolved in single-celled organisms as adefence strategy, and that these mecha-nisms were subsequently adopted for usein development and homeostasis after theevolution of multicellularity.

In metazoans, physiological cell deathoften exhibits a characteristic morphologytermed "apoptosis" (5-7). Cells dyingautonomously, due to lack of growth fac-tor, for example (8), or cells killed non-autonomously, such as those targeted bycytotoxic T cells, display the same mor-phology (9). Apoptosis is often accompa-nied by rapid cleavage of the cell's DNAinto multiples of 180 bp, corresponding tothe internucleosomal spacing (10). Detec-tion of the broken ends of the DNA, the180-bp "ladder" seen upon electrophore-sis ofDNA from apoptotic cells, or reduc-tion of the total DNA content to below 2Call indicate that a dying cell'sDNA is beingdegraded and have become commonlyused experimental techniques for showingthat apoptosis is probably occurring (11,12).

It is convenient to divide the process ofphysiological cell death into phases (Fig.1). The earliest phase is the stimulus thatprovokes the apoptotic response. This maybe an external signal delivered throughsurface receptors or may originate insidethe cell from the action of a drug, toxin, orradiation. The next phase includes detec-tion of this signal or metabolic state andtransduction of the signal. Signal trans-duction pathways send this message to thecell death effector machinery. The effec-tor phase is the third part of the cell deathmechanism and includes the proteasesthat are activated during apoptosis, as wellas their positive and negative regulators(see below). The fourth phase of cell deathis the postmortem phase, in which thecell's chromatin condenses and its DNA is

2239

degraded. In vivo (but not necessarily invitro) dying cells are recognized and en-gulfed by other cells. The nematode Cae-norhabditis elegans has provided an excel-lent model system in which the stages ofphysiological cell death can be observedduring development, and mutants inwhich cell death is abnormal have pro-vided invaluable insight into the genes(ced genes = cell death abnormal) thatimplement it (4).

In C. elegans the cysteine proteaseCed-3 is essential for programmed celldeath to occur (13, 14). It therefore ap-pears that apoptosis is precipitated byproteolytic cleavage of one or more vitalsubstrates. The ability of ced-3 to causecell death can be blocked by the bcl-2homolog ced-9 or, indeed, by human bcl-2itself (15-17). It is currently not knownwhether Ced-9 prevents activation of theCed-3 protease or prevents the active en-zyme from reaching its substrates. Most ofthe ced genes (ced-1, -2, -5, -6, -7, -8, -10)are required for efficient disposal of thedead cell corpse, which is recognized andengulfed by neighboring cells (4). A nu-clease encoded by the nuc-1 gene is notessential for cell death but is required fordegradation of the dead cells' DNA. Inmammalian cells, too, the nuclear eventsassociated with apoptosis are not requiredfor the death-associated changes in otherparts of the cell (18, 19). The mechanismsthat implement cell death exist in thecytoplasm, ready to be activated withoutneeding to be newly synthesized (20).

Interestingly, C. elegans mutants withnonfunctional copies of ced-3, which con-sequently have no programmed celldeaths, age normally, and have a normallife-span (13). This result shows that in theworm life-span and aging are regulated bydifferent genetic processes to those thatregulate programmed cell death. It islikely that this is also true in vertebrates.Aging may represent a process thatevolved after the development of multi-cellular organisms with separate germ andsomatic cell lineages, whereas the celldeath mechanisms used for defence or

Abbreviations: ICE, interleukin-13 convertingenzyme; TNF, tumor necrosis factor; PARP,poly(ADP-ribose) polymerase; IAP, inhibitorof apoptosis protein; CTL, cytotoxic T cells.*To whom reprint requests should be ad-dressed.

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oxygn odd nuclic acidcyci i~~~drv ¢,,,c-mi stimulus

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mortem

scOveltonFIG. 1. A model of apoptosis in mammalian cells. Apoptosis is a physiological cell death

process triggered by internal or external stimuli; the process can be divided into four phases. Manysignal-transduction pathways, of which only a few examples are shown, can independently lead toactivation of effector aspartases resembling the C. elegans cysteine protease Ced-3. Theseproteases may be able to activate themselves and each other, but their ultimate targets areunknown. Depending on their ratio, Bcl-2 and similar proteins can inhibit the action of theproteases, either by blocking their activation or by preventing them from reaching their targets.Arrows do not necessarily indicate direct interactions.

development may have evolved earlier, insingle-celled organisms.

Cysteine Proteases

In mammals six different homologs ofCed-3 have been identified [interleukin 13

converting enzyme (ICE), Nedd2, CPP32,ICErel II/TX/Ich-2, ICErel III, mch2] (21-26), and it has been shown for several ofthem that over-expression can cause apo-ptosis and that this death can be inhibitedby interfering with protease function. Allof these genes encode cysteine proteasesthat are initially translated as inactiveprecursor polypeptides. These precursorscan be found in most, if not all, nucleatedmammalian cells, but in order to becomeactive and cause apoptosis, they must becleaved at aspartate residues and assem-bled into heterotetramers. The active cys-teine is in the middle of a conservedQACRG motif common to all the pro-teases. Mutation of this cysteine inacti-vates these enzymes. These mammalianproteases can be subdivided into threegroups: (i) human CPP32 and Mch2 whichresemble nematode Ced-3 most closely;(ii) Nedd2, which shows intermediate sim-ilarity, and (iii) ICE, ICErel II (TX, Ich-2),and ICErei I,, form a less similar group thatshare a higher level of similarity to eachother.

All of these cysteine proteases cleavetheir substrates after aspartate residues,but their affinity for their substrates also

depends on other residues, so they displaydifferent substrate preferences and havedifferent susceptibilities to protease inhib-itors (26-28). Additional substrate speci-ficities might be generated if mature tet-rameric proteases can be formed fromsubunits of different polypeptides (e.g.,CPP32 plus Nedd2). The requirement forall known Ced-3-like proteases to be pro-cessed at aspartate residues, together withtheir ability to cleave at aspartate residues,suggests that some of these proteases maybe able to activate themselves or act oneach other in a hierarchical cascade. In-deed, some interactions between cysteineproteases have been observed both in invitro and in cellular systems. Thus matureICE can activate pro-ICE as well as pro-CPP32 (24, 29-31).Only a handful of the substrates for

these cell death proteases are known. Itmay be that there are a large number ofsubstrates, no one of which is vital for cellsurvival, or it may be that there is a smallnumber of vital substrates that must becleaved for cell death to occur. As well as

being able to activate CPP32, ICE isknown to liberate bioactive interleukin-1f3from pro-interleukin-1, but interleukin-13does mediate the apoptotic signal-ratherit is a proinflammatory lymphokine thatcan alert neighboring cells, as well as

distant cells of the immune system, thatapoptosis is occurring. This signaling mayallow inflammatory cells to activate andaccumulate and become functionally ac-

tive at a site where cell suicide is beingused as an antiviral defence (32). On theother hand, when apoptosis is to be usedfor morphogenesis or homeostasis andinflammation is undesirable, a proteaseother than ICE may be used.

Protease CPP32 can cleave and inacti-vate poly-(ADP ribose) polymerase(PARP), an enzyme that is used for DNArepair (28, 33, 34). Cleavage of PARP canbe used as a convenient marker of apo-ptosis, but PARP knock-out mice developnormally; thus PARP cleavage is probablydispensable for apoptosis (35). ICE is notable to cleave PARP directly but can do soindirectly by first activating CPP32 (31).Nicholson et al. (28) have shown thatcytoplasmic extracts from apoptotic cellscan cause apoptotic changes in isolatednuclei and can cleave PARP (28). Re-moval of protease CPP32 from the ex-tracts prevented them from causing thesechanges, but the activity was restored withpurified active CPP32 but was not restoredwith ICE. Intriguingly, although activeCPP32 alone can cleave PARP, on its ownit did not cause apoptotic changes to theisolated nuclei, suggesting that CPP32must act on a substrate within the cyto-plasmic extract to precipitate apoptoticchanges in the nuclei (28). Elegant studiessuch as these should reveal the identity ofthis and other substrates.

Activation and Regulation of theProteases

What initiates the proteolytic cascade ofcysteine proteases is not known. In C.elegans Ced-4 is required for cell death,and it may have a role upstream of theCed-3 protease (13). However the mam-malian homolog of Ced-4 has not beenidentified, and its function is not known(36). What is clear is that almost all mam-malian cells express several cell deathproteases, even when they are not under-going apoptosis. The proteases can thenbe activated without having to be synthe-sized anew, and apoptosis can be inducedwithout influencing transcription from theprotease genes (18, 20). Over-expressionof ICE, ICErel II (TX/Ich-2), ICErel III, orNedd2 results in apoptosis of TH1 cells,even though these cells contain easily de-tectable levels of message for ICE andNedd2 (and several of the other cysteineproteases) before transfection (26, 28).Why this procedure induces cell death isobscure. Maybe transient overexpressionleads to levels of protease sufficient toallow autocatalytic activation of the pro-tease precursor. Alternatively, intrinsicactivation signals may exist in cells that areordinarily inadequate to activate sufficientprotease for apoptosis, but increasing thelevel of precursor by transfection mayallow sufficient production of active en-zyme to cause cell death. CPP32 can causeapoptosis of insect cells when expressed at

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very high levels but does not cause apo-ptosis as readily as ICE in mammaliancells (22). This result may reflect a lowertendency of CPP32 to autoactivate com-pared to ICE.Although the final activation of these

proteases is posttranslational, some of thepathways that lead to their activation mayinvolve steps that are transcriptionallycontrolled. For example, DNA damagecaused by irradiation leads to increasedlevels of p53, a transcription factor, thatcan then cause a cell to kill itself (37-39).Moreover, deregulated expression of c-Myc, an oncogenic transcription factor,can also cause apoptosis in certain cells(40, 41). Some signals can provoke apo-ptosis in the presence of cycloheximide oractinomycin D, and these agents them-selves stimulate apoptosis of many celltypes (42). On the other hand, in someinstances apoptosis caused by withdrawalof growth factor can be blocked by cyclo-heximide-for example, when nervegrowth factor is removed from cultures ofprimary neurons or PC-12 cells (43). Thisresult suggests that, in these cells, factorwithdrawal activates transcription fromdeath-signaling genes. In other cell types,such as the interleukin-3-dependent he-mopoietic cell line FDC-P1, cyclohexi-mide does not block apoptosis due tofactor withdrawal but actually enhances it(20). In this situation growth factors maymaintain levels of a labile protein thatprevents cell death. This protein is notBcl-2, or Bcl-2-like, because cells main-tained in growth factor remain highly sus-ceptible to apoptosis triggered by cytotox-ins, whereas bcl-2-transfected cells areprotected from the same agents.

Apoptosis induced by withdrawal ofgrowth factor, irradiation, or myc over-expression can be strongly inhibited byBcl-2 (and some of its relatives, see below)(20, 44-53). This result provides evidencethat different stimuli activate distinct sig-naling pathways that converge upon acommon cell death effector mechanism.The activation or signaling phase of cell

death encompasses a great variety of sig-nal transduction pathways that mediatesignals from outside the cell, as well asothers that originate inside the cell. Theseare likely to include most of the knownsystems, including tyrosine kinases, ste-roid receptors, ceramide, inositol phos-phates and the cytokine receptors them-selves. For example, when apoptosis isinduced in T cells by activation through itsantigen receptor, signals are passed intothe cell, leading to activation of the celland expression of c-myc. The cell thenup-regulates both CD95 (Fas/APO-1)and its ligand and expresses them on thecell surface. These molecules can theninteract with each other (in an autocrineor paracrine manner), initiating the celldeath-inducing signaling pathway (54).

Ligation of CD95 usually causes apo-ptosis. Thymocytes from mice in which theICE-encoding gene has been mutated andcell lines treated with ICE-specific inhib-itors are not killed by this stimulus, indi-cating that ICE is essential for CD95-activation-induced apoptosis of these cells(55-59). Ceramide has been implicated inkilling by CD95 and tumor necrosis factor(TNF), and ceramide itself can provokeapoptosis in several cell types (60-63), butthe exact role for ceramide has not yetbeen determined (64, 65). Several pro-teins that interact with the cytoplasmicdomains of CD95 and the TNF receptors(p55 TNF-R1 and p75 TNF-R2) have re-cently been implicated as signaling inter-mediaries. Some of these proteins(Mortl/FADD, RIP, and TRADD) bear"death domains" resembling those inCD95 and the p55 TNF receptor, andover-expression of these proteins inducesapoptosis (66-70). "Death domains"probably facilitate homotypic protein-protein interaction. It is presumed thatover-expression of proteins bearing deathdomains leads to their association, even inthe absence of CD95 ligand or TNF, andthus sends an apoptotic signal.

Binding of TNF to the TNF-R2 (p75)can also trigger apoptosis, even thoughTNF-R2 does not bear a death domain(71-73). The cytoplasmic domain ofTNF-R2 associates with factors termedTRAF1 and TRAF2 that are required foractivation of transcription factor NF-KBby TNF-R2 (74, 75). Proteins resemblingthe inhibitor of apoptosis proteins (IAPs)from baculoviruses (see below) are able tobind via TRAFM and TRAF2 factors tothe TNF-R2 cytoplasmic domain as partof a multiprotein complex. Some of thesemammalian IAP proteins can inhibit apo-ptosis due to transfection with pro-ICE,indicating that IAP proteins may functionto interfere with death signals activated byTNF (D.L.V., unpublished observations).

Apoptosis triggered by CD95 ligand orTNF, or over-expression of TRADD pro-tein, is efficiently blocked by Bcl-2 in somecells but not in others (65, 68, 76-78),suggesting that alternative pathways maybe activated by these agents in differentcells. Killing by CD95 ligand, TNF, orTRADD over-expression is effectivelystopped by CrmA, the ICE inhibitor fromCowpox virus (refs. 57, 68, and 78;K. G. C. Smith and D.L.V., unpublishedobservations). However, ICE is not essen-tial for developmental cell death (55, 79).Neither is it required for death due toradiation, corticosteroids, or growth factordeprivation, in which apoptosis is efficientlyantagonized by Bcl-2. It therefore appearsthat different apoptosis-triggering stimulimay activate distinct cysteine proteases.The first gene shown to be specifically

involved in the process of physiologicalcell death was bcl-2 (44). Bcl-2 can inhibitapoptosis of many cells triggered by many

different agents (48, 50, 80) and resemblesthe cell death inhibitor from C. elegans,Ced-9, both functionally and structuraily(15, 17). In mammals six genes with struc-tural similarity to bcl-2 have so far beenidentified. Function of Al has yet to bedescribed (81). Mcl-1 acts like Bcl-2 toprotect cells (82, 83). Bax, Bak, and Badantagonize the protection offered by Bcl-2(or Bcl-XL, see below) (84-88). Anothergene (bcl-x) encodes two (or more) pro-teins; the longer protein, Bcl-XL, inhibitsapoptosis while the shorter, Bcl-xS, antag-onizes the protective function of Bcl-2(and Bcl-xL) (89). Members of the Bcl-2family share conserved domains that allowformation ofhomo- and heterodimers (84,90, 91). Exactly how these multiproteincomplexes work and the conformation ofthe functional forms are currently un-known.

Cell Death as an Antiviral Defence

The idea that apoptosis could be used asa host defense against viruses was pro-posed by Clouston and Kerr in 1985 be-cause apoptosis can be seen in the virallyinfected targets of cytotoxic T cells (92).That cell-autonomous apoptosis can be aneffective antiviral strategy was shownmost clearly by Clem and Miller (93).The fact that a growing number of

viruses have been found to encode specificinhibitors of apoptosis supports this idea.CrmA from cowpox virus binds to andinactivates ICE (94); a number of virusesencode proteins resembling Bcl-2, and anumber encode proteins that can antago-nize p53 (for reviews, see refs. 32 and 95).Two novel classes of virus-encoded apo-ptosis inhibitors (p35 and IAP) have beendescribed by Miller and coworkers. Theseproteins are required for full virulence ofbaculoviruses (93, 96, 97); if these genesare mutated, the host insect cells respondto infection by undergoing apoptosis,drastically reducing viral replication andtiter. p35 is believed to act like CrmA as adirect protease inhibitor (98, 99), whereasIAPs may act upstream to prevent activa-tion of the proteases. Both baculovirusgenes can function in heterologous sys-tems to block apoptosis. For example,both IAP and p35 block ICE over-expression-induced apoptosis in mamma-lian cells, and p35 can act in C. elegans toblock programmed cell death mediated byCed-3 (100-102, C. J. Hawkins andD.L.V., unpublished observations). p35can prevent apoptosis in insect cells me-diated by reaper and hid, indicating thatthese two genes also act in the same celldeath pathway in Drosophila (103, 104).Although no genes resembling p35 havebeen found, several genes bearing similarstructural motifs to TAP have been iden-tified in the mammalian genome. Thefinding that mammalian cells have celldeath mechanisms capable of interacting

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with insect IAPs has led to the identifica-tion of mammalian IAP-like genes thatcan also inhibit cell death (A. Uren andD.L.V., unpublished observations).

Cytotoxic T-Cell-Mediated Apoptosis

A major main defense of vertebratesagainst viral infections is cytotoxic T cells(CTL), which can recognize and induceapoptosis of virally infected cells (105).CTL use more than one killing pathway(106-108). CTL express the ligand forCD95 and thus can kill CD95-bearingtarget cells. Secondly, CTL bear granulesthat they can deliver into their targets.One of the proteases in the CTL granules,granzyme B, cleaves after aspartate resi-dues (109); its substrate specificity there-fore resembles that of the Ced-3-like cys-teine proteases (32, 110). Experimentswith spontaneous and genetically engi-neered mutant mice demonstrated thatCD95 ligand, perforin, and granzyme Bare all required for optimal cytolytic func-tion of CTL (106-108, 111-114). Whenadded to cells along with perforin, gran-zyme B causes apoptosis (115), which canbe effectively blocked by Bcl-2 in somecases (116). Bcl-2 does, however, not pro-tect against killing by intact CTL (50, 117),even if CD95 signaling is absent. Thisresult suggests that there are some medi-ators other than granzyme B used by CTLto kill in a perforin-requiring manner.Some physiological cell killing may oc-

cur that does not involve apoptosis. In C.elegans the male linker cell appears to bekilled by its neighbors, and this killing stilloccurs in ced-3 mutant animals (13). Intransgenic mice that constitutively expressbcl-2 in neutrophils, these cells do notundergo apoptosis as they age, the waynormal neutrophils do, but in spite of thisthey are engulfed and degraded by mac-rophages with similar kinetics (118).Blocking apoptosis by Bcl-2 may not pre-vent expression of "eat me" signals on the

.az

aged neutrophils. Maybe a similar phago-cytic cell killing occurs in the bone marrowand thymus to allow clonal deletion ofautoreactive lymphocytes even when apo-ptosis is blocked by transgenic expressionof Bcl-2 and/or mutation of CD95. De-termination of the sequence of the genesrequired for engulfment in C. elegans [ced-1, -2, -5, -6, -7, -8 and -10 (4)] and theirmammalian homologs should help answerthese questions.

Apoptosis Induced by Drugs

Cells do not only commit suicide in re-sponse to physiological stimuli: manydrugs, toxins, and physical insults provokethe same response (119, 120). Even isch-emia, or agents such as azide that deprivea cell of ATP, do not merely cause necro-sis; often the dying cells show morpholog-ical changes typical of apoptosis, includingrapid degradation of the DNA (121, 122).Why do cells respond to so many differentagents, including many anticancer drugs,by killing themselves, rather than attempt-ing to defend themselves against the in-sult? Often cells treated with a cytotoxicdrug commit suicide by apoptosis beforethey are killed by the drug; blocking apo-ptosis with Bcl-2 can inhibit this suicideresponse, but death may still ensue, with apurely necrotic morphology. If the drugtreatment or anoxia is transitory, blockingthe suicide response may prevent the cellfrom dying at all.One possible explanation for these find-

ings is that cells may detect early changescaused by the drug or insult and activatephysiological death mechanisms on theassumption that the changes were due toan infecting virus (123). Thus it would bewrong to conclude that anoxia, azide, di-oxin, or diphtheria toxin act primarily tocause apoptosis; rather the cell respondsto some change they cause by activatingthe apoptotic mechanism. Thus theseagents are part of the initial "stimulus"

time

FIG. 2. The relationship between physiological and pathological cell death. A cell can respondto noxious stimuli by activating its physiological cell death mechanism (- - -). If the physiologicalmechanisms do not activate, the cell may eventually be killed by the agent (-). Cells receivinga transient, or low-level exposure to the agent, may be able to recover (- - -). Preventing activationof physiological cell death mechanisms could allow more cells to recover from a transient ischemicevent, such as a heart attack or stroke. Conversely, activation of physiological cell deathmechanisms in tumor cells could potentiate the effect of cytotoxic drugs or radiation.

phase of cell death, rather than part of thesignaling or effector phase.

Pharmaceutical agents that could blockapoptosis may be useful in treating isch-emic conditions such as heart attacks,strokes, or reperfusion injury, by blockingthe suicide response in cells that had re-ceived sublethal amounts of anoxia (Fig.2) (124). Experiments showing decreasedapoptosis of neurons and a reduction inthe absolute amount of neuronal loss inbcl-2-expressing transgenic mice sub-jected to transient cerebral ischemia sup-port this idea (125). Apoptosis due toinappropriate activation of cell cycle reg-ulators (e.g., c-myc) and to reactive oxygenspecies may also represent a response ofthe cell to a perceived viral threat, ratherthan any of these agents playing any in-trinsic role in physiological cell death.The basic structure of the cell death

mechanism has been preserved throughevolution, but mammals possess severalisoforms of the original pathway that canbe independently regulated. A detailedunderstanding of these mechanisms willhopefully allow design of therapeuticagents to enhance cell death when it failsor block it when it occurs inappropriately.

We thank Drs. G. Hacker, K. G. C. Smith, S.Kumar, and J. Trapani for stimulating discus-sions. D.L.V. is an Investigator with the CancerResearch Institute of New York and is sup-ported by the Anti-Cancer Council of Victoriaand the National Health and Medical ResearchCouncil (NHMRC) (Canberra, Australia). A.S.was supported by the Leukemia Society ofAmerica, the Swiss National Science Founda-tion, and the NHMRC (Canberra, Australia).

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