SUMMARY

1. Apoptosis is an essential process to remove excess,unwanted and harmful cells and maintain homeostasis. One ofthe key steps in apoptosis is activation of a group of proteasestermed caspases.

2. Caspases are cysteine proteases that cleave their substratesafter an aspartate residue. Approximately one dozen such pro-teases have been cloned during the past few years. While somecaspases are largely responsible for the proteolytic processingof proinflammatory cytokines, such as interleukin (IL)-1b,others are directly involved in the execution of apoptosis.

3. Once apoptotic upstream caspases are activated in responseto specific apoptotic stimuli, they can activate the downstreamor effector class of caspases. Most proteins that are cleavedduring apoptosis leading to the characteristic apoptoticmorphology are targeted by the downstream caspases. Thecleavage of these proteins by caspases can be either an activatingor inactivating event for the function of a protein; however, inmost cases, it contributes to the apoptotic phenotype of the cell.

4. Because caspase cleavage is the initiating event in mostforms of apoptosis, it is a tightly controlled process with manychecks and balances. An understanding of the regulation ofcaspases is providing novel ways for therapeutic intervention tomodulate apoptotic behaviour of cells in many diseases that arisedue to inappropriate apoptosis.

5. The present article will endeavour to discuss recentadvances in our understanding of caspase regulation and willelaborate on how this knowledge is being used in the develop-ment of new classes of therapeutic molecules that can be usedfor the treatment of human ailments.

Key words: Apaf-1, Bcl-2, caspase inhibitors, caspases, CED-3, CED-4, CED-9, cell death.

INTRODUCTION

The control of cell numbers during development and in the adultanimal is maintained by a balance between cell proliferation andcell death. It is now generally believed that most, if not all, differ-entiated cells in metazoans carry an intrinsic suicide programme.When activated by a number of different signals, this programmeinitiates a morphologically characteristic form of death called apop-tosis, which is distinct from accidental cell death or necrosis.1

Apoptotic cell death is defined by a number of features, includinga decrease in cell size, condensation of the cytoplasm, blebbing ofthe plasma membrane, collapse of the chromatin and fragmentationof DNA into oligonucleosome-sized pieces.1,2 Eventually, the dyingcell breaks down into membrane-bound apoptotic bodies that arerapidly removed by neighbouring cells by phagocytosis. As a resultof this rapid elimination of the dead cell without a leakage of itscontents, apoptotic cell death, in most cases, does not induce aninflammatory response.

ABERRANT APOPTOSIS AND DISEASE

Because the primary function of apoptosis is to regulate cell numbersand achieve homeostasis, a breakdown in the apoptotic mechanismcan result in either an inappropriate accumulation or a prematureloss of cells and, thus, lead to a pathological condition. Indeed, bothof these situations are seen in a number of human disorders. Manytypes of tumours are characterized by increased survival of cells thatnormally die. A classical example of this is human B cell lymphomacarrying a 14:18 chromosomal translocation. This chromosomalrearrangement juxtaposes the Bcl-2 gene from 18q21 with the immu-noglobulin heavy chain (IgH) locus at 14q32, resulting in increasedtranscription of the Bcl-2 gene and elevated levels of the Bcl-2protein in follicular B cell lymphomas.3,4 Bcl-2 is a survival factorthat negatively regulates cell death and its enhanced expression inB cell lymphomas results in increased survival of the lymphomacells.5 In addition to B cell lymphoma, Bcl-2 expression is alsoshown to be associated with a poor prognosis in prostatic cancer,neuroblastoma and colon cancer.6,7 Many chemotherapeutic agentswork by initiating DNA damage in cancer cells. Cells with damagedDNA are then removed by apoptosis. The product of the p53 tumoursuppressor gene is required for apoptosis in response to genotoxicagents.8 Because the p53 gene is mutated in a variety of tumours,the inability of cells to undergo apoptosis in response to DNA

BRIEF REVIEW

REGULATION OF CASPASE ACTIVATION IN APOPTOSIS:IMPLICATIONS IN PATHOGENESIS AND TREATMENT OF DISEASE

Sharad Kumar

Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia

Correspondence: Sharad Kumar, Hanson Centre for Cancer Research,Institute of Medical and Veterinary Science, PO Box 14, Rundle Mall,Adelaide, SA 5000, Australia. Email: <sharad.kumar@imvs.sa.gov.au>

Received 22 June 1998; revision 30 October 1998; accepted 4 November1998.

Clinical and Experimental Pharmacology and Physiology (1999) 26, 295–303

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damage may underlie the enhanced resistance of these tumours tochemotherapeutic agents and radiation.

A defect in the apoptotic potential of cells is also seen in auto-immune disorders. Mice carrying a mutation in either the Fas ligandor its receptor have autoimmune disease.9 Patients with systemiclupus erythematosus have elevated levels of soluble Fas, whichprobably inhibits Fas ligand–receptor interactions and, thus, Fas-mediated apoptosis.9,10 Disorders such as AIDS and Alzheimer’s andParkinson’s diseases are characterized by the inappropriate loss ofspecific cell types.10 The progression of AIDS is correlated with thedepletion of CD41 T cells, while neurodegenerative disorders arecharacterized by a gradual loss of specific subsets of neurons. Theloss of cells in these disorders is due to aberrant apoptosis. One ofthe genes linked to spinal muscular atrophy has recently been shownto encode a protein that bears similarity with an apoptosis inhibitoryprotein encoded by an insect virus.11

Several recent reviews describe the general features of apoptosisand its role in disease.10 The purpose of the present article is tosummarize some more recent advances in the molecular under-standing of apoptosis, with a particular emphasis on caspases, agroup of proteases that play a key role in apoptosis. I will also discusshow this recently accumulated knowledge is impacting on the waywe think about the onset and therapeutic intervention of varioushuman diseases arising from dysregulated apoptosis.

COMPONENTS OF THE DEATH MACHINERY

Until approximately a decade ago, almost nothing was known aboutthe molecular basis of apoptosis. The intense interest in apoptosisthat ensued owes a great deal to the work on developmental celldeath in the nematode Caenorhabditis elegans. In this worm,approximately a dozen genes regulate the deletion of a definednumber of cells during development. Of these genes, four are centralto the regulation of cell survival and the death of all cells. Three(egl-1, ced-3 and ced-4) have a pro-apoptotic function and arerequired for cell death to occur, while ced-9 counteracts ced-3 andced-4 function and is thus required for cell survival.12 Cloning ofthese genes and isolation of their mammalian counterparts providedevidence that cell death pathways have remained essentially con-served during evolution (Fig. 1). The first such evidence came whenVaux et al. showed that the expression of the human Bcl-2 gene inC. elegans generates a phenotype similar to the ced-9 gain-of-function phenotype.13 Indeed, upon cloning of ced-9, it was foundto encode a protein similar to Bcl-2.14 Cloning of ced-3 in 1993revealed that its gene product is similar to interleukin (IL)-1b-con-verting enzyme,15 a cysteine protease required for the processing ofIL-1b precursor to its mature secreted form,16 and Nedd2, a devel-opmentally regulated protein.17 The first mammalian homologue ofCED-4, called Apaf-1, was reported last year.18 The most recentlycloned gene, egl-1, lies upstream of ced-9 and negatively regulates

ced-9 function.19 Understandably, the mammalian apoptotic appa-ratus is much more complex, with a large family of genes repre-senting each of the C. elegans genes. The mammalian homologuesof CED-9 include both death antagonists (e.g. Bcl-2, Bcl-XL, A1,Mcl-1 and Bcl-w) and death agonists (e.g. Bax, Bad, Bak, Bik, Bidand Harakiri).6 The EGL-1 protein is also partly related to the Bcl-2 family and contains one of the four Bcl-2 homology (BH) do-mains.19 Structurally and functionally, EGL-1 is similar to Bik, Bidand Harakiri, all of which contain a single BH domain, BH3.19 TheCED-3 homologues are now termed caspases (for cysteine proteasesthat cleave following an aspartate residue in their substrates) andinclude 13 members20–22 (Table 1). Although only one mammalianhomologue of CED-4 has been discovered to date,18 several mam-malian adaptor molecules that are functionally similar to CED-4 exist(vide infra). It is now clear that the central death machinery includesfour groups of proteins homologous to EGL-1, CED-3, CED-4 andCED-9, respectively. Following the discovery of CED-3, it was envisaged that the crucial event in the initiation of apoptosis is likelyto be the activation of caspases, perhaps in a CED-4-dependent manner, while CED-9 may function by blocking the activation of caspases. These earlier assumptions turned out to be essentiallytrue, but much more complex than had originally been assumed.

CASPASES

Interleukin-1b-converting enzyme, the first known mammalianhomologue of CED-3, is the prototype for this family of proteasesand, hence, is named caspase-1.16,20,21 Caspase-2 (Nedd2) wasdescribed soon after as a developmentally regulated gene in themouse central nervous system.17,23 Following this, several othermembers of the family have been cloned, mainly by polymerasechain reaction-based methods and searching of the expressedsequence tag (EST) databases.20–22 Of the 13 caspases currentlyknown, not all seem to play a role in apoptosis (Table 1). Forexample, caspase-1 and its close relatives caspase-4, -5 and -11appear to be primarily involved in cytokine processing. The primarydefect in caspase-1 and caspase-11 knockout mice lies in theproduction of IL-1a and IL-1b.24–26 Other caspases, such as caspase-2, -3, -7, -8, -9 and -10, play a direct role in apoptosis.Caspase-2-deficient mice have extra female germ cells and oocytesfrom these animals are resistant to apoptosis induced by the anti-cancer drug doxorubicin.27 Caspase-3 and caspase-9 –/– animalsshow profound developmental aberrations in the brain and mostanimals die soon after birth.28–31 The brain mass in these animals is substantially larger than the wild-type animals, largely due to a lack of neuronal cell death.28–31 Additionally, cells derived from caspase-3 and caspase-9 null mice are resistant to UV-irradiation and cytotoxic drug- and osmotic shock-induced cell death, but have functional CD95 and tumour necrosis factorreceptor (TNFR) pathways.29–31 In contrast, in caspase-8 –/–animals, which exhibit embryonic lethality, apoptosis mediated by

List of abbreviations:

IL InterleukinTNF Tumour necrosis factorTNFR TNF receptorDD Death domain

DED Death effector domainSMA Spinal muscular atrophyCAD Caspase-activated DNaseHD Huntington’s disease

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CD95, TNFR and DR3 is abrogated.32 These results suggest thatparticular caspases may play a role in specific apoptotic pathwaysand cell-specific apoptotic programmes.

MECHANISMS OF CASPASE ACTIVATION

Caspases are synthesized as precursor molecules (zymogens),which require proteolytic processing into two subunits for their acti-vation. The crystal structures of mature caspase-1 and caspase-3suggest that the active caspases are composed of a tetramer consis-ting of two molecules of each subunit.20 Both subunits in the tetramercontribute to catalysis (formation of the substrate-binding site) andmany features that determine the substrate specificity are conservedin closely related caspases.20

The currently available data suggest two modes of caspaseactivation: autoprocessing and processing by other caspases. Whenoverexpressed in Escherichia coli, most caspases show some auto-processing, but whether this occurs in vivo is still a matter of somedebate. However, there is some consensus emerging on how thecaspase cascade may be initiated. The first evidence came from thediscovery that ligation of the death receptor Fas can result in therecruitment of a caspase to the cytoplasmic death-inducing signallingcomplex (DISC). Two groups independently showed that caspase-8 can be recruited to the DISC through binding to the adap-tor molecule FADD/MORT1, which associates with the cytoplasmicdeath domain (DD) of Fas.33,34 This suggested that recruitment ofa caspase to a death complex may be sufficient for its activation.Indeed, caspase-8 is activated by self-processing once it is recruited

Fig. 1 Main components of the apoptotic apparatus are conserved between Caenorhabditis elegans and mammals.

Table 1 Members of caspase family

Caspase* Other names Class† Cleavage Majorspecificity‡ function§

1 ICE I (W/L)EHD Cytokine processing2 NEDD2, ICH-1 I VDxxD Apoptosis3 CPP32, Yama, apopain II DExD Apoptosis4 ICErelII, TX I (W/L)EHD Cytokine processing5 ICErelIII, TY I (W/L)EHD Cytokine processing6 MCH2 II VEHD Apoptosis7 MCH3, ICE-LAP3 II DExD Apoptosis8 FLICE, MACH, MCH5 I LETD Apoptosis9 MCH6, ICE-LAP6 I LEHD Apoptosis

10 MCH4, FLICE2 I ? Apoptosis11 mICH-3 II ? Cytokine processing12 mCASP-12 I ? ?13 ERICE I ? Apoptosis?

*References to the initial cloning of caspase-1 to caspase-11 can be found in recent reviews.23,24 Caspase-12 and caspase-13 are described by Van deCraen et al.97 and Humke et al.,22 respectively.

†Classified on the basis of the length of the prodomain. See text for details.‡Indicates P4–P1 specificity. Derived from Talanian et al.98 and Thornberry et al.99

§Based on cellular, biochemical and gene knockout (caspase-1, -2, -3, -8, -9 and -11) studies. See text for details.

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to the activated Fas receptor35 (Fig. 2). Caspase-10 was also shownto be activated upon Fas and TNFR ligation.36 Another adaptormolecule, RAIDD, was subsequently cloned and shown to associatewith caspase-2 and RIP, a protein that interacts with TNFR throughthe adaptor protein TRADD.37 Thus, caspase-2, -8 and -10 can berecruited through adaptors to death complexes at the cytoplasmicend of the death receptors. The common feature between thesecaspases is that they all contain long amino-terminal prodomains,regions that are cleaved off following the proteolytic activation ofthe caspase molecule. Both caspase-8 and -10 contain two deatheffector domains (DED), similar to those found in adaptor molecules,such as FADD, and which mediate the interaction between the cas-pase and the adaptor.33,34,36 FADD also contains death domains (DD),

which mediate its recruitment to the DD found in the carboxyl cyto-plasmic region of the death receptors. Similarly, the caspase-2prodomain contains a region of homology with the adaptor RAIDD,while RAIDD can, in turn, interact with TRADD, another DED-containing protein that is recruited to the TNFR and can interact withthe Fas death complex through FADD.37 However, direct evidencefor the involvement of caspase-2 in receptor-mediated cell death isstill lacking. Because caspases with a long prodomain appear to bethe proximal effector molecules that link the apoptotic signals tothe central death apparatus, they are often called the upstream cas-pases or class I caspases. The downstream, or class II, caspases, con-sisting of caspase-3, -6 and -7, lack a long prodomain and are thusunlikely to be activated by recruitment to death complexes. Based

Fig. 2 Death receptors mediate apoptosis by direct recruitment of class I caspases. This specific example is based on data with CD95 (Fas/Apo-1). Activationof a death receptor (e.g. CD95) results in the recruitment of a specific adaptor (e.g. FADD) through death domains (DD) found in both the cytoplasmic tailof the receptors and the adaptor molecules. The adaptors, directly or indirectly through additional adaptors, recruit a class I caspase (e.g. caspase-8) to themembrane-associated death complex mediating oligomerization and the activation of the apical caspase. The activated class I caspase can process down-stream (class II) caspases (e.g. caspase-3 and -7) that mediate the cleavage of various cellular proteins during apoptosis. Various viral and cellular proteinscontrol different steps in this pathway. For example, decoy receptors lacking the cytoplasmic tail95,96 can mop up the ligand, preventing receptor activation;viral and cellular FLIP block the recruitment of a class I caspase by sequestering the adaptor and viral and cellular proteins, such as IAP, p35 and CrmA,directly inhibit caspases. DED, death effector domain.

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on in vitro cleavage data, these caspases are likely to be activatedby class I caspases and are responsible for the cleavage of a largenumber of proteins involved in homeostatic regulation and structural alterations in an apoptotic cell.20,21 Upon receiving a deathsignal, activation of the first caspase can be seen as the initiatingevent in a cascade that commits a cell to apoptosis.

How does initial activation of a class I caspase occur? Becausecaspases require processing that occurs after certain Asp residues,it was predicted that they may have intrinsic pro-enzyme activitythat may mediate their autocatalysis.20,21 A number of recent reportshave shown that some class I caspases, such as caspase-2 and -8,can be autoactivated by homodimerization/oligomerization.38–40 Inthe case of caspase-2, the amino-terminal prodomain region wasshown to be essential for dimerization, autoprocessing and activationof the procaspase molecule.38 Class II caspases, such as caspase-3,show poor autoprocessing ability in mammalian cells,41 suggestingthat they require activation by class I caspases. Our recent resultssuggest that it is possible to convert a class II caspase, such ascaspase-3, to an autoactivating caspase simply by fusing a class Icaspase (such as caspase-2) prodomain at the amino terminal ofprocaspase-3.41 Taken together, these observations indicate thatinitial activation of caspases in response to apoptotic stimuliprobably occurs following dimerization and self-processing of theprecursor molecules and dimerization is mediated through theprodomain region. It is therefore reasonable to assume that thecaspase recruitment step, mediated by adaptor molecules, such asFADD and RAIDD, serves a primary function to bring class Iprocaspase molecules into close proximity with each other, thuspromoting their dimerization and processing.

Do all apoptotic pathways follow this direct mechanism of caspaseactivation? The answer to this is much more complex than originallypredicted from the studies in C. elegans. Genetic studies in C. ele-gans indicated that CED-9 lies upstream of CED-4 and CED-3.42

Mammalian homologues of CED-9, Bcl-2 and Bcl-XL, have beenshown to inhibit the activation of downstream caspases, such as cas-pase-3.43 Further investigations showed that CED-4 can physicallyinteract simultaneously with both CED-3 and CED-9.44 A similarscenario appears to occur in mammalian cells where Apaf-1, the onlyknown mammalian homologue of CED-4, has been shown to forma ternary complex with Bcl-XL and caspase-9.45,46 In vitro studiessuggest that caspase-9 requires Apaf-1, dATP and cytochrome c,which is released from mitochondria, for its activation.47 Thus, itcan be envisaged that Bcl-2-like survival factors work by seques-tering Apaf-1, thereby preventing activation of caspase-9. Bcl-2 hasalso been shown to prevent release of cytochrome c from mito-chondria, thereby indirectly affecting activation of caspase-9.48 Thus,caspase-9 is a class I caspase that requires interaction through its prodomain with an adaptor (Apaf-1) for its activation. Oncecaspase-9 is activated, it can activate class II caspases, such ascaspase-3.47 No other class I caspases tested, including caspase-1,-2, -4, -8, -10 and -13, seem to require cytochrome c for activation,49

suggesting that caspase-9 is the apical non-redundant caspase inmany, if not all, non-receptor-mediated apoptotic pathways. Theessential function of Apaf-1 in this pathway is demonstrated bytargeted disruption of the Apaf-1 gene in mice.50,51 Similar tocaspase-3 and caspase-9 mutant mice, Apaf-1-deficient mice showreduced developmental cell death in brain and a lack of activationof caspase-3.50,51 Cells derived from these animals are resistant toa variety of apoptotic stimuli, but are sensitive to CD95-mediated

apoptosis,50,51 confirming that Apaf-1 primarily functions in themitochondrial apoptotic pathways.

The main difference between the pathways involving the tumournecrosis factor (TNF) family of death receptors and mitochondria,appears to be the involvement of Bcl-2-like proteins. While Bcl-2can efficiently block cell death mediated by a number of agents, suchas withdrawal of trophic support, DNA-damaging agents and varioustoxic agents, its effect on apoptosis mediated by Fas is minimal.6,7

Although some cell lines overexpressing Bcl-2 show resistance toFas-mediated apoptosis, in most cases the major pathway of celldeath is apparently independent of Bcl-2.52 Thus, one can predictthat death receptor-mediated apoptosis (Bcl-2 independent) followsa different pathway from apoptosis mediated by other signals (Bcl-2 inhibitable). In both pathways, Bcl-2 or Bcl-XL overexpres-sion can block mitochondrial-related apoptotic processes, such asrelease of cytochrome c, but caspase-8 and caspase-3 activation isunaffected in the Bcl-2-independent pathway.53 It is also likely that,in the case of death receptor-mediated apoptosis, although mitochon-drial involvement is not obligatory, mitochondria may participateto amplify apoptotic signals downstream of caspase-8 activation.50

REGULATION OF CASPASE ACTIVATION

Because caspase activation is a crucial step in the initiation of anapoptotic cascade, it is predictably a very tightly regulated process.Recent data from various laboratories (see below) support this notion. Mammalian cells seem to have developed several ways toprevent inappropriate activation of caspases. Once activated, cas-pases can act rapidly to amplify the death signal by activating sev-eral other caspase family members and perhaps, therefore, mostcontrol mechanisms appear to act at or upstream of the activationof caspases. As discussed earlier, Bcl-2-like proteins are the mainplayers in preventing cell death mediated by most stimuli; however,apoptosis mediated by death receptors, in most circumstances, by-passes the Bcl-2 control mechanisms. Although death receptor-mediated apoptosis, except in the caspase of Fas and TNF, is poorlyunderstood, it may be an important way of deleting unwanted cellsduring development.

Apoptosis is an important cellular defence against viral infection.Some viral proteins, such as cowpox virus cytokine responsemodifier CrmA and baculovirus P35, and IAP proteins inhibit apop-tosis by directly interacting with caspases.20,21 CrmA is a potentinhibitor of caspase-8, a component of the CD95 and TNFR deathpathways, which CrmA is able to inhibit with high affinity.20,21

Baculovirus P35 is a broader inhibitor of caspases and its overexpres-sion has been shown to inhibit apoptosis in a number of systems.54,55

Recently, a new family of viral apoptosis-inhibitory proteins hasbeen identified and designated v-FLIP due to their ability to inhibitcaspase-8.56 Viral FLIP act by impeding recruitment of caspase-8to the Fas death receptor to prevent subsequent apoptotic signalling.The majority of g-herpesviruses encode FLIP in order to successfullyestablish persistent infection through the inhibition of apoptosis.56

Two distinct cellular mechanisms negatively regulate apoptosisby acting upstream of caspase activation. Several groups haverecently characterized a cellular protein that shares significanthomology with the DED-containing caspases (caspase-8 and -10),but lacks features required for protease activity. This protein,variously called FLIP, Casper, Flame, Cash, I-FLICE, MRIT andUsurpin,57–63 functions in a manner similar to v-FLIP by inhibiting

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the recruitment of caspase-8 and -10 to activated death receptors.Several mammalian proteins share similarity with the baculovirusIAP protein. Neuronal apoptosis inhibitory protein (NAIP), mutatedin the severe forms of spinal muscular atrophy (SMA), was the firstIAP-like protein discovered in mammals.11 Overexpression of NAIPprevents apoptosis induced by numerous stimuli, which correspondsto NAIP acting as a negative regulator of motor neuron apoptosis.64

Four other mammalian IAP homologues (XIAP/MIHA/hILP,MIHB/hIAP-1/c-IAP-1, MIHC/hIAP-2/c-IAP2 and survivin) havesubsequently been cloned and shown to inhibit apoptosis.65–68 Recentstudies have shown that mammalian IAP can directly bind tocaspases and block their activity.69 Because IAP can also physicallyinteract with Drosophila cell death proteins HID and GRIM, whichare unrelated to caspases,70 inhibition of caspase activity may notbe the only function of IAP.

CELLULAR TARGETS OF CASPASES

Approximately 40 proteins are now known to be cleaved by classII caspases in cells undergoing apoptosis. Cleavage by caspases caneither functionally inactivate or activate a protein. Although the func-tional significance of all caspase cleavage events is not known, manyof the proteins inactivated by caspase-mediated cleavage play a rolein cellular homeostasis and their cleavage may simply result in shut-ting down the cell cycle and repair machinery, while cleavage ofothers is more directly associated with morphological changescharacteristic of apoptosis. A comprehensive list of the growingnumber of caspase substrates can be found in some recent arti-cles,20,21 but a few need special mention here. Apoptosis has longbeen known to be associated with cleavage of DNA in characteris-tic nucleosomal length fragments.2 The DNAse responsible for this fragmentation has recently been identified and termed CAD (caspase-activated DNAse).71 Caspase-activated DNAse normallyremains bound to a cytoplasmic inhibitor (ICAD/DFF45), which isdegraded by caspase-3 in apoptotic cells.71,72 The free CAD thenenters the nucleus and acts on chromatin.71 Cells lacking caspase-3 can still undergo apoptosis; however, they fail to show charac-teristic DNA fragmentation,29,73 suggesting that caspase-3 has anon-redundant function in activating CAD, but DNA fragmentationis not essential for apoptosis to occur. Bcl-2 and Bcl-XL proteinsare also cleaved during apoptosis and the fragments generated bythe cleavage appear to enhance apoptotic changes in dying cells.74,75

Cleavage of gelsolin, an actin-associated protein, generates a frag-ment that promotes rounding-up of cells.76 PAK2, a serine/threo-nine kinase, is activated by caspase cleavage.77 Because PAK2 isinvolved in the regulation of the actin cytoskeleton, its activationseems to help formation of apoptotic bodies.77 Thus, cleavage ofspecific proteins by caspases is responsible for the ultimate demiseof the condemned cell and, perhaps, also for its removal by phagocytosis.

CASPASES AND THE PATHOGENESIS OFDISEASE

Although no systematic studies to investigate germ line mutationsin caspase genes in human disease have been reported so far,numerous examples indirectly implicating caspases in disease areappearing. A breast carcinoma cell line (MCF-7) carries a 47 b.p.deletion within exon 3 of the caspase-3 gene; however, these cellscan still undergo apoptosis73 and it is not clear whether caspase-3

mutation in this cell line plays any role in tumorigenesis.Usurpin/FLIP, the caspase-8 inhibitor that confers resistance to Fas-induced apoptosis, is expressed in cardiac tissue that is protectedfrom apoptosis following ischaemia–reperfusion injury, but not intissue that undergoes destruction.63 Usurpin levels are also down-regulated from infarcted cardiac tissue following ischaemia,63

suggesting that the normal function of Usurpin is to preventinappropriate caspase activation and its regulation may be impor-tant in pathogenesis of disease. As discussed earlier, mammalianhomologues of IAP are intracellular inhibitors of apoptosis. One pre-dicted role of these molecules is to directly inhibit caspase activationand the catalytic activities of activated caspases. The NAIP gene,which encodes an IAP-like protein, is mutated in some cases ofSMA.11 Because SMA is characterized by spinal cord motor neurondepletion, mutation in NAIP resulting in the abrogation of its normalapoptosis inhibitory function may contribute to more severe formsof the SMA phenotype.11 Another IAP-like molecule, survivin, ishighly expressed in a variety of human cancers, but not in normalcells; thus, it probably promotes increased cell viability of cancercells.68,78,79 These recent results suggest that misregulation of mol-ecules that control apoptosis by blocking caspase activation cancause both degenerative and proliferative disorders. No doubt, asmore studies are undertaken, more links between caspases andhuman diseases are likely to come to light.

Huntington’s disease (HD), another neurodegenerative disorder,is characterized by expansion of a CAG trinucleotide (codon for glu-tamine) repeat in the Hdh gene, which encodes another caspase-3target, huntingtin.80 Five caspase-3 sites in huntingtin lie downstreamof the polyglutamine region (resulting due to the expansion of CAGrepeats) at which cleavage occurs during apoptosis.80 The length ofthe polyglutamine track appears to determine the susceptibility ofhungtingtin to caspase-3 cleavage and the amino-terminal fragmentgenerated by caspase-3 cleavage can itself induce apoptosis. Thus,cleavage of expanded huntingtin protein in HD neurons generatespeptides that can potentially accelerate cell death and, thus, play apossible role in disease pathology.80

MODULATION OF CASPASE ACTIVATION:THERAPEUTIC POTENTIAL

Because caspase activation represents the initiation of the apoptoticcascade, molecules that modulate either the activation of caspasesor affect the activities of already processed caspases are potentiallyexcellent targets for therapeutic intervention to control inappropriateapoptosis characteristics of numerous diseases. Additionally, mol-ecules can be designed that directly activate the caspase cascade toinduce apoptosis in cancer cells that have acquired drug resistanceor to inhibit caspases to prevent excess or premature cell death indegenerative diseases. So far, most work has involved developingmolecules that inhibit caspase action. Synthetic peptide inhibitorsof caspases are potent inhibitors of apoptosis. The three mostextensively used peptides include YVAD, based on the caspase-1cleavage site in proIL-1b, DEVD, designed from the caspase-3cleavage site in poly(ADP) ribose polymerase, and VAD, a broadinhibitor of caspases.20,21 These synthetic peptides are commonlycoupled with a C-terminal aldehyde (e.g. Ac-YVAD-CHO or Ac-DEVD-CHO) or a ketone (e.g. Ac-DEVD-cmk or zVAD-fmk,where -cmk and -fmk denote chloromethyl and fluoromethylketones, respectively), which act as effective competitive, reversible

Activation of caspases in apoptosis 301

and irreversible caspase inhibitors, respectively.20,21 zVAD-fmk, thecell-permeable inhibitor that acts on a number of caspases, has beenextensively used to inhibit apoptosis in cultured cells, tissues andwhole animals.20,21 Earlier studies demonstrated that YVAD-cmk caninhibit apoptosis in motor neurons deprived of trophic factor invitro.81 Systemic injections of this tetrapeptide inhibitor in chickembryos also inhibited naturally occurring motor neuron cell deathand there was a dose-dependent decrease in the number of pyknoticcells within the spinal cord and a corresponding increase in thenumber of motor neurons 15 h post-treatment.81 Although not allneuronal cells were protected by YVAD-cmk, various other cellswere, including interdigital cells of the limbs, which are normallydestined to die to give rise to individual claws within chicks; treat-ment with YVAD-cmk gave rise to webbed-like foot pads.81

Subsequent studies from a number of laboratories, mostly with thecell-permeable inhibitor zVAD-fmk, demonstrated that caspaseinhibition can be very effective in preventing inappropriate celldeath.20,21 For example, zVAD-fmk prevents fulminant liverdestruction and death in mice mediated by administration of anti-Fas antibody or TNF-a.82–84 Peptide inhibitors of caspases also pro-tect against doxorubicin-induced female germ cell apoptosis,85

myocardial reperfusion injury86 and neuronal death followingischaemia.87,88 These findings demonstrate the potential use ofsynthetic caspase inhibitors in preventing apoptosis in vivo and haveobvious therapeutic implications in humans. Perhaps the mostpromising immediate use of peptide inhibitors lies in reducingdelayed neuronal damage that often follows stroke.89

Other useful molecules for drug design are viral and cellularinhibitors of caspases, such as CrmA, P35 and IAP. Recent experi-ments show that transient forebrain ischaemia selectively elevateslevels of NAIP in rat neurons that are resistant to the injurious effectsof this treatment.90 Consistent with this observation, overexpressingNAIP in vivo using an adenovirus vector reduced ischaemic damagein the rat hippocampus.90 Transgenic expression of P35 in the eyeof Drosophila mutants that exhibit age-related retinal degenerationblocks retinal apoptosis and shows significant retention of visualfunction.91 Inhibition of caspases by the expression of catalyticallyinactive caspase molecules in transgenic mice is also reported to slowdown the onset of amyotrophic lateral sclerosis (ALS), a motorneuron disease,92 neuronal cell death induced by trophic factor with-drawal and ischaemic brain injury.93 These experiments clearly showthat inhibition of caspases can rescue premature apoptosis on a long-term basis.

Drug resistance in cancer cells is a common problem and accumu-lating evidence suggests that drug-resistant cells are defective inactivation of caspases. In many cases, drug resistance can be ascribedto an overexpression of Bcl-2 and Bcl-XL,7 which act upstream ofcaspase-9 in the apoptotic pathway. Chemotherapeutic drugs act byinducing apoptosis in target cells so, ideally, drugs that target theexecution machinery of apoptosis (i.e. activate caspases) would beable to bypass many aberrant control mechanisms that confer resis-tance in cancer cells. Alternatively, drugs and antisense therapy thattarget apoptosis inhibitors Bcl-2 and IAP-like proteins could workwell in situations when overexpression of these proteins confersresistance to conventional chemotherapy. A novel approach forkilling cancer cells by chemically induced oligomerization-mediatedactivation of caspases has been proposed recently.94 This approachtakes advantage of an inducible oligomerization domain found inFKBP12, a protein that binds the immunosuppressive drug FK 506.

This domain is fused to a caspase precursor molecule and dimer-ization is induced by AP1903, a small non-toxic cell-permeableanalogue of FK 506. Results show that addition of AP1903 resultsin rapid apoptosis due to the oligomerization and subsequent pro-cessing of the FKBP–caspase chimeric molecule in transfectedcells.94 Although this approach relies on target cells expressing thechimeric FKBP–caspase molecule, it is potentially useful in killingcells that have acquired resistance to apoptosis due to a defectupstream of caspase activation.

CONCLUSIONS

During the past few years, rapid advances in molecular under-standing of apoptotic pathways have identified numerous compo-nents of the basic cell death apparatus. It is now clear that apoptosisis an evolutionarily conserved physiological mechanism in meta-zoans that uses a complex array of effector and regulatory proteins.This complexity in the regulation of cell death provides checksagainst inappropriate initiation of apoptosis. As aberrant expressionand/or function of molecules that regulate apoptosis can result inhuman disorders, drugs that modulate the apoptosis regulatory mech-anisms are potentially useful in the treatment of diseases charac-terized by inappropriate apoptosis. Caspases, being the centralexecutioners of the apoptosis machinery, are obvious targets fortherapeutic intervention and the recent studies described aboveclearly indicate the potential of novel therapies that target caspaseactivation for controlling dysregulated apoptosis.

ACKNOWLEDGEMENTS

I thank Linda Shearwin-Whyatt and Natasha Harvey from my laboratory for comments on the manuscript. I am grateful to theWellcome Trust and the National Health and Medical ResearchCouncil of Australia for providing research support in recent years.The author is a Wellcome Senior Fellow in Medical Science. I apologise to the authors of many primary papers that could not becited due to space limitations.

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