Apoptosis 2006; 11: 889–904C© 2006 Springer Science + Business Media, LLC. Manufactured in The United States.DOI: 10.1007/s10495-006-6712-8

Apoptosis effector mechanisms: A requiem performedin different keys

N. Hail Jr., B. Z. Carter, M. Konopleva and M. Andreeff

Department of Clinical Pharmacy, School of Pharmacy, The University of Colorado at Denver and Health Sciences Center,Denver, CO 80262, USA (N. Hail Jr.); Section of Molecular Hematology and Therapy, Department of Blood and MarrowTransplantation, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA (B. Z. Carter, M.Konopleva, M. Andreeff)

Published online: 13 March 2006

Apoptosis is the regulated form of cell death utilized bymetazoans to remove unneeded, damaged, or potentiallydeleterious cells. Certain manifestations of apoptosis maybe associated with the proteolytic activity of caspases.These changes are often held as hallmarks of apoptosisin dying cells. Consequently, many regard caspases as thecentral effectors or executioners of apoptosis. However, this“caspase-centric” paradigm of apoptotic cell death does notappear to be as universal as once believed. In fact, duringapoptosis the efficacy of caspases may be highly dependenton the cytotoxic stimulus as well as genetic and epigeneticfactors. An ever-increasing number of studies strongly sug-gest that there are effectors in addition to caspases, whichare important in generating apoptotic signatures in dyingcells. These seemingly caspase-independent effectors mayrepresent evolutionarily redundant or failsafe mechanismsfor apoptotic cell elimination. In this review, we will discussthe molecular regulation of caspases and various caspase-independent effectors of apoptosis, describe the potentialcontext and/or limitations of these mechanisms, and ex-plore why the understanding of these processes may haverelevance in cancer where treatment is believed to engageapoptosis to destroy tumor cells.

Keywords: apoptosis; cancer; caspases; mitochondria; pro-teases; reactive oxygen species.

Introduction

“That which has been successfully defined has been success-fully killed” –Christmas Humphreys. The phrases “apoptosis-like cell death” and “caspase-independent cell death” are fre-quently used in the literature to define seemingly regulatedforms of cell loss that do not appear to fulfill the criteriaof apoptosis. While remarkable advances have been madesince the early 1990’s in the understanding of apoptosis, ourcollective knowledge of this process is far form complete,

Correspondence to: M. Andreeff, M.D., Ph.D., Section of Molec-ular Hematology and Therapy, Department of Blood and MarrowTransplantation, Unit 448, The University of Texas M. D. AndersonCancer Center, 1515 Holcombe Boulevard, Houston, TX 77030,USA. Tel.: + 713-792-7260; Fax: + 713-794-4747; e-mail: man-dreef@mdanderson.org

and consequently may only encompass a highly stereotypi-cal series of events that are appropriate for certain cell types.For this reason, we humbly believe it is inaccurate to use aqualitative distinction such as “apoptosis-like cell death” or“caspase-independent cell death” to describe a possible func-tional derivative of apoptosis. Furthermore, “apoptosis-likecell death” and “caspase-independent cell death” are some-what prejudicial because they imply that caspase activityis synonymous with apoptosis, which is not always accu-rate. We will adhere to Ockham’s razor for now by simplyembracing the notion that apoptosis is a distinctive formof cell death with ultrastructural features suggesting an ac-tive, inherently regulated phenomenon.1 This review willexamine the mechanistic underpinnings that are potentiallyresponsible for this phenomenon.

The apoptotic cell death concept

Cell death that culminates in rapid (e.g., occurring withinhours or days) cell loss is prevalent in metazoans. This processcan be regulated or unregulated. The regulated type of rapidcell loss is known as apoptosis, while the unregulated form isreferred to as oncosis.2 Autophagy has also been suggestedto be a regulated mode of cell death.3 However, besidesbeing highly regulated, autophagy is typically reversible,4,5

which suggest a level of cell stress below that required for thecell loss associated with apoptosis or oncosis. Furthermore,recent evidence suggest that autophagy is probably mostrepresentative of a cell survival pathway6–8 that terminatesin apoptosis if unsuccessful.9

Apoptotic cell death has been implicated in embryonicdevelopment, immune system regulation, morphogenesis,and the preservation of tissue homeostasis,1,2 as well as var-ious disease states.10 Morphological and histological studiesof hepatocytes during ischaemic liver injury provided thebackground for the formulation of the apoptotic cell deathconcept, which was proposed by Kerr, Wyllie, and Currie in1972.11 An apoptotic cell typically undergoes shrinkage (i.e.,apoptotic volume decrease), chromatin condensation, kary-orrhexis, and the eventual budding of the plasma membrane

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Figure 1. The morphological features of apoptosis, oncosis, and necrosis. This figure was adapted from Majno and Joris.2 Please refer tothe text for details.

into apoptotic bodies (Figure 1). These morphologicalchanges are considered the gold standard for distinguishingthis type of cell death.12–14 Conversely, oncosis is a passivecatastrophic cellular event where marked swelling, aggre-gate organelle disruption, and plasma membrane blebbingprevail. There is little or no evidence of chromatin remod-eling during oncosis, and the cell rapidly succumbs to cy-tolysis. This cytolysis is end-stage cellular decay that is thedefining feature of necrosis.2 Apoptotic cells will eventu-ally lose plasma membrane integrity and become necroticin vitro. However, this is not believed to occur with highfrequency in vivo because apoptotic cells display signals(e.g., the externalization of phosphatidylserine (PS) on theirplasma membrane) that encourage their expeditious removalby phagocytosis.2,15

Apoptosis was originally viewed as being non-degener-ative in nature.1,11 However, an impressive body of ensuingresearch has proven otherwise. Apoptosis can be dividedinto three phases: initiation, effector, and degradation.16 Theinitiation phase is largely dependent on cell type and theapoptotic stimulus (e.g., oxidative stress, DNA damage, ionfluctuations, and cytokines). In certain instances, as we willdiscuss later, the initiation phase may influence the efficacyof the effector and/or degradation phases. The effector phaseconstitutes the activation of proteases, nucleases, and otherdiffusible intermediaries that participate in the degradationphase.17–20 Together, the effector and degradation phases

promote the ultrastructural features that are suggestive ofapoptosis. Consequently, in most models of apoptosis theinterruption of these phases does not confer cell survival,rather it merely deregulates what was to be regulated celldeath.21,22

Is caspase-mediated apoptosis acontext-dependent phenomenon?

Most of the recent advances in the elucidation of apoptosishave come about through the characterization of the effectormechanisms. The caspases are a family of cysteine proteasesthat are constitutively present in most mammalian cells,and they reside in the cytosol as single chain proenzymes.Over a dozen caspases have been identified, and approxi-mately two-thirds of these enzymes have been suggested tofunction in apoptosis.17,20 Nevertheless, many of these samemolecules also participate in homeostatic cellular functions(i.e., cytokine production, terminal differentiation, andproliferation) that are not associated with cell death.23,24

There are two types of caspases, upstream caspases calledinitiator caspases (e.g., caspases-8, -9, and -10), and theirdownstream targets known as effector or executionercaspases (e.g., caspases-3, -6, and -7).17 Several componentscomprise the “caspase-centric”25 effector model of apop-tosis, and two pathways associated with caspase activation

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Figure 2. The regulation of the intrinsic and extrinsic pathways of caspase activation during apoptosis. Please refer to the text for details.

have been characterized extensively. These include themitochondrial-mediated or intrinsic pathway, and thedeath receptor-mediated (e.g., tumor necrosis factor (TNF)receptor and Fas) or extrinsic pathway26,27 (Figure 2).

The intrinsic pathway relies on mitochondrial membranepermeabilization (MMP) to liberate the electron transportchain intermediate cytochrome c from the mitochondrialintermembrane space. Once released to the cytosol, cy-tochrome c can combine with dATP, APAF-1, and caspase-9to form the apoptosome. This catalytic complex is respon-sible for the activation of effector caspase-3 and -7.16,27 Theintrinsic pathway can be regulated by the Bcl-2 family ofproteins,28 proteases,29–31 as well as agents (e.g., ceramide,reactive oxygen species (ROS), and Ca2+) that promotethe mitochondrial permeability transition (MPT).32–35 Forinstance, during conditions of cell stress, antiapoptoticBcl-2 family members (e.g., Bcl-2 and Bcl-XL) residingin the outer mitochondrial membrane can be destabilizedthrough decreased expression, or by the induction ofproapoptotic Bcl-2 family members (e.g., Bax, Bad, andBak). In the latter scenario, the ratio of proapoptotic familymembers to antiapoptotic family members becomes greaterallowing the formation of proteinaceous outer membranechannels by the proapoptotic Bcl-2 family members, whichcan liberate cytochrome c.28,36

The extrinsic pathway is initiated by the ligation of deathreceptors that subsequently cluster in the plasma membraneand promote the recruitment of adapter proteins (Figure 2).Caspase-8 is the apical caspase in the death receptor path-way. The zymogen of caspase-8 can interact with the adapterproteins to generate the active form of caspase-8. Once ac-tive, caspase-8 can trigger the activation of downstreameffector caspases like caspase-3.17,37,38 In certain cell sys-tems, the activation of caspase-8 is sufficient to commencethe proteolytic cascade required to achieve apoptotic cel-lular degradation.17,20,38 However, the activation of death

receptors has also been shown to promote the recruitmentand downstream action of the intrinsic pathway, which ap-pears to be required to execute apoptosis in certain celltypes.39,40 For example, besides activating effector caspases,another notable target of caspase-8 is the BH3 only Bcl-2relative Bid. In response to Fas ligand or TNF, caspase-8induces the cleavage of Bid to yield a truncated carboxy-terminal fragment that translocates from the cytosol to theouter mitochondrial membrane. Oligomers of the truncatedBid can trigger MMP and cytochrome c-mediated caspaseactivation.39,40 There is also evidence indicating that trun-cated Bid can trigger conformational changes in Bax, whichthen localizes with the voltage-dependant anion channelin the outer mitochondrial membrane to trigger MMP.41

Thus, the recruitment of the intrinsic pathway of apoptosisby caspase-8 activation can serve to initiate and/or amplifyintracellular signals to trigger apoptosis.

Caspases selectively cleave a restricted set of tar-get proteins after an aspartate residue in their primarysequence.17,37 The caspase-mediated cleavage of cellular pro-teins has been implicated in the removal of endogenousapoptosis inhibitors, morphological changes, and DNA frag-mentation. For example, the cleavage of inhibitory regula-tors of MMP like Bcl-2 or Bcl-XL not only inactivates theirinhibitory function, but also produces a protein fragmentthat promotes MMP.28 The cleavage of nuclear lamins andcytoskeletal proteins such as fodrin and gelsolin is associ-ated with morphological changes in apoptotic cells,20 andthe cleavage of the inhibitor of the caspase-activated DNAse(ICAD) releases CAD, which produces nucleosomal DNAfragmentation.42

In many experimental situations, the evidence forcaspase-mediated apoptosis is indirect (e.g., the cleavage ofpoly(ADP-ribose) polymerase (PARP), nucleosomal DNAfragmentation, and/or procaspase-3 processing) demonstrat-ing that the cells could activate caspases during apoptosis

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rather than caspase activity was required for thisphenomenon.22 Nevertheless, caspase activity is consideredby many investigators in the field of cell death to be thedefining feature of apoptosis.17,37,43 In fact, if nucleosomalDNA fragmentation, which is detected by the terminal de-oxyribonucleotide transferase-mediated dUTP nick end la-beling (TUNEL) technique, is deemed to be a hallmarkof both caspase activity and apoptosis, the “caspase-centric”paradigm would appear to be a self-fulfilling prophecy. Thenagain, this presents a problem for classifying cell death thatwould otherwise appear to be apoptotic, albeit perhaps lack-ing in conspicuous nucleosomal DNA fragmentation, in celltypes that express relatively low levels of ICAD and CAD(e.g., neurons, hepatocytes, and fibroblasts44–46). Interest-ingly, TUNEL-positive DNA fragmentation can occur fol-lowing the phagocytosis of thymocytes derived from trans-genic mice that ubiquitously expressed a caspase-resistantform of ICAD.47 Lysosomal nucleases in phagocytic cellsmay account for this DNA degradation.44 Furthermore,non-caspase proteases have also been shown to cleave ICADand release CAD,48,49 suggesting that some features ofcaspase activity may be sufficient but not essential forapoptosis.42,44

The caspase-regulated ICAD-CAD system of nucleoso-mal DNA fragmentation may be required in certain celltypes to maintain context-dependent homeostasis. For ex-ample, the “caspase-centric” effector model of apoptosisappears to be common in lymphoid cells, which have arelatively small cytoplasm,25 and concomitantly very fewmitochondria.50 This would suggest that caspase-mediatednuclear/DNA degradation should predominate in lymphoidcells during apoptosis. Since these cells are part of the cir-culatory and lymphatic systems, they are essentially om-nipresent in an organism. Lymphocytes abundantly expressthe ICAD-CAD system,45 and they show prominent cas-pase activity and nucleosomal DNA fragmentation duringFas-induced apoptosis.42 Consequently, the caspase-regulated ICAD-CAD system may be important for themaintenance of immune system homeostasis, since endoge-nous DNA that escapes nucleosomal DNA fragmentationduring hematopoietic cell apoptosis may be able to acti-vate innate immunity via a Toll-like receptor 9-independentpathway.44

The production of ROS can initiate apoptosis and onco-sis, or result from processes that are associated with cellu-lar degradation.51 Certain cell types (e.g., neurons, hepato-cytes, endothelial cells, cutaneous keratinocytes, and renalepithelial cells) are prone to ROS-induced cell death in vivo,and many in vitro models of apoptosis include ROS pro-duction and/or the disruption of cellular redox homeosta-sis. In fact, antioxidants (e.g., ascorbic acid, α-tocopherol,N-acetylcysteine) are perhaps the most effective agents inpreventing apoptosis induction by a variety of stimuli bothin vivo and in vitro,52,53 suggesting that ROS and/or oxida-tive stress participate, to some extent, as general mediatorsof cytotoxicity. Anomalous ROS production can potentially

inactivate caspases through the oxidation of the cysteineresidues at their active sites.52–54 Similarly, reactive nitrogenspecies (RNS, e.g., nitric oxide and peroxynitrite) have alsobeen reported to inactivate caspases.52,55–57 These observa-tions would suggest that caspase activity might be mutuallyexclusive in many apoptosis scenarios, especially those in-volving anomalous ROS/RNS production and/or oxidativestress.

In certain cell systems, the MMP-mediated release of cy-tochrome c appears to occur in an all-or-nothing fashionwith respect to apoptosis induction.58–61 Furthermore, inaddition to MMP, the oxidation of cardiolipin may also berequired to solubilize sufficient amounts of cytochrome c dur-ing apoptosis.62 As we alluded to previously, these activitiesseem paradoxical with respect to prominent caspase activitybecause they imply the establishment of an oxidizing in-tracellular environment. For example, cytochrome c oxidase(i.e., complex IV) and its primary redox partner cytochromec are typically present in effectively equivalent quantitiesin the electron transport chain.63–65 Hence, a robust exo-dus of cytochrome c from respiring mitochondria would beexpected to inhibit respiration and cause an increase in mi-tochondrial ROS production.66 This ROS production couldcause cardiolipin oxidation and conceivably retard the acti-vation and/or activity of caspases during apoptosis. Finally,an oxidizing intracellular environment is characteristic ofmany tumor cell types,67–69 and a variety of chemothera-peutic agents, as well as radiation therapy, promote ROSproduction and/or oxidative stress as a way of killing ma-lignant cells.70,71 These observations imply that cancer cellspossess an innately inhospitable cellular environment for theimplementation of the effector and/or degradation phases ofapoptosis if caspase activity were pivotal in the events, andthis quality could conceivably be reinforced during radia-tion therapy or chemotherapy. Yet, these therapies are gen-erally believed to eradicate cancer cells via the induction ofapoptosis.72–74

Genetic and epigenetic factors may also affect the abilityof caspases to act as the central effectors of apoptosis. Forexample, the expression of Fas is reportedly decreased inhepatoma cells relative to normal hepatocytes.75 Since Fasligand is a principal initiator of cytotoxic T lymphocyte-mediated apoptosis, it has been suggested that Fas downregulation might contribute to the evasion of immune sys-tem surveillance by transformed hepatocytes during livercarcinogenesis.75 The deregulation of extrinsic pathway ofapoptosis is also reportedly associated with the etiology ofnon-melanoma skin cancers.76 Caspase expression is lostin a variety of tumor cell types via gene mutation ormethylation.77 The caspase-8 gene is apparently silencedthrough DNA methylation, as well as gene deletion, in neu-roblastoma cells.78 It has also been reported that the expres-sion of caspase-7 is markedly diminished in colon carcinomacells,79 and cells from the breast carcinoma cell line MCF-7apparently lack caspase-3 activity due to a point mutationin the gene coding for this protein.80 Alterations in the

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expression and/or function of apoptosome constituents arealso commonly observed in cancer cells.81 The APAF-1 geneis frequently silenced by hypermethylation in melanomas,82

and leukemias,83 and caspase-9 expression is diminished incolon epithelial cells during colon carcinogenesis.79 Further-more, many tumor cell types aberrantly express inhibitorsof apoptosis protein (IAP) family members (e.g., survivin,c-IAP1, and the X-chromosome-linked IAP) that directlyinterfere with caspase activation during apoptosis.84–86

While many characteristics (e.g., the loss of mito-chondrial inner transmembrane potential,87 MMP,37 PSexternalization,88 proteolysis,37 cell shrinkage,89 and DNAdegradation44) of apoptosis may be attributed to caspaseactivity, these events may be highly dependent on the cyto-toxic stimulus as well as genetic and epigenetic factors. Itis increasingly evident that all of the aforementioned eventscan also occur in the absence of caspase activity. This wouldsuggest that backup or alternative processes must exist toregulate apoptosis. Thus, the activity of seemingly caspase-independent effectors may represent evolutionarily redun-dant or failsafe mechanisms for apoptotic cell elimination.

Evolution always provides backups, justin case: Caspase-independent effectorsof apoptosis

If one acquiesces to the notion that apoptosis is a phylo-genetically ancient process that almost certainly evolved tosome extent as a result of a symbiotic relationship betweenan anaerobic archaebacterium (i.e., the host) and an aerobicproteobacterium (i.e., the endosymbiont),32,90,91 it is proba-bly not difficult to also accept that evolution would providecomplementary checks-and-balances mechanisms, caspasesnot withstanding, to assure apoptotic cell elimination.92,93

This tenet is suggested by several lines of evidence. First,caspase inhibition is reportedly unable to block apoptosis incultured cells exposed to a variety of cytotoxic stimuli.94–107

Second, apoptosis can occur in the absence of caspases inmany in vivo cell death models.22,108–114 Finally, it is in-creasingly evident that, in addition to the mitochondria,the endoplasmic reticulum (ER), Golgi apparatus, and lyso-somes can also function as major points of integration fordamage sensing in the cell, and these organelles can gener-ate caspase-independent signaling intermediates that partic-ipate in the effector and/or degradation phases of apoptosis.19

The following sections will consider some of the purport-edly caspase-independent signaling intermediates that canfunction as effectors of apoptosis.

Non-caspase proteases associated with apoptosis

Non-caspase proteases like cathepsins, calpains, and granz-ymes have been implicated as effectors of apoptosis for quitesome time.30 Consequently, perhaps a plausible reason for

the distinction between the activity of caspases and non-caspase proteases during apoptosis is that the caspase bi-ologists have done an outstanding job of promoting theirresearch during the past 10 years. The cathepsin familyof proteases consists of cysteine, aspartate, and serine pro-teases. Cathepsin B and cathepsin L, both cysteine proteases,and cathepsin D, an aspartate protease, are most frequentlylinked to apoptosis.30,115 These proteases are localized inlysosomes and/or endosomes, but they translocate to thecytoplasm during apoptosis.30,116 Cathepsins can cleave anumber of substrates including Bcl-2 family members, p53,cyclin D, c-Fos, and c-Jun.30,115 Furthermore, cathepsin ac-tivity is reportedly associated with MMP,29,103,117,118 chro-matin condensation,119,120 the degradation of the intracel-lular matrix,30,121 the processing of procaspases,122,123 andthe externalization of PS on the plasma membrane of apop-totic cells29,30,117 (Figure 3). One mechanism recently impli-cated in cathepsin-mediated MMP and caspase-independentapoptosis involves the novel lysosome-associated apoptosis-inducing protein LAPF, which promoted lysosomal mem-brane permeabilization and cathepsin release in fibrosarcomacells.124

The calpain family of cysteine proteases resides in thecytosol. Both µ-calpain and m-calpain have been linked toapoptotic processes,30,125,126 and certain human diseases thatare marked by excessive cell loss (e.g., Alzheimer’s127,128 andParkinson’s129 disease) are directly linked to aberrant cal-pain activity. Calpains are activated by anomalous increasesin intracellular free Ca2+.30,125 While some studies sug-gest that caspase activity is absent during calpain-mediatedapoptosis,95,126 there are others that imply enhanced pro-teolytic activity during apoptosis via a feed-forward am-plification loop that involves caspases.130–133 Furthermore,a “calpain-cathepsin cascade”122 has also been proposed tointegrate and enhance proteolytic activity during apopto-sis. Given that cathepsin B, cathepsin L, µ-calpain, andm-calpain are all cysteine proteases, these enzymes may havea limited window of activity during apoptosis if oxidizingconditions are prevalent, much in the same way speculatedpreviously for caspases.

Granzymes are serine proteases that exhibit a structuralsimilarity to chymotrypsin. The specificity of granzymes isunusual because they cleave their substrates on the carboxyside of acidic amino acid residues, especially after aspartateresidues. Granzymes are secreted by exocytosis, which al-lows natural killer cells to induce apoptosis in target cells.30

Granzymes reportedly promoted caspase-independent DNAfragmentation, MMP, and the externalization of PS onapoptotic cells.134,135 For instance, granzymes encouragedMMP by cleaving the proapoptotic Bcl-2 family membersBid134,136 and Bax,134 or by processing the antiapoptoticBcl-2 family member Mcl-1.137,138 Furthermore, granzymeB can directly cleave ICAD allowing CAD to trigger nucle-osomal DNA fragmentation.48,49

The active form of the serine protease Omi/HtrA2 islocalized in the mitochondrial intermembrane space, and

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Figure 3. The cellular signatures associated with caspase-independent effectors of apoptotic cell death. Please refer to the text for de-tails. Abbreviations: AIF, apoptosis-inducing factor; AMID, AIF-homologous mitochondrion-associated inducer of death; MMP, mitochondrialmembrane permeabilization; ROS, reactive oxygen species; RNS, reactive nitrogen species; PS, phosphatidylserine

it is reportedly released to the cytosol following variousapoptotic stimuli.139,140 HtrA2/Omi has a distinct role asan indirect mediator of caspase activation by binding anddegrading IAP family members31,85,141 (e.g., Apollon142),and the development of certain cancers may be dependenton these regulatory interactions in tumor cells. A recentstudy reported that cisplatin resistance in ovarian carcinomacells may be due to the reduced expression and/or activityof HtrA2/Omi. Cytosolic HtrA2/Omi levels appeared to bepartly regulated by IAP proteins in ovarian carcinoma cellsexposed to cisplatin, suggesting that IAP expression may beassociated with chemoresistance in ovarian cancer.143

Omi/HtrA2 also appears to promote apoptosis directlyvia its serine protease activity.144 For example, Omi/HtrA2induced proteolytic degradation and apoptosis in caspase-9-deficient fibroblasts,145 and a recent study demonstratedthat the cytoplasmic expression of Omi/HtrA2 in HeLaand embryonic kidney cells induced caspase-independentMMP, cell rounding, and cell shrinkage.31 Furthermore,mitochondrial Omi/HtrA2 was found to cleave the mi-tochondrial antiapoptotic protein HS1-associated proteinX-1 in situ during apoptosis.144 A crystal structure anal-ysis has revealed that the formation of a homotrimeris a prerequisite for the caspase-independent function ofOmi/HtrA2.146 However, considering that the cellular sub-strates for Omi/HtrA2 are largely unknown, the underlyingcaspase-independent mechanism of Omi/HtrA2-inducedapoptosis remains poorly characterized.31,147,148 In additionto cathepsins, calpains, granzymes, and Omi/HtrA2, pro-teasomal proteases have also been reported to play a pivotalrole in apoptosis by modulating the degradation of various

apoptotic regulators such as members of the Bcl-2 and IAPfamilies.149

And if proteolysis isn’t enough, there are bonusapoptotic signatures triggered by ex-mitochondrialproteins

Apoptosis-inducing factor (AIF) is a mitochondrial flavo-protein that is released from the intermembrane spaceduring apoptosis. Once liberated from the mitochondria,AIF translocates to the nucleus where it induces chro-matin condensation and large-scale (i.e., ∼50 kb) DNAfragmentation.97,150,151 The release, translocation, and/orDNA fragmentation associated with AIF-induced apopto-sis reportedly occurred in a caspase-independent fashion inseveral cell types.106,152–158 Furthermore, the participationof AIF appears to be required for apoptosis induction causedby HIV infection,159 traumatic brain injury,160 retinaldetachment,161 and neuronal degenerative cell loss.153,160

AIF consists of three structural domains: an amino-terminalmitochondrial localization sequence, a spacer sequence, anda carboxy-terminal oxidoreductase domain.162 The carboxy-terminal domain is essential for the DNA fragmentationfunction of AIF, since the deletion of this domain abol-ishes this activity.151 Apparently, this domain promotes aDNA-AIF electrostatic interaction that is required for DNAdegradation.163,164 AIF displays NADH oxidase as well asmonodehydroascorbate reductase activities.165 The oxidore-ductase function of AIF’s carboxy-terminal domain also ap-pears to be linked to its apoptotic effects in the nucleus,

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since the electron donor NADH was particularly efficientin enhancing the generation of higher-order DNA-AIFand RNA-AIF complexes, and compaction of nucleic acidswithin the nucleus of apoptotic cells.166

Although AIF itself does not have intrinsic DNaseactivity,151,152 it may produce this effect by recruiting oractivating an endonuclease.164 In this regard, a recent studyfound that AIF interacts with cyclophilin A to form anactive DNase,167 and an investigation of AIF-induced apop-tosis in nematodes revealed that AIF and the mitochon-drial endonuclease G (EndoG) may work cooperatively topromote DNA degradation.152 The apoptogenic AIF sig-naling between the mitochondria and the nucleus may bebi-directional considering that enhanced ROS productionand the activation of PARP can initiate a nuclear signal(i.e., poly(ADP-ribose) polymers) that targets the mitochon-dria to trigger caspase-independent AIF release, peripheralchromatin condensation, large-scale DNA fragmentation,and, ultimately, apoptosis.150,168 The apoptosis-promotingfunction of AIF can be negatively regulated by the cytopro-tective heat-shock protein 70 (HSP70).169 This regulatoryquality may have a bearing on AIF’s apoptogenic effectsin cancer cells, since many tumor cell types over expressHSP70.170–173 It is also worth mentioning that the redoxfunction of mitochondrial AIF could be important as an an-tioxidant defense mechanism. This has been suggested byaging studies in Harlequin mice, which harbor a mutationthat diminishes AIF expression.174 Furthermore, mitochon-drial AIF reportedly maintained the transformed state ofcolon carcinoma cells through its NADH oxidase activity,which appeared to regulate the function of complex I in themitochondrial electron transport chain. This would suggestthat mitochondrial AIF may represent a novel anticancerdrug target.175

Mitochondrial EndoG is the most abundant nuclease inthe mitochondria of eukaryotic cells, and one of the mostplentiful nucleases in whole-cell extracts. Normally, thisMg2+-dependent endonuclease probably assists with themaintenance of the mitochondrial genome by participat-ing in mitochondrial DNA duplication and repair.176 Nu-merous apoptotic stimuli cause EndoG to be released frommitochondria. The ex-mitochondrial EndoG then translo-cates to the nucleus where it induces DNA fragmen-tation that is somewhat similar to CAD-induced DNAfragmentation.177,178 However, unlike CAD, EndoG doesnot require caspase processing to be activated.177 Giventhat EndoG, CAD, and DNase I each generate nucleoso-mal DNA fragments in vitro, it has been speculated thatthese nucleases may work cooperatively to promote nucleardegradation during apoptosis.179 It is also interesting toconjecture that EndoG could essentially substitute for CADin certain cell types, especially those that have an abundanceof mitochondria and a deficiency of CAD (e.g., hepatocytesand neurons44–46). Furthermore, given that oncocytic tumorcells exhibit a striking number of mitochondria,180 target-ing MMP in these cells may be an effective strategy to

enforce apoptotic cell death via the release apoptogenic mi-tochondrial proteins (e.g., cytochrome c, AIF, Omi/HtrA2,and EndoG).

The list of mitochondrial proteins that appear to havea function as caspase-independent effectors of apoptosisis growing. WOX1 (also known as WWOX or FOR) isanother mitochondrial oxidoreductase that translocates tothe nucleus following apoptotic stimuli.181–183 Once inthe nucleus, WOX1 reportedly activated p53 and JNK1in fibroblast. This activity down regulated antiapoptoticBcl-2 molecules to enhance TNF-induced apoptosis.181,184

The AIF-homologous mitochondrion-associated inducer ofdeath (AMID) is yet another proapoptotic mitochondrialoxidoreductase that localizes to the outer mitochondrialmembrane. AMID can be induced by p53, and its pro-posed disruption of mitochondrial membranes has been as-sociated with caspase-independent apoptosis.185,186 Giventhat AMID promoted peripheral chromatin condensationin embryonic kidney cells185 and exhibits DNA bindingability,187 it would be interesting to determine if AMID,like AIF, can also promote DNA fragmentation. More-over, ex-mitochondrial cytochrome c reportedly accumu-lated in the nucleus of HeLa cells and promoted chromatincondensation,188 caused PS oxidation and externalizationon apoptotic lymphocytes,189–191 and triggered apoptoticvolume decrease in vascular smooth muscle cells via its in-teractions with plasma membrane K+ channels.192 All ofthese apoptotic signatures evidently occurred in the absenceof caspase activity, suggesting that cytochrome c may be re-sponsible for multiple proapoptotic effects in addition to itsfunction as a caspase-activating factor.

ROS, RNS, Ca2+, and sphingolipids as apoptosiseffectors

The mitochondria are the primary source of ROS produc-tion in most cells,193–196 and anomalous ROS generationby these organelles can play a pivotal role in apoptosissignaling.90,197–199 For example, excessive mitochondrialROS generation and/or the disruption of mitochondrial re-dox homeostasis can promote the oxidation of thiols thatregulate the conformation of proteins constituting the MPTpore complex, which can cause MMP via the induction ofthe MPT.35,200 The MPT allows water and solutes up to1500 Da to infiltrate the mitochondrial matrix that resultsin colloidal osmotic swelling of the matrix.16,35 Conspicuousmitochondrial swelling can cause the physical rupture ofthe outer mitochondrial membrane and the liberation ofapoptogenic mitochondrial proteins.27 Similarly, RNS havebeen reported to be stimulators of the MPT in various celltypes.201–203 Aberrant ROS production may also promoteconformational changes in proapoptotic Bcl-2 family mem-bers (e.g., Bax and Bak), which facilitates their participationin MMP.204–209 Hence, during apoptosis induction by cer-tain mechanisms, ROS generation may be rate limiting in

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the regulation of MMP by both the MPT and proapoptoticBcl-2 family members. This scenario may be representativeof a feed-forward amplification loop of mitochondrial degra-dation. It has been suggested that an increase in mitochon-drial ROS could cause MPT in a subset of mitochondria.This mitochondrial disruption would then promote addi-tional ROS production and further modulate the MMP ofvicinal mitochondria by redox-related changes in proapop-totic Bcl-2 family members.205

As we mentioned previously, mitochondrial ROS gener-ation can also facilitate the dispersal of cytochrome c fromthe inner mitochondrial membrane by breaching its elec-trostatic and/or hydrophobic affiliations with cardiolipin.62

Cellular membranes in addition to those associated with themitochondria are also sensitive to ROS and/or alterationsin cellular redox homeostasis.210 The redox-regulated re-lease of proapoptotic constituents such as Ca2+ from theER211 and cathepsins from lysosomes117,212–215 have beenreported, and PS oxidation and externalization on apoptoticcell plasma membranes may result directly from ROS and/oroxidative stress.189–191,216–219 Furthermore, ROS have beenimplicated as mediators of apoptotic volume decrease,220,221

which may occur through the oxidative activation of volume-sensitive Cl– channels in the plasma membrane.220

Sustained elevations in intracellular free Ca2+ canmediate apoptotic cell death in various cell systems.This is potentially regulated via the activation of Ca2+-dependent enzymes like calpains, endonucleases, andphospholipases.125,222 The disruption of intracellular Ca2+homeostasis and/or ER stress can also promote the activa-tion of caspase-12, which is believed to reside on the cy-toplasmic face of the ER.223–225 This process may resultdirectly from the activity of calpains, and is suggestive ofcross talk between the proteolytic activity of calpains andcaspases during apoptosis.125 Caspase-12 has been impli-cated as a potential initiator caspase through its activationof caspase-8, caspase-9, and caspase-3 during apoptosis.226

In addition to the ER, the mitochondria can also contributeto the regulation of intracellular Ca2+ homeostasis. Sus-tained Ca2+ release from the ER can trigger Ca2+ uptakeby the mitochondria.227–230 The ability of mitochondria tobuffer cytoplasmic Ca2+ loads may be highly dependent ontheir energetic capacity,231–233 and if unchecked, this processcould potentially cause the MPT considering that Ca2+ isa prototypical MPT inducing agent.35,231 Conversely, smallamounts of cytochrome c released from a sub-set of mito-chondria in cervical carcinoma and pheochromocytoma cellsreportedly functioned to alter ER Ca2+ handling by bindinginositol (1,4,5) trisphosphate receptors. The cytochrome c-inositol (1,4,5) trisphosphate receptor interaction triggeredER Ca2+ release and apparently activated a feed-forwardapoptotic cycle in these cells causing subsequent mitochon-drial Ca2+ overload, the MPT, and further global cytochromec release from vicinal mitochondria.234 In addition to itsability to trigger the MPT, mitochondrial Ca2+ uptake hasalso been shown to cause cardiolipin oxidation via an ROS-

dependent mechanism.235 Furthermore, sustained increasesin intracellular free Ca2+ was reportedly associated withapoptotic volume decrease and plasma membrane buddingin cutaneous keratinocytes,231 and may cause PS externaliza-tion on apoptotic cells via the activation of plasma membranePS scramblases.236–238

The sphingolipid ceramide has been shown to mediateapoptosis in response to inflammatory cytokines like Fasand TNF,239–241 and/or conditions associated with oxida-tive stress.242,243 The enhancement of ceramide generationby processes associated with ROS production appears tobe independent of caspase activation,244–246 as opposed tothe caspase-dependent production of ceramide by cells ex-posed to certain inflammatory cytokines.239–241 Ceramidecan be generated in cells de novo, or through the hydrolysis ofsphingomyelin.247,248 During conditions of cell stress, thederegulation of ceramide generating and/or utilizing pro-cesses are believed to cause a net increase in cellular ceramidethat is sufficient to trigger apoptosis induction.249 Moreover,aberrant ROS production and/or oxidative stress can activatesphingomyelinases and increase ceramide production.250–252

Since the mitochondria and MPT induction appear to be atarget of ceramide and during apoptosis,250,252–257 the cou-pling between oxidative stress and ceramide generation isprobably bi-directional and amplifying in nature, since bothprocess would be expected to ultimately eliminate ROS over-producing cells.242 Enhanced ceramide generation may alsofunction to sensitize plasma membrane PS scramblases tocytoplasmic free Ca2+, and facilitate the externalization ofPS on apoptotic cells.236,258

In addition to its direct involvement in apoptosis, ce-ramide can also serve as a carbon source for glycosphin-golipid synthesis in the Golgi network. The glycosphin-golipid ganglioside GD3 (GD3) is a metabolite of ceramidethat has also been implicated in apoptosis induction.243,259

GD3 is a minor ganglioside in most normal tissues ex-cept placenta and thymus.259 However, like ceramide, cel-lular levels of GD3 apparently increase in response to apop-totic stimuli,260–262 and the inhibition of GD3 synthase,the enzyme responsible for GD3 synthesis, can block apop-tosis induction by various mechanisms.243,259 Once it isreleased from the Golgi complex, GD3 apparently tar-gets the mitochondria and directly causes MMP to triggerapoptosis.260–263

We have offered several notable examples of effectormechanisms, in addition to caspases, that promote apoptoticsignatures in dying cells. Figuratively speaking, if a wheelwere to represent the process of apoptosis the aforemen-tioned observations would strongly suggest that caspases areprobably more symbolic of a spoke in this wheel, ratherthan its hub. This is also true for the other effectors ofapoptosis described in this review. The rim of the wheelwould represent the continuum of cytotoxic stimuli thatimpinge on the spokes to produce the morphological andbiochemical characteristics of apoptosis, which would bedenoted by the wheel’s hub. Of course, only the knowledge

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that is gained from the continued examination of apopto-sis regulation will be able to substantiate or negate thisparadigm.

Exploiting apoptosis effectors in cancertherapy

If metazoans were to rely on a single effector mechanismfor apoptosis (i.e., caspases) their existence would indeedbe a precarious one. This is especially true with respectto the development of diseases such as cancer.116 Canceris a complex disease that is manifested through the sur-vival advantage inherent to tumor cells. As we mentionedpreviously, apoptosis can be subverted during tumorigenesisthrough the systematic decay of a panoply of regulatory con-trol mechanisms. This ultimately results in the generationof a malignant phenotype and resistance to chemotherapyand radiation therapy.264–266 Yet, cancerous cells can still beeliminated by apoptosis if we decipher their defense strategy.Thus, in order to design the most efficacious therapeutic ap-proaches for cancer, we need to further elucidate the molecu-lar control of the effectors of apoptosis, as well as strengthenour knowledge of the antiapoptotic events that promote tu-morigenesis. Armed with this information, we will be ableto clearly define therapeutic targets, develop cancer treat-ments that correct defective effector pathways associatedwith these targets, or perhaps bypass them altogether in fa-vor of functional effector mechanisms in tumor cells. Theactivation of caspases may be a goal of therapy in certain can-cers. For example, XIAP may be a good therapeutic targetin cancers in which XIAP over expression, and subsequentsuppression caspase activation, perpetuates cell survival.267

Conversely, caspase-independent effectors of apoptosis mayalso be valuable as anticancer targets,121,172,264,268–271 con-sidering that many novel cancer chemopreventive32,272 andchemotherapeutic34 agents appear to engage these effectorsin transformed cells to directly enforce apoptotic cell killingirrespective of caspases.

Conclusions and future perspectives

In this review, we have detailed some mechanistic aspectsassociated with the effectors of apoptosis, and provided somecaveats for these processes as they potentially relate to can-cer. These mechanisms will certainly be a focus of ensuinginvestigations of apoptosis regulation, and the prevention ortreatment of cancer. Most of the compounds currently usedas anticancer agents are believed to target tumor cell elimi-nation via the induction of apoptosis.72–74 For all our optionsin the prevention and/or treatment of cancer, the selectiveactivation of apoptosis in transformed cells remains the es-sential issue to be addressed. This issue will certainly leadto the development of new preventive/therapeutic agentsthat are more active and/or less toxic than the ones em-

ployed today. Furthermore, future patient-specific profilesof apoptosis-related genetic alteration in tumor cells andgenetic comparisons between chemotherapy-sensitive andchemotherapy-resistant tumor cells will potentially pave theway for patient-directed, highly selective apoptosis-basedcancer therapy with fewer adverse effects.273,274

Acknowledgments

The first and second authors have contributed equally to thiswork and should be considered co-first authors. Supportedin part by grants from the National Institutes of Health(PO1 CA55164 and CA16672) and the Paul and Mary HaasChair in Genetics (to M. A.)

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