Apoptosis effector mechanisms: A requiem performed ... - Springer Link

Apoptosis 2006; 11: 889–904


C 2006 Springer Science + Business Media, LLC. Manufactured in The United States. DOI: 10.1007/s10495-006-6712-8

Apoptosis effector mechanisms: A requiem performed in 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 Marrow Transplantation, 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 by metazoans to remove unneeded, damaged, or potentially deleterious cells. Certain manifestations of apoptosis may be associated with the proteolytic activity of caspases. These changes are often held as hallmarks of apoptosis in dying cells. Consequently, many regard caspases as the central effectors or executioners of apoptosis. However, this “caspase-centric” paradigm of apoptotic cell death does not appear to be as universal as once believed. In fact, during apoptosis the efficacy of caspases may be highly dependent on the cytotoxic stimulus as well as genetic and epigenetic factors. An ever-increasing number of studies strongly suggest that there are effectors in addition to caspases, which are important in generating apoptotic signatures in dying cells. These seemingly caspase-independent effectors may represent evolutionarily redundant or failsafe mechanisms for apoptotic cell elimination. In this review, we will discuss the molecular regulation of caspases and various caspaseindependent effectors of apoptosis, describe the potential context and/or limitations of these mechanisms, and explore why the understanding of these processes may have relevance in cancer where treatment is believed to engage apoptosis to destroy tumor cells.

Keywords: apoptosis; cancer; caspases; mitochondria; proteases; reactive oxygen species.

Introduction “That which has been successfully defined has been successfully killed” –Christmas Humphreys. The phrases “apoptosislike cell death” and “caspase-independent cell death” are frequently used in the literature to define seemingly regulated forms of cell loss that do not appear to fulfill the criteria of apoptosis. While remarkable advances have been made since the early 1990’s in the understanding of apoptosis, our collective knowledge of this process is far form complete, Correspondence to: M. Andreeff, M.D., Ph.D., Section of Molecular Hematology and Therapy, Department of Blood and Marrow Transplantation, Unit 448, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: + 713-792-7260; Fax: + 713-794-4747; e-mail: [email protected]

and consequently may only encompass a highly stereotypical series of events that are appropriate for certain cell types. For this reason, we humbly believe it is inaccurate to use a qualitative distinction such as “apoptosis-like cell death” or “caspase-independent cell death” to describe a possible functional derivative of apoptosis. Furthermore, “apoptosis-like cell death” and “caspase-independent cell death” are somewhat prejudicial because they imply that caspase activity is synonymous with apoptosis, which is not always accurate. We will adhere to Ockham’s razor for now by simply embracing the notion that apoptosis is a distinctive form of cell death with ultrastructural features suggesting an active, inherently regulated phenomenon.1 This review will examine the mechanistic underpinnings that are potentially responsible for this phenomenon.

The apoptotic cell death concept Cell death that culminates in rapid (e.g., occurring within hours or days) cell loss is prevalent in metazoans. This process can be regulated or unregulated. The regulated type of rapid cell loss is known as apoptosis, while the unregulated form is referred to as oncosis.2 Autophagy has also been suggested to be a regulated mode of cell death.3 However, besides being highly regulated, autophagy is typically reversible,4,5 which suggest a level of cell stress below that required for the cell loss associated with apoptosis or oncosis. Furthermore, recent evidence suggest that autophagy is probably most representative of a cell survival pathway6–8 that terminates in apoptosis if unsuccessful.9 Apoptotic cell death has been implicated in embryonic development, immune system regulation, morphogenesis, and the preservation of tissue homeostasis,1,2 as well as various disease states.10 Morphological and histological studies of hepatocytes during ischaemic liver injury provided the background for the formulation of the apoptotic cell death concept, which was proposed by Kerr, Wyllie, and Currie in 1972.11 An apoptotic cell typically undergoes shrinkage (i.e., apoptotic volume decrease), chromatin condensation, karyorrhexis, and the eventual budding of the plasma membrane Apoptosis · Vol 11 · No 6 · 2006

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N. Hail Jr. et al. Figure 1. The morphological features of apoptosis, oncosis, and necrosis. This figure was adapted from Majno and Joris.2 Please refer to the text for details.

into apoptotic bodies (Figure 1). These morphological changes are considered the gold standard for distinguishing this type of cell death.12–14 Conversely, oncosis is a passive catastrophic cellular event where marked swelling, aggregate organelle disruption, and plasma membrane blebbing prevail. There is little or no evidence of chromatin remodeling during oncosis, and the cell rapidly succumbs to cytolysis. This cytolysis is end-stage cellular decay that is the defining feature of necrosis.2 Apoptotic cells will eventually lose plasma membrane integrity and become necrotic in vitro. However, this is not believed to occur with high frequency in vivo because apoptotic cells display signals (e.g., the externalization of phosphatidylserine (PS) on their plasma membrane) that encourage their expeditious removal by phagocytosis.2,15 Apoptosis was originally viewed as being non-degenerative in nature.1,11 However, an impressive body of ensuing research has proven otherwise. Apoptosis can be divided into three phases: initiation, effector, and degradation.16 The initiation phase is largely dependent on cell type and the apoptotic stimulus (e.g., oxidative stress, DNA damage, ion fluctuations, and cytokines). In certain instances, as we will discuss later, the initiation phase may influence the efficacy of the effector and/or degradation phases. The effector phase constitutes the activation of proteases, nucleases, and other diffusible intermediaries that participate in the degradation phase.17–20 Together, the effector and degradation phases 890 Apoptosis · Vol 11 · No 6 · 2006

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

Is caspase-mediated apoptosis a context-dependent phenomenon? Most of the recent advances in the elucidation of apoptosis have come about through the characterization of the effector mechanisms. The caspases are a family of cysteine proteases that 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 approximately two-thirds of these enzymes have been suggested to function in apoptosis.17,20 Nevertheless, many of these same molecules also participate in homeostatic cellular functions (i.e., cytokine production, terminal differentiation, and proliferation) that are not associated with cell death.23,24 There are two types of caspases, upstream caspases called initiator caspases (e.g., caspases-8, -9, and -10), and their downstream targets known as effector or executioner caspases (e.g., caspases-3, -6, and -7).17 Several components comprise the “caspase-centric”25 effector model of apoptosis, and two pathways associated with caspase activation

Caspase-independent apoptosis 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 the mitochondrial-mediated or intrinsic pathway, and the death receptor-mediated (e.g., tumor necrosis factor (TNF) receptor and Fas) or extrinsic pathway26,27 (Figure 2). The intrinsic pathway relies on mitochondrial membrane permeabilization (MMP) to liberate the electron transport chain intermediate cytochrome c from the mitochondrial intermembrane space. Once released to the cytosol, cytochrome c can combine with dATP, APAF-1, and caspase-9 to form the apoptosome. This catalytic complex is responsible for the activation of effector caspase-3 and -7.16,27 The intrinsic pathway can be regulated by the Bcl-2 family of proteins,28 proteases,29–31 as well as agents (e.g., ceramide, reactive oxygen species (ROS), and Ca2+ ) that promote the mitochondrial permeability transition (MPT).32–35 For instance, during conditions of cell stress, antiapoptotic Bcl-2 family members (e.g., Bcl-2 and Bcl-XL ) residing in the outer mitochondrial membrane can be destabilized through decreased expression, or by the induction of proapoptotic Bcl-2 family members (e.g., Bax, Bad, and Bak). In the latter scenario, the ratio of proapoptotic family members to antiapoptotic family members becomes greater allowing the formation of proteinaceous outer membrane channels by the proapoptotic Bcl-2 family members, which can liberate cytochrome c.28,36 The extrinsic pathway is initiated by the ligation of death receptors that subsequently cluster in the plasma membrane and promote the recruitment of adapter proteins (Figure 2). Caspase-8 is the apical caspase in the death receptor pathway. The zymogen of caspase-8 can interact with the adapter proteins to generate the active form of caspase-8. Once active, caspase-8 can trigger the activation of downstream effector caspases like caspase-3.17,37,38 In certain cell systems, the activation of caspase-8 is sufficient to commence the proteolytic cascade required to achieve apoptotic cellular degradation.17,20,38 However, the activation of death

receptors has also been shown to promote the recruitment and downstream action of the intrinsic pathway, which appears to be required to execute apoptosis in certain cell types.39,40 For example, besides activating effector caspases, another notable target of caspase-8 is the BH3 only Bcl-2 relative Bid. In response to Fas ligand or TNF, caspase-8 induces the cleavage of Bid to yield a truncated carboxyterminal fragment that translocates from the cytosol to the outer mitochondrial membrane. Oligomers of the truncated Bid can trigger MMP and cytochrome c-mediated caspase activation.39,40 There is also evidence indicating that truncated Bid can trigger conformational changes in Bax, which then localizes with the voltage-dependant anion channel in the outer mitochondrial membrane to trigger MMP.41 Thus, the recruitment of the intrinsic pathway of apoptosis by caspase-8 activation can serve to initiate and/or amplify intracellular signals to trigger apoptosis. Caspases selectively cleave a restricted set of target proteins after an aspartate residue in their primary sequence.17,37 The caspase-mediated cleavage of cellular proteins has been implicated in the removal of endogenous apoptosis inhibitors, morphological changes, and DNA fragmentation. For example, the cleavage of inhibitory regulators of MMP like Bcl-2 or Bcl-XL not only inactivates their inhibitory function, but also produces a protein fragment that promotes MMP.28 The cleavage of nuclear lamins and cytoskeletal proteins such as fodrin and gelsolin is associated with morphological changes in apoptotic cells,20 and the cleavage of the inhibitor of the caspase-activated DNAse (ICAD) releases CAD, which produces nucleosomal DNA fragmentation.42 In many experimental situations, the evidence for caspase-mediated apoptosis is indirect (e.g., the cleavage of poly(ADP-ribose) polymerase (PARP), nucleosomal DNA fragmentation, and/or procaspase-3 processing) demonstrating that the cells could activate caspases during apoptosis Apoptosis · Vol 11 · No 6 · 2006

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rather than caspase activity was required for this phenomenon.22 Nevertheless, caspase activity is considered by many investigators in the field of cell death to be the defining feature of apoptosis.17,37,43 In fact, if nucleosomal DNA fragmentation, which is detected by the terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique, is deemed to be a hallmark of both caspase activity and apoptosis, the “caspase-centric” paradigm would appear to be a self-fulfilling prophecy. Then again, this presents a problem for classifying cell death that would otherwise appear to be apoptotic, albeit perhaps lacking in conspicuous nucleosomal DNA fragmentation, in cell types that express relatively low levels of ICAD and CAD (e.g., neurons, hepatocytes, and fibroblasts44–46 ). Interestingly, TUNEL-positive DNA fragmentation can occur following the phagocytosis of thymocytes derived from transgenic mice that ubiquitously expressed a caspase-resistant form of ICAD.47 Lysosomal nucleases in phagocytic cells may account for this DNA degradation.44 Furthermore, non-caspase proteases have also been shown to cleave ICAD and release CAD,48,49 suggesting that some features of caspase activity may be sufficient but not essential for apoptosis.42,44 The caspase-regulated ICAD-CAD system of nucleosomal DNA fragmentation may be required in certain cell types to maintain context-dependent homeostasis. For example, the “caspase-centric” effector model of apoptosis appears to be common in lymphoid cells, which have a relatively small cytoplasm,25 and concomitantly very few mitochondria.50 This would suggest that caspase-mediated nuclear/DNA degradation should predominate in lymphoid cells during apoptosis. Since these cells are part of the circulatory and lymphatic systems, they are essentially omnipresent in an organism. Lymphocytes abundantly express the ICAD-CAD system,45 and they show prominent caspase activity and nucleosomal DNA fragmentation during Fas-induced apoptosis.42 Consequently, the caspaseregulated ICAD-CAD system may be important for the maintenance of immune system homeostasis, since endogenous DNA that escapes nucleosomal DNA fragmentation during hematopoietic cell apoptosis may be able to activate innate immunity via a Toll-like receptor 9-independent pathway.44 The production of ROS can initiate apoptosis and oncosis, or result from processes that are associated with cellular degradation.51 Certain cell types (e.g., neurons, hepatocytes, endothelial cells, cutaneous keratinocytes, and renal epithelial cells) are prone to ROS-induced cell death in vivo, and many in vitro models of apoptosis include ROS production and/or the disruption of cellular redox homeostasis. In fact, antioxidants (e.g., ascorbic acid, α-tocopherol, N-acetylcysteine) are perhaps the most effective agents in preventing apoptosis induction by a variety of stimuli both in vivo and in vitro,52,53 suggesting that ROS and/or oxidative stress participate, to some extent, as general mediators of cytotoxicity. Anomalous ROS production can potentially 892 Apoptosis · Vol 11 · No 6 · 2006

inactivate caspases through the oxidation of the cysteine residues at their active sites.52–54 Similarly, reactive nitrogen species (RNS, e.g., nitric oxide and peroxynitrite) have also been reported to inactivate caspases.52,55–57 These observations would suggest that caspase activity might be mutually exclusive in many apoptosis scenarios, especially those involving anomalous ROS/RNS production and/or oxidative stress. In certain cell systems, the MMP-mediated release of cytochrome c appears to occur in an all-or-nothing fashion with respect to apoptosis induction.58–61 Furthermore, in addition to MMP, the oxidation of cardiolipin may also be required to solubilize sufficient amounts of cytochrome c during apoptosis.62 As we alluded to previously, these activities seem paradoxical with respect to prominent caspase activity because they imply the establishment of an oxidizing intracellular environment. For example, cytochrome c oxidase (i.e., complex IV) and its primary redox partner cytochrome c are typically present in effectively equivalent quantities in the electron transport chain.63–65 Hence, a robust exodus of cytochrome c from respiring mitochondria would be expected to inhibit respiration and cause an increase in mitochondrial ROS production.66 This ROS production could cause cardiolipin oxidation and conceivably retard the activation and/or activity of caspases during apoptosis. Finally, an oxidizing intracellular environment is characteristic of many tumor cell types,67–69 and a variety of chemotherapeutic agents, as well as radiation therapy, promote ROS production and/or oxidative stress as a way of killing malignant cells.70,71 These observations imply that cancer cells possess an innately inhospitable cellular environment for the implementation of the effector and/or degradation phases of apoptosis if caspase activity were pivotal in the events, and this quality could conceivably be reinforced during radiation therapy or chemotherapy. Yet, these therapies are generally believed to eradicate cancer cells via the induction of apoptosis.72–74 Genetic and epigenetic factors may also affect the ability of caspases to act as the central effectors of apoptosis. For example, the expression of Fas is reportedly decreased in hepatoma cells relative to normal hepatocytes.75 Since Fas ligand is a principal initiator of cytotoxic T lymphocytemediated apoptosis, it has been suggested that Fas down regulation might contribute to the evasion of immune system surveillance by transformed hepatocytes during liver carcinogenesis.75 The deregulation of extrinsic pathway of apoptosis is also reportedly associated with the etiology of non-melanoma skin cancers.76 Caspase expression is lost in a variety of tumor cell types via gene mutation or methylation.77 The caspase-8 gene is apparently silenced through DNA methylation, as well as gene deletion, in neuroblastoma cells.78 It has also been reported that the expression of caspase-7 is markedly diminished in colon carcinoma cells,79 and cells from the breast carcinoma cell line MCF-7 apparently lack caspase-3 activity due to a point mutation in the gene coding for this protein.80 Alterations in the

Caspase-independent apoptosis

expression and/or function of apoptosome constituents are also commonly observed in cancer cells.81 The APAF-1 gene is frequently silenced by hypermethylation in melanomas,82 and leukemias,83 and caspase-9 expression is diminished in colon epithelial cells during colon carcinogenesis.79 Furthermore, many tumor cell types aberrantly express inhibitors of apoptosis protein (IAP) family members (e.g., survivin, c-IAP1, and the X-chromosome-linked IAP) that directly interfere with caspase activation during apoptosis.84–86 While many characteristics (e.g., the loss of mitochondrial inner transmembrane potential,87 MMP,37 PS externalization,88 proteolysis,37 cell shrinkage,89 and DNA degradation44 ) of apoptosis may be attributed to caspase activity, these events may be highly dependent on the cytotoxic stimulus as well as genetic and epigenetic factors. It is increasingly evident that all of the aforementioned events can also occur in the absence of caspase activity. This would suggest that backup or alternative processes must exist to regulate apoptosis. Thus, the activity of seemingly caspaseindependent effectors may represent evolutionarily redundant or failsafe mechanisms for apoptotic cell elimination.

Evolution always provides backups, just in case: Caspase-independent effectors of apoptosis If one acquiesces to the notion that apoptosis is a phylogenetically ancient process that almost certainly evolved to some extent as a result of a symbiotic relationship between an anaerobic archaebacterium (i.e., the host) and an aerobic proteobacterium (i.e., the endosymbiont),32,90,91 it is probably not difficult to also accept that evolution would provide complementary checks-and-balances mechanisms, caspases not 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 in cultured cells exposed to a variety of cytotoxic stimuli.94–107 Second, apoptosis can occur in the absence of caspases in many in vivo cell death models.22,108–114 Finally, it is increasingly evident that, in addition to the mitochondria, the endoplasmic reticulum (ER), Golgi apparatus, and lysosomes can also function as major points of integration for damage sensing in the cell, and these organelles can generate caspase-independent signaling intermediates that participate in the effector and/or degradation phases of apoptosis.19 The following sections will consider some of the purportedly caspase-independent signaling intermediates that can function as effectors of apoptosis.

Non-caspase proteases associated with apoptosis Non-caspase proteases like cathepsins, calpains, and granzymes have been implicated as effectors of apoptosis for quite some time.30 Consequently, perhaps a plausible reason for

the distinction between the activity of caspases and noncaspase proteases during apoptosis is that the caspase biologists have done an outstanding job of promoting their research during the past 10 years. The cathepsin family of proteases consists of cysteine, aspartate, and serine proteases. Cathepsin B and cathepsin L, both cysteine proteases, and cathepsin D, an aspartate protease, are most frequently linked to apoptosis.30,115 These proteases are localized in lysosomes and/or endosomes, but they translocate to the cytoplasm during apoptosis.30,116 Cathepsins can cleave a number of substrates including Bcl-2 family members, p53, cyclin D, c-Fos, and c-Jun.30,115 Furthermore, cathepsin activity is reportedly associated with MMP,29,103,117,118 chromatin condensation,119,120 the degradation of the intracellular matrix,30,121 the processing of procaspases,122,123 and the externalization of PS on the plasma membrane of apoptotic cells29,30,117 (Figure 3). One mechanism recently implicated in cathepsin-mediated MMP and caspase-independent apoptosis involves the novel lysosome-associated apoptosisinducing protein LAPF, which promoted lysosomal membrane permeabilization and cathepsin release in fibrosarcoma cells.124 The calpain family of cysteine proteases resides in the cytosol. Both µ-calpain and m-calpain have been linked to apoptotic processes,30,125,126 and certain human diseases that are marked by excessive cell loss (e.g., Alzheimer’s127,128 and Parkinson’s129 disease) are directly linked to aberrant calpain activity. Calpains are activated by anomalous increases in intracellular free Ca2+ .30,125 While some studies suggest that caspase activity is absent during calpain-mediated apoptosis,95,126 there are others that imply enhanced proteolytic activity during apoptosis via a feed-forward amplification loop that involves caspases.130–133 Furthermore, a “calpain-cathepsin cascade”122 has also been proposed to integrate and enhance proteolytic activity during apoptosis. Given that cathepsin B, cathepsin L, µ-calpain, and m-calpain are all cysteine proteases, these enzymes may have a limited window of activity during apoptosis if oxidizing conditions are prevalent, much in the same way speculated previously for caspases. Granzymes are serine proteases that exhibit a structural similarity to chymotrypsin. The specificity of granzymes is unusual because they cleave their substrates on the carboxy side of acidic amino acid residues, especially after aspartate residues. Granzymes are secreted by exocytosis, which allows natural killer cells to induce apoptosis in target cells.30 Granzymes reportedly promoted caspase-independent DNA fragmentation, MMP, and the externalization of PS on apoptotic cells.134,135 For instance, granzymes encouraged MMP by cleaving the proapoptotic Bcl-2 family members Bid134,136 and Bax,134 or by processing the antiapoptotic Bcl-2 family member Mcl-1.137,138 Furthermore, granzyme B can directly cleave ICAD allowing CAD to trigger nucleosomal DNA fragmentation.48,49 The active form of the serine protease Omi/HtrA2 is localized in the mitochondrial intermembrane space, and Apoptosis · Vol 11 · No 6 · 2006

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

it is reportedly released to the cytosol following various apoptotic stimuli.139,140 HtrA2/Omi has a distinct role as an indirect mediator of caspase activation by binding and degrading IAP family members31,85,141 (e.g., Apollon142 ), and the development of certain cancers may be dependent on these regulatory interactions in tumor cells. A recent study reported that cisplatin resistance in ovarian carcinoma cells may be due to the reduced expression and/or activity of HtrA2/Omi. Cytosolic HtrA2/Omi levels appeared to be partly regulated by IAP proteins in ovarian carcinoma cells exposed to cisplatin, suggesting that IAP expression may be associated with chemoresistance in ovarian cancer.143 Omi/HtrA2 also appears to promote apoptosis directly via its serine protease activity.144 For example, Omi/HtrA2 induced proteolytic degradation and apoptosis in caspase9-deficient fibroblasts,145 and a recent study demonstrated that the cytoplasmic expression of Omi/HtrA2 in HeLa and embryonic kidney cells induced caspase-independent MMP, cell rounding, and cell shrinkage.31 Furthermore, mitochondrial Omi/HtrA2 was found to cleave the mitochondrial antiapoptotic protein HS1-associated protein X-1 in situ during apoptosis.144 A crystal structure analysis has revealed that the formation of a homotrimer is a prerequisite for the caspase-independent function of Omi/HtrA2.146 However, considering that the cellular substrates for Omi/HtrA2 are largely unknown, the underlying caspase-independent mechanism of Omi/HtrA2-induced apoptosis remains poorly characterized.31,147,148 In addition to cathepsins, calpains, granzymes, and Omi/HtrA2, proteasomal proteases have also been reported to play a pivotal role in apoptosis by modulating the degradation of various 894 Apoptosis · Vol 11 · No 6 · 2006

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

And if proteolysis isn’t enough, there are bonus apoptotic signatures triggered by ex-mitochondrial proteins Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that is released from the intermembrane space during apoptosis. Once liberated from the mitochondria, AIF translocates to the nucleus where it induces chromatin condensation and large-scale (i.e., ∼50 kb) DNA fragmentation.97,150,151 The release, translocation, and/or DNA fragmentation associated with AIF-induced apoptosis reportedly occurred in a caspase-independent fashion in several cell types.106,152–158 Furthermore, the participation of AIF appears to be required for apoptosis induction caused by HIV infection,159 traumatic brain injury,160 retinal detachment,161 and neuronal degenerative cell loss.153,160 AIF consists of three structural domains: an amino-terminal mitochondrial localization sequence, a spacer sequence, and a carboxy-terminal oxidoreductase domain.162 The carboxyterminal domain is essential for the DNA fragmentation function of AIF, since the deletion of this domain abolishes this activity.151 Apparently, this domain promotes a DNA-AIF electrostatic interaction that is required for DNA degradation.163,164 AIF displays NADH oxidase as well as monodehydroascorbate reductase activities.165 The oxidoreductase function of AIF’s carboxy-terminal domain also appears to be linked to its apoptotic effects in the nucleus,

Caspase-independent apoptosis

since the electron donor NADH was particularly efficient in enhancing the generation of higher-order DNA-AIF and RNA-AIF complexes, and compaction of nucleic acids within the nucleus of apoptotic cells.166 Although AIF itself does not have intrinsic DNase activity,151,152 it may produce this effect by recruiting or activating an endonuclease.164 In this regard, a recent study found that AIF interacts with cyclophilin A to form an active DNase,167 and an investigation of AIF-induced apoptosis in nematodes revealed that AIF and the mitochondrial endonuclease G (EndoG) may work cooperatively to promote DNA degradation.152 The apoptogenic AIF signaling between the mitochondria and the nucleus may be bi-directional considering that enhanced ROS production and the activation of PARP can initiate a nuclear signal (i.e., poly(ADP-ribose) polymers) that targets the mitochondria to trigger caspase-independent AIF release, peripheral chromatin condensation, large-scale DNA fragmentation, and, ultimately, apoptosis.150,168 The apoptosis-promoting function of AIF can be negatively regulated by the cytoprotective heat-shock protein 70 (HSP70).169 This regulatory quality may have a bearing on AIF’s apoptogenic effects in cancer cells, since many tumor cell types over express HSP70.170–173 It is also worth mentioning that the redox function of mitochondrial AIF could be important as an antioxidant defense mechanism. This has been suggested by aging studies in Harlequin mice, which harbor a mutation that diminishes AIF expression.174 Furthermore, mitochondrial AIF reportedly maintained the transformed state of colon carcinoma cells through its NADH oxidase activity, which appeared to regulate the function of complex I in the mitochondrial electron transport chain. This would suggest that mitochondrial AIF may represent a novel anticancer drug target.175 Mitochondrial EndoG is the most abundant nuclease in the mitochondria of eukaryotic cells, and one of the most plentiful nucleases in whole-cell extracts. Normally, this Mg2+ -dependent endonuclease probably assists with the maintenance of the mitochondrial genome by participating in mitochondrial DNA duplication and repair.176 Numerous apoptotic stimuli cause EndoG to be released from mitochondria. The ex-mitochondrial EndoG then translocates to the nucleus where it induces DNA fragmentation that is somewhat similar to CAD-induced DNA fragmentation.177,178 However, unlike CAD, EndoG does not require caspase processing to be activated.177 Given that EndoG, CAD, and DNase I each generate nucleosomal DNA fragments in vitro, it has been speculated that these nucleases may work cooperatively to promote nuclear degradation during apoptosis.179 It is also interesting to conjecture that EndoG could essentially substitute for CAD in certain cell types, especially those that have an abundance of mitochondria and a deficiency of CAD (e.g., hepatocytes and neurons44–46 ). Furthermore, given that oncocytic tumor cells exhibit a striking number of mitochondria,180 targeting MMP in these cells may be an effective strategy to

enforce apoptotic cell death via the release apoptogenic mitochondrial proteins (e.g., cytochrome c, AIF, Omi/HtrA2, and EndoG). The list of mitochondrial proteins that appear to have a function as caspase-independent effectors of apoptosis is growing. WOX1 (also known as WWOX or FOR) is another mitochondrial oxidoreductase that translocates to the nucleus following apoptotic stimuli.181–183 Once in the nucleus, WOX1 reportedly activated p53 and JNK1 in fibroblast. This activity down regulated antiapoptotic Bcl-2 molecules to enhance TNF-induced apoptosis.181,184 The AIF-homologous mitochondrion-associated inducer of death (AMID) is yet another proapoptotic mitochondrial oxidoreductase that localizes to the outer mitochondrial membrane. AMID can be induced by p53, and its proposed disruption of mitochondrial membranes has been associated with caspase-independent apoptosis.185,186 Given that AMID promoted peripheral chromatin condensation in embryonic kidney cells185 and exhibits DNA binding ability,187 it would be interesting to determine if AMID, like AIF, can also promote DNA fragmentation. Moreover, ex-mitochondrial cytochrome c reportedly accumulated in the nucleus of HeLa cells and promoted chromatin condensation,188 caused PS oxidation and externalization on apoptotic lymphocytes,189–191 and triggered apoptotic volume decrease in vascular smooth muscle cells via its interactions with plasma membrane K+ channels.192 All of these apoptotic signatures evidently occurred in the absence of caspase activity, suggesting that cytochrome c may be responsible for multiple proapoptotic effects in addition to its function as a caspase-activating factor.

ROS, RNS, Ca2+ , and sphingolipids as apoptosis effectors The mitochondria are the primary source of ROS production in most cells,193–196 and anomalous ROS generation by these organelles can play a pivotal role in apoptosis signaling.90,197–199 For example, excessive mitochondrial ROS generation and/or the disruption of mitochondrial redox homeostasis can promote the oxidation of thiols that regulate the conformation of proteins constituting the MPT pore complex, which can cause MMP via the induction of the MPT.35,200 The MPT allows water and solutes up to 1500 Da to infiltrate the mitochondrial matrix that results in colloidal osmotic swelling of the matrix.16,35 Conspicuous mitochondrial swelling can cause the physical rupture of the outer mitochondrial membrane and the liberation of apoptogenic mitochondrial proteins.27 Similarly, RNS have been reported to be stimulators of the MPT in various cell types.201–203 Aberrant ROS production may also promote conformational changes in proapoptotic Bcl-2 family members (e.g., Bax and Bak), which facilitates their participation in MMP.204–209 Hence, during apoptosis induction by certain mechanisms, ROS generation may be rate limiting in Apoptosis · Vol 11 · No 6 · 2006

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the regulation of MMP by both the MPT and proapoptotic Bcl-2 family members. This scenario may be representative of a feed-forward amplification loop of mitochondrial degradation. It has been suggested that an increase in mitochondrial ROS could cause MPT in a subset of mitochondria. This mitochondrial disruption would then promote additional ROS production and further modulate the MMP of vicinal mitochondria by redox-related changes in proapoptotic Bcl-2 family members.205 As we mentioned previously, mitochondrial ROS generation can also facilitate the dispersal of cytochrome c from the inner mitochondrial membrane by breaching its electrostatic and/or hydrophobic affiliations with cardiolipin.62 Cellular membranes in addition to those associated with the mitochondria are also sensitive to ROS and/or alterations in cellular redox homeostasis.210 The redox-regulated release of proapoptotic constituents such as Ca2+ from the ER211 and cathepsins from lysosomes117,212–215 have been reported, and PS oxidation and externalization on apoptotic cell plasma membranes may result directly from ROS and/or oxidative stress.189–191,216–219 Furthermore, ROS have been implicated as mediators of apoptotic volume decrease,220,221 which may occur through the oxidative activation of volumesensitive Cl– channels in the plasma membrane.220 Sustained elevations in intracellular free Ca2+ can mediate apoptotic cell death in various cell systems. This is potentially regulated via the activation of Ca2+ dependent enzymes like calpains, endonucleases, and phospholipases.125,222 The disruption of intracellular Ca2+ homeostasis and/or ER stress can also promote the activation of caspase-12, which is believed to reside on the cytoplasmic face of the ER.223–225 This process may result directly from the activity of calpains, and is suggestive of cross talk between the proteolytic activity of calpains and caspases during apoptosis.125 Caspase-12 has been implicated as a potential initiator caspase through its activation of caspase-8, caspase-9, and caspase-3 during apoptosis.226 In addition to the ER, the mitochondria can also contribute to the regulation of intracellular Ca2+ homeostasis. Sustained Ca2+ release from the ER can trigger Ca2+ uptake by the mitochondria.227–230 The ability of mitochondria to buffer cytoplasmic Ca2+ loads may be highly dependent on their energetic capacity,231–233 and if unchecked, this process could potentially cause the MPT considering that Ca2+ is a prototypical MPT inducing agent.35,231 Conversely, small amounts of cytochrome c released from a sub-set of mitochondria in cervical carcinoma and pheochromocytoma cells reportedly functioned to alter ER Ca2+ handling by binding inositol (1,4,5) trisphosphate receptors. The cytochrome cinositol (1,4,5) trisphosphate receptor interaction triggered ER Ca2+ release and apparently activated a feed-forward apoptotic cycle in these cells causing subsequent mitochondrial Ca2+ overload, the MPT, and further global cytochrome c release from vicinal mitochondria.234 In addition to its ability to trigger the MPT, mitochondrial Ca2+ uptake has also been shown to cause cardiolipin oxidation via an ROS896 Apoptosis · Vol 11 · No 6 · 2006

dependent mechanism.235 Furthermore, sustained increases in intracellular free Ca2+ was reportedly associated with apoptotic volume decrease and plasma membrane budding in cutaneous keratinocytes,231 and may cause PS externalization on apoptotic cells via the activation of plasma membrane PS scramblases.236–238 The sphingolipid ceramide has been shown to mediate apoptosis in response to inflammatory cytokines like Fas and TNF,239–241 and/or conditions associated with oxidative stress.242,243 The enhancement of ceramide generation by processes associated with ROS production appears to be independent of caspase activation,244–246 as opposed to the caspase-dependent production of ceramide by cells exposed to certain inflammatory cytokines.239–241 Ceramide can be generated in cells de novo, or through the hydrolysis of sphingomyelin.247,248 During conditions of cell stress, the deregulation of ceramide generating and/or utilizing processes are believed to cause a net increase in cellular ceramide that is sufficient to trigger apoptosis induction.249 Moreover, aberrant ROS production and/or oxidative stress can activate sphingomyelinases and increase ceramide production.250–252 Since the mitochondria and MPT induction appear to be a target of ceramide and during apoptosis,250,252–257 the coupling between oxidative stress and ceramide generation is probably bi-directional and amplifying in nature, since both process would be expected to ultimately eliminate ROS overproducing cells.242 Enhanced ceramide generation may also function to sensitize plasma membrane PS scramblases to cytoplasmic free Ca2+ , and facilitate the externalization of PS on apoptotic cells.236,258 In addition to its direct involvement in apoptosis, ceramide can also serve as a carbon source for glycosphingolipid synthesis in the Golgi network. The glycosphingolipid ganglioside GD3 (GD3) is a metabolite of ceramide that has also been implicated in apoptosis induction.243,259 GD3 is a minor ganglioside in most normal tissues except placenta and thymus.259 However, like ceramide, cellular levels of GD3 apparently increase in response to apoptotic stimuli,260–262 and the inhibition of GD3 synthase, the enzyme responsible for GD3 synthesis, can block apoptosis induction by various mechanisms.243,259 Once it is released from the Golgi complex, GD3 apparently targets the mitochondria and directly causes MMP to trigger apoptosis.260–263 We have offered several notable examples of effector mechanisms, in addition to caspases, that promote apoptotic signatures in dying cells. Figuratively speaking, if a wheel were to represent the process of apoptosis the aforementioned observations would strongly suggest that caspases are probably more symbolic of a spoke in this wheel, rather than its hub. This is also true for the other effectors of apoptosis described in this review. The rim of the wheel would represent the continuum of cytotoxic stimuli that impinge on the spokes to produce the morphological and biochemical characteristics of apoptosis, which would be denoted by the wheel’s hub. Of course, only the knowledge

Caspase-independent apoptosis

that is gained from the continued examination of apoptosis regulation will be able to substantiate or negate this paradigm.

Exploiting apoptosis effectors in cancer therapy If metazoans were to rely on a single effector mechanism for apoptosis (i.e., caspases) their existence would indeed be a precarious one. This is especially true with respect to the development of diseases such as cancer.116 Cancer is a complex disease that is manifested through the survival advantage inherent to tumor cells. As we mentioned previously, apoptosis can be subverted during tumorigenesis through the systematic decay of a panoply of regulatory control mechanisms. This ultimately results in the generation of a malignant phenotype and resistance to chemotherapy and radiation therapy.264–266 Yet, cancerous cells can still be eliminated by apoptosis if we decipher their defense strategy. Thus, in order to design the most efficacious therapeutic approaches for cancer, we need to further elucidate the molecular control of the effectors of apoptosis, as well as strengthen our knowledge of the antiapoptotic events that promote tumorigenesis. Armed with this information, we will be able to clearly define therapeutic targets, develop cancer treatments that correct defective effector pathways associated with these targets, or perhaps bypass them altogether in favor of functional effector mechanisms in tumor cells. The activation of caspases may be a goal of therapy in certain cancers. For example, XIAP may be a good therapeutic target in cancers in which XIAP over expression, and subsequent suppression caspase activation, perpetuates cell survival.267 Conversely, caspase-independent effectors of apoptosis may also be valuable as anticancer targets,121,172,264,268–271 considering that many novel cancer chemopreventive32,272 and chemotherapeutic34 agents appear to engage these effectors in transformed cells to directly enforce apoptotic cell killing irrespective of caspases.

Conclusions and future perspectives In this review, we have detailed some mechanistic aspects associated with the effectors of apoptosis, and provided some caveats for these processes as they potentially relate to cancer. These mechanisms will certainly be a focus of ensuing investigations of apoptosis regulation, and the prevention or treatment of cancer. Most of the compounds currently used as anticancer agents are believed to target tumor cell elimination via the induction of apoptosis.72–74 For all our options in the prevention and/or treatment of cancer, the selective activation of apoptosis in transformed cells remains the essential issue to be addressed. This issue will certainly lead to the development of new preventive/therapeutic agents that are more active and/or less toxic than the ones em-

ployed today. Furthermore, future patient-specific profiles of apoptosis-related genetic alteration in tumor cells and genetic comparisons between chemotherapy-sensitive and chemotherapy-resistant tumor cells will potentially pave the way for patient-directed, highly selective apoptosis-based cancer therapy with fewer adverse effects.273,274

Acknowledgments

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

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