Apoptosis regulators and their role in tumorigenesis

Biochimica et Biophysica Acta 1551 (2001) F1^F37 www.bba-direct.com Review Apoptosis regulators and their role in tumorigenesis Martin Zo«rnig a; *, Anne-Odile Hueber b, Wiebke Baum a, Gerard Evan c b a Georg-Speyer-Haus, Paul-Ehrlich-StraMe 42^44, 60596 Frankfurt, Germany Institute of Signaling, Developmental Biology and Cancer Research CNRS UMR 6543, Centre A. Lacassagne, 33 Avenue Valombrose, 06189 Nice, France c UCSF Cancer Center, 2340 Sutter Street, San Francisco, CA 94143-0128, USA Received 9 May 2001; received in revised form 12 July 2001; accepted 25 July 2001 Abstract It has become clear that, together with deregulated growth, inhibition of programmed cell death (PCD) plays a pivotal role in tumorigenesis. In this review, we present an overview of the genes and mechanisms involved in PCD. We then summarize the evidence that impaired PCD is a prerequisite for tumorigenesis, as indicated by the fact that more and more neoplastic mutations appear to act by interfering with PCD. This has made the idea of restoration of corrupted `death programs' an intriguing new area for potential cancer therapy. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Tumorigenesis; Programmed cell death 1. Introduction * Corresponding author. Tel.: +49-69-63395115; Fax: +49-69-63395297. E-mail address: zoernig@em.uni-frankfurt.de (M. Zo«rnig). For many biologists it came as a surprise to realize that the death of a cell is not necessarily a bad thing. Indeed, in metazoans cell death is required for development, maintenance and survival of the organism. Physiological cell death has been observed in di ering tissues and in various organisms for more than 100 years [1]. Cell death occurs throughout the life span of multicellular organisms and arguably represents the only irreversible cell fate decision. Prominent examples of physiological apoptosis include the hormonally regulated involution of the tadpole tail during development, negative selection of lymphocytes to delete autoreactive or non-reactive cells, widespread cell death of neuronal cells during the self-assembly of the central nervous system, and the formation of digits by involution of interdigital cells in the primitive limb paddle (a more extensive survey of literature describing apoptosis occuring in vivo can be found in [2]). Apoptosis is, by far, the most predominant form of physiological cell death. In contrast, unambiguous examples of physiological cell necrosis are few. Because it is a regulated process, controlled by a diversity of extracellular and intracellular signals, apoptosis is used for the coordinated death of excess, hazardous or damaged somatic cells. Moreover, the apoptotic process includes mechanisms that organize both packaging and disposal of cell corpses, thereby preventing in ammation of the surrounding tissue. This is an essential requirement in metazoans which, for obvious reasons, need to be able to distinguish cells that die as part of the normal process of main- 0304-419X / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S0304-419X(01)00031-2

F2 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 taining tissue homeostasis from cells that die as a result of trauma. Severe disturbance of homeostasis of any particular cell population or lineage can cause major pathologies in multicellular organisms. Not surprisingly, therefore, substantial evidence indicates that alterations in control of cell death/survival contribute to the pathogenesis of many human diseases [3]. Diseases linked with suppression of apoptosis include cancer, autoimmune disorders (e.g. systemic lupus erythematosus) and viral infections (e.g. herpesviruses, poxviruses, adenoviruses); diseases in which increased apoptosis is an element include AIDS [4], neurodegenerative disorders [5], myelodysplastic syndromes, ischemic injury (e.g. stroke, myocardial infarction), toxin-induced liver disease (e.g. alcohol) and some autoimmune disorders [6]. In many cases, it is unclear whether perturbations in cell death mechanisms are causal or merely a consequence of the disease process. Nonetheless, e orts aimed at treating these diseases by manipulating cell suicide would seem to have great potential, although they are thus far at a relatively early stage. 2. Cell death: de nition and signi cance Historically, apoptosis was de ned on a morphological basis by contrast with another type of cell death, necrosis ([7,8]. The necrotic cell swells and its chromatin takes on the appearance of a occulent mass that eventually disappears to leave a nuclear ghost. Cell DNA is non-speci cally degraded and characteristically appears as a smear when size fractionated on an agarose gel. One of the most prominent features of apoptosis involves the nucleus. Chromatin condenses and forms aggregates near the nuclear membrane which, in turn, becomes convoluted, whilst the nucleolus becomes enlarged and appears abnormally granular. Chromatin is also subject to the actions of di erent activated endonucleases that cleave the DNA initially into 300^50 kb fragments and subsequently into 180 bp fragments [9^11]. Also during apoptosis, the cell visibly shrinks, adherent cells round up, and distinct protuberances or membrane blebs become discernible (see Fig. 1). Blebbing cells still exclude vital dyes indicating membrane integrity. Organelles within the Fig. 1. Cells undergoing apoptosis. (A, B) Rat-1 broblasts expressing c-myc in the presence (A) or absence (B) of serum. (A) Normal growing cells. (B) Rat-1 cell in a late stage of apoptotic cell death. Cell shrinkage, nuclear condensation and nal fragmentation of the whole cell are obvious. shrunken apoptotic cytoplasm retain a largely normal appearance save for some dilation of the endoplasmic reticulum (ER) and swelling of the mitochondria. The transition from normal to shrunken and blebbing is rapid, typically taking only some 10^30 min, and it is at this point that apoptotic cells are probably phagocytosed in vivo, either by their nearest neighbors or by professional macrophages [12]. Recognition and phagocytosis of apoptotic cells is mediated by a variety of independent receptor^ligand interactions which will not be discussed in detail further and have been reviewed elsewhere [13]. However, one feature of apoptotic cells involved in their phagocytosis is commonly used as a marker for apoptosis: activation of a ippase in apoptotic cells leads them to express externalized phosphatidylserine, usually present only in the internal lea et of the plasma membrane [14]. The rapidity of the apo-

M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 F3 ptotic program and of the clearance of apoptotic cells within the soma largely explains why apoptosis was, until recently, largely overlooked as a major homeostatic process. Another morphologically distinct form of programmed cell death (PCD) is the process of autophagy, or bulk degradation of cellular proteins through an autophagosomic^lysosomal pathway. Autophagy is important in normal growth control, regulated by steroids during development and may be defective in tumor cells [15,16]. 3. Evolution of PCD: the nematode Caenorhabditis elegans as an invertebrate model Physiological PCD has been described in all multicellular organisms so far studied, including plants, slime molds, nematodes, insects and vertebrates [17]. While the physiological role of apoptosis in the shaping and rebuilding of complex tissues of multicellular organisms is plain to see, the biological rationale for PCD is less clear for single cell organisms. Furthermore, unicellular PCD seems not to involve the action of caspases ^ the cysteine proteases that are the hallmarks of metazoan apoptosis. Nonetheless, there is growing evidence that some form of PCD does exist in unicellular organisms like Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Dictyostelium discoideum or Tetrahymena thermophila [18] and perhaps even in bacteria [19]. Possibly, PCD arose in unicellular organisms as a way of ensuring survival of at least some members of a clonal colony during periods of privation. An alternative idea is that unicellular PCD evolved as a defense against the spread of virus infection. Indeed, many metazoan viruses actively suppress apoptosis in order to ensure their propagation, indicating that host cell suicide is an e ective way of forestalling virus spread. The various known anti-apoptotic viral genes possess a variety of di ering structures and modes of action. C. elegans is well suited for the study of cell death at the cellular, genetic, and molecular levels because it is both transparent and developmentally invariant. This has permitted the complete lineage description of every one of the 1090 cells born during development of the hermaphrodite form. During C. elegans development, 131 cells die to leave a nal total of 959 in the adult. Such detailed knowledge of developmental cell deaths enabled easy identi cation of mutants with aberrant patterns of cell death [20]. Their analysis has identi ed genes controlling four aspects of the cell death process: (1) a determination step, (2) the execution of cell death, (3) engulfment of the dying cell, and (4) degradation of the engulfed cell DNA. Three genes are involved in the critical cell death execution step. Two of these, ced (cell death defective)-3 and ced-4 are required for each cell death [21]: if inactivated by mutation, none of the 131 normal cell deaths occur. The other gene, ced-9, acts to antagonize the killing activity of ced-3 and -4: gain of function ced-9 mutants show absence of cell death whereas mutations that inactivate ced-9 lead to widespread and lethal embryonic death [22]. In the last few years, many of the molecular functions of the proteins encoded by the ced-3, -4 and -9 genes have been deduced [23]. The killer gene ced-3 encodes the Ced-3 protein, a member of a class of cysteine protease that cleave after aspartate residues ^ hence their name `caspase' (cysteine aspartyl protease [24]). Caspases are synthesized as inactive zymogens that are activated by cleavage at sites that themselves conform to canonical caspase cleavage sites. Activation abscises an N-terminal prodomain and cleaves the remaining polypeptide into small and large subunits that then assemble as an K 2 L 2 active tetramer. The Ced-4 protein physically interacts with both Ced-3 [25,26] and Ced-9 [26^29] proteins and appears to act as an adapter protein that facilitates Ced-3 autoactivation [30]. Ced-4 has a putative ATPase (nucleotide binding) domain that is required for its ability to activate Ced-3. The interaction of Ced-4 with the death-inhibiting protein Ced-9 inhibits its ability to activate Ced-3. As discussed below, the Ced-3, -4 and -9 basal apoptotic machinery is highly conserved amongst the metazoa. The Ced-3 caspase is homologous to similar enzymes identi ed in insects and in vertebrates ^ the prototypical mammalian homologue being the eponymous interleukin-1l converting enzyme (ICE) [31], although some 14 other mammalian caspases are now known [32,33]. The mammalian homologues of Ced-9 are the Bcl- 2 proteins [34,35] ^ key regulators of cell survival rst identi ed by the oncogenic activity of Bcl-2 in

F4 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 human follicular B cell lymphoma. Recent evidence suggests that Ced-9 prevents apoptosis in C. elegans by two distinct mechanisms [36]: it may directly inhibit the Ced-3 protease by an interaction involving Ced-3 cleavage sites within Ced-9, or Ced-9 may also directly or indirectly inhibit Ced-3 by means of a protective mechanism similar to that used by mammalian Bcl-2: cleavage of Ced-9 by Ced-3 generates a carboxy-terminal product that resembles Bcl-2 in sequence and in function. A mammalian homologue of Ced-4 has been identi ed as Apaf-1 (apoptotic protease activating factor), a large protein implicated in regulating caspase activity through mediating cytochrome c-dependent activation of caspase-9 [37]. Such tremendous evolutionary conservation of cell death machinery amongst metazoans has the bene t that analysis of cell death in `simple' genetic model systems like C. elegans is very informative in developing our understanding of the control of apoptosis in mammalian cells. For example, the C. elegans egl-1 encodes a small protein containing a nine amino acid stretch similar to the BH3 domain, a domain found in various pro-apoptotic Bcl-2-like mammalian cell death regulators. Experimental analysis showed that egl-1 probably acts upstream of ced-3 and ced-4, and direct interaction between the Egl-1 and Ced-9 proteins was also demonstrated [38]. This led to the suggestion that Egl-1 may act by interfering with Ced-9 so that it can no longer inhibit Ced-4, which is then free to activate Ced-3, leading to cell death. Further genes required for removal of apoptotic cells have been identi ed in C. elegans [20] resulting in mammalian homologues being identi ed and characterized. The recently cloned human homologue of the C. elegans engulfment protein Ced-6, for example, speci cally promotes phagocytosis of apoptotic cells [39,40]. 4. Molecular pathways of cell death 4.1. Central e ectors: caspases It is widely accepted that caspases, the family of related Ced-3-like cysteine proteases, are common e ectors of classical metazoan apoptosis. The rst mammalian homologue of Ced-3 identi ed in 1993 was ICE [31], now called caspase-1. Caspase-1 and - 11 (and possibly -4 and -5) are principally involved in proteolytic maturation of cytokines such as interleukin-1l [41^43] and may have little to do with apoptosis. However, subsequent studies have identi ed a further 10 ICE family members of which caspases-2, -3, -6, -7, -8, -9, -10, -12, -13 and -14 are all implicated in regulation and/or execution of apoptosis: Nedd-2/ICH-1 (caspase-2), Yama/CPP32/apopain (caspase-3), T x /ICH-2/ICErel II (caspase-4), ICErel III (caspase-5), Mch-2 (caspase-6), ICE-Lap-3/Mch-3/ CMH-1 (caspase-7), FLICE/MACH (caspase-8), ICE-LAP-6 (caspase-9), Mch-4/FLICE-2 (caspase- 10), ICH-3 (caspase-11), caspase-12, ERICE (caspase-13) and MICE (caspase-14) [32,44]. These apoptotic caspases undergo activating cleavage during apoptosis (either through autoactivation, as part of a caspase cascade or by other non-caspase proteinases such as granzyme B) and between them they cut a range of substrate proteins whose cleavage either mediates or attends the apoptotic process. Furthermore, caspase inhibitors, whether virus proteins such as cowpoxvirus CrmA or baculovirus p35, or aldehydes or uoromethyl ketone-derivatized synthetic peptide inhibitors based on preferred substrate sequences (e.g. ZVAD-fmk), suppress many aspects of mammalian apoptosis. Caspases share many common structural and catalytic features. All contain an active site pentapeptide sequence with the general structure QACXG (where X is R, Q or G): the cysteine (together with a distant histidine) is directly involved in catalysis. In the main, caspases recognize a tetrameric primary sequence in their substrates with a distinctive requirement for an aspartic acid residue in the substrate P 1 position. As tetrapeptides corresponding to the substrate P 4^P 1 residues are su cient for speci c recognition by caspases [45], such substrates have provided the basis for design of a range of inhibitors [46]. Phylogenetic analysis of the caspases shows they fall into three subfamilies, each with signi cantly di ering substrate speci city which generally correlates with caspase function: the ICE subfamily of cytokine processors (caspases-1, -4, -5 and -11; because of sequence homology to caspase-1 the caspases-12, -13 and -14 are also grouped with the cytokine processors), the Ced-3/CPP32 subfamily of apoptotic executioners (caspases-3, -6, and -7) and

M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 F5 the ICH-1/Nedd-2 subfamily of apoptotic initiators (caspase-2, -8, -9 and -10). Caspases are synthesized as precursor proenzymes which are proteolytically processed to their active forms. Active caspases are composed of a heterodimer comprising a large subunit (P20 for caspase- 1, P17 for caspase-3) that contains the catalytic cysteine residue, and a smaller subunit (P10 for caspase- 1, P12 for caspase-3) that contains determinants which govern substrate speci city. The X-ray crystal structure of activated caspase-1 indicates that two independent functional P20/P10 heterodimers are intimately associated to form a (P20/P10) 2 tetramer in which the two active sites reside at opposite ends of the complex [47,48]. Procaspases are activated by cleavage at critical aspartate residues that themselves conform to the substrate consensus for caspases. This indicates that caspases exist within hierarchies of families that undergo auto- and trans-cleavage. For example, evidence supports the idea that caspase-8 autoactivates itself upon recruitment to a death receptor signaling complex (the zymogene full length caspase-8 molecule obviously possesses some residual proteolytic activity su cient for this selfcleavage) and the active enzyme then in turn cleaves and activates the e ector caspases-3 and -7 that execute the apoptotic program. Interestingly, caspase-9 does not necessarily require proteolytic processing, but instead requires binding to APAF-1 with which it forms an active holoenzyme [49,50]. All procaspases have an N-terminal prodomain that is also removed during activation. For some caspases (caspase-3, -6, -7 and -14) the prodomain is short (10^40 residues) whilst for the other caspases it is extensive and contains recognizable domains. The extensive prodomains play important roles in caspase regulation and function as signal integrators for apoptotic or pro-in ammatory signals [51,52]. For example, the prodomains of caspase-8 and -10 each contain two so-called death e ector domains that mediate the proenzymes' interaction, via an adapter molecule, with the cytoplasmic tail of members of the TNF-R1/CD95 receptor family. This allows receptor-induced activation of the caspases in response to ligand binding. Caspases-1, -2, -4, -9 and Ced-3 possess a distinct prodomain structure termed CARD (caspase recruitment domain). CARD domains (also found in the adapter protein APAF-1) are presumed to mediate speci c intermolecular interactions that regulate caspase activation. Indeed, one strongly inactivating mutation of Ced-3 is located within its prodomain [53], indicating the importance of this region for Ced-3 activation. CARDs and death e ector domains as well as death domains, all contain six anti-parallel K-helices arranged in a similar three-dimensional fold and associate via like^like interactions [32]. Genetic knockout studies in mice have been used to investigate the measure of redundancy amongst caspases [54]. Available data suggest that apoptosis triggered by di ering stimuli frequently employ different `subsets' of activated caspases. Thus, mice de- cient in caspase-1 develop normally, are fertile, appear healthy and exhibit no apparent abnormalities, suggesting absence of any gross defects in normal physiological processes involving apoptosis [41,55]. However, thymocytes from the caspase-1 null mice exhibit partial resistance to CD95-induced apoptosis, implying a role for caspase-1 in implementing apoptosis in response to that particular trigger. Caspase- 11 knockout mice also show defective interleukin-1l production but develop normally and have minimal apoptotic defects [43]. Mice lacking caspase-3 are smaller than their wildtype littermates and die at 1^3 weeks of age [56]. Analysis shows that the development of the brain in these animals is markedly a ected: a variety of central nervous system hyperplasias are observed from embryonic day 12 on, indicative of defective apoptosis. However, thymocytes from caspase-3 null mice exhibit normal sensitivity to apoptosis induced by a number of di erent stimuli and the rather tissue-restricted phenotype in caspase-3-de cient mice again demonstrates that other caspases can substitute for caspase-3 in most tissue or cell types. Animals de cient in caspase-8 or caspase-9 die perinatally because of profound defects in developmental cell deaths [57^59]. Caspase-2-de cient mice develop normally, but cells from these animals show diminished or enhanced apoptosis, depending on their tissue of origin [60]. Caspase-12 is localized to the ER and becomes activated by ER stress. Mice that are de cient in caspase-12 are resistant to ER stress-induced apoptosis, but their cells undergo apoptosis in response to other death stimuli [61]. Caspase substrates [62,63] can be grouped into dif-

F6 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 ferent classes according to their (putative) function. Amongst these are pro- and anti-apoptotic proteins, components of the apoptotic machinery, structural proteins, homeostatic proteins and proteins important for signaling, cellular repair and macromolecular synthesis [32]. The following examples represent these di erent classes of caspase substrates. (1) Transactivation of procaspases by already activated caspases could generate su cient proteolytic activity to overwhelm endogenous caspase inhibitors such as inhibitor of apoptosis proteins (IAPs). (2) Caspase-3 cleaves Bcl-2 and Bcl-x L which destroys the anti-apoptotic function of these proteins and releases C-terminal fragments that are pro-apoptotic [64,65]. (3) Caspase-8 cleaves Bid, a pro-apoptotic Bcl-2 family member, generating a C-terminal fragment that induces release of mitochondrial cytochrome c [66,67]. (4) Caspase-3 cleaves ICAD/DFF45 allowing the ICAD-bound nuclease CAD to translocate to the nucleus and to cut DNA [68^70]. (5) Caspase-3 also cleaves and activates Gelsolin, a protein that regulates actin dynamics and promotes both cytoplasmic and nuclear apoptosis, including DNA fragmentation [71]. (6) Lamins are major structural proteins within the nuclear envelope, and their cleavage by caspase-6 may be responsible for some of the observed nuclear changes [72]. (7) Cleavage of L-catenin and FAK might interrupt cell^cell contacts and cell^matrix focal adhesions thereby promoting cellular packaging and phagocytosis [73,74]. (8) Poly(- ADP-ribose) polymerase (PARP) cleavage may interfere with its key homeostatic function as a DNA double-strand break repair enzyme [75] which might facilitate or allow the DNA degradation characteristic of apoptosis [33]. However, it is worth noting that PARP null mice seem to develop normally [76]. Unfortunately, in the case of many caspase substrates it is not always obvious what, if any, mechanistic role their cleavage plays in apoptosis. Many caspase substrates are `plausible' candidate e ectors for apoptosis, but so far no `key' target has been found whose cleavage appears to provide the ultimate `killer' cut for the cell. Nonetheless, given the central role of caspases in the apoptotic program, these proteases o er obvious therapeutic targets for the control of inappropriate apoptosis [46]. Caspase over-reactivity promotes cellular suicide, and this may be the basis for degenerative disorders such as Huntington's disease and Alzheimer's disease: caspase-3 and -12 seem to be involved in proteolytic cleavage of Alzheimer amyloid-l precursor protein and formation of the apoptosis-inducing amyloidogenic AL peptide [61,77]. The polyglutamine repeats associated with Huntington's disease induce neuronal cell death via caspase-8 [78]. Caspase-10 loss of function mutations have been linked to defective cell death in autoimmune lymphoproliferative syndrome type II [79]. Caspase inactivation may also promote oncogenesis [80]. However, there is one caveat: some triggers of apoptosis retain the ability to kill cells even when caspases are inhibited [81^84], although it is not clear what mechanisms are involved in such caspase-independent cell death. The CD95/ Fas/Apo-1 receptor is able to kill activated primary T-cells in the absence of active caspases (see below); this cell death involves necrotic morphological changes and depends on the kinase Rip as e ector molecule [85]. 4.2. The ancestral pathway: the role of mitochondria, cytochrome c and Apaf-1 in mammalian apoptosis Ideas concerning the mechanism of activation and control of apoptosis have been greatly in uenced by the recent discovery that cytochrome c is released from mitochondria during cell death and is involved in triggering the e ector machinery of apoptosis [23,86^89]. Cytochrome c normally resides in the space between the outer and inner membranes of mitochondria where it participates in the process of oxidative phosphorylation. Upon exposure of cells to apoptotic stimuli, cytochrome c is released from mitochondria into the cytosol, where it is one of several factors implicated in the proteolytic activation of caspase-3 by caspase-9 [90]. Biochemical analysis has identi ed two cytosolic proteins, Apaf-1 and Apaf-3, that form the complex with cytochrome c that activates caspase-3. Apaf-1 shares limited homology with the product of the C. elegans ced-4 gene product [37], although it is a larger and more complex protein, while Apaf-3 is caspase-9 [91]. The Ced-4-like domain in Apaf-1 is anked on one side by a region with strong homology to the CARD motif within the prodomains of Ced-3 and mammalian caspases-2 and -9 and on the other side by several WD-40 repeats believed to me-

M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 F7 diate protein^protein interactions. The CARD domains in Apaf-1 and the prodomain of caspase-9 interact and, in the presence of cytochrome c and either ATP or ADP, this induces autocatalytic activation of the caspase which then activates the downstream caspase e ector cascade involving caspases-2, -3, -6, -7, -8 and -10 [90]. Apaf-1 dimerization is repressed by its own C-terminus containing the WD-40 repeats. It is speculated that cytochrome c binding to the WD repeats then induces a conformational change that allows Apaf-1 to oligomerize and by promoting clustering of this caspase to activate caspase-9 [92]. The Apaf-1/caspase-9 interaction is clearly reminiscent of the mechanism of Ced-3 activation by Ced-4 in C. elegans. This similarity extends to the involvement of the mammalian anti-apoptotic Bcl-2 and Bcl-x L, proteins which are homologues of Ced-9. Bcl-2/Bcl-x L reside in the outer mitochondrial membrane, where they function to suppress apoptosis in both or either of two ways: blocking cytochrome c release and binding to Apaf-1 to prevent its activating caspase-9. The mammalian pro-apoptotic Bcl-2 family members, such as Bax, Bak and Bik, may promote apoptosis by displacing Apaf-1 from Bcl- 2/x L. Nevertheless, although a direct binding of Bcl-x L to Apaf-1 (shown by in vitro experiments) has been reported [93,94], these data (or at least their physiological relevance) have been questioned recently [95]. The anti-apoptotic protein Aven, which was identi ed in a yeast two-hybrid screen, has been shown to bind to both Bcl-x L and Apaf-1 and this molecule might link the two molecules and target the Bcl-2 family member to the apoptosome [96]. The fact that direct Bcl-x L or Bcl-2 binding to Apaf-1 could not be observed in vivo might also indicate that other Ced-4 homologues exist which could bind to anti-apoptotic Bcl-2 family members. The idea of further Ced-4 family members is supported by the murine Apaf-1 knockout which leads to an impairment of apoptosis in some, but not all circumstances and cell types [97,98]. Targeted inactivation of Apaf-1 in mice nevertheless leads to profound developmental abnormalities in cell number regulation in the brain as well as in other tissues such as the peripheral nervous system, resulting in embryonic lethality. By using green uorescent protein (GFP)-tagged cytochrome c transfected into HeLa cells it was recently demonstrated that the release of cytochrome c-gfp always precedes exposure of phosphatidylserine at the cell surface and the loss of plasma membrane integrity [99]. The time interval between death stimulus and cytochrome c release can vary in individual cells (and depending on the apoptotic insult) but once initiated, cytochrome c is released from all mitochondria in individual cells within 5 min. This study also showed that the drop in the mitochondrial membrane potential typically seen in apoptotic cells occurs later than cytochrome c release from mitochondria and that this process is dependent on caspase activation, whereas cytochrome c release is not. These results suggest a speci c permeability of the outer mitochondrial membrane without alteration of the inner mitochondrial membrane. So far, several competing models exist to explain exactly how permeabilization of mitochondrial membranes is mediated during apoptosis (see Fig. 2) [88]: the outer mitochondrial membrane might rupture as a result of swelling of the mitochondrial matrix. In one model swelling is postulated to result from opening of a megachannel called the permeability transition pore (PTP). The adenine nucleotide translocator (ANT; located in the inner mitochondrial membrane) and the voltage-dependent anion channel (VDAC, found in the outer mitochondrial membrane) are major components of the PTP which is proposed to span both the inner and the outer mitochondrial membranes at sites at which the two membranes are opposed. According to the PTP model, PTP openers, including the pro-apoptotic Bcl-2 family member Bax, cause permeabilization of the inner membrane and mitochondrial depolarization by binding to the ANT [100] (although this is not supported by the data mentioned above). This process allows entry of water and solutes into the matrix and leads to mitochondrial swelling. Another model postulates that swelling is due to a defect in mitochondrial ATP/ADP exchange as a result of closure of the VDAC thus leading to hyperpolarization of the inner mitochondrial membrane and subsequent matrix swelling. Again such a scenario contradicts reports stating that the drop in membrane potential, at least in some cell types, follows the release of cytochrome c. Other models do not predict damage of the outer

F8 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 Fig. 2. Di erent models explain the release of cytochrome c from mitochondria during apoptosis. The outer mitochondrial membrane might rupture as a result of swelling of the mitochondrial matrix. This could be explained with opening of the permeability transition pore (PTP) or with the closure of the voltage-dependent anion channel (VDAC). Other models relate cytochrome c release to pore formation allowing the passage of cytochrome c into the cytosol. mitochondrial membrane but rather the formation of a pore in this membrane allowing the passage of cytochrome c (and other mitochondrial proteins) into the cytosol. Bax is a candidate for the formation of this pore. Bax oligomers can form large conductance channels in lipid planar bilayers [88]. Addition of Bax directly to isolated mitochondria triggers release of cytochrome c through a mechanism that is insensitive to PTP blockers and does not involve mitochondrial swelling. Yet another model involves Bax cooperating with the VDAC to form a cytochrome c-conducting channel [101]. Nevertheless, direct evidence for the formation of such pore structures in mitochondria during apoptosis is still missing. An interesting link between death receptor-activated apical caspases such as caspase-8 and mitochondrial cytochrome c release has been established in the form of the BH3-domain-only protein Bid: caspase-8 ^ initially activated at the death-inducing signaling complex (DISC) of cell surface death receptors ^ cleaves the pro-apoptotic Bcl-2 family member Bid [66,67]. Cleaved Bid (tbid) then binds to Bax leading to Bax oligomerization and integration into the outer mitochondrial membrane where it triggers cytochrome c release [102]. Similarly, tbid binds to and oligomerizes another pro-apoptotic Bcl-2 homologue, Bak, resulting in cytochrome c release [103]. While studies in bak knockout cells show that tbid does not require Bak for mitochondrial targeting, Bak proved necessary for tbid-induced cytochrome c release. Consequently, bax3/3bak3/3 double knockout cells are resistant to a wide range of apoptotic stimuli [104]. Several proteins in addition to cytochrome c are released from mitochondria in cells induced to undergo apoptosis. Among them is the recently identi ed Smac/Diablo molecule which binds to, and inactivates, IAPs [105,106]. IAPs inhibit cell death by binding to procaspases and activated caspases, thereby blocking their processing and their activity. Smac/ Diablo is released from the mitochondria along with

M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 F9 cytochrome c during apoptosis and relieves inhibition of caspase-9 activation by IAP inactivation [107]. It is also possible that in some cells (type II cells) Smac/Diablo is required to inactivate an IAP preventing direct caspase-3 activation by caspase-8. In this scenario cytochrome c release might not be relevant for the death process, but rather Smac/Diablo liberation into the cytosol. Smac/Diablo and the pro-apoptotic Drosophila proteins Reaper, Grim and Hid seem to function in a similar way (by inhibiting IAP activity) and a sequence similarity among these proteins (restricted to their N-terminal 14 amino acids) has been reported [108] suggesting that Smac/Diablo and the insect apoptosis-inducing proteins might be structural as well as functional homologues. Mitochondrial integrity is important not only for sequestering cytochrome c and Smac/Diablo but also for other ways to regulate caspase activation and apoptosis [87]. A fraction of both caspase-9 and caspase-3 has been localized to the mitochondrial intermembrane space in some cell types, and caspase-2 has also been reported to reside in mitochondria. These caspases ^ like cytochrome c ^ can be released from the mitochondria to the cytosol during apoptosis induction. Another protein, AIF (apoptosis-inducing factor), also redistributes from mitochondria and induces some of the nuclear morphology associated with apoptosis in a caspase-independent manner [109]. Genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation and disables PCD during caviation of embryoid bodies in early mouse morphogenesis [110]. 4.3. The death receptor pathway Recently, a direct mechanistic link between a particular apoptotic stimulus and activation of the basal caspase apoptotic machinery has been forged: activation of a speci c group of transmembrane receptors of the tumor necrosis factor (TNF) receptor superfamily, either by ligand or (experimentally) by binding an agonistic antibody, can lead to direct activation of caspases. The list of TNF receptor family members is growing and includes TNF-R1 (P55), TNF-R2 (P75), TNF-R3 (TNF-RP), LT-KR, Ox- 40, CD27, CD28, CD30, CD40, 4-1BB, p75 NGF- R (low a nity nerve growth factor receptor), GIT-R [111], Rank, CD95, DR6 [112] and the newly discovered TRAIL receptors TRAIL-R1 (DR4), -R2 (DR5), -R3 (DcR-1) and DcR-2 [113]. Activation of members of this receptor family triggers a variety of cellular responses depending on cell type and context, amongst which are (T-cell) activation and stimulation, proliferation, di erentiation, survival and apoptotic cell death [114^116]. Mammalian TNF-R family members are type I membrane proteins characterized by conserved extracellular cysteine-rich domains. A functional TNF superfamily receptor is typically a trimeric or multimeric complex stabilized by disul de bonds, although some, such as CD95, TNF-R1 and TNF- R2, also exist in a soluble form generated by proteolytic cleavage [117]. The receptors' ligands comprise another related family that includes TNF, LT-K (lymphotoxin-k), CD95 ligand (FasL/CD95L), TRAIL, OX40L, CD27L, CD30L, CD40L, 4-1BBL and LT-L. Each of the ligands is synthesized as a nascent type II membrane-associated protein and shares a characteristic 150 amino acid region towards the C-terminus by which each ligand interacts with its cognate receptor. For the most part, these ligands exist as trimeric or multimeric membrane-bound proteins that may function to induce receptor aggregation. However, a few members, such as TNF and CD95L, are also functional in soluble form. Interestingly, a domain N-terminal to the ligand binding domain in the extracellular region of TNF-R1, TNF-R2 and CD95 was recently identi ed that mediated receptor self-association before ligand binding [118]. This pre-ligand binding assembly domain (PLAD) is critical for assembly of functional receptor complexes on the cell surface. Thus, TNF receptor family members might function as preformed complexes rather than as individual receptor subunits that oligomerize after ligand binding. A detailed discourse on the multiple pleiotropic cellular and physiological activities provoked by ligation of TNF receptor family members in various cell types is beyond the scope of this article. We will con ne ourselves to those receptors whose ligation has been shown to induce apoptosis ^ namely TNF-R1, CD27 [119], CD30, CD40, LT-LR, CD95, DR3, DR4, DR5 and DR6 [120]. A subgroup of these receptors shares a common intracellular protein^protein interaction domain, the so-called death

F10 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 domain. These receptors are referred to as death receptors and they include TNF-R1, CD95, DR3, DR4, DR5, and DR6. In particular, we shall summarize current knowledge concerning the CD95, TNF-R1 and the TRAIL receptors and the attendant molecules mediating their death signal transduction. 4.3.1. CD95/FAS/Apo-1 CD95/Fas/Apo-1, henceforth called CD95, is expressed in activated lymphocytes as well as in all other tissues such as the liver, heart and lung. Ligation of CD95, whether by its ligand CD95L or, experimentally, by agonistic antibody can induce apoptosis in several cell types. CD95L is predominantly expressed on activated lymphocytes, NK cells, erythroblasts and immune privilege tissues, but also on certain tumors. The CD95/CD95L apoptotic pathway also functions to maintain homeostasis in various tissues ^ the liver being a particularly well documented example [121]. However, the biological role of CD95 is probably best understood in the immune system, where it is implicated in peripheral clonal deletion of T-lymphocytes, activation-induced suicide of mature T-cells, cytotoxic response and induction of apoptosis in B-cells. Constitutive cell surface expression of CD95L also seems to contribute to immunological privilege of certain organs by killing in ltrating lymphocytes and in ammatory cells expressing CD95 receptor [122]. Mice carrying mutations in the genes for CD95 (lpr for lymphoproliferation) and CD95L (gld for generalized lymphoproliferative disease) have been identi ed. Mice homozygous for either of these mutations accumulate an excess of non-malignant CD3 B220 CD4 3 CD8 3 T-cells in their spleens and lymph nodes and also su er from an autoimmune systemic lupus erythematosus-like condition. This demonstrates that CD95/ CD95L signaling ful ls an important function in deleting autoreactive lymphocytes and maintaining peripheral tolerance. Mutations in the human CD95 gene cause a similar lympho-accumulative syndrome [123]: patients with autoimmune lymphoproliferative syndrome type 1A have heterozygous CD95 germline mutations and their lymphocytes are resistant to CD95-induced apoptosis [124]. No identi able catalytic motifs are present in the cytoplasmic domains of the CD95/TNF-R1 receptors. Rather, signal transduction is mediated via direct recruitment of, and intermolecular association with, various downstream signaling e ector molecules [51]. In this regard, a key intracellular interaction domain present in the cytoplasmic tail of all death receptors is the 65 amino acid `death domain' (DD), a name deriving from its ability to recruit downstream e ectors that can induce apoptosis [125]. However, the term `death domain' is somewhat unfortunate, since it implies that cell death is the generic function of this type of motif. In fact, DDs are domains that mediate homo- and heterotypic protein^protein interactions in order to propagate signals, and they have since been found in signaling pathways that have no obvious link with cell death [126]. The DDs in the ligated TNF-R1/CD95 receptors recruit the C-terminal DD of the cytoplasmic adapter FADD/MORT-1. At its N-terminus, FADD/MORT possesses a di erent protein binding domain, a `death e ector domain', that mediates interaction with the N-terminal prodomain of caspase-8 [127,128]. The recruitment of caspase-8 by FADD/ MORT to the activated CD95 receptor generates a DISC [129] that leads to proteolytic autoactivation of caspase-8. Caspase-8 then activates other caspases, including caspase-1 and caspase-3, which then are presumed to execute the apoptotic dissolution of the cell [130]. Members of the TNF receptor family which lack a death domain (e.g. TNF-R2, CD27, CD30, CD40) are also under certain circumstances able to induce cell death via alternative mechanisms [120]. CD95, through recruitment of the DISC, appears to provide a direct link between external ligand and the basal e ector machinery of apoptosis. However, it has recently become clear that this direct molecular cantilever only seems to operate in certain cell types ^ type 1 cells [131]. In other (type 2) cells, CD95 leads to changes in mitochondria that activate downstream caspases in a di erent way. The amount of receptor-activated caspase-8 in type 2 cells is much lower than in type 1 cells [132] and probably insu cient to induce downstream e ector caspase cleavage. It nevertheless is enough to cleave Bid, a BH3 domain-only member of the Bcl-2 family [66,67]. Truncated Bid then translocates to the mitochondria where it induces cytochrome c release and conse-

M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 F11 quently further caspase activation and nally cell death. The DDs of both CD95 and TNF-R1 interact with the C-terminal DD of a second receptor-associated protein, designated RIP [133]. The RIP N-terminus resembles a tyrosine kinase domain which is intriguing because experimental data implicate a tyrosine kinase in CD95-mediated signal transduction. Pharmacologic inhibitors of protein kinases block, in a concentration-dependent manner, CD95-induced DNA fragmentation and prolong cell survival [134]. The DD of RIP also binds to the C-terminal DD of another `death adapter protein', RAIDD (for RIPassociated ICH-1/Ced-3 homologous protein with a death domain). At its N-terminus, RAIDD is homologous to, and oligomerizes with, the prodomain of caspase-2 (Ich-1). Thus, caspase-2 can be recruited to the CD95 receptor through sequential interactions of RAIDD, RIP, FADD and CD95 [115]. A close relative of RAIDD is CRADD, which also interacts with both RIP and caspase-2 [135]. RIP is required for TNF-induced NF-UB activation. Cleavage of RIP by caspase-8 results in the blockage of NF-UB-mediated anti-apoptotic signals [136]. Recently, another CD95 binding protein, Daxx, has been described [137]. Daxx also binds to the CD95 death domain but lacks a death domain of its own. Overexpression of Daxx activates Jun N-terminal kinase (JNK) and potentiates CD95-induced apoptosis. On this basis, it has been proposed that CD95 engages two independent pathways that induce cell death: one pathway via FADD/caspase-8/ 2 and the other via Daxx/JNK activation. Interestingly, Daxx is a nuclear protein that interacts and colocalizes with the tumor-suppressive promyelocytic leukemia protein PML in nuclear bodies [138]. Reporter gene assays show that DAXX is able to repress basal transcription; SUMO-1-modi ed PML sequesters DAXX to the nuclear bodies and inhibits Daxx-mediated transcriptional repression. How precisely CD95 activation acts on Daxx localization, and its in uence on transcription, is presently unclear. Strangely enough, rather than showing decreased apoptosis, inactivation of Daxx results in extensive apoptosis and embryonic lethality in mice [139]. Among the proteins which have been shown to bind to the cytosolic domain of the CD95 receptor is the Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase FIST/HIPK3 [140]. FIST not only binds to CD95 but also interacts with FADD in a trimolecular complex composed of CD95, FADD and FIST. FIST kinase induces FADD phosphorylation and inhibits CD95- mediated JNK kinase activation. It is localized both in the cytoplasm and in the nucleus and is capable of binding to Daxx in a kinase activity-dependent manner. In addition to activation of caspases and JNKs, both CD95 and TNF-R1 trigger other signaling effectors. CD95-generated apoptotic signals activate acidic sphingomyelinase causing accumulation of ceramide [141] which is observed in both CD95- and TNF-R1-induced apoptosis. Whether ceramide production is a major determinant of the apoptotic decision is still a matter of debate. Naturally occurring inhibitors of the CD95/TNF- R1 death signaling pathways exist in the guise of the FLIPs (Fas-associated death domain-like ICE inhibitory proteins) which interfere with recruitment of caspases to the CD95/TNF-R1 signaling complexes. A number of viruses encode FLIPs as part of their strategy for manipulating host cell suicide and viability. For example, the Q-herpesviruses encode FLIPs that comprise two death e ector domains which interact with FADD/MORT and inhibit its recruitment and activation of caspase-8 [142]. Recently, a cellular homologue of v-flip was identi ed by di erent groups [143^150]. c-flip is structurally similar to caspase-8 since it contains two death e ector domains and an inactive caspase-like domain lacking the conserved functional cysteine residue. c-flip is expressed in two isoforms (long and short form), both of which are recruited to the CD95 DISC in a stimulation-dependent fashion. c-flip blocks caspase-8 activation at the DISC and thereby inhibits CD95-mediated apoptosis [151]. During this process, both caspase-8 and c-flip undergo cleavage between the p18 and p10 subunits, generating two stable intermediates of 43 kda that stay bound to the DISC. B- and T-cells downregulate c-flip upon activation in vitro [152], providing a possible explanation for the observation that resting peripheral T-cells are resistant to CD95-induced apoptosis and become susceptible only after their activation. By inhibiting

F12 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37 death receptor-mediated cell death, c-flip has been identi ed as a tumor progression factor in mouse models [153,154]. Several groups found a pro-apoptotic function of c-flip in transient overexpression studies [146], the physiological relevance of which is presently unclear [151]. Another way to inhibit death ligand-induced apoptosis is to quench the signal via decoy receptors. A soluble CD95 decoy receptor (DcR3) has been discovered that binds to CD95L and inhibits CD95Linduced apoptosis [155,156]. The physiological importance of such signal inhibition is underlined by the nding that the DcR3 gene was ampli ed in about half of the primary lung and colon tumors studied. Knowledge of the downstream e ectors involved in CD95 death signaling has facilitated analysis of the role of CD95 in vivo. As discussed above, mice with inactivating mutations in the genes for either CD95 (lpr) or CD95L (gld) exhibit generalized lymphoproliferative disease. The cowpoxvirus caspase inhibitor CrmA, which e ciently blocks caspase-8 (as well as other caspases such as caspase-1), has been expressed transgenically in peripheral T-lymphocytes via the CD2 promoter. Such CD2-crmA transgenic mice exhibit resistance to CD95-induced apoptosis equivalent to that seen in lpr mice [157] although neither Q-radiation- nor corticosteroid-induced cell death is suppressed. However, in contrast to lpr mice, CD2-crmA transgenic mice develop neither T-cell hyperplasia nor serum autoantibodies, implying that the lpr phenotype is not merely due to failure of CD95 to trigger caspase-dependent T-cell apoptosis. Expression of a dominant negative mutant of FADD in T-lymphocytes also severely repressed CD95 killing yet failed to cause accumulation of peripheral T-cells as seen in lpr and gld mice [158,159]. Mice with a deletion in the FADD gene die at day 11.5 of embryogenesis; their phenotype suggests that FADD is essential for embryo development and signaling from some (but not all) inducers of apoptosis [160]. Interestingly, inactivation of FADD by expression of a FADD dominant negative molecule or by gene targeting leads to impairment of activation-induced T-cell proliferation [158,159,161,162]. CD95 is also interesting in another aspect of tumor therapy: several anticancer drugs have been shown to sensitize certain cell types to apoptosis by upregulating CD95 or CD95L [163,164] although the generality of this concept has been questioned [165,166]. 4.3.2. TNF receptors TNF is well recognized as a cytokine produced by activated T-cells and macrophages that orchestrates aspects of the host in ammatory response. It does so by in uencing the proliferation, di erentiation and apoptosis of cells involved in in ammation. TNF (together with the lymphotoxin LT) is the ligand for two receptors ^ TNF-R1 and TNF-R2. TNF- R1 alone appears to be able to mediate most, if not all, of the biological responses engendered by TNF, although TNF-R2 may provide an auxiliary function in cooperating in the binding of TNF to TNF-R1 [167]. Genetic deletions of both receptors have demonstrated the di erences in biological functionality of TNF-R1 and TNF-R2 in vivo [168^170]. Although both act to potentiate in ammation/host defense and share the common ability to activate the pleiotropic transcription factor NF-UB [171], TNF-R1 alone can clearly trigger apoptosis whereas TNF-R2 mainly seems to promote cell survival, although it was shown to kill certain cells when overexpressed [120]. However, substantial evidence indicates that TNF-R1 can also promote cell survival under certain circumstances, although this anti-apoptotic activity, unlike activation of the caspase cascade, appears to be indirect and require de novo synthesis of survival proteins. TNF-R1 signaling, like CD95, is able to activate the proteolytic caspase cascade by recruiting caspase- 8 via FADD/MORT. Although the FADD/MORT adapter molecule does not bind directly to TNF-R1, it is recruited to the activated receptor via an intermediary cytoplasmic DD adapter called TRADD (TNF-R-associated death domain). TRADD also binds RIP, thereby linking TNF-R1 to caspase-2 activation via RAIDD and CRADD. Both TNF-R1 and TNF-R2 recruit another class of signaling adapter molecules, called TRAFs (TNFR-associated factors) of which six are currently identi ed [172]. Certain of the TRAFs mediate activation of JNK or NF-UB [173], the latter by interaction with the downstream signaling kinase NIK. NIK, in turn, activates the IUB kinases which phosphorylate and inactivate IUB, the endogenous cellular inhibitor of NF-UB