key: cord-020757-q4ivezyq
authors: Saikumar, Pothana; Kar, Rekha
title: Apoptosis and Cell Death: Relevance to Lung
date: 2010-05-21
journal: Molecular Pathology of Lung Diseases
DOI: 10.1007/978-0-387-72430-0_4
sha: 
doc_id: 20757
cord_uid: q4ivezyq

In multicellular organisms, cell death plays an important role in development, morphogenesis, control of cell numbers, and removal of infected, mutated, or damaged cells. The term apoptosis was first coined in 1972 by Kerr et al.1 to describe the morphologic features of a type of cell death that is distinct from necrosis and is today considered to represent programmed cell death. In fact, the evidence that a genetic program existed for physiologic cell death came from the developmental studies of the nematode Caenorhabditis elegans.2 As time has progressed, however, apoptotic cell death has been shown to occur in many cell types under a variety of physiologic and pathologic conditions. Cells dying by apoptosis exhibit several characteristic morphologic features that include cell shrinkage, nuclear condensation, membrane blebbing, nuclear and cellular fragmentation into membrane-bound apoptotic bodies, and eventual phagocytosis of the fragmented cell (Figure 4.1).

In multicellular organisms, cell death plays an important role in development, morphogenesis, control of cell numbers, and removal of infected, mutated, or damaged cells. The term apoptosis was fi rst coined in 1972 by Kerr et al. 1 to describe the morphologic features of a type of cell death that is distinct from necrosis and is today considered to represent programmed cell death. In fact, the evidence that a genetic program existed for physiologic cell death came from the developmental studies of the nematode Caenorhabditis elegans. 2 As time has progressed, however, apoptotic cell death has been shown to occur in many cell types under a variety of physiologic and pathologic conditions. Cells dying by apoptosis exhibit several characteristic morphologic features that include cell shrinkage, nuclear condensation, membrane blebbing, nuclear and cellular fragmentation into membrane-bound apoptotic bodies, and eventual phagocytosis of the fragmented cell (Figure 4 .1).

Cell death is central to the normal development of multicellular organisms during embryogenesis and maintenance of tissue homeostasis in adults. 3 During development, sculpting of body parts is achieved through selective cell death, which imparts appropriate shape and creates required cavities in particular organs. In adults, cell death balances cell division as a homeostatic mechanism regulating constancy of tissue mass. Deletion of injured cells because of disease, genetic defects, aging, or exposure to toxins is also achieved by apoptosis. In essence, apoptotic cell death has important biologic roles not only in development and homeostasis but also in the pathogenesis of several disease processes.

Dysregulation of apoptosis is found in a wide spectrum of human diseases, including cancer, autoimmune diseases, neurodegenerative diseases, ischemic diseases, viral infections, 4 and lung diseases. 5 Our knowledge of cell death and the mechanisms of its regulation increased dramatically in the past two decades with the discovery Nevertheless, necrosis has been shown to occur in cells having defects in apoptotic machinery or upon inhibition of apoptosis, 7 and this form of cell death is emerging as an important therapeutic tool for cancer treatment. 8 Autophagy Autophagy, which is also referred to as type II programmed cell death, is characterized by sequestration of cytoplasm and organelles in double or multimembrane structures called autophagic vesicles, followed by degradation of the contents of these vesicles by the cell's own lysosomal system (see Figure 4 .1). The precise role of autophagy in cell death or survival is not clearly understood. Autophagy has long been regarded as a cell survival mechanism whereby cells eliminate long-lived proteins and organelles. In this regard, it is argued that autophagy may help cancer cells survive under nutrientlimiting and low-oxygen conditions and against ionizing radiation. 9,10 However, recent observations that there is There is early membrane damage with eventual loss of plasma membrane integrity and leakage of cytosol into extracellular space. Despite early clumping, the nuclear chromatin undergoes lysis (karyolysis). Apoptosis: Cells die by type I programmed cell death (also called apoptosis); they are shrunken and develop blebs containing dense cytoplasm. Membrane integrity is not lost until after cell death. Nuclear chromatin undergoes striking condensation and fragmentation. The cytoplasm becomes divided to form apoptotic bodies containing organelles and/or nuclear debris. Terminally, apoptotic cells and fragments are engulfed by phagocytes or surrounding cells. Autophagy: Cells die by type II programmed cell death, which is characterized by the accumulation of autophagic vesicles (autophagosomes and autophagolysosomes). One feature that distinguishes apoptosis from autophagic cell death is the source of the lysosomal enzymes used for most of the dying-cell degradation. Apoptotic cells use phagocytic cell lysosomes for this process, whereas cells with autophagic morphology use the endogenous lysosomal machinery of dying cells. Paraptosis: Cells die by type III programmed cell death, which is characterized by extensive cytoplasmic vacuolization and swelling and clumping of mitochondria, along with absence of nuclear fragmentation, membrane blebbing, or apoptotic body formation. Autoschizis: In this form of cell death, the cell membrane forms cuts or schisms that allow the cytoplasm to leak out. The cell shrinks to about one-third of its original size, and the nucleus and organelles remain surrounded by a tiny ribbon of cytoplasm. After further excisions of cytoplasm, the nuclei exhibit nucleolar segregation and chromatin decondensation followed by nuclear karyorrhexis and karyolysis. decreased autophagy during experimental carcinogenesis and heterologous disruption of an autophagy gene, Beclin 1 (Bcn1), in cancer cells 11, 12 suggest that breakdown of autophagic machinery may contribute to development of cancer. Other interesting studies have shed some light on the relationship between autophagy and apoptosis. These investigations have shown prevention of caspase inhibitor z-VAD-induced cell death in mouse L929 cells by RNA interference directed against autophagy genes atg7 and Bcn1 13 and protection of Bax −/− , Bak −/− murine embryonic fi broblasts against staurosporine-or etoposide-induced cell death by RNA interference against autophagy genes atg5 and Bcn1. 14 However, both of these studies were done in cells whose apoptotic pathways had been compromised. Thus, it remains to be seen whether cells with intact apoptotic machinery can also die by autophagy and whether apoptotic-competent cells lacking autophagy genes will be resistant to different death stimuli.

Paraptosis has recently been described as a form of cell death characterized by extensive cytoplasmic vacuolation (see Figure 4 .1) caused by swelling of mitochondria and endoplasmic reticulum. This form of cell death does not involve caspase activation, is not inhibited by caspase inhibitors, but is inhibited by the inhibitors of transcription and translation, actinomycin D, and cycloheximide, respectively, 15 suggesting a requirement for new protein synthesis. The tumor necrosis factor receptor family TAJ/TROY and the insulin-like growth factor I receptor have been shown to trigger paraptosis. 16 Paraptosis appears to be mediated by mitogen-activated protein kinases and inhibited by AIP1/Alix, a protein interacting with the calcium-binding death-related protein ALG-2. 16 Autoschizis Autoschizis is a recently described type of cell death that differs from apoptosis and necrosis and is induced by oxidative stress. 17 In this type of death, cells lose cytoplasm by self-morsellation or self-excision (see Figure  4 .1). Autoschizis usually affects contiguous groups of cells both in vitro and in vivo but can also occasionally affect scattered individual cells trapped in subcapsular sinuses of lymph nodes. 18 The nuclear envelope and pores remain intact while the cytoplasm is reduced to a narrow rim surrounding the nucleus. The chromatin marginates along the nuclear membrane, and mitochondria and other organelles around the nucleus aggregate as a result of cytoskeletal damage and condensation of the cytosol. Interestingly, the rough endoplasmic reticulum is preserved until the late stages of autoschizis, in which cells fragment and the nucleolus becomes condensed and breaks into smaller fragments. 19 Eventually, the nuclear envelope and the remaining organelles dissipate with cell demise.

Genetic studies in the nematode worm C. elegans led to the characterization of apoptosis. Activation of specifi c death genes during the development of this worm results in death of exactly 131 cells, leaving 959 cells intact. 2 Further studies revealed that apoptosis can be divided into three successive stages: (1) commitment phase, in which death is initiated by specifi c extracellular or intracellular signals; (2) execution phase; and (3) clean-up phase, in which dead cells are removed by other cells with eventual degradation of the dead cells in the lysosomes of phagocytic cells. 20 The apoptotic machinery is conserved through evolution from worm to human. 21 In C. elegans, execution of apoptosis is mediated by CED-3 and CED-4 proteins. Commitment to a death signal results in the activation of CED-3 by CED-4 binding. The CED-9 protein prevents activation of CED-3 by binding to CED-4. 22, 23 Mechanisms of Apoptosis Caspases Studies over the past decade have indicated that two distinct apoptotic pathways are followed in mammalian systems: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. The executioners in both intrinsic and extrinsic pathways of cell death are the caspases, 24 which are cysteine proteases with specifi city to cleave their substrates after aspartic acid residues. The central role of caspases in apoptosis is underscored by the observation that apoptosis and all classic changes associated with apoptosis can be blocked by inhibition of caspase activity. To date, 12 mammalian caspases (caspase-1 to -10, caspase-14, and mouse caspase-12) have been identifi ed. 25 Caspase-13 was later found to represent a bovine homolog and caspase-11 appears to be a murine homolog of human caspases-4 and -5, respectively.

Caspases are normally produced as inactive zymogens containing an N-terminal prodomain followed by a large and a small subunit that constitute the catalytic core of the protease. They have been categorized into two distinct classes: initiator and effector caspases. The upstream initiator caspases contain long N-terminal prodomains and one of the two characteristic protein-protein interaction motifs: the death effector domain (DED; caspase-8 and -10) and the caspase activation and recruitment domain (caspase-1, -2, -4, -5, -9, and -12). The downstream effector caspases (caspase-3, -6, and -7) are characterized by the presence of a short prodomain. Apart from the structural differences, a prominent difference between initiator and effector caspases is their basal state. Both the zymogen and the activated forms of effector caspases exist as constitutive homodimers, whereas initiator caspase-9 exists predominantly as a monomer both before and after proteolytic processing. 26 Initiator caspase-8 has been reported to exist in an equilibrium between monomers and homodimers. 27 Although the initiator caspases are capable of autocatalytic activation, the activation of effector caspases requires formation of oligomeric complexes with their adapter proteins and often intrachain cleavage within the initiator caspase.

Caspases have also been divided into three categories based on substrate specifi city. 28 Group I members (caspase-1, -4, and -5) have a substrate specifi city for the WEHD sequence with high promiscuity; group II members (caspase-2, -3, and -7 and CED-3) prefer the DEXD sequence and have an absolute requirement for aspartate (D) at P4; and members of group III (caspase-6, -8, and -9 and the "aspase" granzyme B) have a preference for (I/ L/V)EXD sequences. Several reports have suggested a role for group I members in infl ammation and that of group II and III members in apoptotic signaling events.

The extrinsic pathway involves binding of death ligands such as tumor necrosis factor-α (TNF-α), CD95 ligand (Fas ligand), and TNF-related apoptosis-inducing ligand (TRAIL) to their cognate cell surface receptors TNFR1, CD95/Fas, TRAIL-R1, TRAIL-R2, and the DR series of receptors, 29 resulting in the activation of initiator caspase-8 (also known as FADD-homologous ICE/CED-3-like protease or FLICE) and subsequent activation of effector caspase-3 ( Figure 4 .2). 30 The cytoplasmic domains of death receptors contain the "death domain," which plays a crucial role in transmitting the signal from the cell's surface to intracellular signaling molecules. Binding of the ligands to their cognate receptors results in receptor trimerization and recruitment of adapter proteins to the cell membrane, which involves homophilic interactions between death domains of the receptors and the adapter proteins. The adapter protein for the receptors TNFR1 and DR3 is TNFR-associated death domain protein (TRADD) 31 and that for Fas, TRAIL-R1, TRAIL-R2, and DR4 is Fas-associated death domain protein (FADD). 32 The receptor/ligand and FADD complex in turn recruits caspase-8 to the activated receptor, resulting in the formation of death-inducing signaling complex (DISC) and subsequent activation of caspase-8 through oligomerization and self-cleavage. Depending on the cell type and/or apoptotic stimulus, caspase-8 can also be activated by caspase-6. 33 Activated caspase-8 then activates effector caspase-3. In some cell types, cleavage of caspase-3 by caspase-8 also requires a mitochondrial amplifi cation loop involving cleavage of proapoptotic protein Bid by caspase-8 and its translocation to the mitochondrial membrane, triggering the release of apoptogenic proteins from mitochondria into cytosol (see Figure 4 .2). In these cell types, overexpression of Bcl-2 and Bcl-xL can block CD95-induced apoptosis. 34 Tumor necrosis factor-α is produced by T cells and activated macrophages in response to infection. Although TNF-α-mediated signaling can be propagated through either TNFR1 or TNFR2 receptors, the majority of biologic functions are initiated by TNFR1. 35 Binding of TNF-α to TNFR1 causes release of inhibitory protein silencer of death domain protein (SODD) from TNFR1, which enables recruitment of adapter protein TRADD. Signaling induced by activation of TNFR1 or DR3 diverges at the level of TRADD. In one pathway, nuclear translocation of the transcription factor nuclear factor-κB (NF-κB) and activation of c-Jun N-terminal kinase (JNK) are initiated, which results in the induction of a number of proinfl ammatory and immunomodulatory genes. 36 In another pathway, TNF-α signaling is coupled to Fas signaling events through interaction of TRADD with FADD. 37 The TNFR1-TRADD complex can alternatively engage TRAF2 protein, resulting in activation of transcription factor c-Jun, which is involved in survival signaling. Furthermore, binding of receptor interaction protein to TNFR1 through TRADD results in activation of transcription factor NF-κB, which suppresses apoptosis through transcriptional upregulation of antiapoptotic molecules such as TRAF1, TRAF2, cIAP1, cIAP2, and FLIP. The FLICE-associated huge protein was identifi ed to be a CED-4 homolog interacting with the DED of caspase-8 and was shown to modulate Fas-mediated activation of caspase-8. 38 Another class of protein, FLIP (FLICE inhibitory protein), was shown to block Fasinduced and TNF-α-induced DISC formation and subsequent activation of caspase-8. 39 Cytotoxic T cells play a major role in vertebrate defense against viral infection. 40 They induce cell death in infected cells to prevent viral multiplication and spread of infection. 41 Cytotoxic T cells can kill their targets either by activating the Fas ligand/Fas pathway or by injecting granzyme B, a serine protease, into target cells. Cytotoxic T cells carry Fas ligand on their surface but also carry granules containing the channel-forming protein perforin and granzyme B. Upon recognizing the infected cells, the lymphocytes bind and secrete granules onto the surface of infected cells. Perforin then assembles into transmembrane channels to allow the entry of granzyme B into the target cell. Upon entry, granzyme B, which cleaves after aspartate residues in proteins ("aspase"), activates one or more of the apoptotic proteases (caspase-2, -3, -7, -8, and -10) to trigger the proteolytic death cascade (see Figure  4 .2). Fas ligand/Fas and perforin/granzyme B systems are the main apoptotic machinery that regulates homeostasis in immune cell populations.

Cells can respond to various stressful stimuli and metabolic disturbances by triggering apoptosis. Drugs, toxins, heat, radiation, hypoxia, and viral infections are some of The TNF-α TNFR1 complex can also elicit an antiapoptotic response by recruiting TRAF2, which results in NF-κBmediated upregulation of antiapoptotic genes. In cytotoxic T lymphocyte-induced death, granzyme B, which enters the cell through membrane channels formed by the protein perforin, activates caspases by cleaving them directly or indirectly. Intracellular Pathways: Lack of survival stimuli (withdrawal of growth factor, hypoxia, genotoxic substances, etc.) is thought to generate apoptotic signals through ill-defi ned mechanisms, which lead to translocation of proapoptotic proteins such as Bax to the outer mitochondrial membrane. In some cases, transcription mediated by p53 may be required to induce proteins such as Bax. Translocated Bax undergoes conformational changes in the outer membrane to form oligomeric structures (pores) that leak cytochrome c from mitochondria into the cytosol. Formation of a ternary complex of cytochrome c, the adapter protein Apaf-1, and the initiator caspase-9 results in the activation of caspase-9 followed by sequential activation of effector caspase(s) such as caspase-3 and others. The action of caspases, endonucleases, and possibly other enzymes leads to cellular disintegration. For example, the endonuclease CAD (caspase activated DNAse) becomes activated when it is released from its inhibitor ICAD upon cleavage of ICAD by an effector caspase. Antiapoptotic proteins such as Bcl-2 and Bcl-xL inhibit the membrane-permeabilizing effects of Bax and other proapoptotic proteins. Cross-talk between extra-and intracellular pathways occurs through caspase-8-mediated Bid cleavage, which yields a 15 kDa protein that migrates to mitochondria and releases cytochrome c, thereby setting in motion events that lead to apoptosis via caspase-9.

the stimuli known to activate death pathways. Cell death, however, is not necessarily inevitable after exposure to these agents, and the mechanisms determining the outcome of the injury are a topic of active interest. The current consensus appears to be that it is the intensity and the duration of the stimulus that determine the outcome. The stimulus must go beyond a threshold to commit cells to apoptosis. Although the exact mechanism used by each stimulus may be unique and different, a few broad patterns can be identifi ed. For example, agents that damage DNA, such as ionizing radiation and certain xenobiotics, lead to activation of p53-mediated mechanisms that commit cells to apoptosis, at least in part through transcriptional upregulation of proapoptotic proteins. 42 Other stresses induce increased activity of stress-activated protein kinases, which result ultimately in apoptotic commitment. 43 These different mechanisms converge in the activation of caspases.

A cascade of caspases plays the central executioner role by cleaving various mammalian cytosolic and nuclear proteins that play roles in cell division, maintenance of cytoskeletal structure, DNA replication and repair, RNA splicing, and other cellular processes. This proteolytic carnage produces the characteristic morphologic changes of apoptosis. Once the caspase cascade is initiated, the process of cell death has crossed the point of no return.

The roles of various caspases in apoptotic pathways and their relative importance for animal development have been examined in genetic studies involving knockout of different caspase genes. A caspase-1 (interleukin [IL]-1b converting enzyme [ICE]) knockout study suggested that ICE plays an important role in infl ammation by activating cytokines such as IL-1b and IL-18. However, caspase-1 was not required to mediate apoptosis under normal circumstances and did not have a major role during development. 44 Surprisingly, ischemic brain injury was signifi cantly reduced in caspase-1 knockout mice compared with wild-type mice, 45 suggesting that infl ammation may contribute to ischemic injury. Caspase-3 deficiency leads to impaired brain development and premature death. Also, functional caspase-3 is required for some typical hallmarks of apoptosis such as formation of apoptotic bodies, chromatin condensation, and DNA fragmentation in many cell types. 46 Lack of caspase-8 results in the death of embryos at day 11 with abnormal formation of the heart, 47 suggesting that caspase-8 is required for cell death during mammalian development. In support of this fi nding, knockout of FADD, which is required for caspase-8 activation, resulted in fetal death with signs of abdominal hemorrhage and cardiac failure. 48 Moreover, caspase-8-defi cient cells did not die in response to signals from members of the TNF receptor family. 47 However, cells lacking either FADD or caspase-8, which are resistant to TNF-α-mediated or CD95-mediated death, are susceptible to chemotherapeutic drugs, serum depriva-tion, ceramide, γ-irradiation, and dexamethasone-induced killing. 48 In contrast, caspase-9 has a key role in apoptosis induced by intracellular activators, particularly those that cause DNA damage. Deletion of caspase-9 resulted in perinatal lethality, apoptotic failure in developing neurons, enlarged brains, and craniofacial abnormalities. 49 In caspase-9-defi cient cells, caspase-3 was not activated, suggesting that caspase-9 is upstream of caspase-3 in the apoptotic cascade. As a consequence, caspase-9-defi cient cells are resistant to dexamethasone or irradiation, whereas they retain their sensitivity to TNF-α-induced or CD95-induced death 49 because of the presence of caspase-8, the initiator caspase involved in death receptor signaling that can also activate caspase-3. Overall, these observations support the idea that different death signaling pathways converge on downstream effector caspases (see Figure 4 .2). Indeed, caspase-3 is regarded as one of the key executioner molecules activated by apoptotic stimuli originating either at receptors for exogenous molecules or within cells through the action of drugs, toxins, or radiation.

In C. elegans, biochemical and genetic studies have indicated a role for CED-4 upstream of CED-3. 50 Upon receiving death commitment signals, CED-4 binds to pro-CED-3 and releases active CED-3. 50 However, when overexpressed, CED-9 can inhibit the activation of pro-CED-3 by binding to CED-4 and sequestering it away from pro-CED-3. Therefore, CED-3 and CED-4 are involved in activation of apoptosis, and CED-9 inhibits apoptosis. After the discovery of caspases as CED-3 homologs, a search for activators and inhibitors analogous to CED-4 and CED-9 led to the discovery of diverse mammalian regulators of apoptosis. The plethora of these molecules and their functional diversity allowed them to be classifi ed into four broad categories: (1) adapter proteins, (2) the Bcl-2 family of regulators, (3) inhibitors of apoptosis (IAPs), and (4) other regulators.

As stated earlier, two major pathways of apoptosis, involving either the initiator caspase-8 or the initiator caspase-9 (see Figure 4 .2), have been recognized. Signaling by death receptors (CD95, TNFRI) occurs through a well-defi ned process of recruitment of caspase-8 to the death receptor by adapter proteins such as FADD. Recruitment occurs through interactions between the death domains that are present on both receptor and adapter proteins. Receptorbound FADD then recruits caspase-8 through interactions between DEDs common to both caspase-8 and FADD forming a DISC. In the DISC, caspase-8 activation occurs through oligomerization and autocatalysis. Activated caspase-8 then activates downstream caspase-3, culminating in apoptosis. The inhibitory protein, FLIP was shown to block Fas-induced and TNF-α-induced DISC formation and subsequent activation of caspase-8. 39 Of particular interest is cellular FLIP, which stimulates caspase-8 activation at physiologically relevant levels and inhibited apoptosis upon high ectopic expression. 51 Cellular FLIP contains two DEDs that can compete with caspase-8 for recruitment to the DISC. This limits the degree of association of caspase-8 with FADD and thus limits activation of the caspase cascade. It also forms a heterodimer with caspase-8 and caspase-10 through interactions between both the DEDs and the caspase-like domains of the proteins, thus activating both caspase-8 and caspase-10. 52 Apoptotic protease activating factor-1 (Apaf-1), a CED-4 homolog in mammalian cells, affects the activation of initiator caspase-9. 53 This factor binds to procaspase-9 in the presence of cytochrome c and 2′deoxyadenosine 5′-triphosphate (dATP) or adenosine triphosphate (ATP) and activates this protease, which in turn activates a downstream cascade of proteases (see Figure 4 .2). 54 By and large, Apaf-1 defi ciency is embryonically lethal and the embryos exhibit brain abnormalities similar to those seen in caspase-9 knockout mice. 55 These genetic fi ndings support the idea that Apaf-1 is coupled to caspase-9 in the death pathway. Unlike CED-4 in nematodes, Apaf-1 requires the binding of ATP and cytochrome c to activate procaspase-9. The multiple WD40 repeats in the C-terminal end of Apaf-1 have a regulatory role in the activation of caspase-9. 56

The CED-9 homolog in mammals is the Bcl-2 protein. Bcl-2 was fi rst discovered in B-cell lymphoma as a protooncogene. Overexpression of Bcl-2 was shown to offer protection against a variety of death stimuli. 57 The Bcl-2 protein family includes both proapoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Nr13, and A1/Bfl -1) and antiapoptotic proteins (Bax, Bak, Bok, Diva, Bcl-Xs, Bik, Bim, Hrk, Nip3, Nix, Bad, and Bid). 58 These proteins are characterized by the presence of Bcl-2 homology (BH) domains: BH1, BH2, BH3, and BH4 (Figure 4.3) . The proapoptotic members have two subfamilies: a multidomain and a BH3-only group (see Figure 4 .3). The relative ratio of pro-and antiapoptotic proteins determines the sensitivity of cells to various apoptotic stimuli.

The best-studied proapoptotic members are Bax and Bid. Exposure to various apoptotic stimuli leads to translocation of cytosolic Bax from the cytosol to the mitochondrial membrane. 59 Bax oligomerizes on the mitochondrial membrane along with another proapoptotic protein, Bak, leading to the release of cytochrome c from the mitochondrial membrane into the cytosol. 60 Other proapoptotic proteins, mainly the BH3-only proteins, are thought to aid in Bax-Bak oligomerization on the mitochondrial membrane. The antiapoptotic Bcl-2 family members are known to block Bax-Bak oligomerization on the mitochondrial membrane and subsequent release of cytochrome c into the cytosol. 60, 61 After release from the mitochondria, cytochrome c is known to interact with the WD40 repeats of the adaptor protein Apaf-1, resulting in the formation of the apoptosome complex.

Seven molecules of Apaf-1, interacting through their N-terminal caspase activation and recruitment domain, form the central hub region of the symmetric wheel-like structure, the apoptosome. Binding of ATP/dATP to Apaf-1 triggers the formation of the apoptosome, which subsequently recruits procaspase-9 into the apoptosome complex, resulting in its activation 62 . Activated caspase-9 then activates executioner caspases, such as caspase-3 and caspase-7, eventually leading to programmed cell death.

The IAPs, fi rst discovered in baculoviruses and then in insects and Drosophila, inhibit activated caspases by directly binding to the active enzymes. 63 These proteins contain one or more baculovirus inhibitor of apoptosis repeat domains, which are responsible for the caspase inhibitory activity. 64 To date, eight mammalian IAPs have been identifi ed. They include X-linked IAP (XIAP), c-IAP1, c-IAP2, Melanoma IAP (ML-IAP)/Livin, IAPlike protein-2 (ILP-2), neuronal apoptosis-inhibitory protein (NAIP), Bruce/Apollon, and Survivin. In mammals, caspase-3, -7, and -9 are inhibited by IAPs. 62 There are reports suggesting aberrant expression of IAPs in many cancer tissues. For example, cIAP1 is overexpressed in esophageal squamous cell sarcoma 65 ; cIAP2 locus is translocated in mucosa-associate lymphoid lymphoma 66 and Survivin has been shown to be upregulated in many cancer cells. 67 

The caspase inhibitory activity of IAPs is inhibited by proteins containing an IAP-binding tetrapeptide motif. 62 The founding member of this family is SMAC/DIABLO, which is released from the mitochondrial intermembrane space into the cytosol during apoptosis. In the cytosol, it interacts with several IAPs and inhibits their function. The other mitochondrial protein, Omi/HtrA2, is also known to antagonize XIAP-mediated inhibition of caspase-9 at high concentrations. 68 A serine protease, Omi/HtrA2 can proteolytically cleave and inactivate IAP proteins and thus is considered to be a more potent suppressor of IAPs than SMAC. 69 It has been reported that the heat shock proteins Hsp90, Hsp70, and Hsp27 can inhibit caspase activation by cytochrome c either by interacting with Apaf-1 or other players in the pathway. [70] [71] [72] A high-throughput screen identifi ed a compound called PETCM (α-[trichloromethyl]-4-pyridineethanol) as a caspase-3 activator. Further work with PETCM revealed its involvement in apoptosome regulation. 73 This pathway also includes oncoprotein prothymosin-α and tumor suppressor putative HLA-DR-associated proteins. These proteins were shown to promote caspase-9 activation after apoptosome formation, whereas prothymosin-α inhibited caspase-9 activation by inhibiting apoptosome formation.

In an apoptotic cell, the regulatory, structural, and housekeeping proteins are the main targets of the caspases. The regulatory proteins mitogen-activated protein/extracellular signal-regulated kinase kinase-1, p21-activated kinase-2, and Mst-1 are activated upon cleavage by caspases. 74 Caspase-mediated protein hydrolysis inactivates other proteins, including focal adhesion kinase, phosphatidylinositol-3 kinase, Akt, Raf-1, IAPs, and inhibitors of caspase-activated DNAse (ICAD). Caspases also convert the antiapoptotic protein Bcl-2 into a proapoptotic protein such as Bax upon cleavage. There are many structural protein targets of caspases, which include nuclear lamins, actin, and regulatory proteins such as spectrin, gelsolin, and fodrins. 75 Degradation of nuclear DNA into internucleosomal chromatin fragments is one of the hallmarks of apoptotic cell death that occurs in response to various apoptotic stimuli in a wide variety of cells. A specifi c DNase, CAD (caspase-activated DNase), that cleaves chromosomal DNA in a caspase-dependent manner, is synthesized with the help of ICAD. In proliferating cells, CAD is always found to be associated with ICAD in the cytosol. When cells are undergoing apoptosis, caspases (particularly caspase-3) cleave ICAD to release CAD and allow its translocation to the nucleus to cleave chromosomal DNA. Thus, cells that are ICAD defi cient or that express caspase-resistant ICAD mutant do not exhibit DNA fragmentation during apoptosis.

Apoptosis plays a critical role in the postnatal lung. 76 Regulated removal of infl ammatory cells by apoptosis helps in the resolution of infl ammation in the lung. 77 Recent evidence also supports a role for apoptosis in the remodeling of lung tissue after acute lung injury 78 and in the pathogenesis of chronic pulmonary hypertension, 79 idiopathic pulmonary fi brosis, and chronic obstructive pulmonary disease. 80, 81 Acute Lung Injury/Acute Respiratory Distress Syndrome Acute lung injury, which clinically manifests itself as the acute respiratory distress syndrome (ARDS), involves disruption of the alveolar epithelium and endothelium, increased vascular permeability, and edema. Two main hypotheses link the pathogenesis of ARDS to apoptosis, namely, the "neutrophilic hypothesis" and the "epithelial hypothesis." These two hypotheses are not mutually exclusive, and both could play important roles in the pathogenesis of ARDS.

The neutrophilic hypothesis suggests that neutrophil apoptosis plays an important role in the resolution of infl ammation and that the inhibition of neutrophil apoptosis or the inhibition of clearance of apoptotic neutrophils is deleterious in ARDS. 82, 83 Studies in humans showed that bronchoalveolar lavage fl uids from patients with early ARDS inhibit the rate at which neutrophils develop apoptosis in vitro. 84 The inhibitory effect of bronchoalveolar lavage fl uids on neutrophil apoptosis is mediated by granulocyte/macrophage colony-stimulating factor, and possibly by IL-8 and IL-2. 85,86 A membrane surface molecule, CD44, has been shown to play an important role in the clearance of apoptotic cells in vivo and in vitro. 87 In a model of bleomycin-induced lung injury, CD44-defi cient mice failed to clear apoptotic neutrophils, which was associated with worsened infl ammation and increased mortality. 87 Activation of phagocytic cells inhibits production of proinfl ammatory cytokines, including IL-1β, IL-8, IL-10, granulocyte/ macrophage colony-stimulating factor, and TNF-α and increases release of anti-infl ammatory mediators such as transforming growth factor-β, prostaglandin E 2 , and platelet-activating factor. 88, 89 The net effects of these changes could favor resolution of infl ammation.

The epithelial hypothesis suggests that the apoptotic death of alveolar epithelial cells, in response to soluble mediators such as Fas ligand, contributes to the prominent alveolar epithelial injury characteristic of ARDS. Several lines of evidence suggest a role for the Fas/Fas ligand system in epithelial cell apoptosis. 90 Fas is expressed on alveolar and airway epithelial cells, 91, 92 and its expression increases in response to infl ammatory mediators such as lipopolysaccharide. Fas-mediated lung cell apoptosis is modulated by surfactant protein A, which inhibits apoptosis in vivo. 93 Chronic Obstructive Pulmonary Disease Chronic obstructive pulmonary disease, caused primarily by smoking, generally refers to chronic bronchitis and emphysema. Several factors, including protease/antiprotease imbalance, oxidative stress, cigarette smokederived toxins, and infl ammation mediated by neutrophils, macrophages, and CD8 + T cells, have been shown to contribute to the disease process. Furthermore, matrix metalloproteinase 94 and vascular endothelial growth factor receptor inhibition, 95, 96 but not Fas/Fas ligand, have been shown to play role in the development of emphysema. Asthma Allergic asthma is characterized by intermittent or persistent bronchoconstriction and has been linked to airway remodeling and chronic infl ammation, with increased numbers of eosinophils, CD4 + T cells, and mast cells. Although at present a role for apoptosis in asthma is not confi rmed, studies ex vivo have shown reduced apoptosis of circulating peripheral CD4 + T cells and eosinophils in asthma, which might contribute to infl ammation. Corticosteroids used to reduce infl ammation in asthma have been shown to induce eosinophil apoptosis. 97 

Pulmonary fi brosis is characterized by epithelial damage, fi broblast proliferation, and deposition of collagen. Although the mechanism of alveolar epithelial cell apoptosis in pulmonary fi brosis is not known, several reports have suggested Fas pathway, 98 angiotensin pathway, 99 activated T cell-derived perforin, 100 IL-13 stimulation, 101 and transforming growth factor-β1 activation 102 to play critical roles.

Because insuffi cient apoptosis is often associated with tumorigenesis, modulation of apoptotic and antiapoptotic targets seems to be an attractive approach to cancer therapy. Lung cancers can be divided into small cell lung cancers (SCLCs) and non-small cell lung cancers (NSCLCs). 103 The SCLCs are relatively more sensitive to anticancer drugs and irradiation than are the NSCLCs, 104 but the molecular basis for this difference is not clearly known. Evaluation of apoptosis-associated substances has shown that caspase-8, Fas, and Fas ligand are often downregulated in SCLCs but not in NSCLCs. 105 An investigation of the basis for these differences revealed that there were no differences in the levels of Bax and Bcl-xL, but the expression of Bcl-2 was found to be signifi cantly higher in SCLC than in NSCLC cell lines. The observation that in some cases Bcl-2 can be converted into a proapoptotic Bax-like death molecule may offer an explanation for the paradoxic expression of Bcl-2 in SCLC. 106 The lack of expression of procaspase-1, -4, -8, and -10 107 reported in SCLC suggests that these caspases probably do not contribute to spontaneous apoptosis in these cells. Apoptosis regulators Apaf-1 and procaspase-3 are overexpressed and are functional in NSCLC cell lines. In both types of lung cancer, apoptotic stimuli result in cytochrome c release and activation of caspase-9 and caspase-3, but only SCLC cell lines showed a relocalization of caspase-3 into the nucleus 108 ; this suggests that the resistance of NSCLC cell lines is probably due to defective relocalization of caspase-3. The expression of caspase-9 and caspase-7 in NSCLCs was found to be similar to normal lung tissue. 109 However, these cell lines express the apoptosis inhibitor and splice variant of caspase-9 CASP9b. In vitro, chemotherapy-resistant NSCLC cell lines exhibit decreased caspase-9 and caspase-3 expression, 110 which suggests an inhibition of apoptosis induction via apoptosome formation in NSCLC.

Additionally, both NSCLC and SCLC cells express high and almost equal levels of Survivin. 107 The resistant NSCLC cells showed higher expression of c-IAP2, and the radiosensitive SCLC cells exhibited increased expression of XIAP. 111 These results suggest no correlation between the level of expression of the IAPs and the difference in the radiosensitivity between NSCLC and SCLC cells.

Cell death has become an area of intense interest and investigation in science and medicine because of the recognition that cell death, in general, and apoptosis, in par-ticular, are important features of many biologic processes. Involvement of many genes in the death process suggests that cell death is a complex phenomenon with many redundant mechanisms to ensure defi nitiveness. The realization that defective cell death plays a central role in the pathogenesis of diseases has stimulated work on therapies targeted to these processes, and this work will undoubtedly continue in the future.

Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics

Genetic control of programmed cell death in the nematode C. elegans

Programmed cell death in animal development

Apoptosis: defi nition, mechanisms, and relevance to disease

Apoptosis as a therapeutic target for the treatment of lung disease

Four deaths and a funeral: from caspases to alternative mechanisms

Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways

Alkylating DNA damage stimulates a regulated form of necrotic cell death

A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles

Autophagy: in sickness and in health

Tissue protein turnover during liver carcinogenesis

Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells

Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8

Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes

An alternative, nonapoptotic form of programmed cell death

Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix

Autoschizis: a novel cell death

Inhibition of the development of metastases by dietary vitamin C:K3 combination

Autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate/menadione treatment. Nuclear and DNA degradation

The molecular biology of apoptosis

Evolutionary conservation of a genetic pathway of programmed cell death

Interaction between the C. elegans cell-death regulators CED-9 and CED-4

Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9

Caspases: enemies within

Vital functions for lethal caspases

Mechanism of XIAPmediated inhibition of caspase-9

Insights into the regulatory mechanism for caspase-8 activation

A combinatorial approach defi nes specifi cities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis

Signalling by CD95 and TNF receptors: not only life and death

Apoptosis control by death and decoy receptors

The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation

FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis

Caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain

Two CD95 (APO-1/Fas) signaling pathways

Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF

Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2. Nuclear factor kappaB (NFkappaB)-inducing kinase requirement for activation of activating protein 1 and NFkappaB but not of c-Jun Nterminal kinase/stress-activated protein kinase

Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1-and TNF receptor-induced cell death

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Viral FLICEinhibitory proteins (FLIPs) prevent apoptosis induced by death receptors

Memory and distribution of virus-specifi c cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection

Fasdependent CD4 + cytotoxic T-cell-mediated pathogenesis during virus infection

Transcriptional regulation during p21WAF1/CIP1-induced apoptosis in human ovarian cancer cells

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/ SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma

Characterization of mice defi cient in interleukin-1 beta converting enzyme

Reduced ischemic brain injury in interleukin-1 beta converting enzyme-defi cient mice

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis

Targeted disruption of the mouse caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally

FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis

Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9

The ins and outs of programmed cell death during C. elegans development

c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis

The fl ip side of FLIP

Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3

An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9

Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development

Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization

bcl-2 inhibits death of central neural cells induced by multiple agents

Bcl-2 family proteins

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release

Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane

Mechanisms of caspase activation and inhibition during apoptosis

DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9

IAP family proteins-suppressors of apoptosis

Identifi cation of cIAP1 as a candidate target gene within an amplicon at 11q22 in esophageal squamous cell carcinomas

The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue lymphomas

A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma

A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death

Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90

Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway

Caspase-dependent cleavage of signaling proteins during apoptosis. A turn-off mechanism for anti-apoptotic signals

Caspasemediated proteolysis during apoptosis: insights from apoptotic neutrophils

Programmed cell death contributes to postnatal lung development

Granulocyte apoptosis and its role in the resolution and control of lung infl ammation

Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury

Mechanisms of structural remodeling in chronic pulmonary hypertension

Induction of apoptosis and pulmonary fi brosis in mice in response to ligation of Fas antigen

Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fi brosis

Granulocyte apoptosis and the control of infl ammation

Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary infl ammation in vivo

Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome

G-CSF and IL-8 but not GM-CSF correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome

Interleukin-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear neutrophil apoptosis and patient survival

Resolution of lung infl ammation by CD44

Macrophages that have ingested apoptotic cells in vitro inhibit proinfl ammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF

Phosphatidylserinedependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of infl ammation

Recombinant human Fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits

Fas expression in pulmonary alveolar type II cells

Expression of Fas (CD95) and FasL (CD95L) in human airway epithelium

Natural protection from apoptosis by surfactant protein A in type II pneumocytes

Upregulation of gelatinases A and B, collagenases 1 and 2, and increased parenchymal cell death in COPD

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade

Glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action

Increased circulating levels of soluble Fas ligand are correlated with disease activity in patients with fi brosing lung diseases

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo

The perforin mediated apoptotic pathway in lung injury and fi brosis

Interleukin-13 induces tissue fi brosis by selectively stimulating and activating transforming growth factor beta(1)

Early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fi brosis

United States lung carcinoma incidence trends: declining for most histologic types among males, increasing among females

Progress in understanding the molecular pathogenesis of human lung cancer

Loss of expression of death-inducing signaling complex (DISC) components in lung cancer cell lines and the infl uence of MYC amplifi cation

Conversion of Bcl-2 to a Bax-like death effector by caspases

Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma

Defective caspase-3 relocalization in non-small cell lung carcinoma

Increased expression of Apaf-1 and procaspase-3 and the functionality of intrinsic apoptosis apparatus in non-small cell lung carcinoma

Rescue of death receptor and mitochondrial apoptosis signaling in resistant human NSCLC in vivo

Expression of inhibitor of apoptosis proteins in small-and non-small-cell lung carcinoma cells