Apoptosis: Programmed cell death at a molecular level

Apoptosis: Programmed Cell Death at a Molecular Level Duane R. Schultz and William J. Harrington, Jr. Objectives: To characterize cell surface receptors, their ligands, and their proteins in the 2 major pathways of apoptosis; the components that promote/ suppress these interactions; the noninflammatory removal of apoptotic bodies by dendritic cells; and methods of assay in studies of cell death. To describe: how deregulation of apoptosis may contribute to autoimmunity, cancer, and neurodegenerative disorders and strategies some viruses have evolved that interfere with the host’s apoptotic pathways. Methods: The authors reviewed and compiled literature on the extrinsic (tumor necrosis factor [TNF] receptor superfamily and ligands) and intrinsic (mitochondria-associated) apoptotic pathways, the pro- and antiapoptotic proteins of the B-cell follicular lymphoma (Bcl)-2 family, the nuclear factor (NF)-␬B family of proteins, commonly used laboratory methods to distinguish apoptosis from necrosis, the recognition and removal by phagocytosis of apoptotic cells by dendritic cells, and viral strategies to avoid a host’s apoptotic response. Results: The 2 major pathways of apoptosis are (1) FasL and other TNF superfamily ligands induce trimerization of cell-surface death receptors and (2) perturbated mitochondria release cytochrome c, the flavoprotein apoptosisinducing factor, and second mitochondria-derived activator of caspases/ DIABLO (a protein that directly neutralizes inhibitors of apoptotic proteins and activates proteases). Catalytically inactive cysteine proteases, called caspases, and other proteases are activated, ultimately leading to cell death with characteristic cellular chromatin condensation and DNA cleavage to fragments of approximately 180 bp. The inhibitory/promoting action of Bcl-2 family members is involved in the release of cytochrome c, an essential factor for the mitochondrial-associated pathway. A balance between inhibition/promotion determines a cell’s fate. The NF-␬B family in the cytoplasm of cells activates various genes carrying the NF-␬B response element, such as members of the inhibitor of apoptotic proteins family. A few of the more common methods to detect apoptotic cell death are described, which use immunochemical, morphologic and flow cytometric methods, and genetic markers. Exposed phosphatidylserine at the outer leaflet of the plasma membrane of the apoptotic cell serves as a possible receptor for phagocytosis by immature dendritic cells. These cells phagocytize both apoptotic and necrotic cells, but only the latter induce maturation to become fully functional antigen-presenting cells. Viral inhibitors of apoptosis allow increased virus replication in cells, possibly resulting in their oncogenicity. Conclusions: Balanced apoptosis is crucial in development and homeostasis, and all multicellular organisms have a physiologically programmed continuum From the Department of Medicine, University of Miami School of Medicine, Miami, FL. Duane R. Schultz, PhD: Professor of Medicine, Division of Rheumatology & Immunology, Department of Medicine, University of Miami School of Medicine, Miami, FL; William J. Harrington, Jr., MD: Professor of Medicine, Division of Hematology/Oncology, Department of Medicine, University of Miami School of Medicine, Miami, FL. Address reprint requests to Duane R. Schultz, PhD, Division of Rheumatology & Immunology (R-102), Department of Medicine, University of Miami School of Medicine, PO Box 016960, Miami, FL 33101. © 2003 Elsevier Inc. All rights reserved. 0049-0172/03/3206-0001$30.00/0 doi:10.1053/sarh.2003.50005 Seminars in Arthritis and Rheumatism, Vol 32, No 6 (June), 2003: pp 345-369

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of pathways to apoptotic cell death. Further studies of the control at the molecular level of key components and promoters/suppressors of apoptosis may provide better approaches to treatment of autoimmune diseases, malignancies, and neurodegenerative disorders. Many important questions remain regarding the advantages of modifying apoptotic programs in clinical situations. Semin Arthritis Rheum 32:345-369. © 2003 Elsevier Inc. All rights reserved. KEY WORDS: Programmed cell death; caspases; tumor necrosis factor; mitochondria; dendritic cells.

A

POPTOSIS IS A continuous physiologic process for noninflammatory, programmed cell death, and is one of today’s most active fields of biomedical research (1-4). The importance of this research was recognized when the Nobel Prize in Medicine was awarded on October 7, 2002, for investigations into organ growth and apoptosis of the tiny nematode Caenorhabditis elegans. The prize was shared by an American (H. Robert Horvitz, Massachusetts Institute of Technology) and by 2 Britons (Sydney Brenner of the Salk Institute for Biological Studies in San Diego and the Molecular Sciences Institute, Berkeley, CA; and John E. Sulston, retired from the Wellcome Trust Sanger Institute in Cambridge, England). The pathways of apoptosis play an integral part in many biologic events, including morphogenesis, cell turnover, and the removal of harmful cells, and balanced apoptosis is crucial to ensuring good health. Cell death via apoptosis follows the activation of effector proteases called caspases, which participate in enzymic cascades that terminate in cellular disassembly (5,6). This action can occur through several routes, including mitochondriaindependent and -dependent pathways. Death by the terminal pathways of apoptosis is frequently compared with death by necrosis, but it is distinguished from necrotic death in that apoptosis is a closely regulated process induced by a specific stimulus, and occurs without the release of inflammatory mediators. Death by necrosis occurs because of failure to control cellular homeostasis after undergoing damage. Cells undergoing apoptosis commit suicide in an orderly fashion by cutting themselves into mem-

For a complete glossary of terms and definitions, please see the Appendix after the reference section.

brane-packaged parcels after cleavage of their chromosomal DNA, and then they are removed by phagocytic cells. Two major cells for clearing and degrading apoptotic bodies and necrotic material are dendritic cells (DCs) and macrophages. Immature DCs exist in peripheral tissues and in secondary lymphoid organs. They are derived from bone marrow leukocytes, and migrate as precursors via the vasculature to tissues in which they become residents. They are called Langerhans cells in the epidermis and interstitial cells in the dermis. In blood, the DC subsets are called plasmacytoid and myeloid DCs, respectively, and in lymph nodes, they are known as interdigitating cells. DCs function as professional antigen-capturing, -processing, and -presenting cells for initiation of a primary immune response. After phagocytosis of necrotic cells, debris, and exogenous factors, immature DCs initiate an irreversible maturation process associated with phenotypic changes to become mature antigen-presenting cells. These cells eventuate in stimulation of CD4(⫹) T helper cells, and activation of CD8(⫹) T cells into cytotoxic T lymphocytes. The latter are capable of destroying virusinfected cells. Thus, DCs are indispensable for adaptive immunity (7,8). There is a different picture after phagocytosis of apoptotic cells by immature DCs, and the maturation process is significantly reduced with noninflammatory clearance of the apoptotic material. This pathway may be important for down-regulation of the response to apoptotic self-antigens (eg, nucleic acids) and the maintenance of self-tolerance. The highly orchestrated form of cell death and clearance is critical to the health of many organisms during development, and also for maintaining the normal function of the immune system. These activities include peripheral deletion of activated mature T cells at the end of an immune response,

APOPTOSIS

killing virus-infected or malignant cells by cytotoxic T cells and by natural killer cells, and killingof inflammatory cells at immune-privileged sites, such as the eye, although the latter activity has been disputed (9-11). The importance of regulation of cell numbers and/or the removal of aged, damaged, or autoimmune cells to minimize inflammatory or immune reactions is discussed (4). At least 16 members of the tumor necrosis factor (TNF) ligand family have been identified, and most are synthesized as type II transmembrane precursors. The membrane-bound and soluble TNF ligands bind to receptors belonging to the TNF receptor gene superfamily. The cell surface receptors that transmit apoptotic signals initiated by ligation (eg, specific antibody) or by the previously mentioned natural ligands are called death receptors (DRs) and play a central role in instructive apoptosis (12). These DRs transmit signals via a death domain (DD) in their cytoplasmic tail and activate cytosolic cysteine caspases—14 of these proteases have been described in association with the cell death program (5). The cathepsins (lysosomal cysteine proteases), calpains, granzymes, and the proteasome are additional proteases that participate in the apoptotic pathways (13-15). Intracellularly, there is a recruitment of specific adaptor proteins to the oligomerized receptor to initiate the apoptotic pathway via DDs, eventually leading to activation by proteolysis of caspase precursors, membrane blebbing, and nuclear condensation; the final result is the demise of a cell. Two well-characterized DRs are Fas (CD95 or Apo 1) and TNF receptor-1 (TNFR1), also called p55 or CD120a. Studies of genetic and microbial-induced malfunctions in animals and humans suggest the potential role of apoptotic mechanisms in autoimmune diseases, cancer, and nervous system and neurodegenerative disorders. For example, synovial hyperplasia leading to destruction of cartilage and bone in rheumatoid arthritis may be caused, at least in part, by an imbalance of cell proliferation and cell death (2). The lpr or gld mutations bred into strains of mice not prone to autoimmunity show that Fas/Fas L deficiency results in autoantibody production and lymphoproliferation (3,4). Suggestive evidence also has indicated that excessive cell death may lead to brain damage caused by a stroke or by the progressive neurodegenerative dementia of Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (16).

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In each of the 6 sections, we have focused on currently used terminology, and because many of the molecules are described as acronyms, we have compiled a comprehensive glossary of terms (see Appendix). After reviewing many of the numerous reactants that interact in the major pathways and the proteins that promote or suppress the molecular interactions, it will be apparent that control of these pathways may provide answers to many questions in cellular regulation and approaches, and may lead to a better understanding and treatment of malignant and autoimmune diseases. Although progress has been made in this direction, control at the molecular level of these life and death processes is still in its infancy, but holds great promise for the future. APOPTOTIC PATHWAYS

The Extrinsic Pathway Activation of apoptotic pathways and programmed cell death are initiated by the binding of a specific protein ligand to a cell surface transmembrane receptor (Fig 1). Apoptotic signals are transmitted to target cells via the TNF superfamily of DRs, the members characterized by a conserved extracellular cysteine-rich motif. Six different DRs are known: Fas, TNFR1, DR3, TNF-related apoptosis-inducing ligand (TRAIL) (TRAIL-R1 or DR4, and TRAIL-R2 or DR5), and DR6. These receptors, generally composed of 3 identical polypeptide chains, have binding and signaling features in common as well as unique, individual characteristics (12,17). TRAIL, a member of the TNF/nerve growth factor superfamily, can interact with 5 different receptors, but only DR4 and DR5 are capable of transmitting a death signal (18). Each of the 6 DRs has an intracellular DD that function as a protein-protein binding module after recruiting various cytosolic signaling molecules that comprise the specific apoptotic pathways. CD40, another member of the TNFR family, lacks a DD on its cytoplasmic tail. It is involved in macrophage and B-cell activation. One of the best-characterized surface receptors is Fas, a 319-amino acid type 1 transmembrane glycoprotein, with broad distribution on both lymphoid and nonlymphoid cells (19). There are 3 extracellular region cysteine-repeat domains and an 80-amino acid region of the intracellular domain termed the DD located in the C-terminal region that is functionally conserved among DRs

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Fig 1. The 2 major pathways of apoptosis—the extrinsic (Fas and other TNFR superfamily members and ligands) and the intrinsic (mitochondria-associated) pathways. Both pathways lead to activation of caspase-3, giving rise to apoptotic cell death. Only a few examples of proteins that directly affect cell death and the resulting DNA fragmentation are shown in the simplified diagram. Most of the effector and control proteins are written as acronyms, and are defined in the Appendix.

(Fas, TNFR1, DR3, DR4, DR5, DR6, and the more distantly related p75 neurotrophin receptor). This DR is crucial for the signaling of apoptosis as well as the activation of the transcription factor nuclear factor (NF)-␬B (12,20). NF-␬B protects cells from apoptosis by promoting expression of survival factors, such as members of the inhibitor of apoptosis (IAP) family. An apoptotic signal is initiated in target cells when Fas is engaged by its natural ligand FasL or by agonistic antibodies. The interaction may occur on the effector cell, or in some cases, on the same cell. FasL, a trimeric type 2 transmembrane protein, is primarily expressed by activated CD4(⫹) and CD8(⫹) T cells. It is released in soluble form after cleavage from its membrane site by metalloproteinases before binding Fas (21,22). Unlike Fas and TNFR1, TNFR2 has no DD. However, Pimentel-Muinos and Seed (23) showed

that, in a human T-cell line, an alternative pathway involving TNFR2 required a receptor-interacting protein (also called ring finger–interacting protein), a serine-threonine kinase, to promote apoptosis. The Fas-FasL interaction causes receptor oligomerization (12,24). The DD of the receptor then recruits so-called adaptor proteins that also have DDs. One such protein is Fas-associated DD (FADD) protein, which has a DD at its C-terminus and a second protein-protein interaction domain, called a death-effector domain, at its N-terminus (25,26). The death effector domain of the adaptor protein binds to the death-effector domain, or prodomain, of caspase-8, and a complex termed the death-inducing signaling complex (DISC) is formed which subsequently signals proteolysis and endonucleolytic cleavage in both T and B lymphocytes as well as nonlymphoid target populations.

APOPTOSIS

Therefore, FADD recruits procaspase-8 to the Fassignaling complex by homotypic interactions between the death-effector domains of each protein (25,26). Activated caspase-8 then activates a series of downstream caspases that result in cleavage of structural and regulatory intracellular proteins, ultimately leading to apoptosis. The DRs have both unique features and characteristics in common. For example, TNF-␣ interacts with 2 receptors, TNFR1 (CD120a, or p55) and TNFR2 (CD120b, or p75) to stimulate multiple biologic responses critical to the host (12). However, although TNFR2 has no DD (23), intracellularly, TNFR1 associates with TNFR1-associated DD, which, via its DD, binds to FADD, and the result is transmission of a death signal. Nuclear magnetic resonance studies have shown the importance of the electrostatic charge conferred by solvent-exposed basic residues in mediating the interactions between the DDs of TNFR1 and TNFRassociated DD (27). On the other hand, FADD is neither required for TRAIL-induced apoptosis (28) nor for the role of TRAIL in directly influencing T-cell function (29). The overall result after recruitment to the DISC is the autocatalytic processing that leads to activation of caspase-8 (25,26). The activated protease in turn triggers the cleavage of downstream caspases, such as caspase-3 (its target is the inhibitor of caspase-activated deoxyribonuclease (CAD), which is an endogenous endonuclease) (30). Inhibitor of CAD and CAD exist as an inactive complex, and the inhibitor of CAD is cleaved by activated caspases to release CAD (31). The latter now enters the nucleus and degrades the cell’s chromosomal DNA, leading to DNA fragmentation and cell death. Apoptosis also can be initiated by numerous agents that can damage DNA, including ultraviolet irradiation, X-irradiation, growth factor withdrawal, and chemotherapeutic drugs. In fact, many anticancer agents induce apoptosis (32). Thus, there are DR-independent induction pathways of cell death, and these may be active and work in parallel to the DR-activated mechanisms. A 55-kd inhibitor of this pathway is termed the cellular Fas-associated DD-like interleukin (IL)-1– converting enzyme inhibitory protein (c-FLIP). cFLIP is an enzymatically inactive homologue of caspase-8; it contains 2 death effector domains, and it binds to the death effector domains of FADD and caspase-8, thereby blocking transmission of

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the Fas-induced death signal and prolonging the survival of the cell (33-36). There is also a viral FLIP (v-FLIP) that is capable of modulating DRinitiated apoptotic pathways that involve caspase-8, and this molecule has homology with mammalian c-FLIP (see Regulation of Apoptosis by Viruses). The inhibitory action of v-FLIP on apoptosis prolongs residence in cells, eventually leading to viral propagation and spreading to other cells. In some cell types, Fas-induced apoptosis is regulated by mitochondrial-associated caspases and apoptosispromoting members of the B-cell follicular lymphoma (Bcl)-2 family (37). The Mitochondria-Associated (Intrinsic) Pathway Mitochondria are the site of eukaryotic oxidative metabolism, and provide adenosine triphosphate (ATP) via a pathway of oxidative phosphorylation, and cytochrome c. In the context of cell death, they play a central role in some of the apoptotic pathways. Mitochondrial function appears to be critical in some cells for executing a death program, and activation of these cellular organelles is a crucial step for coordinating and integrating several upstream and downstream apoptotic pathways. As described previously in more detail, after a ligand of the TNF superfamily binds to its cell receptor, there is receptor multimerization, the intracellular DD recruits cytosolic signaling proteins, and a DISC is formed with the following components: (1) the DD of the receptor; (2) the DD of an adaptor molecule (eg, FADD); and (3) the inactive precursor of caspase-8, which is then activated by proteolysis. In the presence of caspase-3, the quantity of active caspase-8 produced at the DISC determines whether a mitochondrial-dependent (low quantity, type II) or -independent (high quantity, type I) apoptotic pathway is used (37). Caspase-8 also cleaves and activates a promoter of apoptosis in the Bcl-2 family termed Bid (Table 1) (38), which is thought to be 1 of the factors responsible for releasing cytochrome c from the mitochondria (tBid is translocated and activated Bid). Ceramide is another factor that induces cytochrome c release from mitochondria, generated by hydrolysis of sphingomyelin, followed by activation of acidic sphingomyelinase (39). Normally, cytochrome c is localized in the intermembrane space, loosely attached to the outer surface of the inner mitochondrial membrane (38,39). The caspases are activated by perturbance

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Table 1: The Mammalian Bcl-2 Family of Inhibitors or Promoters of Apoptosis

Protein Bcl-2 Bcl-XL Mcl-1

Action on Apoptosis

Bcl-2 Homology (BH) Domains

Transmembrane Anchor

Inhibit

BH1, BH2, BH3, BH4

(⫹)

Inhibit

BH1, BH2, BH3, BH4 Weak homology to Bcl-2 in BH4 region

(⫹)

Bax Bak Bok

Promote

BH1, BH2, BH3, BH4(?)

(⫹)

Bcl-XS

Promote

BH3, BH4

(⫹)

Promote

BH3 only

(⫹)

BH3 only

(⫺) Exhibit diffuse cytoplasmic distribution

A1 Bcl-w

BiK Hrk Bim Blk Bad Bid

NOTE. Space limitations preclude comprehensive referencing for all the work discussed. Abbreviations: Bad, B-cell follicular lymphoma-2–associated protein ␣; Bax, B-cell follicular lymphoma-2–associated protein X; Bcl, B-cell follicular lymphoma; BH, B-cell follicular lymphoma homology.

of the mitochondria, and this may lead to the opening of the mitochondrial permeability transition pore complex with organelle swelling, and then rupture of the outer membrane. This results in release of the apoptosis-stimulating molecules cytochrome c and apoptosis-inducing factor (AIF), a nuclear-encoded (Xq25-26) flavoprotein similar to bacterial oxidoreductases. Another recently identified factor that is released is second mitochondriaderived activator of caspases (Smac)/DIABLO (human Smac/murine DIABLO; see Appendix), a protein that eliminates the inhibitory effect of IAPs and interferes with their protective effect against ultraviolet radiation–induced cell death (40). Smac/DIABLO does not appear to induce apoptosis in healthy cells. A second mechanism allows direct release of cytochrome c and the other factors without evidence that the permeability transition pore complex has been opened to the environment. Bcl-2, a protein inhibitor of programmed cell death, blocks both the permeability transition pore opening and the release of cytochrome c (41). Released cytochrome c induces multimerization of apoptosis

protease-activating factor (Apaf)-1 to activate procaspases-9 and -3, and Smac/DIABLO eliminates the inhibitory effect of IAPs. Although apoptosisinducing factor is only partially characterized, it not only activates caspases but also acts directly on nuclei to induce DNA fragmentation (41,42). Thus, a major role for mitochondria as an initiator of apoptosis has been firmly established in many cells (43-44). Mitochondria appear to remain intact during apoptosis, in contrast to the gross swelling and rupture of the internal cristae that occur in necrosis (45). The Bcl-2 family of proteins, located in the outer mitochondrial membrane, is important in preventing and permitting apoptosis, and is instrumental in controlling the release of cytochrome c (46-48). In fact, all mitochondrial activities in apoptosis can be blocked by overexpression of Bcl-2 or Bcl-XL (see The Bcl-2 Family: Pro- and Antiapoptotic Proteins), but overexpression is an experimental phenomenon that may not occur under normal physiologic conditions. Cytochrome c is a required cofactor and forms a complex with Apaf-1, procaspase-9, and deoxyadenosine monophosphate (dATP) (49). The large complex is called the

APOPTOSIS

apoptosome. Apaf-1 is an adaptor protein that binds procaspase-9 through the caspase activation and recruitment domain, resulting in caspase-9 autoactivation in the presence of cytochrome c and ATP or dATP. After caspases-9 and -8 are activated, cleavage and activation of additional upstream caspases in amplifying loops may occur, or this action allows caspase-9 to activate downstream caspases such as caspase-3, leading to endonuclease-fragmentation of the cell’s DNA. THE BCL-2 FAMILY: PRO- AND ANTIAPOPTOTIC PROTEINS

Human bcl-2 was the first protooncogene identified to function by protecting cells from programmed cell death (50-55). It was recognized early that Bcl-2 was a mammalian homologue of the antideath protein cell death abnormal (ced)-9 of the nematode C elegans, and was mainly located on the outer membrane of mitochondria, but was also on the endoplasmic reticular membrane and outer nuclear envelope (50-55). Mutations of ced-9 that decreased or eliminated its function caused cells that would normally survive to instead undergo apoptosis (56). Additionally, overexpression of the apoptotic inhibitory protein Bcl-2 in B cells resulted in B-cell follicular lymphoma; B cells survived and proliferated for unusually long time periods (57). The proapoptotic Bcl-2–associated protein x (Bax) was the first cell death promoter to be identified in the group as a protein coimmunoprecipitating with Bcl-2. It was later determined that family members interact with one another, forming heterodimers and, occasionally, homodimers. Additional related members were discovered and their expression at the gene and/or protein level has been detected in many cell types—this has resulted in the classification of the Bcl-2 protein family (Table 1). Family members are regulated by cytokines and other death-survival signals at different levels of the apoptotic pathways. Three family members are found in myeloid leukocytes: A1 and Mcl-1 occur in neutrophils (58-62), whereas A1 and Bcl-XL can both be up-regulated in macrophages by proinflammatory stimuli in vitro (63,64). Hockenberg et al (65) found that Bcl-2 blocked cell membrane blebbing, volume contraction, nuclear condensation, and endonucleolytic cleavage of DNA, the phenomenon that was initially called apoptosis in 1987 by Wyllie (66). Members of this

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family either inhibit or promote cell death, and together with mitochondria, cytochrome c (a required cofactor for Apaf-1), AIF, intracellular balances of dATP or ATP, and caspases, are involved in the initiation and execution of the intrinsic pathway (67,68). It is well accepted that Bcl-2 controls mitochondrial permeability transition, and overexpression of the protein or of close family members (Table 1) blocks the release of cytochrome c and AIF; this action blocks the mitochondrial apoptotic pathway (69-71). The balance between pro- and antiapoptotic Bcl-2 family members expressed in the outer mitochondrial membrane probably determines whether programmed cell death is initiated or whether the cell will survive (72,73). In addition, cell death–suppressor molecules, such as Bcl-2, may have radically different effects when targeted to different intracellular membranes (74). The Bcl-2 family shares homology within specific regions, called Bcl-2 homology (BH) domains, designated BH1, BH2, BH3, and BH4 (75,76). However, the overall amino acid sequence homology among members is low (77-83). Family members are classified based on function and BHdomain organization. The proteins, via their BH domains, interact specifically to form either homoor heterodimers, ultimately expressing their pro- or antiapoptotic influence. On heterodimerization, there appears to be a titration of one another’s functional activity, suggesting that relative concentration of family members acts as a gauge for cell death. The family is divided into 2 groups, either inhibiting or promoting apoptosis (Table 1). The first, inhibitors of apoptosis, include Bcl-2, BclXL, and Mcl-1—they have 4 conserved BH motifs and all closely resemble Bcl-2. Two additional inhibitory proteins, A1 and Bcl-w, also have 4 homology domains, but have weak homology to Bcl-2 in the BH4 region. The BH4 domain binds to several other proteins in addition to Bcl-2 family members, including calcineurin, a calcium-dependent protein phosphatase (84). Calcineurin is 1 of the proteins in the T-cell activation pathway that increases the expression of CD40L on the surface of T cells, leading to increased production of antibody by the interacting B cell. In addition to the extracellular domains, family members also have a transmembrane domain that localizes the proteins to intracellular membranes.

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The group characterized as having 3 BH domains (BH1, BH2, BH3) include Bax, Bak, and Bok, but the presence of a BH4 domain is uncertain; they promote apoptosis. All Bcl-2 family members contain a BH3 domain, but it appears to be functionally important for the proteins that promote death. For example, Bax opposes the action of Bcl-2 and accelerates cell death when overexpressed in mammalian cells (85). The BH3 domain alone suffices, but is not required, for regulating cytochrome c release and activation of caspases. Another apoptosis promoting protein, Bcl-Xs, contains 2 domains, BH3 and BH4. The next group shares only the BH3 domain, is also proapoptotic, and includes Bik, Hrk, Bim, Blk, Bcl-2–associated protein d (Bad), and Bid. In the latter 2 members, a transmembrane tail has not been identified (75,76). The BH3-only family members appear to function as death promoters primarily on the basis of forming heterodimers with survival counterparts (eg, Bcl-2 and Bcl-XL) within the family. A study by Ayllon et al (86) with the BH3-only Bad, a proapoptotic member of the Bcl-2 family, showed that the protein was attached to lipid rafts in IL-4 –stimulated cells and thymocytes, but associated with mitochondria in IL-4 – deprived cells (86). Disruption of the lipid rafts by treatment with methyl-␤-cyclodextrin induced separation of Bad from the rafts, which correlated with apoptosis. Thus, Bad was active in controlling apoptotsis. Lipid rafts differ from plasma membranes in that they contain cholesterol and glycosphingolipid-containing microdomains that are enriched in a number of receptors and signaling molecules (87). Lipid rafts are thought to function as platforms that orchestrate the induction of various signaling pathways and protein trafficking processes. Fluorescent probe methods have been valuable for characterizing their structure and functions. In reference to proapoptotic Bad (86), it was suggested that the sequestration in rafts avoided both the association with partners (eg, Bcl-2) and the availability to play a role in promoting apoptosis. Also of interest, Cottin et al (88) showed that amino acid sequences located within the DD of TNFR1 promote the appropriate location of this receptor to lipid rafts in addition to its role in receptor signaling. Bcl-2 family members associated with the outer mitochondrial membrane not only have receptorligand activity via their BH domains but also may

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contribute to mitochondrial integrity by affecting ATP or dATP trafficking, membrane potential, and the channel or pore through which cytochrome c is released to the cytosol (89). Mutations that change or disrupt the domains destroy both the ability to heterodimerize and to promote cell death. The BH3 domains alone of Bcl-2 family members are sufficient for regulating cytochrome c release and caspase activation. Cytochrome c release is accompanied by a drop in the mitochondrial membrane potential (⌬␺), the symbol used for the electrostatic membrane potential when importing material through membranes, and requires energy of ATP hydrolysis. Charge differences across a biologic membrane generate an electric potential difference, ⌬␺ ⫽ ⌬(in) ⫺ ␺(out) where ⌬␺ is called the membrane potential. These metabolically generated membrane potentials extend across many biologic membranes, in addition to mitochondria. One currently proposed mechanism is that the drop in potential and the leakage of mitochondrial proteins are attributed to opening of the permeability transition pore complex whose biochemical composition is unknown, but it is thought to be composed of several proteins. Further information on the Bcl-2 family proteins that describes their roles in programmed cell death and survival, cytochrome c release, and the significance of the Bcl-2 homology domains (eg, BH3) in protein-protein interactions, has been published (68-70,73,89). Kelekar and Thompson (75) have comprehensively reviewed domain organization. REGULATION OF APOPTOSIS BY TRANSCRIPTION FACTOR NF-␬B

NF-␬B is a collective term that refers to a family of proteins involved in inflammatory reactions, lymphoid organ development, and innate and adaptive immunity. For purposes of this review, the action of NF-␬B in pro- and antiapoptotic cellular responses is the focus. NF-␬Bs are dimeric transcription factors in the Rel family that are regulated by shuttling within cells from the cytoplasm to the nucleus in response to various stimuli (90). Maintained as an inactive form in the cytoplasm, on activation, the NF-␬B translocates to the nucleus to up-regulate target gene expression. There are 5 members of the NF-␬B family: p50/p105 (NF-␬B1), p52/p100 (NF-␬B2), c-Rel,

APOPTOSIS

RelA, and RelB (91). Another set of proteins known as inhibitors of ␬B (I␬Bs), bind hetero- and homodimerized NF-␬B proteins in the cytoplasm, preventing their entry to the nucleus. Bcl-3, an I␬B member, interacts with NF-␬B1 and NF-␬B2 and promotes their nuclear localization (91). After a variety of stimuli, including cytokines and viral infections, I␬B is phosphorylated in response to inflammatory signals by another set of proteins known as the I␬B kinase complex (IKK). The complex contains 2 kinases, IKK-1 and IKK-2, as well as several scaffolding proteins. The IKKs are activated by signals derived from the TNFR. The I␬B is then degraded via the cytosolically based, ATP-dependent proteolytic ubiquitin proteosome pathway. This allows for translocation of NF-␬B to the nucleus to influence transcription of a wide range of immune response genes. It has become clear that activation of NF-␬B is an important antiapoptotic mechanism in cancer (92). Evidence supporting the oncogenic role of NF-␬B first arose from the observation that v-Rel, a retroviral homologue of c-Rel, causes tumors in chickens (92). More recent work on some human tumors showed that coding regions of NF-␬B proteins are involved in chromosomal rearrangements and gene amplification (93). Therefore, the products of specific NF-␬B target genes inhibit cell death. The constitutive nuclear activity of NF-␬B is an important marker that predicts a poor prognosis in diffused large B-cell lymphomas. Diffused large B-cell lymphomas may be classified into 2 major subtypes: germinal center and activated B cell. The activated B-cell–type lymphomas manifest an elevated expression of NF-␬B, with a target of antiapoptotic genes. These tumors (compared with germinal center types) are resistant to chemotherapy and are usually fatal (94). NF-␬B suppresses apoptosis through various mechanisms, including activation of TNFR-associated factor-1 and -2, cIAP-1 and -2, xIAP, Bcl-x, and A1 (95). NF-␬B also may compete for transcriptional coactivators with p53, a nuclear protein suppressor of human cancer, thereby inhibiting its function (93). p53 also is a regulator of the expression of DR genes in apoptosis induction. In addition, DR/DR ligand signaling also may recruit antiapoptotic elements such as TNRF-associated factors and the I␬B kinase complex. These data indicate that signaling through DRs may paradoxically activate antiapo-

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ptotic signals mediated by NF-␬B (96). The NF-␬B also may oppose apoptosis by activating target genes that stimulate proliferation and the cell cycle such as the CD40 ligand, granulocyte-macrophage colony–stimulating factor, and cyclin D, and other oncogenic genes that influence migration and angiogenesis (97). It is evident from this brief discussion that the antiapoptotic function of NF-␬B is a major impedance to successful cancer therapy. METHODS FOR DETECTION OF APOPTOTIC CELL DEATH

Table 2 tabulates, briefly describes, and references some of the more commonly used methods to detect apoptotic cell death. Only a small number of the dozens of methods in the literature that use immunochemical, morphologic, flow cytometric methods, and genetic markers are listed for these assays. Many of the commercially available test products cannot distinguish apoptosis from necrosis. Because apoptosis is now linked to a variety of autoimmune, malignant, and neurodegenerative disorders, it is imperative to be critically aware of the positive and negative points of each assay. Additionally, it is important to obtain positive results from more than 1 independent method (preferably several methods) before concluding that apoptotic pathways, their activators, or inhibitors are involved in experimental results and conclusions. The problem is complex because of the profusion of new signaling, modulating, and effector molecules now described in apoptosis. The methods also should be selected to study apoptosis at different stages (early, intermediate, late) of the pathway(s) under investigation. RECOGNITION AND REMOVAL OF APOPTOTIC CELLS

Of the phospholipids distributed in membranes of viable cells, anionic phosphatidylserine is totally located in the inner leaflet (cytoplasmic side), and is not normally in contact with blood components. After the death pathways described previously have been activated in these cells, a specific sequence of highly regulated events takes place in which phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane (ie, the cell surface) and is exposed to the extracellular environment (133,134). Furthermore, there is characteristic apoptotic nuclear morphology as well as fragmentation of the cell’s genomic DNA

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Table 2: Some Published Methods for Detection of Apoptotic Cells Distinguishes Apoptosis From Necrosis

Method*

Description

Gel electrophoresis to detect DNA fragmentation in apoptotic cells†

DNA ladders (200-5000 bp fragments): visualized after staining gels with fluorescent or chromogenic reagents In situ—end labeling of free 3⬘ ends of DNA fragments by using fluorescent or chromogenic agents Denatured DNA detected by monoclonal antibody reactive with single-stranded DNA Testing for histones: quantitative detection of DNA fragmentation in ruptured cells The change in ADP/ATP ratios is used to differentiate apoptosis from necrosis In vitro detection of membrane PS is achieved with annexin V; measurement of binding is combined with a dye exclusion test to rule out necrosis Assessed with voltage-sensitive dyes

TUNEL methods

Formamide-induced DNA denaturation ELISA

ADP/ATP ratio

Annexin-V binding

Changes in membrane potential: release of cytochrome c from mitochondria and activation of caspase-9 Caspase cleavage

Caspase-3 assay Flow cytometry Test signal proteins, Fas, TNFR1, Bcl-2

Western blots, immunochemistry, detection of proteolytic cleavage products Functional or immunologic methods Multiparameter; monoclonal antibodies, fluorescent conjugates Western blots, immunochemistry, flow cytometry

Reference

Yes

98-100

No

101-112

Yes

113-115

Yes

116

Yes

117

Yes

118-120

Yes

121

Early apoptosis

122-124

Early apoptosis Yes

125-127 128-129

Yes

130-132

Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Bcl-2, B-cell follicular lymphoma-2; ELISA, enzymelinked immunosorbent assay; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. *There are 4 general divisions that are helpful in categorizing the methods: DNA changes, membrane alterations, enzymic activity, and apoptosis-regulating genes. †Not all cells that die by apoptosis show DNA fragmentation that can be visualized by DNA laddering.

by endonuclease action into fragments of 180 to 200 bp (DNA laddering) (135), with the release of membrane-bound fragments called apoptotic blebs and bodies (136,137). The apoptotic cells and bodies are eliminated by phagocytic cells, suggesting

that receptors specifically recognize not only translocated phosphatidylserine but also uncharacterized ligands unique to the surface of the apoptotic cells (133). In vitro, the anticoagulant Annexin V conjugated to a fluorescent dye binds to the ex-

APOPTOSIS

posed phosphatidylserine, allowing the detection of apoptotic cell death by flow cytometry or fluorescent microscopy. In vivo, apoptotic bodies may undergo secondary necrosis if not cleared rapidly. For this action, the dead or dying cells are rapidly cleared by proximal DCs, macrophages, and scavenger cells, eliminating potentially inflammatory cell components without inducing an inflammatory response and preventing the formation of selfreactive responses and autoimmunity (138,139). Therefore, 1 important function of DCs is clearance of dead or dying cells and extracellular debris, and they acquire functionally distinct characteristics for this action. In published comparative studies, it was found that phagocytosis of certain necrotic cells induced DC maturation, but ingestion of apoptotic or primary tissue cells failed to activate DCs—an observation that may contribute to down-regulation of the immunostimulatory response to apoptotic cell-derived self-antigens and to the maintenance of self-tolerance (140,141). Both immature and mature DCs, and their role in the fate of necrotic and apoptotic cells, are described below. DCs DCs (dendritic means branched) are a group of potent antigen-presenting cells of the immune system that have an extraordinary capacity to interact with B and T cells in response to both new and recall antigens, and can modulate their responses. B cells can directly recognize antigens, whereas CD4(⫹) and CD8(⫹) T cells recognize antigenic pieces bound to major histocompatibility complex (MHC) class I and II molecules expressed on DCs. Bone marrow– derived DCs and their precursors are found as a loose cellular network in the skin (142), mucosa (143), intestines (144), liver (145), lung (146), thymus (147), and secondary lymphoid tissues (148); small numbers circulate in the peripheral blood as immature DCs where they capture and process exogenous antigens, and when necessary, pathogenic microorganisms (141). An example is the lymphoid follicular DC, a specialized stromal cell that is essential in sustaining the viability, growth, and differentiation of activated B cells for eventual recirculation (148). These cells have a characteristic spindled/stellate shape with long cytoplasmic processes. A second type captures foreign antigens from many sources in peripheral tissue for transportation to lymphoid or-

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gans to optimize clonal selection of rare CD4(⫹) and CD8(⫹) T cells. DCs play a significant role in orchestrating the adaptive immune system, functioning not only in the induction of immunity but also in the maintenance of tolerance (148,150153). The ingestion of apoptotic cells and antigen uptake by DCs is restricted to an immature stage of development, and at this stage, they express low levels of MHC and costimulatory molecules necessary for the stimulation of resting T cells. Several receptors expressed by immature DCs belong to the C-type lectin superfamily, including langerin (CD207), the mannose receptor (CD206), and DEC-205 (154,155). Subsets of DCs that differ in localization, phenotype, and function express distinct C-type lectins. The C-type lectins are characterized as having either a single carbohydrate recognition domain or multiple carbohydrate recognition domains, which interact with proteins with either mannose or galactose side chains in a calcium-dependent reaction (155). These lectins function in capturing antigens by recognizing, for example, carbohydrate profiles on microorganisms: a binding that depends on arrangements of sugar residues and their branching. Binding to 1 of these lectins may lead to internalization of the antigen(s) and intracellular processing into lysosomal compartments for MHC class II–mediated antigen presentation to T lymphocytes. Albert et al. (140), in a study of DCs and phagocytosis of apoptotic cells, characterized the developmental stage and receptors. They found that the immature phase was 4 to 5 times more efficient than mature cells for efficient phagocytosis. The immature stage of DCs also expressed a unique profile of receptors, the ␣v␤5 integrin and CD36 (␣v␤3/thrombospondin receptor), which appears to facilitate phagocytosis of apoptotic material (156). The studies indicated that immature DCs readily phagocytose apoptotic cells and the action is noninflammatory, but they are not sufficiently efficient for cross-presenting antigens unless they receive a maturation stimulus (140,156). The state of activation of DCs is of utmost importance for the efficient priming of T-cell responses, and the term maturation has been applied for this property. Many different antigens and stimuli trigger maturation, including lipopolysaccharides, bacteria and viruses, contact allergens, cell products, TNF-␣, IL-1␤, prostaglandin E2, inter-

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feron (IFN)-␥, immunostimulatory unmethylated cytodine phosphoguanosine oligonucleotides, and polyinosine:cytosine (157-165). In addition, receptor-mediated events, such as engagement of the CD40 antigen located on the surface of DCs, trigger maturation (166,167). Mature DCs migrate to secondary lymphoid organs where they optimally act as potent antigen-presenting cells and display large quantities of MHC-peptide complexes at their surface (140,141). DCs are the major human or mouse type of cell that can present exogenous (extracellular) antigens to CD8⫹ cells (168), suggesting that, in vivo, DCs constitute the primary cross-presenting antigen-presenting cell. This ability for cross-priming is of great importance for the generation of antitumor cytotoxic T lymphocytes, and for the immune response to viruses, allografts, and intracellular pathogens. Additional studies by Sauter et al. (141) showed great differences in DCs in manipulating dying tumor or transformed cell lines, which depended on the mechanism of cell death. The DCs phagocytosed both apoptotic and necrotic cells, but only necrotic tumor or transformed cell line exposure selectively induced maturation of DCs to become fully functional antigen-presenting cells. This observation also was reported with murine DCs (169). DCs appeared to distinguish between cells that underwent necrosis or apoptosis. In summary, (1) immature DCs efficiently phagocytose many different apoptotic and necrotic tumor cells and (2) only exposure to necrotic tumor cells, but not to apoptotic cells, induces maturation (151). Fascinating subjects for future investigations will be which signals initiate migration and whether differences in immunogenic signals exist. The mature DCs, after acquisition of restricted markers and costimulatory molecules such as CD80 and CD86, cell adhesion molecules, and cytokine production (151), produce antigenic peptides for MHC class I and class II molecule presentation; then they became potent CD4(⫹) and CD8(⫹) stimulators. The importance of costimulatory molecules such as CD80, for example, is undisputed in the induction of effective antitumor immune responses (170). Another maturation marker for DCs is CD83, a member of the immunoglobulin superfamily (171). Along with costimulatory molecules CD80 and CD86, CD83 is thought to be an important regulator of immune responses. In addition, the production of proinflammatory

SCHULTZ AND HARRINGTON

IL-12 by fully functional and mature DCs, a central immunoregulatory cytokine, is a major Th1-driving cytokine, and promotes activated cytotoxic T-lymphocyte effector cells (172-174). This cytokine is critical for the production of IFN-␥ during innate and adaptive immune responses to many different parasitic, bacterial, and viral infections. Furthermore, expression/secretion of IL-12 may establish an autocrine signal to DCs, sustaining the potential of DCs to maintain ongoing T-cell priming (169,175). As discussed by Stuart et al (176), the apoptotic cell inhibits activation of DCs; thus, it may contribute to down-regulation of the response to apoptotic cell– derived self-antigens and to maintain self-tolerance. DCs also have the capacity to undergo rapid apoptotic death (177,178). Mature DCs are hypothesized to undergo rapid apoptosis to ensure that the host inflammatory response does not become excessive and result in tissue damage (177). In addition, a fast turnover is an important factor in the maintenance of tolerance. Other studies of interest with DCs and rapid apoptosis have involved TRAIL, the 40-kd type II transmembrane protein that is structurally related to the TNF family of proteins (179,180). The binding of TRAIL by its receptors DR4 (TRAILR1) and DR5 (TRAIL-R2) rapidly induces apoptosis of TRAIL-sensitive cells. Cell death by this mechanism is largely independent of Bcl-2 reactions because it bypasses the mitochondrial pathway in favor of a direct caspase activation pathway (181-183). Activation of apoptosis through TRAIL has shown that DCs are especially sensitive to death via this route, but not via FasL or TNF (184). A long persistence of DCs could cause an excessive stimulation of T and B cells, generating an inappropriate T-cell accumulation and a chronic inflammatory response (147,185). The preparation of ex vivo differentiated DCs for immunization purposes has been used for the treatment of cancer and other diseases. Individuals with neoplastic growth were immunized with specific tumor antigen–loaded DCs prepared in vitro with varying degrees of success. The method used in animal models has resulted in protective immunity and the rejection of established tumors (186,187). The clinical use of peptide- or tumorpulsed DCs for vaccination has met with variable success in some cancer patients (188-190), and this approach to therapy may hold future promise after

APOPTOSIS

optimizing the protocols. The introduction of agents into tumors via DCs with the idea of either inducing the cancer cells back to normal apoptotic cell death or selectively enhancing this process, but having no or minimal effects on normal cells, is currently a goal of many investigators (191,192). Studies by Sombroek et al (193), showing the role of cyclooxygenase-1–and-2–regulated prostanoids in the tumor-associated inhibition of DC differentiation, indicate at least 1 mechanism of escaping immunosurveillance. REGULATION OF APOPTOSIS BY VIRUSES

Many viruses have evolved molecules that regulate apoptosis in cells of the host. For example, crmA is expressed by the cowpox virus, and is an antiapoptotic protein that potently blocks Fas- and TNF-induced apoptosis by inhibiting caspase activation (194). The inhibitory action by the cowpox factor prolongs replication in host cells. Another protein (p35) encoded by baculoviruses also inhibits caspases (195). The IAP family of proteins, of which there are several cellular homologues, blocks activation of caspases (196). A large family of viral antiapoptotic proteins homologous to c-FLIP has been cloned from several viruses, including the human oncogenic gamma herpesvirus-8 (HHV-8) or Kaposi sarcoma herpesvirus, a lymphotropic virus. Like c-FLIP, these viral FLIPs (v-FLIPs) inhibit apoptosis by interfering with the death effector domain of the DISC and activation of procaspase-8 (197) (Fig 1). B cells expressing v-FLIP are also tumorgenic in a murine model, and block cytotoxic T-lymphocyte– mediated receptor-induced apoptosis (198). Although there is evidence that c-FLIP is regulated by NF-␬B, whether this occurs with v-FLIP is unknown (199). The 2 known human oncogenic gamma herpesviruses, HHV-8 and Epstein-Barr virus (EBV), encode genes that are homologues to cellular growth factors, growth factor receptors, and antiapoptotic genes that permit the growth and replication of the infected host cells (200). Survival of virus-infected cancers such as EBV-associated immunoblastic lymphomas and HHV-8 associated primary effusion lymphomas (PEL), a rare distinct subtype of non-Hodgkin lymphoma, is dependent on constitutive NF-␬B activity. Blockade of NF-␬B induces apoptosis in both of these tumor types (201,202). Keller et al (201) used an irrevers-

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ible inhibitor of I␬B␣ phosphorylation (Bay 117082) to specifically inhibit NF-␬B binding to DNA in PEL cells to induce apoptosis, whereas Cahir-McFarland and Kieff (202) used a tetracycline-regulated expression of a deletion mutant of I␬B␣ (⌬N-I␬B␣) in IB4 cells to greatly diminish NF-␬B activity and induce apoptosis. Also of interest, these investigators (202, 203) showed that caspase activity was not required, indicating that multiple apoptotic pathways are activated after blocking NF-␬B activation. In the case of EBV-associated lymphoproliferative disease, constitutive NF-␬B activity and DNA– binding site activator protein-1 induction occur in response to EBV oncoprotein latent membrane protein (LMP)-1 expression. The latter is considered an oncogene because of its ability to transform rodent fibroblasts and to drive proliferation of lymphoblastoid cell lines (204). Oncoprotein LMP-1 engages TNRF-associated factors and the TNFR-associated DD, and is critical for transforming primary B lymphocytes and suppressing apoptosis in B lymphocytes, possibly via the upregulation of Bcl-2 (205,206). The mechanism whereby NF-␬B is constitutively activated in HHV-8 –associated PEL is unclear. A report by Liu et al (207) indicated that HHV-8 v-FLIP was capable of activating IKK. The viral protein latency–associated nuclear antigen, which is expressed in all HHV-8 tumors, associates with the activator protein-1 site and has a variety of functional domains, 1 of which represses p53 activity (208). In comparison to gamma herpesviruses, the human oncogenic RNA virus human T-cell leukemia virus type-I is small and has relatively few genes (209). Human T-cell leukemia virus type-I encodes a nuclear phosphoprotein called Tax, which transactivates viral and gene cellular expression. Tax transactivates cyclic adenosine monophosphate response elements and NF-␬B transcription factors, events thought to be critical in the development of T-cell transformation, leading to adult T-cell leukemia (210). Constitutive NF-␬B also appears to be a hallmark of adult T-cell leukemia, although Tax expression is absent in primary adult T-cell leukemia cells (211). Therefore, the oncoprotein Tax is likely to be necessary for the evolution of adult T-cell leukemia, but not required once the malignancy is fully established. Gamma herpesviruses encode a variety of other

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anti-apoptotic proteins. EBV BHRF1 (messenger RNA coding an early lytic cycle protein) has sequence homology to cellular Bcl-2 and inhibits apoptosis and delays cell death (212). Like EBV, HHV-8 also encodes a Bcl-2 that reportedly does not heterodimerize with Bax, and is not cleaved by caspases as in the case with cellular Bcl-2 (213). Susceptibility or resistance to apoptosis varies according to the histologic subtype in EBV-associated lymphomas. The 2 main categories of lymphomas in immunocompromised patients, Burkitt lymphoma and immunoblastic lymphoma, differ in their association and expression pattern of EBV. EBV-positive Burkitt lymphoma exhibits a type I pattern: Epstein-Barr– encoded poly RNA(⫹), LMP(⫺), and Epstein-Barr nuclear antigen-2(⫺); large cell varieties usually express a type III pattern: Epstein-Barr– encoded poly RNA(⫹), LMP(⫹), and Epstein-Barr nuclear antigen(⫹). Because LMP elicits a cytotoxic T-cell response, it has been postulated that its presence and the subsequent up-regulation of Bcl-2 may be a reflection of the profound immunodeficiency seen in patients with acquired immunodeficiency syndrome (214). An interesting antiapoptotic mechanism has been reported in Hodgkin disease. In cell lines and in clinical samples derived from Hodgkin disease (an EBV-associated lymphoma), nonfunctional I␬B that results in constitutive NF-␬B activity has been shown (90). HHV-8 contains several open reading frames that encode interferon regulatory factors (IRFs), called vIRFs, which regulate the expression of interferon-responsive elements. IRFs are a family of cellular transcription proteins composed of 10 distinct members that regulate expression of genes involved in pathogen response, cell proliferation, and immune modulation (215). The vIRF-1 blocks type I and II IFNs, and blocks IRF-1–mediated transcription, apparently by competing with host

IRFs for binding to coactivators cyclic adenosine monophosphate responsive element– binding protein and p300 (216). Overexpression of vIRF-1 is capable of inducing tumors in nude mice (217). The vIRF-2 blocks IFN-␣–mediated inhibition of vesicular stomatitis virus replication, presumably by binding and inhibiting protein kinase R. The latter, which is induced by IFN-␣, is an important host defense factor that inhibits protein synthesis and activates cellular antiviral mechanisms (218). Toomey et al (219) have shown a unique proapoptotic effect of the antiviral combination azidothymidine and IFN-␣. IFN-␣ induces the HHV-8 – associated DR ligand TRAIL in PEL. Interferon ␣–mediated apoptosis in PEL was greatly potentiated in the presence of azidothymidine (219). Further investigation showed that IFN-␣ also activated NF-␬B nuclear translocation, thereby blunting the proapoptotic effect of TRAIL (220). Azidothymidine blocks this effect by inhibiting IKK kinase and NF-␬B, allowing for an unimpeded apoptotic signal through TRAIL. IFN-␣ and azidothymidine blocked tumor (PEL) formation in a severe combined immunodeficiency mouse model and induced a complete remission in a patient suffering from acquired immunodeficiency syndrome–related PEL. From these experiments, it appears that some HHV-8 –mediated lymphomas may actually be susceptible to antiviral therapy. In contrast to these results, IFN-␣ exhibited no activity in EBV(⫹) Burkitt lymphoma cells, and did not upregulate TRAIL expression. Recent data from other investigators indicate that resistance to IFN␣–mediated apoptosis in EBV(⫹) Burkitt lymphoma is caused by viral-encoded poly (A)-RNA that bind to protein kinase R, thereby inhibiting phosphorylation (221). This shows yet another mechanism used by viruses to blunt the host cell apoptotic response, presumably to ensure a site for replication.

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Appendix Alphabetized Glossary of Terms Activation-induced cell death: In T cells, this type of cell death appears to be an important mechanism for the termination of the immune response, especially in killing autoreactive T cells and in preventing autoimmune responses. The interaction between Fas and FasL is the major mechanism in AICD. Annexin V: Annexins are ubiquitous homologous proteins that bind phospholipids in the presence of calcium. At the onset of apoptosis, phosphatidylserine is translocated from the internal membrane surface to the external portion of the membrane. Because the movement of phosphatidylserine from the internal membrane surface to the external portion is an early indicator of apoptosis, annexin V interacts strongly and specifically with phosphatidylserine, and is used to detect apoptosis. AP-1: DNA binding site activator protein–1. Apaf-1: Apoptosis protease-activating factor-1. An adaptor protein associated with the intrinsic pathway of apoptosis that binds to procaspase-9 through the caspase activation and recruitment domain, resulting in caspase-9 activation in the presence of cytochrome c and dATP, leading to an apoptotic protease cascade. Apoptosis: A continuous physiologically programmed cell death that is dependent on protease activation, and in which cell chromatin is condensed and the DNA is rapidly degraded—a hallmark of apoptosis. It is a form of cell death that is used to remove unwanted cells during embryonic development, metamorphosis, immune system maturation, and cell turnover. Apoptosis includes DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage, and disassembly into membrane-enclosed vesicles (apoptotic bodies). In vivo, the process terminates with engulfment of apoptotic bodies by phagocytic cells and removal to minimize inflammatory or immune reactions that could result from the release of cellular components. The apoptotic cells are cleared silently by phagocytosis, and recent evidence suggests that apoptotic bodies inhibit the expression of proinflammatory cytokines from some phagocytes. Apoptosis-inducing factor: A mitochondrial flavoprotein with homology to bacterial oxidoreductases, it induces apoptotic morphologic changes of the nucleus, independent of caspases. It may represent a minor pathway for apoptotic nuclear changes. Apoptotic cell–associated molecular patterns: The exposure of proteins, phospholipids, and sugars by apoptotic cells. Bad: A proapoptotic member of the Bcl-2 family of proteins. Bad shares identity with Bcl-2 only in the BH3 domain and forms heterodimers with Bcl-2 and Bcl-XL to counter the survival-promoting action of Bcl-2 and Bcl-XL and to promote cell death. B-cell activating factor belonging to the tumor necrosis factor family: A tumor necrosis factor ligand family member that binds to B cells and stimulates their proliferation via receptors such as transmembrane activator, a member of the tumor necrosis factor receptor family that does not bear a death domain. It is constitutively expressed by monocytes and macrophages. Increased serum levels have been found in mice strains susceptible to systemic lupus erythematosus. B-cell maturation protein: A member of the tumor necrosis factor receptor family, it is a specific receptor for BAFF, and is expressed only by B lymphocytes. Bcl-2 protein family and subfamilies: Members have either anti- or proapoptotic properties. All members possess at least 1 of 4 conserved motifs termed Bcl-2 homology (BH) domains (BH1 to BH4), which mediate protein interactions. The apoptosis inhibitor members include Bcl-2, Bcl-XL Mcl-1 A1, and Bcl-w. The proapoptosis Bax subfamily, sharing 3 BH domains (BH1, BH2, BH3) includes Bax, Bak, Bok, and Bcl-XS; the BH3-only subfamily, the second proapoptotic subfamily, includes Bik, Hrk, Bim Blk, Bad, and Bid. The proapoptotic BH3-only domain proteins promote apoptosis by binding to and antagonizing antiapoptotic Bcl-2 and Bcl-XL members. The ratio of Bcl-2 to Bax in the cell can determine whether or not the cell initiates apoptosis or survives. Antiapoptotic members (eg, Bcl-2), after transgenic expression in B cells, can increase B-cell numbers, B-cell lifespan, and serum antibody levels. BH: Bcl-2 homology domains in the Bcl-2 family of inhibitors and promoters of apoptosis. CAD: Caspase-activated deoxyribonuclease. The endogenous endonuclease that fragments the cellular DNA, used as a marker for apoptosis.

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Alphabetized Glossary of Terms Caspase activation and recruitment domain: A global homophilic interaction domain found in several caspases with large prodomains. Caspases 1-14: Cysteine proteases that exist as inactive precursors, which can be activated by proteolysis. The protein-clipping enzymes orchestrate the cell death program. After caspases are activated, 1 of their targets is the inhibitor of the caspase-activated deoxyribonuclease. Caspase-actived deoxyribonuclease is an endogenous endonuclease that degrades the cell’s chromosomal DNA. Thus, 1 role of caspases is to inactivate proteins that protect living cells from apoptosis. CD40 and CD40L (CD154): CD40 is the receptor for CD40L, the latter a trimeric, type II transmembrane protein belonging to the tumor necrosis factor family. Interaction between the receptor CD40 and CD40L leads to the recruitment of tumor necrosis factor receptor–associated factor family of protein members and to the subsequent activation of the nuclear factor–␬B signaling pathway. CD95: Fas or Apo-1. Death receptor and a member of the tumor necrosis factor receptor superfamily. It is constitutively expressed in many cells and may be induced in many tissues by appropriate stimuli. Deficiency predisposes individuals toward autoimmunity, cancer, and other disorders. Spontaneous recessive mutations of CD95 in lpr mice and CD95L in gld mice describe an apoptosis gene defect characterized by a lymphoproliferative syndrome caused by the inability to delete long-term activated T cells. CD95L: CD95 ligand, a homotrimeric molecule and tumor necrosis factor family member. CD95 and CD95L may be present in the same cell after stimulation, leading to autocrine suicide or paracrine death by binding of the membrane-bound or soluble ligand to its cognate receptor. CD95L is predominantly expressed in activated T lymphocytes and natural killer cells, but also constitutively in the tissues of immune-privilege sites such as the testis and eye. Cysteine-repeat domain: Or carbohydrate-recognition domain. Cytochrome c: Normally associated with mitochondria and oxidative phosphorylation. It is released from mitochondria into the cytosol during apoptosis, and becomes a component of the intrinsic apoptotic pathway. After release from permeabilized mitochondria, it binds to Apaf-1, inducing it to associate with procaspase-9, followed by caspase-9 activation, which culminates in apoptosis. The release of cytochrome c is regulated by members of the pro- and the antiapoptotic proteins of the Bcl-2 family. Cytokine-response modifier A: An orthopoxvirus-encoded protein that blocks death receptor–mediated apoptosis by preventing the activation of caspase-8. Daxx: A Fas binding protein that binds to the Fas death domain but activates a Fas-associated death domain–independent death pathway to enhance apoptosis that involves the stress-activated c-Jun N-terminal kinase pathway. It links autoimmune disease to the major histocompatibility complex and to apoptosis. DD: Death domain. Critical for apoptosis signaling. Death effector domain: An 80–amino acid region of a intracellular domain, the death domain. Death receptor–independent cytotoxic agents: Glucocorticoids (dexamethasone), ionizing irradiation, and chemotherapeutic drugs. Death signals can be integrated directly or indirectly via the intrinsic apoptotic pathway, which involves mitochondria, leading to the eventual activation of caspase proteolytic pathways and disruption of cell integrity. Deoxyribonuclease: Enzymes that cleave DNA. DISC: Death-inducing signaling complex. Composed of the death domains of the receptor (ie, Fas), caspase-8, and Fas-associated death domain adaptor protein. DR: Death receptors. They are members of the tumor necrosis factor receptor superfamily. EBV: Epstein-Barr virus. FADD: Fas-associated death domain, an adaptor protein. The death domains of Fas ⫹ FADD ⫹ caspase-8 combine to produce the death-inducing signal complex. The molecule is essential for apoptosis induction by Fas (CD95).

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Alphabetized Glossary of Terms Fas: Same as CD95 or Apo-1, and is a cell-surface protein that mediates apoptosis. It is a member of the tumor necrosis factor receptor family, and can trigger apoptosis when it is bound by CD95L. The cytoplasmic domain of Fas, which has a death domain, associates with the death domain of Fas-associated death domain, causing Fas-associated death domain to recruit procaspase-8, which is then activated, initiating the apoptotic pathway. Fas apoptosis-inhibitor molecule: Used by cells to counteract the death pathways. It is a survival protein, originally isolated from inducibly Fas-resistant B lymphocytes. Fas-associated death domain–like interleukin-1␤–converting enzyme: FLICE; now designated caspase-8. FasL: Same as CD95L, the ligand for Fas. FLIP: FLICE (caspase-8)–inhibitory survival protein. Cellular FLIP (Fas-associated death domain–like interleukin-1–converting enzyme inhibitory protein) can prevent Fas-associated death domain from activating caspase-8. It is an enzymatically inactive homologue of caspase-8, which can bind to the death effector domains of Fas-associated death domain and caspase-8. It contains 2 death effector domains that bind to the death effector domain of Fas-associated death domain and FLICE, thereby blocking transmission of the Fas-induced death signal. Viral FLIPs bind to the death effector domain of Fas-associated death domain and interfere with the Fas-associated death domain–caspase-8 interaction, thereby inhibiting the recruitment and activation of caspase-8 by Fas, and prolonging the survival of the cell. Granzyme B: A serine protease found in the granules of natural killer cells and cytotoxic T lymphocytes. Granzyme B is the most prominent granzyme in a family of 11 found in the cytotoxic granules, and cleaves after aspartic acid residues, inducing cell death by various caspase-induced pathways. It also induces cytochrome c release from mitochondria independent of caspases. The cytotoxic T lymphocytes recognize virus-containing cells and cause them to undergo apoptosis by transferring enzyme-bearing granules (eg, granzyme B) through pores in their surface formed with perforin. Apoptosis caused by cytotoxic T lymphocytes has an identical appearance to apoptosis caused by other agents. HHV-8: Human oncogenic gamma herpesvirus-8. IAP: Inhibitor of apoptotic proteins. A family of survival proteins, the expression promoted by nuclear factor-␬B in the nucleus of the cell. IBL: Immunoblastic lymphoma. ICAD: Inhibitor caspase-activated deoxyribonuclease. It appears to function as a chaperone for caspase-activated deoxyribonuclease during its synthesis, remaining complexed with caspase-activated deoxyribonuclease to inhibit its deoxyribonuclease activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing caspase-activated deoxyribonuclease into the nucleus to degrade chromosomal DNA. I␬B: Inhibitors of kappa B. They bind homo- and heterodimerized nuclear factor-␬B proteins in the cytoplasm, preventing their entry to the nucleus. Nuclear factor-␬B is kept in an inactive form in the cytoplasm through interaction with a family of inhibitory proteins called I␬Bs (I␬B␣, I␬B␤, and I␬B␧). IKK: I␬B kinase complex that phosphorylates I␬Bs in response to proinflammatory signals. The IKK complex contains 2 kinases, IKK-1 and IKK-2, as well as several scaffolding proteins. The IKKs are activated by signals derived from the tumor necrosis factor receptor. IL-1␤: A cytokine synthesized by activated mononuclear phagocytes sharing many properties in common with tumor necrosis factor. It is found free in the circulation and is a signaling molecule of the immune system that triggers inflammation. Immune deviation: Preferential activation of 1 Th cell subset over another. Interleukin-1␤–converting enzyme: Presently called caspase-1, a mammalian cysteine protease. It resides in the cytoplasm, and converts prointerleukin-1␤ to active interleukin-1␤. The latter alerts neighboring cells and the immune system that it is destroying itself in response to a viral threat. IRF: Interferon regulatory factor. Kaposi sarcoma–associated herpesvirus: Also called human oncogenic gamma herpesvirus-8.

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Alphabetized Glossary of Terms Latency-associated nuclear antigen: The viral protein expressed in all human oncogenic gamma herpesvirus-8 tumors. LMP-1: An oncogene, latent membrane protein-1. Necrosis: Possibly a caspase-independent signaling pathway that may be distinct or interrelated with the apoptotic cell-death pathway. Four common mechanisms are associated with cell injuries that induce necrosis: adenosine triphosphate depletion (hypoxia/ischemia-inhibited aerobic respiration), free radicals (generation of reduced oxygen species), membrane damage (defects in membrane permeability), and calcium influx (disruption of calcium homeostasis). The cell contents leak out in necrosis, leading to inflammation and, ultimately, cell disintegration. NF-␬B: Nuclear factor-␬B. Consists of 2 subunits (p50 and p65), and exists as a complex with I␬B in the cytoplasm of resting cells. The I␬B is 1 of a class of several related inhibitory proteins. It can be activated by proinflammatory stimuli, such as tumor necrosis factor or interleukin-1, and results in phosphorylation of I␬B; the released NF-␬B enters the nucleus and activates various genes carrying the NF-␬B response element. The target genes for NF-␬B encode survival factors, such as members of the inhibitor of apoptosis and the Bcl-2 homologues Bfl-1/A1 and Bcl-XL. Thus, NF-␬B protects many cells from apoptotic signals. However, the protection or induction of apoptosis has to be considered in the context of stimuli, cell types, and differentiation stages of the cells. Acetylsalicylic acid (aspirin) inhibits the NF-␬B pathway by binding to IKK␤ (a protein kinase complex), thus preventing I␬B degradation and the nuclear translocation of NF-␬B. p53: A nuclear protein suppressor of human cancer, it serves as a critical regulator of cell survival and proliferation, and is a major determinant of chemo- and radio-sensitivity. This 393–amino acid nuclear phosphoprotein regulates the expression of death receptor genes in apoptosis induction. These include both Fas and tumor necrosis factor–related apoptosis-inducing ligand death receptor-5 receptors. At least in some tumor types (eg, breast cancer), abnormal p53 function is associated with an adverse prognosis. Studies of murine cells have suggested that p53 loss leads to resistance to apoptosis induced by exposure to ionizing radiation or to a variety of chemotherapeutic drugs. The introduction of functional p53 into cells can induce growth arrest and/or cell death. Pattern recognition receptors: On macrophages and dendritic cells. PEL: Primary effusion lymphoma, a rare distinct subtype of non-Hodgkin lymphoma. Perforin: A 70-kd glycoprotein from cytolytic T lymphocytes and natural killer cell granules that produces target cell lylsis. Perforin, released from cytotoxic T-cell granules, mediates apoptosis through the release of granzyme B. Permeability transition pore complex in mitochondria: A reversible Ca2⫹-induced permeabilization of the mitochondrial inner membrane through the opening of the mitochondrial permeability transition pore. Prostaglandin (PG) E2: A biologically active lipid derived from arachidonic acid through the action of the enzyme cyclooxygenase. It blocks major histocompatibility complex class II molecule expression in T cells and in macrophages, and inhibits T-cell growth. It also prevents aggregation of platelets. PS: Phosphatidylserine. Receptor-interacting protein: Or ring finger–interacting protein. A serine-threonine kinase containing a death domain that binds to tumor necrosis factor receptor-1–associated death domain protein via interactions between their death domains, which is a direct downstream mediator of Fas-triggered cell death. Fas, tumor necrosis factor–related apoptosis–inducing ligand, and tumor necrosis factor receptors initiate death by 2 alternative pathways: (1) rely on caspase 8 and (2) depend on the kinase RIP. Smac/DIABLO: Second mitochondria-derived activator of caspases. Human Smac, second mitochondriaderived activator of caspases. Murine DIABLO, direct inhibitor of apoptotic protein binding protein with low isoionic point. After release from mitochondria, it eliminates the inhibitory effect of many inhibitors of apoptotic proteins. Smac appears to be a master regulator of apoptosis in mammals.

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Alphabetized Glossary of Terms TNF: Tumor necrosis factor ligand family. At least 16 members have been identified. Most ligand members are synthesized as type II transmembrane precursors. Their extracellular domains can be cleaved by metalloproteinases to form soluble cytokines. Soluble and membrane-bound TNF ligand family members bind to receptors of the TNF receptor family. TNFR1: A member of the tumor necrosis factor receptor superfamily that has a death domain. Other human TNFR superfamily death receptor members with death domains include: Fas, death receptor-3, death receptor-4 (also called tumor necrosis factor–related apoptosis-inducing ligand receptor 1, or TRAIL R1), death receptor-5 (TRAIL R2) and death receptor-6. Each of the receptors can directly initiate apoptosis. TRADD: Tumor necrosis factor receptor-1–associated death domain protein. It binds to tumor necrosis factor receptor-1, but unlike Fas-associated death domain, TRADD does not carry a death effector domain, and its death domain is responsible for mediating apoptosis. It functions as a platform adaptor that recruits several signaling molecules to the activated receptor. TRAF: Tumor necrosis factor receptor–associated factor family of proteins. TRAIL: Tumor necrosis factor–related apoptosis-inducing ligand, also known as Apo-2 ligand. A type II transmembrane protein belonging to the tumor necrosis factor/nerve growth factor superfamily, which preferentially induces apoptotic cell death in a wide variety of transformed cells but not in normal cells. Two cell death–inducing receptors (TRAIL-R1/death receptor-4 and TRAIL-R2/death receptor-5) and two non-cell-death-inducing receptors (TRAIL-R3/decoy receptor 1 and TRAIL-R4/decoy receptor 2) have been identified. It has an important role in suppressing tumor metastasis, and inhibits T-cell activation, cell cycle progression, and cytokine production in human autoreactive and foreign antigen-specific T cells. Transmembrane activator and calcium-modulator and cyclophilin ligand interactor: A member of the tumor necrosis factor receptor family that does not bear a death domain; it is a receptor for B-cell activating factor. Tumor necrosis factor–related activation protein: A cell surface protein almost exclusively expressed on CD4⫹ T lymphocytes. It is the ligand for CD40. Interaction of TRAP with CD40 plays an important role in T-cell help for B cells; it is critical for immunoglobulin class switching. Tumor necrosis factor–like weak inducer of apoptosis: A possible ligand for death receptor-3. TUNEL: Terminal deoxyribonucleoside transferase–mediated deoxyuridine triphosphate nick-end labeling. A method to detect fragmented DNA. The method does not discriminate among apoptosis, necrosis, and autolytic cell death. Ubiquitin: Named because it is ubiquitous and abundant in eukaryotes; it is the most highly conserved protein described in the literature. It is a cytosolically based adenosine triphosphate–dependent proteolytic system that degrades both normal and abnormal proteins, independent of the lysosomal system. vIRF: Viral interferon–regulated factors.