Major apoptotic mechanisms and genes involved in apoptosis

IAP Antagonists Induce Autoubiquitination of c-IAPs, NF-κB Activation, and TNFα-Dependent Apoptosis

To study the relationship between the expression levels of cytokine/receptor genes in placenta and the pathogenesis of pre-eclampsia. The study was performed to compare the mRNA contents of cytokine (receptor) superfamily genes in placentas from 5 patients with pre-eclampsia and 5 strictly matched normal pregnancies. A complementary DNA microarray representing over 220 cytokine-associated genes was employed to complete the detection. It was shown that, among the 221 kinds of cytokine-associated genes, 162 of those including 22 interleukin/interleukin receptor genes presented with a difference of over two times in pre-eclamptic placentas compared to normal placentas. Most of the 22 interleukin/interleukin receptor genes were shown to be highly expressed in preeclamptic placenta, while the expression of IL-2 receptor (IL-2 R alpha, GenBank: X01057) gene in preeclamptic placenta was comparatively lower than that in normal placenta. Furthermore, some tumor necrosis factor (TNF)/receptor superfamily genes, including TNF (GenBank: X02910), TNF ligands (GenBank: U03398, U37518, AF053712, AF055872) and TNF receptors (GenBank: X60592, X63717, M83554, AF016266, AF016267, U81232) were also shown to be highly expressed in pre-eclamptic placenta. Besides interleukin and tumor necrosis factor (receptor) gene superfamily, the mRNA levels of another 39 cytokine and 15 cytokine receptor genes showed a two-fold difference between pre-eclamptic and normal placental tissues. Additionally, most of the genes were up-regulated in pre-eclamptic placenta. The up-regulation of cytokine-associated genes including interleukin and TNF (receptor) superfamily expression in placenta might be intensively related to the pathogenesis of pre-eclampsia.

In the past decade, it became apparent that immune mediated cell death in a number of autoimmune endocrine diseases was due to the induction of apoptosis in target organ cells. This was conclusively demonstrated for thyroid follicular cells in Hashimoto’s (destructive autoimmune) thyroiditis, but the mechanisms underlying this cell death were not clear. Several hypotheses were put forth involving the role of deathsignalling molecules expressed on thyroid cells. While many of these hypotheses did not hold up under close scrutiny, this stimulated work on the molecular mechanisms of thyroid destruction. Several apoptosis signalling pathways, initiated by molecules such as Fas ligand (FASL) and tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. In this review we will attempt to sort out the inconsistencies in published data on the mechanisms of death-receptor mediated thyroid destruction. We will also review recently proposed models of these mechanisms, and outline directions for research that we feel might lead to discoveries of benefit to the clinician in the treatment and prevention of destructive autoimmune thyroiditis.

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.

APOPTOSIS: PHYSIOLOGICAL CELL DEATH Apoptosis, as a biological phenomenon, is readily identifiable by several characteristic features. It characteristically affects scattered single cells, not groups of contiguous cells as in necrosis, with the dying cell undergoing a relatively ordered form of cell death. This physiological cell death is characterized by cell shrinkage, cellular crenation, cytoplasmic and chromatin condensation, and internucleosomal DNA fragmentation (1). Changes in membrane glycosylation and lipid profiles, and alteration in expression of surface receptors have been observed. The apoptotic cells are rapidly phagocytosed and degraded by neighbouring cells or resident macrophages without an inflammatory response. This mechanism prevents the release of the phlogistic contents of cells and avoids the possibility of neighbouring host cell injury. The process differs significantly from cell death by necrosis or lysis where cells release their contents into the surrounding tissues and perpetuate the local inflammatory response. A glossary of terms pertinent to this review is included as an appendix. APOPTOSIS: MORPHOLOGICAL EVENTS During apoptosis, the dying cell undergoes a series of profound structural changes. The earliest event observed by electron microscopy is condensation of chromatin to form sharply circumscribed, uniformly dense, cresentic masses that abut the nuclear envelope (2). Nucleolar changes include the dispersal of peripheral nucleolar chromatin to form aggregates in the centre of the nucleus. Simultaneously with the nuclear changes, apoptotic cells detach from neighbouring cells, and specialized surface structures such as microvilli appear. Cell volume decreases, cell density increases, cytoplasmic organelles compact, and convolution of the cell and nuclear outline becomes evident (1) (Fig. 1). Cytoplasmic changes include cytoskeletal filament aggregation, clumping of ribosomal particles, and rearrangement of rough endoplasmic reticulum to form a series of concentric whorls (3). Cytoplasmic and nuclear condensation is followed by the production of numerous membrane protuberances at the plasma membrane that subsequently separate with sealing of the plasmalemma to form membrane-bound apoptotic bodies of varying sizes with condensed cytoplasm and crowded, intact cytoplasmic organelles. The production of apoptotic bodies is a late occurrence in the apoptotic process and is observed extensively in vitro, but less commonly in vivo. This observation emphasises the rapidity of the apoptotic process whereby apoptotic cells are rapidly phagocytosed in vivo prior to apoptotic body formation (4). Phagocytosis is mediated by adjacent epithelial cells, mononuclear phagocytes, or tumor cells. Once phagocytosed, apoptotic bodies are degraded by lysosomal enzymes derived from the ingesting cell. Rapid phagocytosis of apoptotic cells in vivo before their secondary degeneration helps explain the absence of inflammation associated with apoptosis. Apoptotic bodies that escape phagocytosis lose their integrity after an hour or so, resulting in swelling, loss of density, membrane rupture, and organelle disruption and dispersal referred to as secondary necrosis (Fig. 1).Fig. 1: Morphological aspects of cell death by oncosis and apoptosis. Necrosis can occur after both forms of cell death. A normal cell is shown at the top. 1a, Swelling. 1b, Blebbing, vacuolization, and increased permeability. 2a, Shrinkage and pyknosis. 2b, Budding and karyohexis. 2c, Apoptotic bodies. 3, Necrotic changes (shrinkage, coagulation, and karyolysis) occurring after rupture of a cell surface bleb in oncosis, or secondarily due to failure of apoptotic bodies to be phagocytosed in apoptosis. Adapted from Majno and Joris, 1995.APOPTOSIS: BIOCHEMICAL EVENTS Cytoskeletal and membrane alterations Cell shrinkage and apoptotic body formation require significant changes in both the cytoskeleton and plasma membrane lipid bilayer. Cytoskeletal changes include tissue transglutaminase activation, microtubule disruption, α-fodrin (non-erythroid spectrin) and actin cleavage, and a requirement for actin polymerization. These changes facilitate membrane budding and play a role in the maintenance of plasma membrane integrity in apoptotic cells (5). Membrane changes include redistribution of phosphatidylserine from its normal location on the inner leaf of the plasma membrane lipid bilayer to the outer leaf, exposure of surface sugar residues from loss of membrane sialic acid, and loss of expression of surface markers such as FcγRIII (CD16), complement regulatory molecules (CD45 and CD59), and adhesion molecules (CD11/CD18). These membrane changes are believed to play a role in the recognition and eventual phagocytosis of apoptotic cells (6). Cell shrinkage Condensation of the cytoplasmic space resulting in cell shrinkage appears to be a universal characteristic of apoptosis (7). This change is thought to be consequent to net movement of water out of the cell due to vesicles budding from the endoplasmic reticulum and Golgi apparatus fusing with the plasma membrane with release of their contents into the extracellular space. A role for active ion efflux has been implicated in cell shrinkage with active efflux of Na+ and K+ ions through the Na+,K+ ATPase pump and Ca2+-dependent channel (8,9). Apoptotic DNA degradation Apoptotic cells display dramatic changes in the nucleus, including chromatin condensation and margination. A further nuclear-associated event during apoptosis is the degradation of DNA into 180- to 200-bp oligonucleosomal fragments. These fragments form a ladder-type pattern when subjected to agarose gel electrophoresis, a feature that is now one of the biochemical hallmarks of apoptosis (10). However, it should be noted that apoptotic internucleosomal DNA fragmentation is not universal, although higher molecular weight fragments (50–300 Kbp) have been reported in certain cell types immediately proceeding or in the absence of oligonucleosomal fragmentation (11). A number of putative endonucleases have been proposed, including, DNase I, DNase II, NUC-18, as well as other novel endonucleases including the caspase-activated deoxyribonuclease (12). The fact that mRNA of these endonucleases is expressed in only a limited number of human tissues suggests that other enzymes may participate in the degradation of DNA during apoptosis. Regulators of apoptosis It is well established that cell proliferation and differentiation are highly regulated processes; however, it is now emerging that regulation of cell death is just as complex and equally important in the maintenance of tissue homeostasis (13). Apoptotic cell death is regulated by genetic factors, and the intrinsic death program can be modulated by exogenous "survival" factors. Despite Kerr's seminal work (14) revealing that most physiological forms of cell death share a common set of morphological features, and the assumption that a predictable developmental and morphological event implies genetic regulation, evidence for the genetic regulation of cell death was not revealed until the 1980s following studies on developmental mutants of the nematode Caenorhabditis elegans by Ellis and Horvitz (15). Genetic studies of the nematode identified two genes, ced-3 and ced-4, which were required for normal developmental cell death, and a third, ced-9, which appeared to act as a negative regulator of cell death (Fig. 2). The discovery of mammalian homologues of these genes initiated an intense search for new genes involved in the regulation or execution of cell death pathways. Many new cell death regulators have been identified, and a number of regulators have been shown to be previously identified oncogenes or supressor genes (e.g., bcl-2, myc, ras, and p53). Thus, the genetic regulation of apoptosis is controlled by the activation of genes whose actions are to kill the cell and the corresponding deactivation of genes whose actions are to maintain cell homeostasis (Table 1).Table 1: Positive and negative genetic regulators of apoptosisFig. 2: Homology between cell death pathways in C. elegans and mammals. The ced 9/bcl-2 family consists of pro-apoptotic (egl-1 and bax) and anti-apoptotic (ced-9 and bcl-2) protein members. The ced-9/bcl-2 family integrates positive and negative apoptotic signals and arbitrates whether apoptosis should occur; activation of ced-4/apaf-1 commits a cell to apoptosis and the ced3/caspase family mediates the proteolytic destruction of the cell. Adapted from Adams and Cory, 1998.However, the genetic regulation of a death program can be modulated by exogenous stimuli from the cells immediate environment. The concept that certain mammalian cells are under social controls with extrinsic cellular events regulating endogenous apoptotic programmes was first expounded by Raff (16): individual cells are programmed to commit suicide unless they receive signals for survival. In the presence of a limited supply of extracellular growth factors, cell numbers are maintained relatively constant as a result of competition for growth factors, thereby maintaining a balance between division and cell death. A review of the important positive and negative genetic and environmental regulators of apoptosis follows. Positive genetic regulators c-Myc gene family The myc family of protooncogenes (Myc, Mad, Max, and Mxi-1) encode short-lived nuclear proteins with DNA-binding properties, which can heterodimerize to from transcriptional activators or repressors (17). c-myc is a `Janus gene' involved in both cell proliferation and apoptosis (18). This apparent contradiction is reconciled through an understanding of the different responses exacted by survival signals from this gene. In the presence of a survival signal such as anti-apoptotic cytokines [e.g., insulin-like growth-factor-1 (19)] or overexpression of a negative regulator of apoptosis [e.g., bcl-2, (20)], Myc drives proliferation, and in its absence, the default program induced by Myc results in apoptosis. It is now generally believed that oncoprotein-induced apoptosis may reflect the fact that the pathways mediating growth and apoptosis are coupled processes: the dual signal model (21,22). In this model, activation of cell proliferation necessarily primes the apoptotic program that, unless countermanded by appropriate survival signals, automatically removes the affected cell. Survival signals are normally provided by neighbouring cells, and this ensures that somatic cells remain mutually interdependent for survival and so limits the possible proliferative autonomy of any individual cell. This has direct implications for malignant progression, as generally two or more mutations are required to initiate and promote cellular transformation. Thus, the combination of deregulated Myc and survival signals promotes cell proliferation in the absence of apoptosis and provides a rationale for oncogene cooperation in tumorogenesis (Fig. 3) (23).Fig. 3: Model of the relationship between oncogenes and death signals. In this model, oncoproteins do not trigger apoptosis directly, but they act as a sensitizer to apoptotic triggers (death receptor activation/hypoxia, etc.). In the absence of survival signals, myc sensitises the cell to an apoptotic trigger; however, the combination of deregulated Myc and survival signals act to promote cell proliferation. P53 may sensitize cells in part through upregulation of Fas, although other mechanisms likely exist. Adapted from Evan and Littlewood, 1998.p53 tumor supressor gene The p53 protein is a transcription transactivator that plays a central role in mediating the cellular response to DNA damage, helping to maintain genomic stability (24). Inactivation or loss of p53 are the most common aberrations in human cancers and they indicate that inactivation of tumor supressor genes is as equally important as activation of oncogenes like c-myc in tumorogenesis. Following sublethal DNA damage, p53 directs a G1 cell cycle arrest, allowing DNA repair to occur prior to further replication (reviewed in Ref. 25). In the event of excessive DNA damage, p53 initiates execution of the apoptotic program (26). Although the mechanism whereby p53 induces apoptosis is controversial, several studies have suggested that p53 regulates apoptosis by transcriptional suppression of anti-apoptotic proteins such as bcl-2 and induction of proapoptotic proteins such as bax, insulin-like growth factor binding protein 3 (IGF-BP3), and upregulation of the Fas receptor (Fig. 3). However, apoptosis can proceed by p53-independent pathways (e.g., glucocorticoid-mediated apoptosis of thymocytes), and is not required for developmental cell death (27,28). In sepsis, both p53-dependent and -independent pathways of apoptotic cell death have been reported (29). Overall, these findings suggest that the main role of p53 may be as a sensor of DNA damage and the mediation of the appropriate cellular response, cell cycle arrest, or apoptosis. Death receptor and death factor expression Death receptors belong to the tumor necrosis factor receptor (TNFR) gene superfamily, which is defined by similar cysteine-rich extracellular domains (30). A number of mammalian death receptors belonging to the TNFR family have been identified, including Fas, TNFR1, DR-3 (death receptor-3), DR-4, DR-5, and cytopathic avian leukosis-sarcoma virus receptor 1 (CAR1). In addition, this subfamily of TNFR contains a homologous cytoplasmic 80-amino acid domain termed the `death domain' (DD). DDs enable death receptors to engage the cell's apoptotic machinery (31). Aggregation of these receptors by a trimeric ligand induces apoptosis by recruiting adaptor proteins. The adaptor proteins also contain a DD that interacts with the DD of the receptor. The ligands that activate the death receptors are structurally related molecules belonging to the TNF gene superfamily (30). Fas ligand binds to Fas; TNF and lymphotoxin á bind to TNFR1; Apo3 ligand (Apo3L, also called TWEAK) binds DR-3;, and TRAIL (also called Apo2 ligand) binds to DR-4 and DR-5. The ligand for CAR1 is unknown. Following ligand-receptor binding, further protein-protein interactions are involved in the signalling of the death pathway. Homotypic domain interactions between proteins is a common theme in apoptosis. The recent identification of death factor-receptor pairs that regulate apoptosis brings a new level of complexity to the understanding of apoptotic cell death. It indicates that an external killer can control apoptosis in certain instances, and that it may trigger the death pathway through an autocrine, paracrine, or systemic fashion. Furthermore, alterations in death factor-receptor signalling may have important pathogenic roles in mediating inappropriate cell death in human diseases. Caspase [interleukin-1β-converting enzyme (ICE)-like protease] family Two genes were found to be essential in mediating developmental cell death in C. elegans: ced-3 and ced-4. The cloning and characterization of the ced-3 death-promoting gene revealed significant homology to the mammalian ICE, and provided the first indication that proteases may play a central role in apoptosis (Fig. 2) (32). Alnemri et al. (33) proposed a "caspase" nomenclature (for cysteine proteases that cleave after aspartate residues) for human members of this family (Table 2). Further support for a central role of caspases as effectors of apoptosis came from the fact that overexpression of caspases induced apoptosis (34), and the ability of selective caspase inhibitors such as viral cowpox-encoded protein CrmA prevented apoptosis (35). In sepsis, caspase inhibitors have been shown to improve survival by preventing lymphocyte apoptosis, leading to enhanced immunity (36). However, caspase-1-null mice display an apparently normal phenotype (37), suggesting that there may be functional redundancy in the caspase system, allowing a fail-safe mechanism through which the apoptotic process can be completed. However, evidence is now emerging that suggests that although caspase inhibitors may prevent certain characteristic biochemical and morphological features of apoptosis, cells that have sustained a cytotoxic insult and have been treated with caspase inhibitors have lost their replicative or clonogenic potential and all are destined to die, albeit by a slower mechanism not readily identifiable as classical apoptosis (38).Table 2: The human caspase familyNegative genetic regulators Bcl-2 gene family The intrinsic susceptibility of a cell to undergo apoptosis is determined by members of the protooncogene bcl-2 gene family, the mammalian homologue of ced-9 (Fig. 2). The prototypic regulator of cell death is bcl-2. Bcl-2 sets the basic apoptotic resistance threshold of cells (39), and overexpression of bcl-2 has been shown to prolong cellular survival by blocking apoptosis induced by a broad range of signals, including ultraviolet irradiation, cytokines, growth factor deprivation, and heat shock (reviewed in Ref. 40). In addition, overexpression of the bcl-2 gene has been shown to improve survival in sepsis, with a decrease bcl-2 expression found in peripheral monocytes in patients not surviving a septic insult (41,42). Bcl-2 belongs to a growing family of apoptosis regulatory gene products. Several homologues of the bcl-2 gene family have been recognised, including apoptotic antagonists (bcl-2, bcl-w, bcl-xL, bf1-1, brag-1, mcl-1, and A1) and apoptotic agonists [bax (bcl-2-associated protein x), bak, bik, bad, bcl-xs, bid, and hrk] (43). Many members of the bcl-2 protein family are capable of directly interacting with each other through a network of homo- and heterodimers. A dynamic equilibrium is established, with the ratio of death antagonists to agonists determining a cell's life or death response to an apoptotic stimulus (44). Just how bcl-2 blocks cell death is incompletely understood. Bcl-2 appears to have a number of functions in modulating the cellular response to an apoptotic stimulus, including acting as an ion channel, a mitochondrial membrane stabilizer, and as an adaptor or docking protein (Fig. 4) (43,45).Fig. 4: A model of the putative mechanisms of action of the bcl-2 family in regulating apoptosis. Anti-apoptotic bcl-2 family members such as bcl-xL appear to function at multiple levels to block apoptosis. bcl-xL may form discrete ion channels and regulate transmembrane ion fluxes; bcl-xL appears to act as an adaptor or docking protein by pulling other apoptotic regulating proteins out of the cytosol, functioning to either inactivate them to allow them to interact with other sequestered proteins; bcl-xL may act as a mitochondrial membrane stabilzer by inhibiting the opening of the permeability transition (PT) pore and preventing loss of the mitochondrial membrane potential (Δψm). A death signal may, for example, result in heterodimerizing of pro-apoptotic bcl-2 family members such as bax with bcl-xL and block its anti-apoptotic function. Bax may act to either block or alter bcl-xL's ion channel function, to prevent its adaptor function, to open the PT pore, or all three. In addition, bax appears to have intrinsic ion channel activity and may fulfil its pro-apoptotic role by promoting the loss of (Δψm).Ras gene family Ras gene family members (Ha, Ki, and N-ras) encode an almost identical 21-kD membrane-associated GTP-binding protein that has been associated with both proliferation and apoptosis (46). Ras proteins are key transducers of mitogenic signals, a fact attested by the high frequency of mutations in human (46). Ras proteins their potential through activation of the promoting cells to through G1 of the cell cycle the In to cellular overexpression of has been shown to apoptosis The protooncogene is a protein thought to play a role during cell cycle The forms of the family of and have been reported to of their through suppression of apoptosis between on to on the oncogene and a of the a of activity by and can growth cell from apoptosis induced by growth factor has been shown to be involved in activation In to intrinsic genetic factors, extracellular also regulate the susceptibility of cells to undergo apoptosis. Many factors, previously referred to as growth (e.g., insulin-like growth factor and growth are capable of apoptosis and maintaining cell in the absence of proliferation. are now referred to as control of regulation has support the number of and that all cells the of the a default apoptotic program and undergo apoptosis unless they are by the presence of survival This suggests that cell types be to the tissues their survival and of the survival factor Thus, of survival may be a common pathway to apoptosis. example, cells and cells have been shown to be on survival such as factor factor and (reviewed in Ref. The requirement of cells for survival signals may be in appropriate and of cells during and after an response. Following a systemic cytokines such as and are both and at found in have been shown to and apoptosis in and in the inflammatory response have also been shown to and suggesting that both and mechanisms may cell survival. Following of a local decrease in cytokines be to apoptosis in the cell with a to the regulatory controls evident in apoptosis, it is not that a complex and range of signalling molecules are following of an apoptotic response. molecular have been implicated in the apoptotic signalling including and The of morphological and biochemical events observed between different and cell types in the of apoptosis has to that these signals may a central or of the apoptotic process The of a common pathway may explain how negative regulators of apoptosis such as bcl-2 can apoptosis following by a broad of Several have the as the central of the apoptotic process acting to the of apoptosis. the apoptotic process can be into several but the induction in which the apoptotic process is and common are the central execution or in which the and anti-apoptotic signals are and the is in which the characteristic morphological and biochemical features of apoptosis The division of apoptosis, a complex process with multiple regulatory into although highly allow to in a important signalling events in the apoptotic and common signals in apoptosis Death receptor signalling for apoptosis In the number of there has been an in understanding of the death and regulator proteins involved in the release of apoptotic initiated following of the death receptor by its This has provided with at the complexity and of the death signal pathway. is now is that there multiple signalling pathways to cell death. or on the death signal the cell and the balance between apoptotic and survival signals. in the of growth regulation, multiple signalling pathways allow for multiple and regulation of apoptosis. and apoptotic pathways are the most and the binding of to Fas induces of the Fas receptor and activation of its cytoplasmic death recruiting a set of proteins into a signalling complex apoptotic pathways have been proposed (Fig. The characterized pathway the adaptor protein death domain which interacts with Fas its death domain and its death activation of the domain of caspase Caspase caspases such as caspase the mammalian functional homologue of the cell to Fas apoptotic pathways. 1 is by 3 with to form a complex that caspase Following of the apoptotic a series of signalling are which include activation of protein These enzymes can result in a of signalling including activation of and changes, including activation of protein and activation of transcription The of signalling in apoptosis is both cell and on the activation of the cell. of has been shown during apoptosis of peripheral suggesting that of proteins regulates apoptosis in these cells. In activation of in and prevents apoptosis and has been observed during The of on apoptosis is a number of factors, including the cell involved the activation and functioning of and the of inactivation of has been shown to be involved in A event in apoptosis in cell is the of resulting in a sustained in ions appear to play a central role in mediating apoptosis in certain cell with of extracellular or inhibiting apoptosis in these cells has been shown to have multiple potential of action in the apoptotic including activation of enzymes such as tissue and chromatin and gene However, in the of alteration in cellular levels not appear to be essential for the induction of apoptosis, the possibility that alterations in in certain cells occur as a of apoptosis. Furthermore, apoptotic cell death following is not of has been shown to apoptosis in certain cells such as in potential a has been observed cell following the induction of apoptosis by a range of stimuli and has been proposed as a apoptotic However, the requirement for production is not as production is not evident during induction of apoptosis in all cell indicate that the of the cell may be during apoptosis without of through a mechanism In addition, proapoptotic stimuli can apoptosis in the absence, or absence, of which implies that are not the of apoptosis important in the mediating in apoptosis have been production

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.

Objective To explore how docosahexenoic acid (DHA) changes apoptosis signaling pathways to further lead to apoptosis of prostate cancer cells. Methods RT2 Profiler PCR Arrays were used to screen the targets of DHA-induced prostate cancer cells apoptosis (10 apoptosis genes were up-regulated and 5 apoptosis genes were down-regulated). Real-time quantitative polymerase chain reaction (Real-time PCR) was used as reliable tools to validate the results. Results As compared with bovine serum albumin (BSA), after incubation with DHA for 24 h, the transcriptional level of Caspase family: Caspase-1, Caspase-3 and Caspase-9 in DU145 cells were increased 2.06, 4.88 and 12.10 times respectively; The transcriptional level of pro-apoptotic genes B cell lymphoma/leukemia-2 associated X protein (bax) was increased 2.93 times and B cell lymphoma/leukemia-2 (bcl-2)/bax ratio increased. Cell death inducing related genes CIDEA and DFFA were increased 2.34, 3.21 times respectively; Tumor necrosis factor related genes LTA and tumor necrosis factor (TNF) were increased 2.04 and 2.24 times respectively; The expression of tumor protein P53 (TP53) gene mRNA increased 2.97 times. In addition, as compared with the BSA control group, after incubation with DHA for 24 h, the transcriptional levels of apoptosis inducing factor 1 (AIFM1), Akt1, BH3 locus death inducing gene (BID), BIRC6 and X-linked inhibitor of apoptosis protein (XIAP) were decreased. After DHA treatment for 24 h, the mRNA expression of Caspase-1, Caspase-3, Caspase-9 and Caspase-12 was increased respectively. Moreover, the expression of Caspase-3, Caspase-9 and Caspase-12 had a dose-dependent relationship with DHA concentration. The mRNA expression of bax, CIDEA, DFFA, TP53 and TNF normalized with TATA box binding protein (TBP) in 24 h DHA-treated DU145 cells was increased obviously, and the expression of bax, CIDEA and TNF had a dose-dependent relationship with DHA concentration. The mRNA expression of AIFM1, protein kinase B (Akt1), BID, BIRC6 and XIAP was decreased obviously after incubation with different concentrations of DHA for 24 h, and the expression of bax, CIDEA and TNF had a dose-dependent relationship with DHA concentration. Conclusion DHA can change the related genes expression of apoptosis signaling pathways in Caspase family, by this way it can induce the apoptosis of prostate cancer cells. Key words: Prostatic carcinoma; Androgen resistance prostate cancer; Polyunsaturated fatty acids; Docosahexenoic acid

Evasion of programmed cell death is a hallmark of human cancers. Inhibitor of apoptosis (IAP) proteins such as X-chromosome– linked IAP (XIAP) and cellular IAP (cIAP) proteins play an important role in supporting cell survival by blocking cell death. IAP proteins comprise at least one of the signature baculoviral IAP repeat domains, a protein-protein motif critical for their binding to and inhibition of caspases to block the implementation of cell death. Some IAP proteins also contain the really interesting new gene domain with E3 ubiquitin ligase activity responsible for ubiquitination, leading to proteasomal degradation or altered signaling functions of substrates. Because IAP proteins are expressed at high levels in human cancers and contribute to tumor progression, treatment resistance, and poor prognosis, they have attracted considerable attention as therapeutic targets for drug development. To this end, smallmolecule inhibitors have been developed that mimic the N-terminal portion of second mitochondria-derived activator of caspases (Smac), an endogenous IAP antagonist that is released from mitochondria into the cytosol during apoptosis. IAP inhibitors, also called Smac mimetics, are composed of one (monovalent) or two (bivalent) Smac-mimicking units. Smac mimetics induce apoptosis in cancer cells by binding to and neutralizing XIAP, thereby releasing caspases from the inhibitory functions of XIAP (Fig 1). In addition, the binding of Smac mimetics to cIAP proteins stimulates the E3 ligase activity of cIAP1 and cIAP2, leading to their autoubiquitination and degradation via the proteasome (Fig 1). Because cIAP proteins constitutively mediate ubiquitination and degradation of nuclear factor kappa B (NFB) –inducing kinase, a key component of the noncanonical NFB pathway, the Smac mimetic–mediated depletion of cIAP proteins results in accumulation of NFB–inducing kinase, activation of the noncanonical NFB pathway, and upregulation of NFB target genes, including inflammatory cytokines such as tumor necrosis factor (TNF ). TNF , a member of the death receptor ligand family, then triggers cell death on binding to its cognate cell surface receptor TNF receptor 1 in an autocrine/paracrine manner. The concomitant Smac mimetic– triggered loss of cIAP proteins is critical for TNF -induced cell death by shutting off ubiquitination of receptor-interacting protein 1, thereby promoting its interaction with Fas-associated protein with death domain and caspase-8 to form a cytosolic cell death complex that drives caspase-8 activation and apoptosis. Smac mimetics have repeatedly been shown to either directly induce apoptosis or to sensitize cancer cells for apoptosis in response to additional cytotoxic treatments. More than a decade ago, a proof-of-concept study used a preclinical in vivo cancer model to demonstrate the feasibility of targeting IAP proteins by Smac mimetics for cancer therapy. Currently, five distinct Smac mimetic compounds have been evaluated in early clinical trials, including LCL161, a monovalent, orally bioavailable drug. The article by Infante et al that accompanies this Understanding the Pathway reports on the first-inhuman, phase I dose-escalation study of LCL161 in patients with advanced solid tumors. Objectives of the study were to determine the maximum-tolerated dose, dose-limiting toxicities (DLTs), TNFα

Immunoregulatory role of TNFα in inflammatory kidney diseases

The revolution in molecular biology leads to new understanding of the clinical expression of immunodeficiencies

Apoptosis or programmed cell death is a physiologic process that determines tissue homeostasis and provides an effective way to remove unwanted cells, such as those that have accumulated oncogenic mutations. Inhibition of apoptosis disrupts the balance between cell proliferation and cell death and has been recognized as one of six key mechanisms that are essential for the generation of fully transformed malignant cells [1]. Cells that undergo apoptosis are characterized by morphologic changes that include cytoplasmic shrinkage, plasma membrane blebbing, and chromatin condensation in the nucleus, which facilitate the efficient inflammation-free removal of apoptotic cells by macrophages [2]. At the molecular level, proteolytic enzymes such as caspases play an important role as the executors of apoptosis leading to cell death. Apoptosis is distinct from passive nonregulated cell death (necrosis), and, in general, is referred to as caspase-dependent cell death. The term “classical apoptosis” has been coined to distinguish it from other forms of programmed cell death that display a mixture of morphology or molecular features or both representing caspaseor noncaspase-dependent cell death. We focus on the therapeutic exploitation of the core apoptotic machinery that regulates caspase-dependent cell death. Two main caspase activation pathways have been identified (see also Fig. 12-1). One route, known as the intrinsic or mitochondrial pathway, is triggered upon disruption of mitochondria, e.g., because of DNA damage induced by cytotoxic agents, and causes the release of cytochrome c into the cytoplasm [3, 4]. Together with dATP, cytochrome c is a cofactor for the assembly of the apoptosome, which contains Apaf-1 and procaspase-9, and leads to the processing and activation of caspase-9. The second route, the so-called extrinsic or death receptor pathway, is initiated through specific cell membrane receptors, such as Fas/CD95 and tumor necrosis factor (TNF) family receptors, that upon ligand binding recruit the cytosolic death-domain-containing protein FADD (Fas-associated protein with death domain), which is able to bind and activate procaspase-8 in a complex named the death-inducing signaling complex (DISC). Both caspase-8 and -9 can activate the effector caspases-3, -6, and -7 provided that the caspase inhibitory effect of the inhibitor of apoptosis proteins (IAP) is relieved by Smac/Diablo, a proapoptotic protein that is also released from the mitochondria. The IAP family comprises proteins that contain one or more baculovirus IAP repeat (BIR) domains, which mediate in some IAP the interaction with caspases [5, 6]. The most potent caspase-inhibitory IAP is X-linked IAP (XIAP). When released in the cytosol, Smac binds to XIAP facilitating caspase activation. The activation of the effector caspases leads to the cleavage of various substrates, which results in the characteristic morphologic features of apoptotic cell death. An important class of regulators of apoptosis are the BCL-2 family proteins [7–9], comprising both antiapoptotic members, such as BCL-2, BCL-X L , and MCL-1, as well as proapoptotic members such as BAX and BAK, that share homology throughout four or three BCL-2 homology domains, respectively. Their primary mode of action has been assumed to be the regulation of mitochondrial integrity; however, they also appear to be involved in maintaining the integrity of other intracellular membrane structures, such as the endoplasmatic reticulum. Upon apoptosis activation, BAX and BAK translocate from the cytoplasm to the mitochondrial membrane where they oligomerize to form porelike structures, thereby causing mitochondrial outer membrane permealization (MOMP) and the release of apoptogenic factors, such as cytochrome c and Smac. The BH3-only proteins constitute a third class of proapoptotic BCL-2 proteins, which includes BID, BAD, BIK, PUMA, NOXA, BMF, and HRK. These proteins share homology in only one region, the BH3 domain. The BH3-only proteins appear to function as sentinels for the detection of cellular damage or aberrations; for example, BIM is activated by microtubule disarray, whereas

Involvement of tumor necrosis factor (TNF) receptors p55 and p75 in TNF responses of acute myeloid leukemia blasts in vitro

Differential Functions of Tumor Necrosis Factor Receptor 1 and 2 Signaling in Ischemia-Mediated Arteriogenesis and Angiogenesis

The absence of the thyroid hormone (TH) could impair testicular function, but its mechanism is still rudimentary. This study aims to explore the roles of estrogen receptor (ER α, β) and G protein-coupled receptor 30 (GPR30), extracellular signal regulated kinase (ERK1/2) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in apoptosis in testes of hypothyroidism rats. Male Wistar rats were randomly divided into control (C), low-(L) and high-hypothyroidism (H) groups [1 mL per 100 g BW per day normal saline, 0.001% and 0.1% propylthiouracil (PTU), respectively] by intragastrical gavage for 60 days. The levels of triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) in serum were measured. Expressions of ERα, ERβ and GPR30, pathway related protein expressions of ERK1/2 and PI3 K/AKT and apoptosis were detected in testicular homogenates. The results showed that T3 and T4 levels were decreased, and the TSH level was increased significantly in the H group. Protein expressions of ERα, ERβ and GPR30 decreased significantly in the H group. Significantly decreased protein expressions of p-ERK1/2, p-PI3K p85, p-AKT Ser473, Ras, p-Raf-1 Ser259, p-Raf-1 Ser338 and cyclin D1 in L and H groups as well PI3K p85, p-AKT and Thr308 in the H group were observed. Moreover, there was a significant increase in the Bad protein expression in L and H groups. In addition, there was a significant increase in the expression of Bax/Bcl-2, caspase 9 and cleaved caspase 3 and a significant decrease in the total caspase 3 protein expression in the H group. These results suggested that ERK1/2 and PI3K/AKT signaling pathways could be suppressed by hypothyroidism via inhibiting the expressions of ERs and could finally induce apoptosis in testes.

CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-kappaB and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27 revealed that the C-terminal PIQEDYR motif was indispensable for both NF-kappaB and SAPK/JNK activation and was also required for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-kappaB activation by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-kappaB and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-kappaB-inducing kinase (NIK). A kinase-inactive mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated NF-kappaB and SAPK/JNK activation. These results indicate that TRAF2 and TRAF5 are involved in NF-kappaB and SAPK/JNK activation by CD27, and NIK is a common downstream kinase of TRAF2 and TRAF5 for NF-kappaB and SAPK/JNK activation.