CED-4, the C. elegans Apaf-1/Ark homolog
Examining the effects of overexpressing cell-death-related genes in specific C. elegans neurons that normally live, it was demonstrated that the cell-death genes ced-3 (a caspase), ced-4 (an Apaf-1 homolog), and ced-9 (a BCL-2 homolog) all can act cell autonomously to control programmed cell death. Not only the protective activity of ced-9 but also the killer activities of ced-3 and ced-4 are likely to be present in cells that normally live. Killing by overexpression of ced-3 does not require endogenous ced-4 function, whereas killing by overexpression of ced-4 is at least in part dependent on endogenous ced-3 function. These results suggest either that ced-4 acts upstream of ced-3 and ced-4 function can be bypassed by high levels of ced-3 activity or that ced-3 and ced-4 act in parallel, with ced-3 perhaps having a greater ability to kill. The finding that ced-4 appears to facilitate the inhibition of ced-3 by ced-9 suggests that ced-9 acts to negatively regulate ced-4. It is proposed that both in C. elegans and in other organisms a competition between antagonistic protective and killer activities determines whether specific cells will live or die. These results suggest a genetic pathway for programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel with ced-3, and ced-9 negatively regulates the activity of ced-4 (Shaham, 1996a).
ced-4 encodes two transcripts; whereas the major transcript can cause programmed cell death, the minor transcript can act oppositely and prevent programmed cell death, thus defining a novel class of cell death inhibitors. That ced-4 has both cell-killing and cell-protective functions is consistent with previous genetic studies. The dual protective and killer functions of the C. elegans bcl-2-like gene ced-9 are mediated by inhibition of the killer and protective ced-4 functions, respectively. It is proposed that a balance between opposing ced-4 functions influences the decision of a cell to live or to die by programmed cell death and that both ced-9 and ced-4 protective functions are required to prevent programmed cell death (Shaham, 1996b).
Three principal genes are involved in developmental programmed cell death in C. elegans: ced-3 and ced-4 genes are both required for PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes. ced-9 is homologous to the Bcl-2 family, whose role in protecting PCD is illusive; no vertebrate homolog of ced-4 is known. This paper describes the effect of expression of C. elegans ced-4 in yeast. Induction of wild type ced-4 results in rapid focal chromatin condensation and lethality. Mutation of a putative nucleotide binding P-loop motif of CED-4 eliminates the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggests that the phenotype may result from an intrinsic ability of CED-4 to interact with chromatin. Co-expression of ced-9 prevents CED-4-induced chromatin condensation and lethality, and causes the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. It is concluded that CED-4 has a direct role in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. Further studies are required to establish whether the CED-9/CED-4 interaction is required for the activation of CED-3, the Caspase cysteine protease (James, 1997).
Genetic studies of the nematode C. elegans have identified three important components of the cell death machinery. CED-3 and CED-4 function to kill cells, whereas CED-9 protects cells from death. In this study, CED-9 and its mammalian homolog Bcl-xL (a member of the Bcl-2 family of cell death regulators) were both found to interact with and inhibit the function of CED-4. In addition, analysis has revealed that CED-4 can simultaneously interact with CED-3 and its mammalian counterparts interleukin-1beta-converting enzyme (ICE) and FLICE. Thus, CED-4 plays a central role in the cell death pathway, biochemically linking CED-9 and the Bcl-2 family to CED-3 and the ICE family of pro-apoptotic cysteine proteases (Chinnaiyan, 1997).
Genetic studies suggest that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that in vivo CED-9 forms a multimeric protein complex with CED-4 and CED-3. Expression of wild-type CED-4 promotes the ability of CED-3 in mammalian cells to induce apoptosis otherwise inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans (Wu, 1997).
CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. CED-4 acts as a positive regulator of caspases by enhancing the processing of procaspases to their mature forms. In mammalian 293 cells and insect SF21 cells, the processing of the proform of caspase CED-3 to its mature form is facilitated by CED-4, resulting in the acceleration of CED-3-induced cell death. CED-4 can directly activate CED-3 and ATP hydrolysis associated with the ATP binding site (P-loop) of CED-4 is required for CED-3 processing. Additionally, Apaf-1, a mammalian homolog of CED-4 that also has a P-loop motif, activates caspase-9 in the presence of cytochrome c (see Drosophila Cytochrome c proximal and Cytochrome c distal) and dATP, resulting in the sequential activation of caspase-3 in vitro. The CED-3/CED-4 complex has been shown to be activated by the oligomerization of CED-4. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induces massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in CED-4's ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis has shown that in S2 cells, both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation. Furthermore, CED-4 (K165R) partially prevents cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases (Kanuka, 1999a).
Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. This study describes pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals, there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway (Joshi, 2004).
Isolation and characterization of mammalian Apaf-1 family proteins
Apaf-1 (apoptotic protease activating factor-1), a novel 130 kd protein from HeLa cell cytosol, participates in the cytochrome c-dependent activation of caspase-3. The release of cytochrome c from mitochondria in response to apoptotic stimuli is blocked in cells overexpresssing Bcl-2. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. However, Apaf-1 does not seem to be a caspase, since the conserved active site pentapeptide that is present in all identified caspases is not present in Apaf-1. The caspase homologous region is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis (Zou, 1997).
The deduced amino acid sequences have been reported of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box [Mg(2+)-binding site]. Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: (1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; (2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; (3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone, and (4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery (Hlaing, 2001).
Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. A human Apaf-1-related protein, Ipaf, has been identified that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. These results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli (Poyet, 2001).
Is the short form of apaf-1 also present in mammals? The exon/intron boundary was found at a similar position to that of Drosophila. If apaf-1 short (apaf-1S) is conserved through evolution, a similar type of splicing variant could be expected to be present in mammals. Semiquantitative RT-PCR was performed using specific primers that could differentiate apaf-1 (281 bp PCR product) and apaf-1S (242 bp PCR product). As in Drosophila, the expression of apaf-1S was found to be present in embryonic brain but was hard to detect in adult brain. These results suggest that splicing variant apaf-1S is present through evolution (Kanuka, 1999b).
Apaf-1 plays an essential role in apoptosis. In the presence of cytochrome c and dATP, Apaf-1 assembles into an oligomeric apoptosome; this is responsible for the activation of procaspase-9 and the maintenance of the enzymatic activity of the processed caspase-9. Regulation of apoptosome assembly by other cellular factors is poorly understood. This study reports that physiological concentrations of calcium ion negatively affect the assembly of apoptosome by inhibiting nucleotide exchange in the monomeric, autoinhibited Apaf-1 protein. Consequently, calcium blocks the ability of Apaf-1 to activate caspase-9. These observations suggest an important role of calcium homeostasis on the Apaf-1-dependent apoptotic pathway (Bao, 2007).
Mutation of Apaf-1
The cytosolic protein APAF1, a human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. A null allele of the murine Apaf1 was generated by a gene trap. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. These findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals (Cecconi, 1998).
Apoptosis is essential for the precise regulation of cellular homeostasis and development. The role in vivo of Apaf1, a mammalian homolog of C. elegans CED-4, was investigated in gene-targeted Apaf1-/- mice. Apaf1-deficient mice exhibit reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells are resistant to a variety of apoptotic stimuli, and the processing of Caspases 2, 3, and 8 is impaired. However, both Apaf1-/- thymocytes and activated T lymphocytes are sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicate that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development (Yoshida, 1998).
The forebrain overgrowth mutation (fog) was originally described as a spontaneous autosomal recessive mutation mapping to mouse chromosome 10 that produces forebrain defects, facial defects, and spina bifida. Although the fog mutant has been characterized and available to investigators for several years, the underlying mutation causing the pathology has not been known. Because of its phenotypic resemblance to apoptotic protease activating factor-1 (Apaf-1) knockout mice, the possibility that the fog mutation is in the Apaf-1 gene was investigated. Allelic complementation, Western blot analysis, and caspase activation assays indicate that fog mutant mice lack Apaf-1 activity. Northern blot and reverse transcription-PCR analysis show that Apaf-1 mRNA is aberrantly processed, resulting in greatly reduced expression levels of normal Apaf-1 mRNA. These findings are strongly suggestive of the fog mutation being a hypomorphic Apaf-1 defect and implicate neural progenitor cell death in the pathogenesis of spina bifida, a common human congenital malformation. Because a complete deficiency in Apaf-1 usually results in perinatal lethality and fog/fog mice more readily survive into adulthood, these mutants serve as a valuable model with which apoptotic cell death can be studied in vivo (Honarpour, 2001).
Transcriptional regulation of Apaf-1
Loss of function of the retinoblastoma protein, pRB, leads to lack of differentiation, hyperproliferation and apoptosis. Inactivation of pRB results in deregulated E2F activity, which in turn induces entry to S-phase and apoptosis. Induction of apoptosis by either the loss of pRB or the deregulation of E2F activity occurs via both p53-dependent and p53-independent mechanisms. The mechanism by which E2F induces apoptosis is still unclear. E2F1 directly regulates the expression of Apaf-1, the gene for apoptosis protease-activating factor 1. These results provide a direct link between the deregulation of the pRB pathway and apoptosis. Furthermore, because the pRB pathway is functionally inactivated in most cancers, the identification of Apaf-1 as a transcriptional target for E2F might explain the increased sensitivity of tumor cells to chemotherapy. Independently of the pRB pathway, Apaf-1 is a direct transcriptional target of p53, suggesting that p53 might sensitize cells to apoptosis by increasing Apaf-1 levels (Moroni, 2001).
Translational regulation of Apaf
Polypyrimidine tract binding protein 1 (PTB: Drosophila homolog Hephaestus) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr; a single-stranded RNA binding protein which contains five cold shock domains, Drosophila homolog CG7015) is prebound. The Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation (Mitchell, 2003).
The regulatory mechanisms controlling cell death are complex, and in addition to control of transcription, the expression of proteins that are involved in apoptosis is regulated by control of translation. Indeed, many mRNAs whose protein products are involved in apoptosis are translated by the alternative mechanism of internal ribosome entry. This process is mediated by a complex RNA structural element located in the 5' untranslated region (UTR) of the mRNA termed an internal ribosome entry segment (IRES). During apoptosis cap-dependent translation initiation is very much reduced, yet expression of certain key proteins required for this process is maintained by internal ribosome entry. Thus c-myc, DAP5, and XIAP IRESes function to maintain expression of these proteins following apoptosis. Apaf-1 translation is solely initiated by internal ribosome entry, but to date the only situation where a small increase in Apaf-1 IRES function has been observed is following genotoxic stress. Given the importance of Apaf-1 during brain development, it is possible that the Apaf-1 IRES is required for expression of this protein in the developing brain. In this regard the FGF-2 IRES has been shown to be active in adult brain while in developing embryos both the FGF-2 and c-myc IRESes are active. This suggests that certain IRES trans-acting factors (ITAFs) are not present in the fully differentiated cell types, and these and additional studies have demonstrated that the function of certain cellular IRESes varies considerably with cell type. Most cellular IRESes are inactive in vitro, again suggesting an absolute requirement for ITAFs that are not present in these systems. However, very few ITAFs have been identified for cellular IRESes although the auto-antigen La has been shown to interact with the XIAP IRES and hnRNPC has been shown to interact with the PDGF IRES. The Apaf-1 IRES requires both polypyrimidine tract binding protein (PTB; a protein that has a role in regulating splicing as well as aiding internal ribosome entry of certain viral IRESes and upstream of N-ras). PTB only binds to the Apaf-1 IRES RNA if unr is prebound suggesting that unr is required to attain the correct structural conformation of the Apaf-1 IRES (Mitchell, 2003).
Apaf-1 IRES is most active in cell lines of neural origin and this activity correlates with the neuronal enhanced version of PTB, nPTB. The PTB/nPTB binding sites on the Apaf-1 RNA have been located, and by chemical and enzymatic probing of the RNA a structural model for the IRES has been derived in the presence of these binding factors. To test this model, specific structural alterations were made to the Apaf-1 IRES, and these abolished the binding of trans-acting factors while retaining and even increasing IRES activity. The region in which the ribosome lands has been localized, and it would appear that in the presence of unr and nPTB a region of single-stranded RNA becomes accessible to the ribosome. This would suggest that unr and nPTB act as RNA chaperones, changing the structure of the Apaf-1 IRES into one that is functionally competent for 48S formation (Mitchell, 2003).
Apaf-1 interaction with cytochrome
In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. This study shows, through fluorescence polarization experiments, that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c.Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c.Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. The cytochrome c.Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins (Purring-Koch, 2000).
As components of the apoptosome, a caspase-activating complex, cytochrome c (Cyt c) and Apaf-1 are thought to play critical roles during apoptosis. Due to the obligate function of Cyt c in electron transport, its requirement for apoptosis in animals has been difficult to establish. 'Knockin' mice were generated expressing a mutant Cyt c (KA allele), which retains normal electron transfer function but fails to activate Apaf-1. Most KA/KA mice displayed embryonic or perinatal lethality caused by defects in the central nervous system, and surviving mice exhibited impaired lymphocyte homeostasis. Although fibroblasts from the KA/KA mice were resistant to apoptosis, their thymocytes were markedly more sensitive to death stimuli than Apaf-1(-/-) thymocytes. Upon treatment with gamma irradiation, procaspases were efficiently activated in apoptotic KA/KA thymocytes, but Apaf-1 oligomerization was not observed. These studies indicate the existence of a Cyt c- and apoptosome-independent but Apaf-1-dependent mechanism(s) for caspase activation (Hao, 2005).
Apoptosis in metazoans is executed intracellular caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. This study reports the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation (Kim, 2005; full text of article).
Apaf-1 generated in insect cells contains dATP as a cofactor. The bound dATP then undergoes one round of hydrolysis to dADP, a process that is stimulated by cytochrome c. This hydrolysis appears to serve two roles: (1) it provides energy for the conformational change need for Apaf-1 to transient from the inactive monomeric state to the oligomeric state, and (2) it allows exogenous dATP (or ATP) to exchange for the dADP that has lower binding affinity for Apaf-1, a critical step for Apaf-1 to form functional apoptosome rather than nonfunctional aggregates. This hydrolysis only happens in one round. Exogenously added dATP will then bind Apaf-1, but remains unhydrolyzed during apoptosome formation (Kim, 2005).
The formation of the nonfunctional Apaf-1 aggregate is also induced by cytochrome c binding to Apaf-1. Cytochrome c was actually reisolated as an inhibitor of Apaf-1 activity when dATP or ATP was not included in the caspase-3-activating reaction. Such aggregates may be identical to the large, inactive Apaf-1 complex first observed in human monocytic tumor cells (Kim, 2005).
Interestingly, the crystal structure of a WD-40-truncated Apaf-1 revealed Apaf-1 in an inactive configuration with an ADP bound to it. Because WD-40 repeats serve as an autoinhibitory role, Apaf-1 without this region should be active, yet may easily become inactive if the exogenous dATP or ATP level is low. This finding is consistent with the observation that WD-40-less Apaf-1 is indeed active in promoting procaspase-9/3 activation but is rather unstable and the only stable configuration might be the dADP or ADP binding form. It is also interesting that this form of Apaf-1 expressed in bacteria contains an ADP but not dADP, whereas full-length Apaf-1 expressed in insect cells contains exclusively dATP. The measured difference between dATP and ATP in binding affinity to Apaf-1 is 10-fold, not enough to explain this exclusive binding of dATP because intracellular ATP level is several orders of magnitude higher than dATP. One possibility is that dATP level in E. coli is very low. Another possibility could be that mammalian and insect cells contain a dATP-specific loading factor for Apaf-1 that is absent in bacteria. Such a factor could potentially function in conjunction with prothymosin- and PHAPI, two proteins that regulate apoptosome activity (Kim, 2005).
The role of cytochrome c in apoptosome formation also becomes clear through the current study. Upon binding to Apaf-1, cytochrome c releases the autoinhibition imposed by the WD-40 repeats and allows Apaf-1 to hydrolyze the bound dATP. This role of cytochrome c also suggests that its simple release from mitochondria and binding to Apaf-1 may not necessarily result in the activation of caspase-9/3. Without exogenous dATP or ATP exchange, cytochrome c binding to Apaf-1 will irreversibly deplete the Apaf-1 protein in cells without activating caspases. Consistently, when intracellular ATP is depleted, cells undergo necrosis in response to stimuli that normally induce apoptosis, and when the cellular ATP levels are restored, the response shifts back to apoptosis. Therefore, it is hypothesized that the nucleotide exchange on Apaf-1 may provide another regulatory step for apoptosis (Kim, 2005).
The question remains whether endogenous Apaf-1 in mammalian cells also exclusively binds dATP. Addressing this question requires improvement on LC-MS method so that the nucleotide bound to endogenous Apaf-1 can be identified by using much less available material (Kim, 2005).
Apaf-1 and the activation of caspases
This paper reports the purification of Apaf-3, a third protein factor that participates in caspase-3 activation in vitro. Apaf-3 is a member of the caspase family: specifically, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. In turn, activated caspase-9 cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminishes caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade triggered by cytochrome c and dATP. Several caspases, including caspase-9, have long prodomains at their NH2 termini. This domain has been proposed to function as a caspase recruitment domain (CARD), allowing proteins with such domains to interact with each other. Although Apaf-1 is not a caspase, its NH2-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs. The data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD (P. Li, 1997).
Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. A mammalian homolog of CED-4, Apaf-1, can associate with several death proteases in mammalian cells, including caspase-4, caspase-8, caspase-9, and nematode CED-3. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhances the killing activity of caspase-9, which requires the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promotes the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, has been shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL is mediated through both domains of Apaf-1: the CED-4-like domain and the C-terminal domain, which contains WD-40 repeats. Expression of Bcl-XL inhibits the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibits Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL fails to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans (Hu, 1998).
The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).
The reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9 is reported. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3 (Zou, 1999).
Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. A novel Apaf-1 cDNA encoding a protein of 1248 amino acids has been identified, containing an insertion of 11 residues between the CARD and ATPase domains and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activates procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. This Apaf-1 was used to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) is unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enables Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibits greater ability to induce apoptosis compared with the wild-type Apaf-1. Procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associates with and activated procaspase-9, but fails to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis (Hu, 1999).
Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large (on the order of approximately 1.4 MDa). dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of approximately 1.4 MDa and approximately 700 kDa. Kinetic analysis demonstrates that in vitro the approximately 700-kDa complex is produced more rapidly than the approximately 1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the approximately 700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processes effector caspases. Thus, the approximately 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis (Cain, 2000).
During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome has been examined. A multi-step process is proposed, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex (Bratton, 2001).
Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (1) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9, (2) a CED-4-like region enabling self-oligomerization, and (3) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Different apoptotic stimuli induce the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment is obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 is cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site is located within the WD-40 repeats and enables the oligomerization of p84 into a approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer is devoid of caspase-9 or other caspase activity. Thus, these data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis (Lauber, 2001).
Bcl-2 family members as regulators of the cell death hierarchy: Bcl-2 interacts with Apaf-1
The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, the in vivo association studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 can be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain is necessary and sufficient for their interaction. However, Apaf-1 does not bind to any of the six known mammalian prosurvival family members [Bcl-2, Bcl-x(L), Bcl-w, A1, Mcl-1, or Boo], or their viral homologs adenovirus E1B 19K and Epstein-Barr virus BHRF-1. Endogenous Apaf-1 also fails to coimmunoprecipitate with endogenous Bcl-2 or Bcl-x(L), or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli do not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly (Moriishi, 1999).
The C. elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localize to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells have been induced to die, CED-4 assumes a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 (EGL-1 protein contains a Bcl-2 homology 3 domain and can physically interact with CED-9) is blocked by a gain-of-function mutation in ced-9 but is not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death (Chen, 2000).
The death-promoting proteins Bax and BAD, which like EGL-1 contain BH3 domains, translocate to mitochondria and bind anti-apoptotic Bcl-2 family members in response to apoptotic signals. Whether and how this translocation promotes cell death is unknown. The results presented here suggest that Bax and BAD may act to release Apaf-1 or another CED-4-like protein, allowing it to activate caspase processing. Some caspase precursors, specifically procaspases-2, and -3, are present in mitochondria and upon activation translocate to nuclei. It is possible that this movement of caspases involves the translocation of a complex that includes a CED-4-like protein. By analogy, the translocation of a CED-4-CED-3 complex from mitochondria to the nuclear envelope could provide access for the active caspase to both the nucleus and the cytosol, thereby fulfilling the roles of the multiple, differentially localized mammalian caspases (Chen, 2000).
Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L) that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibits the proteolytic activation of caspases in a cell-free extract and suppresses apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator (Chau, 2000).
Apaf-1 interaction with heat-shock proteins
The release of cytochrome c from mitochondria results in the formation of an Apaf-1-caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of procaspase-9. Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90-Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade (Pandey, 2000).
Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. The documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, it is proposed that resistance to apoptosis exhibited by stressed cells and some tumors, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70 (Saleh, 2000).
The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Using a cell-free system, it has been shown that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome (Beere, 2001).
Miscellaneous Apaf-1 interactions
Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation (Chu, 2001).
Apaf-1, apoptosis and development
Caspase 9 (Casp9)/Apaf3, a 45 kDa protein (also known as ICE-LAP-6 or Mch6) forms a multiprotein complex containing Apaf1 and cytochrome c. It has been proposed that cytochrome c initiates apoptosis by inducing the formation of the Casp9/Apaf1 complex. Physical association of Casp9 and Apaf1 is mediated by the interaction of their respective caspase recruitment domains (CARDs). CARDs are also found in other caspases with large prodomains, such as Casp4 and Casp8, that can associate with Apaf1 in mammalian cells. The antiapoptotic protein Bcl-XL has also been shown to interact with Casp9 and Apaf1, resulting in the inhibition of Casp9 activation. The association of Casp9 with antiapoptotic as well as proapoptotic proteins suggests a major role for Casp9 in the control of apoptosis in vivo. Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. The absence of Casp9 leads to a dramatic disturbance of telencephalic development that apparently results from decreased apoptosis in this region. The gross morphological features observed in Casp9-/- mice are remarkably similar to those observed in mice lacking Casp3. Both mutants exhibit a profound disturbance of cortical morphology, an expanded germinal zone, and hydrocephaly, suggesting that these mutations affect a common cellular apoptotic pathway dependent on both Casp9 and Casp3. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. The Casp9-dependent, Casp3-independent apoptotic pathway is preferentially triggered in thymocytes in response to dexamethasone. However, the fact that Casp3 is still processed in dexamethasone-treated Casp9-/- thymocytes suggests that dexamethasone also activates a Casp9-independent and Casp3-dependent apoptotic pathway in these cells. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of multiple apoptotic pathways in mammalian cells (Hakem, 1998).
During inner ear development, programmed cell death occurs in specific areas of the otic epithelium but the significance of this death and the molecules involved have remained unclear. An analysis was undertaken of mouse mutants in which genes encoding apoptosis-associated molecules have been inactivated. Disruption of the Apaf1 gene leads to a dramatic decrease in apoptosis in the inner ear epithelium, severe morphogenetic defects and a significant size reduction of the membranous labyrinth, demonstrating that an Apaf1-dependent apoptotic pathway is necessary for normal inner ear development. This pathway most probably operates through the apoptosome complex because caspase 9 mutant mice suffer similar defects. Inactivation of the Bcl2-like (Bcl2l) gene leads to an overall increase in the number of cells undergoing apoptosis but does not cause any major morphogenetic defects. In contrast, decreased apoptosis is observed in specific locations that suffer from developmental deficits, indicating that proapoptotic isoform(s) produced from Bcl2l might have roles in inner ear development. In Apaf1-/-/Bcl2l-/- double mutant embryos, no cell death could be detected in the otic epithelium, demonstrating that the cell death regulated by the anti-apoptotic Bcl2l isoform (Bcl-XL) in the otic epithelium is Apaf1-dependent. Furthermore, the otic vesicle fails to close completely in all double mutant embryos analyzed. These results indicate important roles for both Apaf1 and Bcl2l in inner ear development (Cecconi, 2004).
Apaf-1 and oncogenesis
The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 are essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, are resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substitutes for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development (Soengas, 1999).
Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic program in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. It is concluded that Apaf-1 is inactivated in metastatic melanomas: this leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumor type (Soengas, 2001).
Apoptosis via the mitochondrial pathway requires release of cytochrome c into the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, 10 ovarian cancer cell lines were examined for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, it was found that cytosolic extracts from all cell lines had diminished cytochrome c-dependent caspase activation, compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because no dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation was detected. Lack of APAF-1 activity correlates in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers (Wolf, 2001).
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