Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1

We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.     

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

We report here the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. 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.     

Ca(2+)-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor-1 (APAF-1)

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro-apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease-activating factor-1 (APAF-1). This complex activates procaspase-9 in the presence of dATP, resulting in caspase-mediated execution of apoptotic neuron death. Many forms of Ca(2+)-mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca(2+) induced prominent degradation of APAF-1 in human SH-SY5Y neuroblastoma cells and in a neuronal cell-free apoptosis system. Loss of APAF-1 correlated with a reduced ability of cytochrome c to activate caspase-3-like proteases. Ca(2+) induced the activation of calpains, monitored by the cleavage of full-length alpha-spectrin into a calpain-specific 150-kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF-1 degradation also occurred via calpain-independent pathways. Our data suggest that Ca(2+) inhibits caspase activation during Ca(2+)-mediated neuron death by triggering the degradation of the cytochrome c-binding protein APAF-1.     

Analysis of apoptosis in cell-free systems

Cell-free systems have been instrumental in the identification of several important components of the cell death machinery such as cytochrome c, APAF-1, ICAD/CAD (DFF45/DFF40) and Smac/Diablo. Such systems have also proved invaluable for the detailed analysis of caspase activation mechanisms, caspase activation cascades, proteolysis of caspase substrates, apoptosis-associated chromatin condensation and internucleosomal DNA fragmentation. Here, we describe a cell-free system that we have used routinely in our laboratory for the analysis of caspase activation and associated events. Caspase activation in this system can be triggered either through assembly of the APAF-1 apoptosome by addition of cytochrome c/dATP, or alternatively, by addition of the cytotoxic lymphocyte protease, granzyme B. In both cases, the order of caspase activation events has been established and the relative importance of individual caspases to apoptosis-associated nuclear events, as well as substrate proteolysis, is known. Cell-free systems are therefore very useful for screening potential caspase-inhibitory compounds or other agents that may positively or negatively affect caspase-dependent events in apoptosis.     

Dual hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by Orf17 (NtpA), a MutT-type enzyme

To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5'-triphosphates tested, 8-hydroxy-2'-deoxyadenosine 5'-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2'-deoxyadenosine 5'-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP.     

Study of the efficacy of a pronucleotide of 2-chloro-2'-deoxyadenosine in deoxycytidine kinase-deficient lymphoma cells

2-Chloro-2 '-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 '-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.     

Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity.

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, we examined 10 ovarian cancer cell lines for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, we 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 we did not detect dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation. Lack of APAF-1 activity correlated 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.     

Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein.

To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides. Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein. These damaged deoxynucleotides elicit transversion mutations in E. coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H. (1998) J. Biol. Chem. 273, 11069-11074). These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides.     

Effects of 2‐Chloro‐2′‐Deoxyadenosine on the Cell Cycle in the Human Leukemia EHEB Cell Line

To explain why 2‐chloro‐2′‐deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA‐resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA‐induced increase in G1 to S phase progression.     

Study of the Efficacy of a Pronucleotide of 2-Chloro-2′-Deoxyadenosine in Deoxycytidine Kinase-Deficient Lymphoma Cells

2-Chloro-2 ′-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 ′-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.     

Characterization of caspase processing and activation in HL-60 cell cytosol under cell-free conditions. Nucleotide requirement and inhibitor profile.

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 degrees C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitro and in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenediphosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn(2+), aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin, N(alpha)-p-tosyl-L-phenylalanine chloromethyl ketone, N(alpha)-p-tosyl-L-lysine chloromethyl ketone, and N-(N(alpha)-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.     

Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.     

Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in the human leukemia EHEB cell line

To explain why 2-chloro-2'-deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA-resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA-induced increase in G1 to S phase progression.     

Differential regulation of the intrinsic pathway of apoptosis in brain and liver during ageing

The intrinsic (mitochondria-dependent) pathway of apoptosis is one of the major pathways leading to cell death. We evaluated cytochrome c/apoptotic protease activation factor-1 (Apaf-1)-dependent activation of caspase-3 in brain and liver of different strains of rodents at different stages of development. In cell-free extracts from brain and liver of Sprague-Dawley rats, caspase was activated by cytochrome c/2'-deoxyadenosine 5'-triphosphate at both neonatal and adult stages. In adult brain extracts from Wistar rats, no activation of caspase was observed while extracts from neonatal brain and liver and from adult liver were activated. In CD-1 mouse, only neonatal extracts were activated. Alteration in levels of endogenous inhibitors of apoptosis were not responsible for the lack of activation observed. Instead, decrease in the content of Apaf-1 and caspase-3 and some degradation of caspase-9 during brain ageing were observed. These results suggest that a decrease in apoptosis activation during ageing is not tissue-specific, but rather displays a complex dependence on species and strains of animals.     

Proapoptotic histone H1.2 induces CASP-3 and -7 activation by forming a protein complex with CYT c, APAF-1 and CASP-9

Cytochrome c (CYT c) is a protein that employs the caspase recruitment domain (CARD)-containing proteins APAF-1 and CASP-9 to activate effectors CASP-3 and -7. By using affinity labeling techniques and mass spectrometry analysis, we show that histone H1.2 is a regulator of caspases upon UV irradiation. We demonstrated that histone H1.2 forms a protein complex with APAF-1, CASP-9 and CYT c upon UV irradiation. In cell-free systems, we show that histone H1.2 triggers activation of CASP-3 and -7 via APAF-1 and CASP-9. We therefore conclude that upon DNA damage histone H1.2 acts as a positive regulator of apoptosome formation.     

A chemical inhibitor of Apaf-1 exerts mitochondrioprotective functions and interferes with the intra-S-phase DNA damage checkpoint

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.     

Selective increase of dATP pools upon activation of deoxycytidine kinase in lymphocytes: implications in apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction--presumably by supplying dATP or its analogues for the apoptosome formation--deserves consideration. Two-hour exposure of human tonsillar lymphocytes to 2-chloro-deoxyadenosine (CdA) led to a two-fold activation of dCK. This activation process was inhibited by pifithrin-alpha, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide-treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.     

Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation.

To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochrome c in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.     

A novel biotinylated adenylate analogue derived from pyrazolo[3,4-d]pyrimidine for labeling DNA probes.

A novel dATP analogue, 3-[5-[(N-biotinyl-6- amiocaproyl)amino]pentyl]-1-(2-deoxy-beta-D-erythro-pentofuranosyl )-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5'-triphosphate (9, bio-13-dAPPTP), which is modified at the 3-position with a flexible linker arm bearing a terminal biotin moiety, has been synthesized. This nucleotide is readily incorporated into DNA probes by nick translation. These probes hybridize to complementary targets as well as probes labeled with bio-dUTP, as judged by slot blot. When incorporated into oligonucleotides, they do not cause the loss of hybridization efficiency that an N-6-substituted adenine nucleotide does when incorporated into the same sites in the oligonucleotide.