Celastrol, a Triterpene, Enhances TRAIL-induced Apoptosis through the Down-regulation of Cell Survival Proteins and Up-regulation of Death Receptors

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.     

PHAPI, CAS, and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1

During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.     

Involvement of protective autophagy in TRAIL resistance of apoptosis-defective tumor cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.     

阿司匹林诱导人恶性睾丸生殖细胞瘤NTera-2细胞凋亡

阿司匹林,又称乙酰水杨酸,已证实有抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡和抑制血管生成等多种抗癌功能.除已知对环氧合酶COX-2的活性有抑制外,阿司匹林抗癌分子机制尚不十分清楚.已报道阿司匹林可以降低多种癌症发生风险,但应用于人类睾丸肿瘤治疗的研究报道很少.本文研究了阿司匹林对人恶性睾丸肿瘤NTera-2细胞凋亡的机制.通过MTT方法检测细胞活力,发现阿司匹林以时间和剂量依赖方式抑制NTera-2细胞增殖.不同浓度阿司匹林处理NTera-2细胞后,采用Hoechest 33258染色方法和Annexin V-FITC/PI流式法分别检测NTera-2细胞的形态学变化、凋亡小体形成、细胞凋亡水平;RT-PCR结果显示,NTera-2细胞中Fas和caspase-8的表达以阿司匹林剂量依赖性上升;蛋白印迹结果显示,FasL的蛋白表达水平下降并活化caspase-8、caspase-3蛋白表达,PARP出现剪切体. 进一步的实验证明,caspase广谱抑制剂Z-VAD-FMK能够减弱阿司匹林诱导NTera-2细胞凋亡. 结果显示,阿司匹林能明显抑制NTera-2细胞活力,并通过激活caspase 通路诱导NTera-2细胞的凋亡,为进一步利用阿司匹林治疗人类睾丸肿瘤的研究奠定基础.     

Delay in Apoptosome Formation Attenuates Apoptosis in Mouse Embryonic Stem Cell Differentiation

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis

The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin–proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.     

Inhibition of Methyltransferases Accelerates Degradation of cFLIP and Sensitizes B-Cell Lymphoma Cells to TRAIL-Induced Apoptosis

Non-Hodgkin lymphomas (NHLs) are characterized by specific abnormalities that alter cell cycle regulation, DNA damage response, and apoptotic signaling. It is believed that cancer cells are particularly sensitive to cell death induced by tumor necrosis factor α–related apoptosis-inducing ligand (TRAIL). However, many cancer cells show blocked TRAIL signaling due to up-regulated expression of anti-apoptotic factors, such as cFLIP. This hurdle to TRAIL’s tumor cytotoxicity might be overcome by combining TRAIL-based therapy with drugs that reverse blockages of its apoptotic signaling. In this study, we investigated the impact of a pan-methyltransferase inhibitor (3-deazaneplanocin A, or DZNep) on TRAIL-induced apoptosis in aggressive B-cell NHLs: mantle cell, Burkitt, and diffuse large B-cell lymphomas. We characterized TRAIL apoptosis regulation and caspase activation in several NHL-derived cell lines pre-treated with DZNep. We found that DZNep increased cancer cell sensitivity to TRAIL signaling by promoting caspase-8 processing through accelerated cFLIP degradation. No change in cFLIP mRNA level indicated independence of promoter methylation alterations in methyltransferase activity induced by DZNep profoundly affected cFLIP mRNA stability and protein stability. This appears to be in part through increased levels of cFLIP-targeting microRNAs (miR-512-3p and miR-346). However, additional microRNAs and cFLIP-regulating mechanisms appear to be involved in DZNep-mediated enhanced response to extrinsic apoptotic stimuli. The capacity of DZNep to target cFLIP expression on multiple levels underscores DZNep’s potential in TRAIL-based therapies for B-cell NHLs.     

Bistability in apoptosis: roles of bax, bcl-2, and mitochondrial permeability transition pores

We propose a mathematical model for mitochondria-dependent apoptosis, in which kinetic cooperativity in formation of the apoptosome is a key element ensuring bistability. We examine the role of Bax and Bcl-2 synthesis and degradation rates, as well as the number of mitochondrial permeability transition pores (MPTPs), on the cell response to apoptotic stimuli. Our analysis suggests that cooperative apoptosome formation is a mechanism for inducing bistability, much more robust than that induced by other mechanisms, such as inhibition of caspase-3 by the inhibitor of apoptosis (IAP). Simulations predict a pathological state in which cells will exhibit a monostable cell survival if Bax degradation rate is above a threshold value, or if Bax expression rate is below a threshold value. Otherwise, cell death or survival occur depending on initial caspase-3 levels. We show that high expression rates of Bcl-2 can counteract the effects of Bax. Our simulations also demonstrate a monostable (pathological) apoptotic response if the number of MPTPs exceeds a threshold value. This study supports our contention, based on mathematical modeling, that cooperativity in apoptosome formation is critically important for determining the healthy responses to apoptotic stimuli, and helps define the roles of Bax, Bcl-2, and MPTP vis-à-vis apoptosome formation.     

Hepatitis C virus core protein modulates TRAIL-mediated apoptosis by enhancing Bid cleavage and activation of mitochondria apoptosis signaling pathway

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease, which leads to cirrhosis of liver and hepatocellular carcinoma. The HCV core protein, a viral nucleocapsid, has been shown to affect various intracellular events, including cell proliferation and apoptosis. However, the precise mechanisms of the effects are not fully understood. In this study, we show that HCV core protein sensitizes human hepatocellular carcinoma cell line, Huh7, conferred sensitivity to TRAIL-, but not Fas ligand-mediated apoptosis. Huh7 cells are resistant to TRAIL, despite the induction of caspase-8 after TRAIL engagement. However, HCV core protein induces TRAIL apoptosis signaling via sequential induction of caspase-8, Bid cleavage, activation of mitochondrial pathway, and effector caspase-3. HCV core protein also induces activation of caspase-9 after TRAIL engagement, and the induction of TRAIL sensitivity by HCV core protein could be reversed by caspase-9 inhibitor. Therefore, the HCV core protein-induced TRAIL-mediated apoptosis is dependent upon activation of caspase-8 downstream pathway to convey the death signal to mitochondria, leading to activation of mitochondrial signaling pathway and breaking the apoptosis resistance. These results combined indicate that the HCV core protein enhances TRAIL-, but not Fas ligand-mediated apoptotic cell death in Huh7 cells via a mechanism dependent on the activation of mitochondria apoptosis signaling pathway. These results suggest that HCV core protein may have a role in immune-mediated liver cell injury by modulation of TRAIL-induced apoptosis.     

Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells

We evaluated the antitumor activity of tocotrienol (T3) on human hepatoma Hep3B cells. At first, we examined the effect of T3 on the proliferation of human hepatoma Hep3B cells and found that gamma-T3 inhibited cell proliferation at lower concentrations and shorter treatment times than alpha-T3. Then, we examined the effect of gamma-T3 apoptosis induction and found that gamma-T3 induced poly (ADP-ribose) polymerase (PARP) cleavage and stimulated a rise in caspase-3 activity. In addition, gamma-T3 stimulated a rise in caspase-8 and caspase-9 activities. We also found that gamma-T3-induced apoptotic cell death was accompanied by up-regulation of Bax and a rise in the fragments of Bid and caspase-8. These data indicate that gamma-T3 induced apoptosis in Hep3B cells and that caspase-8 and caspase-9 were involved in apoptosis induction. Moreover, these results suggest that Bax and Bid regulated apoptosis induction by gamma-T3.     

Acrolein sensitizes human renal cancer Caki cells to TRAIL-induced apoptosis via ROS-mediated up-regulation of death receptor-5 (DR5) and down-regulation of Bcl-2

TRAIL resistance in many cancer cells is one of the major problems in TRAIL-based cancer therapy. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are strictly needed for the improvement of anti-cancer effect of TRAIL. Acrolein is a byproduct of lipid peroxidation, which has been involved in pulmonary, cardiac and neurodegenerative diseases. We investigated whether acrolein, an α,β-unsaturated aldehyde, can potentiate TRAIL-induced apoptosis in human renal cancer cells. The combined treatment with acrolein and TRAIL significantly induced apoptosis, and stimulated of caspase-3 activity, DNA fragmentation, and cleavage of PARP. We found that acrolein down-regulated the protein level of Bcl-2 and Bcl-2 overexpression inhibited the cell death induced by the combined treatment with acrolein and TRAIL. In addition, acrolein up-regulated C/EBP homologous protein (CHOP) and TRAIL death receptor 5 (DR5) and down-regulation of CHOP or DR5 expression using the respective small interfering RNA significantly attenuated the apoptosis induced by acrolein plus TRAIL. Interestingly, pretreatment with an antioxidant, N-acetylcysteine (NAC), inhibited not only CHOP and DR5 up-regulation but also the cell death induced by acrolein plus TRAIL. Taken together, our results demonstrated that acrolein enhances TRAIL-induced apoptosis in Caki cells through down-regulation of Bcl-2 and ROS dependent up-regulation of DR5.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

A key step in the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known; however, the physiology of the assembly of the apoptosome complex at the cellular level is still poorly defined. The aim of this work was to study the subcellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations in which the quality and the number of pituitary cells are determined by cell proliferation and apoptosis. We show herein that 2 h after triggering apoptosis with rotenone, Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of colocalization between Apaf-1 and fluorescently labeled caspase-9 significantly increased during the same period. Furthermore, we show herein for the first time in single cells that the colocalization between Apaf-1 and cytochrome c increases only transiently, indicating a transient interaction between cytochrome c and Apaf-1 during the activation of apoptosis in these cells. cytochrome c; caspase-9; apoptosis; apoptosome complex     

Apoptosis induced by Semliki Forest virus is RNA replication dependent and mediated via Bak

The RNA alphavirus Semliki Forest (SFV) triggers apoptosis in various mammalian cells, but it has remained controversial at what infection stage and by which signalling pathways host cells are killed. Both RNA synthesis-dependent and -independent initiation processes and mitochondrial as well as death receptor signalling pathways have been implicated. Here, we show that SFV-induced apoptosis is initiated at the level of RNA replication or thereafter. Moreover, by expressing antiapoptotic genes from recombinant SFV (replicons) and by using neutralizing reagents and gene-knockout cells, we provide clear evidence that SFV does not require CD95L-, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)- or tumor necrosis factor-mediated signalling but mitochondrial Bak to trigger cytochrome c release, the fall in the mitochondrial membrane potential, apoptotic protease-activating factor-1/caspase-9 apoptosome formation and caspase-3/-7 activation. Of seven BH3-only proteins tested, only Bid contributed to effective SFV-induced apoptosis. However, caspase-8 activation and Bid cleavage occurred downstream of Bax/Bak, indicating that truncated Bid formation serves to amplify rather than trigger SFV-induced apoptosis. Our data show that SFV sequentially activates a mitochondrial, Bak-mediated, caspase-8-dependent and Bid-mediated death signalling pathway that can be accurately dissected with gene-knockout cells and SFV replicons carrying antiapoptotic genes.     

Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.     

Characterization of the Role of Tumor Necrosis Factor Apoptosis Inducing Ligand (TRAIL) in Spermatogenesis through the Evaluation of Trail Gene-Deficient Mice

TRAIL (TNFSF10/Apo2L) is a member of the tumor necrosis factor (TNF) superfamily of proteins and is expressed in human and rodent testis. Although the functional role of TRAIL in spermatogenesis is not known, TRAIL is recognized to induce apoptosis via binding to its cognate receptors; DR4 (TRAIL-R1/TNFRSF10A) and DR5 (TRAIL-R2/TNFRSF10B). Here, we utilize Trail gene-deficient (Trail ^−/−) mice to evaluate the role of TRAIL in spermatogenesis by measuring testis weight, germ cell apoptosis, and spermatid head count at postnatal day (PND) 28 (pubertal) and PND 56 (adult). Trail^−/− mice have significantly reduced testis to body weight ratios as compared to wild-type C57BL/6J at both ages. Also, Trail ^−/− mice (PND 28) show a dramatic increase in basal germ cell apoptotic index (AI, 16.77) as compared to C57BL/6J (3.5). In the testis of adult C57BL/6J mice, the AI was lower than in PND 28 C57BL/6J mice (2.2). However, in adult Trail ^−/− mice, the AI was still higher than that of controls (9.0); indicating a relative high incidence of germ cell apoptosis. Expression of cleaved caspase-8 (CC8) and cleaved caspase-9 (CC9) (markers of the extrinsic and intrinsic apoptotic pathway, respectively) revealed a two-fold increase in the activity of both pathways in adult Trail ^−/− mice compared to C57BL/6J. Spermatid head counts in adult Trail ^−/− mice were dramatically reduced by 54% compared to C57BL/6J, indicating these animals suffer a marked decline in the production of mature spermatozoa. Taken together, these findings indicate that TRAIL is an important signaling molecule for maintaining germ cell homeostasis and functional spermatogenesis in the testis.     

Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c

The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.     

Apoptosome dependent caspase-3 activation pathway is non-redundant and necessary for apoptosis in sympathetic neurons

Although sympathetic neurons are a well-studied model for neuronal apoptosis, the role of the apoptosome in activating caspases in these neurons remains debated. We find that the ability of sympathetic neurons to undergo apoptosis in response to nerve growth factor (NGF) deprivation is completely dependent on having an intact apoptosome pathway. Genetic deletion of Apaf-1, caspase-9, or caspase-3 prevents apoptosis after NGF deprivation, and importantly, allows these neurons to recover and survive long-term following readdition of NGF. The inability of caspase-3 deficient sympathetic neurons to undergo apoptosis is particularly striking, as apoptosis in dermal fibroblasts and cortical neurons proceeds even in the absence of caspase-3. Our results show that in contrast to dermal fibroblasts and cortical neurons, sympathetic neurons express no detectable levels of caspase-7. The strict requirement for an intact apoptosome, coupled with a lack of effector caspase redundancy, provides sympathetic neurons with a markedly increased control over their apoptotic pathway.