All-trans-retinoic acid-mediated modulation of p53 during neural differentiation in murine embryonic stem cells

All-trans-retinoic acid (RA) plays an important physiological role in embryonic development and is teratogenic in large doses in almost all species. p53, a tumor suppressor gene encodes phosphoproteins, which regulate cellular proliferation, differentiation, and apoptosis. Temporal modulation of p53 by retinoic acid was investigated in murine embryonic stem cells during differentiation and apoptosis. Undifferentiated embryonic stem cells express a high level of p53 mRNA and protein followed by a decrease in p53 levels as differentiation proceeds. The addition of retinoic acid during 8–10 days of differentiation increased the levels of p53 mRNA and protein, accompanied by accelerated neural differentiation and apoptosis. Marked increase in apoptosis was observed at 10–20 h after retinoic acid treatment when compared with untreated controls. Retinoic acid-induced morphological differentiation resulted in predominantly neural-type cells. Maximum increase in p53 mRNA in retinoic acid-treated cells occurred on day 17, whereas maximum protein synthesis occurred on days 14–17, which coincided with increased neural differentiation and proliferation. Increased p53 levels did not induce p21 transactivation, interestingly a decrease in p21 was observed on day 17 on exposure to retinoic acid. The level of p53 declined by day 21 of differentiation. The results demonstrated that retinoic acid-mediated apoptosis preceded the changes in p53 expression, suggesting that p53 induction does not initiate retinoic acid-induced apoptosis during development. However, retinoic acid accelerated neural differentiation and increased the expression of p53 in proliferating neural cells, corroborated by decreased p21 levels, indicating the importance of cell type and stage specificity of p53 function. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of G proteins of the Rho family in the regulation of Bcl-2-like protein expression and caspase 3 activation by Gastrins

Gastrins, including amidated gastrin (Gamide) and glycine-extended gastrin (Ggly), are known to accelerate the growth of gastric and colorectal cancer cells by stimulation of proliferation and inhibition of apoptosis. Gamide controls apoptosis by regulation of proteins of the Bcl-2 family and by regulation of the activation of caspases. However the interactions between Ggly and proteins of the Bcl-2 family and caspases are not known. Since in other systems G proteins of the Rho family inhibit apoptosis via interaction with proteins of the Bcl-2 family, leading to changes in caspase activities, we have compared the role of Rho family G proteins in regulation of Bcl-2-like (Bad/Bax/Bcl-xl) protein expression and caspase 3 activation by Ggly and Gamide. The effects of the specific inhibitors C3 (for Rho) and Y-27632 (for ROCK), and of dominant negative mutants of Rac, Cdc42 and PAK, were investigated in the gastric epithelial cell line IMGE-5. Apoptosis was induced by serum starvation and confirmed by annexin V staining and caspase 3 activation. Ggly inhibits caspase 3 activation via a Bcl-2-like protein-mediated pathway which requires activation of both Rho/ROCK and Rac/Cdc42/PAK. Gamide inhibits caspase 3 activation via redundant Bcl-2-like protein-mediated pathways which involve alternative activation of Rac/Cdc42/PAK and Rho/ROCK. Gamide and Ggly differentially activate members of Rho family G proteins which in turn regulate different proteins of the Bcl-2 family leading to changes in caspase 3 activity. The findings offer potential targets for blocking the growth-stimulating effects of these gastrins.     

Bcl-2 controls caspase activation following a p53-dependent cyclin D1-induced death signal

MCF-7 and ZR-75 breast cancer cells infected with an adenovirus constitutively expressing high levels of cyclin D1 demonstrated widespread mitochondrial translocation of Bax and cytochrome c release that was approximately doubled after the addition of all-trans retinoic acid (RA) or Bcl-2 antisense oligonucleotide. By comparison, the percentage of cells in Lac Z virus-infected cultures containing translocated Bax and cytoplasmic cytochrome c was markedly less even after RA treatment. Despite this, RA-treated Lac Z and untreated cyclin D1 virus-infected cultures contained similarly low proportions of cells with active caspase or cells that were permeable to propidium iodide. Bax activation was p53-dependent and accompanied by arrest in G(2) phase. Although constitutive Bcl-2 expression prevented Bax activation, it did not alter cyclin D1-induced cell cycle arrest, illustrating the independence of these events. Both RA and antisense Bcl-2 oligonucleotide decreased Bcl-2 protein levels and markedly increased caspase activity and apoptosis in cyclin D1-infected cells. Thus amplified cyclin D1 expression initiates an apoptotic signal inhibited by different levels of cellular Bcl-2 at two points. Whereas high cellular levels of Bcl-2 prevent mitochondrial Bax translocation, lower levels can prevent apoptosis by inhibition of caspase activation.     

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.     

The apoptotic process of human bladder carcinoma T24 cells induced by retinoid

Breakdown of the cytoskeletal network and redistribution of cytoplasmic organelles are early events of programmed cell death. Previous studies showed that retinoic acid induces programmed cell death in many tumor cell lines and that cytokeratins, particularly cytokeratin 18, are affected in the early events of apoptosis. In this study, patterns of cytoplasmic intermediate filaments (cytokeratin 18), actin filaments, and microtubules, as well as Bax and Bcl-2 proteins in human bladder carcinoma T24 cells were examined before and after retinoic acid treatment by immunocytochemistry and conventional electron microscopy. Our results demonstrate that the redistribution of Bax and Bcl-2 proteins in the subcellular compartment of T24 cells is correlated with reorganization of the cytoplasmic intermediate filament network and that cytokeratins are cleaved by caspases, as revealed by the M30 antibody which recognizes a specific caspase cleavage site within cytokeratin 18. The cytoskeletal architectures of microtubules are not significantly affected in the early apoptotic process, from our observations. We suggest that the breakdown in the intermediate filament network associated with the aggregation of mitochondria and lysosome may be a crucial event in the apoptotic process and that aggregation of cytoplasmic Bax may accelerate apoptotic death.     

Effects of diallyl disulfide (DADS) on expression of apoptosis associated proteins in androgen independent human prostate cancer cells (PC-3)

Prostate cancer is a leading cause of death among the aging men. Surgical or radiotherapy is effective when the cancer is confined to the prostate gland but once the cancer spreads beyond the pelvis even chemotherapy and hormonal ablation therapy fails in curing this disease. Our previous studies have shown that diallyl disulfide (DADS) induces cell cycle arrest and also induces apoptosis in PC-3 cells. And now the present study is focused to see whether there is an activation of caspase cascade pathway. Hence, in the present study the apoptotic effect of DADS is studied by Western blot analysis of caspase-3, -9, -10 and Bcl-2, Bad, and Bax protein. The Apoptotic cells were assessed by Hoechst 33342 staining with 25 and 40 microM concentrations of DADS for 24 h. The results have shown that DADS at 25 and 40 microM concentrations has induced the activation of caspases. There is a significant increase in the expression of caspases (3, 9, and 10). The proapoptotic protein Bax has significantly increased at 40 microM of DADS treatment and there is significant increase of Bad protein at both the concentration. Bcl-2 protein has significantly decreased in DADS treated cells. Therefore, the present investigation serves as evidence that DADS may be a therapeutic drug in the treatment of prostate cancer.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone

To elucidate the biochemical pathways leading to spontaneous apoptosis in primary cultures of human and rat hepatocytes, we examined the activation of the caspase cascade, the expression of Bcl-2-related-proteins and heat shock proteins. Comparisons were made before and after dexamethasone (DEX) treatment. We show that DEX inhibited spontaneous apoptosis in a dose-dependent manner. DEX increases the expression of anti-apoptotic Bcl-2 and Bcl-x(L) proteins, decreases the expression of pro-apoptotic Bax and inhibits Bad translocation thereby preventing the release of cytochrome c, the activation of caspases, and cell death. Although, the expression of Hsp27 and Hsp70 proteins remained unchanged, the oncogenic protein c-Myc is upregulated upon DEX-treatment. These results indicate that DEX mediates its survival effect against spontaneous apoptosis by acting upstream of the mitochondrial changes. Thus, the mitochondrial apoptotic pathway plays a major role in regulating spontaneous apoptosis in these cells. Blocking this pathway therefore may assist with organ preservation for transplant, drug screening, and other purposes.     

Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment

The regulation of chondrocyte apoptosis in articular cartilage may underlay age-associated changes in cartilage and the development of osteoarthritis. Here we demonstrate the importance of Bcl-2 in regulating articular chondrocyte apoptosis in response to both serum withdrawal and retinoic acid treatment. Both stimuli induced apoptosis of primary human articular chondrocytes and a rat chondrocyte cell line as evidenced by the formation of DNA ladders. Apoptosis was accompanied by decreased expression of aggrecan, a chondrocyte specific matrix protein. The expression of Bcl-2 was downregulated by both agents based on Northern and Western analysis, while the level of Bax expression remained unchanged compared to control cells. The importance of Bcl-2 in regulating chondrocyte apoptosis was confirmed by creating cell lines overexpressing sense and antisense Bcl-2 mRNA. Multiple cell lines expressing antisense Bcl-2 displayed increased apoptosis even in the presence of 10% serum as compared to wild-type cells. In contrast, chondrocytes overexpressing Bcl-2 were resistant to apoptosis induced by both serum withdrawal and retinoic acid treatment. Finally, the expression of Bcl-2 did not block the decreased aggrecan expression in IRC cells treated with retinoic acid. We conclude that Bcl-2 plays an important role in the maintenance of articular chondrocyte survival and that retinoic acid inhibits aggrecan expression independent of the apoptotic process. J. Cell. Biochem. 71:302–309, 1998. © 1998 Wiley-Liss, Inc.     

BMRP is a Bcl-2 binding protein that induces apoptosis

Members of the Bcl-2 family of proteins play important roles in the regulation of cell death by apoptosis. The yeast Two-Hybrid system was utilized to identify a protein that interacts with the anti-apoptotic protein Bcl-2, designated BMRP. This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41). Binding experiments confirmed the interaction of BMRP to Bcl-2 in mammalian cells. Subcellular fractionation by differential centrifugation studies showed that both Bcl-2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria). Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues. Additionally, a larger 2.2 kb mRNA species was also observed in some tissues. Western blot analysis showed that endogenous BMRP runs as a band of 16-17 kDa in SDS-PAGE. Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells. Transfection of BMRP showed similar effects to those observed by overexpression of the pro-apoptotic proteins Bax or Bad. BMRP-stimulated cell death was counteracted by co-expression of Bcl-2. The baculoviral caspase inhibitor p35 also protected cells from BMRP-induced cell death. These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl-2 and caspases.     

Bcl-w forms complexes with Bax and Bak, and elevated ratios of Bax/Bcl-w and Bak/Bcl-w correspond to spermatogonial and spermatocyte apoptosis in the testis

Bcl-w, a prosurvival member of the Bcl-2 family, is essential for spermatogenesis. However, the mechanisms by which Bcl-w participates in the regulation of apoptosis in the testis are largely unknown. To explore the potential role of Bcl-w in the regulation of apoptosis in the testis, the expression of Bcl-w mRNA and protein during testicular development and spermatogenesis, the dimerization with the proapoptosis members of the Bcl-2 family, and the responses to hormonal stimulation in vitro and apoptosis-inducing signals in vivo were investigated. Both Bcl-w mRNA and protein were detected in Sertoli cells, spermatogonia, and spermatocytes, as well as in Leydig cells. The steady-state levels of Bcl-w mRNA and protein were much higher in Sertoli cells than in spermatogonia and spermatocytes. In the adult rat testis, both Bcl-w mRNA and protein in Sertoli cells displayed a stage-specific expression pattern. Bcl-w could form complexes with Bax and Bak but not with Bad. Bax and Bak were immunohistochemically localized to the same cell types as Bcl-w, but with higher expression levels in spermatocytes and spermatogonia than in Sertoli cells. FSH could up-regulate Bcl-w mRNA levels in the seminiferous tubules cultured in vitro, whereas no effect was observed when testosterone was applied. Three animal models that display spermatogonial apoptosis induced by blockade of stem cell factor/c-kit interaction by a function-blocking anti-c-kit antibody, spermatocyte apoptosis induced by methoxyacetic acid, and apoptosis of spermatogonia, spermatocytes, and spermatids induced by testosterone withdrawal after ethylene dimethane sulfonate treatment were employed to check the changes of Bcl-w, Bax, and Bak protein levels during apoptosis of specific germ cells. In all three models, the ratios of Bax/Bcl-w and Bak/Bcl-w were significantly elevated. The present study suggests that Bcl-w is an important prosurvival factor of Sertoli cells, spermatogonia, and spermatocytes and participates in the regulation of apoptosis by binding proapoptotic factors Bax and Bak. The ratios of Bax/Bcl-w and Bak/Bcl-w may be decisive for the survival of Sertoli cells, spermatogonia, and spermatocytes.     

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cells but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad.     

Effect of all-trans-retinoic acid on the differentiation, maturation and functions of dendritic cells derived from cord blood monocytes

We investigated the effects of all-trans-retinoic acid on dendritic cells derived from human cord blood monocytes to clarify how vitamin A affects immune function in children. Monocytes were separated from 18 cord blood samples, and dendritic cells were differentiated by culture. The percentage of dendritic cells was markedly lower in all-trans-retinoic acid treated cells than in untreated cells. After exposure to tumour necrosis factor-alpha for 3 days, all-trans-retinoic acid treated dendritic cells showed a reduced capacity to activate alloreactive T cells compared to untreated cells. In addition, all-trans-retinoic acid-treated dendritic cells could drive T cells towards T-helper cell type 2 responses with decreased secretion of interleukin-12, interferon-gamma, and increased production of interleukin-10 and interleukin-4. However, when Ro 41-5253, a selective retinoic acid receptor alpha antagonist, was add to culture, all the above actions were reversed. Thus, all-trans-retinoic acid may act at the first step of the immune response by inhibiting the differentiation of dendritic cells, maturation and induction of the T-helper cell type-2 response. The actions of all-trans-retinoic acid on dendritic cells were mediated through retinoic acid receptor alpha.     

Apoptosis in the developing visual system

Programmed cellular death is a widespread phenomenon during development of the nervous system. Two classes of molecules are particularly important in the context of apoptosis control in the nervous system: intracellular effectors homologous to the Caenorhabditis elegans Ced-3, -4, and -9 proteins, which in mammals correspond to the proteases of the caspase family, Apaf-1, and the members of the Bcl-2 protein family, and neurotrophic factors. Retinal ganglion cells lend a convenient model system with which to investigate apoptosis in central neurons during development as well as after injury. In this review, we discuss the role of these molecules in the control of programmed cellular death in the retinotectal system. Transgenic animal models and expression studies have shown that caspases, Bcl-2, Bax, and possibly Bcl-X are necessary players for the control of programmed cellular death in retinal ganglion cells. Bax and caspase 3 expression in retinal ganglion cells is upregulated after injury, and inhibition of Bax or caspase 3 increases the survival of injured retinal ganglion cells. Neurotrophins can support the survival of injured retinal ganglion cells, but this effect is transient. The physiological role of neurotrophins in the development of the retinocollicular system seems more related to the topographic refinement of retinocollicular projections, a process that is mediated, at least partially, by selective elimination of retinal ganglion cells making inappropriate topographic projections.     

PESV 对K562 细胞BCR/ABL 融合基因及凋亡调控因子Bcl-2、Bad 表达的影响

目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.     

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation.

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation.1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.     

N-acetylphytosphingosine-induced apoptosis of Jurkat cells is mediated by the conformational change in Bak

N-acetylphytosphingosine (NAPS), a sphingolipid derivative, is one of the well-known signal molecules that mediates various cellular functions, including cell growth, differentiation, and apoptosis. In this study, we demonstrated that NAPS induces apoptosis of Jurkat cells by activating Bak, but not Bax, which are both members of a proapoptotic subfamily of the Bcl-2 proteins. NAPS activated caspase-8 in a FADD-independent manner, but the lack of caspase-8 did not suppress the activation of caspase-3 and -9 and cell death, indicating that caspase-8 activation does not play an important role in NAPS-induced cell death. The overexpression of Bcl-x_L, an anti-apoptotic protein, completely inhibited the activation of the caspases and apoptosis, assuming that NAPS-induced apoptosis was initiated by the mitochondria. The expression levels of pro- and anti-apoptotic Bcl-2 family members were not changed by the NAPS treatment. However, Bad was translocated from the cytosol into the mitochondria, where it bound to Bcl-x_L, and Bak was dissociated from Bcl-x_L and conformationally changed. Taken together, these findings indicate that NAPS induced apoptosis of Jurkat cells in a mitochondria-dependent manner that was controlled by the translocation of Bad and the conformational change in Bak. These authors contributed equally to this paper