5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells

In the present study, we investigated the signaling pathways implicated in the induction of apoptosis by two modified nucleosides, 5-phenylselenyl-methyl-2′-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2′-deoxyuridine (MeSe-T), using human cancer cell lines. The induction of apoptosis was associated with proteolytic activation of caspase-3 and -9, PARP cleavage, and decreased levels of IAP family members, including c-IAP-1 and c-IAP-2, but had no effect on XIAP and survivin. PhSe-T and MeSe-T also enhanced the activities of caspase-2 and -8, Bid cleavage, and the conformational activation of Bax. Additionally, nucleoside derivative-induced apoptosis was inhibited by the selective inhibitors of caspase-2, -3, -8, and -9 and also by si-RNAs against caspase-2, -3, -8, and -9; however, inhibition of caspase-2 and -3 was more effective at preventing apoptosis than inhibition of caspase-8 and -9. Moreover, the inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk or by the knockdown of protein expression using siRNA suppressed nucleoside derivative-induced caspase-3 activation, but not vice versa. PhSe-T and MeSe-T also induced a Δψ_m loss via a CsA-insensitive mechanism, ROS production, and DNA damage, including strand breaks. Moreover, ROS scavengers such as NAC, tiron, and quercetin inhibited nucleoside derivative-induced ROS generation and apoptosis by blocking the sequential activation of caspase-2 and -3, indicating the role of ROS in caspase-2-mediated apoptosis. Taken together, these results indicate that caspase-2 acts upstream of caspase-3 and that caspase-2 functions in response to DNA damage in both PhSe-T- and MeSe-T-induced apoptosis. Our results also suggest that ROS are critical regulators of the sequential activation of caspase-2 and -3 in nucleoside derivative-treated cancer cells.     

Titanium dioxide nanoparticles induce apoptosis through the JNK/p38-caspase-8-Bid pathway in phytohemagglutinin-stimulated human lymphocytes

We investigated the signaling pathways underlying nano-TiO₂-induced apoptosis in cultured human lymphocytes. Nano-TiO₂ increased the proportion of sub-G1 cells, activated caspase-9 and caspase-3, and induced caspase-3-mediated PARP cleavage. Nano-TiO₂ also induced loss of mitochondrial membrane potential, which suggests that nano-TiO₂ induces apoptosis via a mitochondrial pathway. A time-sequence analysis of the induction of apoptosis by nano-TiO₂ revealed that nano-TiO₂ triggered apoptosis through caspase-8/Bid activation. We also observed that inhibition of caspase-8 by z-IETD-fmk suppressed the caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and apoptosis. Nano-TiO₂ activated two MAPKs, p38 and JNK. In addition, the selective p38 inhibitor SB203580 and selective JNK inhibitor SP600125 suppressed nano-TiO₂-induced apoptosis and caspase-8 activation to moderate and significant extents, respectively. Knockdown of protein levels of JNK1 and p38 using an RNA interference technique also suppressed caspase-8 activation. Our results suggest that nano-TiO₂-induced apoptosis is mediated by the p38/JNK pathway and the caspase-8-dependent Bid pathway in human lymphocytes.     

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.     

Role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis

A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol. 77:11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome c release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome c release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.     

TNF-alpha signals apoptosis through a bid-dependent conformational change in Bax that is inhibited by E1B 19K

The adenovirus E1B 19K gene product is an inhibitor of apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) during viral infection. We report that E1B 19K inhibited neither caspase-8 activation nor caspase-8-dependent Bid cleavage by TNF-alpha. Rather, TNF-alpha induced a tBid-dependent conformational change in Bax that allowed an interaction between E1B 19K and conformationally altered Bax, which caused inhibition of cytochrome c release and caspase-9 activation. E1B 19K expression interrupted caspase-3 processing, permitting cleavage to remove the p12 subunit but not the prodomain consistent with caspase-8 and not caspase-9 enzymatic activity. Thus, E1B 19K blocks TNF-alpha-mediated death signaling by inhibiting a specific form of Bax that interrupts caspase activation downstream of caspase-8 and upstream of caspase-9.     

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.     

Destabilization of CARP mRNAs by aloe-emodin contributes to caspase-8-mediated p53-independent apoptosis of human carcinoma cells

Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.     

Bid is cleaved upstream of caspase-8 activation during TRAIL-mediated apoptosis in human osteosarcoma cells

TRAIL induces apoptosis in many malignant cell types. In this study, we used the human papilloma virus (HPV) 16 E6 protein as a molecular tool to probe the TRAIL pathway in HCT116 colon carcinoma cells and U2OS osteosarcoma cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells expressing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAIL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8. Cathepsin B activity in U2OS cells was significantly activated shortly after exposure to TRAIL, and the cathepsin B inhibitor, CA074Me, inhibited both TRAIL- and anti-DR5-mediated apoptosis and delayed the cleavage of Bid. These findings suggest that TRAIL activates a pathway dependent on Bid, but largely independent of FADD and caspase-8, in U2OS cells.     

CD437, a synthetic retinoid, induces apoptosis in human respiratory epithelial cells via caspase-independent mitochondrial and caspase-8-dependent pathways both up-regulated by JNK signaling pathway

The synthetic retinoid-related molecule CD437-induced apoptosis in human epithelial airway respiratory cells: the 16HBE bronchial cell line and normal nasal epithelial cells. CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by caspase-8 inhibitor z-IETD-fmk which preserved S-phase cells but was weakly inhibited by others selective caspase-inhibitors, indicating that caspase-8 activation was involved. z-VAD and z-IETD prevented the nuclear envelope fragmentation but did not block the chromatin condensation. The disruption of mitochondrial transmembrane potential was also induced by CD437 treatment. The translocation of Bax to mitochondria was demonstrated, as well as the release of cytochrome c into the cytosol and of apoptosis-inducing factor (AIF) translocated into the nucleus. z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cytochrome c and AIF from mitochondria. These results suggest that CD437-induced apoptosis is executed by two converging pathways. AIF release is responsible for chromatin condensation, the first stage of apoptotic cell, via a mitochondrial pathway independent of caspase. But final stage of apoptosis requires the caspase-8-dependent nuclear envelope fragmentation. In addition, using SP600125, JNK inhibitor, we demonstrated that CD437 activates the JNK-MAP kinase signaling pathway upstream to mitochondrial and caspase-8 pathways. Conversely, JNK pathway inhibition, which suppresses S-phase apoptosis, did not prevent cell cycle arrest within S phase, confirming that these processes are triggered by distinct mechanisms.     

Effects of Bcl-2 levels on Fas signaling-induced caspase-3 activation: molecular genetic tests of computational model predictions

Fas-induced apoptosis is a critical process for normal immune system development and function. Although many molecular components in the Fas signaling pathway have been identified, a systematic understanding of how they work together to determine network dynamics and apoptosis itself has remained elusive. To address this, we generated a computational model for interpreting and predicting effects of pathway component properties. The model integrates current information concerning the signaling network downstream of Fas activation, through both type I and type II pathways, until activation of caspase-3. Unknown parameter values in the model were estimated using experimental data obtained from human Jurkat T cells. To elucidate critical signaling network properties, we examined the effects of altering the level of Bcl-2 on the kinetics of caspase-3 activation, using both overexpression and knockdown in the model and experimentally. Overexpression was used to distinguish among alternative hypotheses for inhibitory binding interactions of Bcl-2 with various components in the mitochondrial pathway. In comparing model simulations with experimental results, we find the best agreement when Bcl-2 blocks the release of cytochrome c by binding to both Bax and truncated Bid instead of Bax, truncated Bid, or Bid alone. Moreover, although Bcl-2 overexpression strongly reduces caspase-3 activation, Bcl-2 knockdown has a negligible effect, demonstrating a general model finding that varying the expression levels of signal molecules frequently has asymmetric effects on the outcome. Finally, we demonstrate that the relative dominance of type I vs type II pathways can be switched by varying particular signaling component levels without changing network structure.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.     

Caspase-2 can function upstream of bid cleavage in the TRAIL apoptosis pathway

In many mammalian cell types, engagement of the TRAIL/Apo2L death receptors DR4 and DR5 alters mitochondrial physiology, thereby promoting the release of pro-apoptotic proteins normally contained within this organelle. A contemporary view of this process is that in so-called type II cells death receptor-activated caspase-8 cleaves the Bcl-2 family member Bid, which generates a truncated Bid fragment that collaborates with Bax, another Bcl-2 relative, to promote the release of mitochondrial factors necessary for activation of executioner caspases and apoptosis. Here we show that in some type II cells caspase-2 is necessary for optimal TRAIL-mediated cleavage of Bid. Down-regulation of caspase-2 using RNA interference significantly inhibited TRAIL-induced apoptosis. Analysis of the TRAIL proteolytic cascade following gene silencing of specific pathway components revealed that caspase-2 is necessary for efficient cleavage of Bid; however, caspase-2 proteolytic processing, which occurs downstream of Bax, is not necessary for its role in Bid cleavage.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

p38 mitogen-activated protein kinase mediates bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide

p38 mitogen-activated protein kinase is activated and involved in cleavage of caspase-3 during apoptosis induced by a number of stimuli. However, the signaling events triggered by p38 that result in caspase-3 activation are still unknown. In human leukemia cells, two reactive oxygen species, singlet oxygen and hydrogen peroxide (H(2)O(2)), selectively stimulated the phosphorylation of p38. Preincubation of cells with SB203580, a specific inhibitor of p38, dose dependently inhibited DNA fragmentation induced by singlet oxygen but not by H(2)O(2). Protection from apoptosis by SB203580 correlated with inhibition of caspase-3, and several events that are associated with caspase-3 activation, including Bid cleavage, decrease in mitochondrial transmembrane potential and release of cytochrome c from mitochondria, whereas caspase-8 cleavage was not affected by this inhibitor. In contrast, blockade of caspase-8 with Ile-Glu-Thr-Asp-fluoromethyl ketone is sufficient to prevent formation of DNA fragments and to inhibit all the above signaling events, with exception of p38 phosphorylation, in both singlet oxygen- and H(2)O(2)-treated cells. These data suggest that caspase-3 activation is regulated through redundant signaling pathways that involve p38 and caspase-8 acting upstream of Bid during singlet oxygen-induced apoptosis, whereas the activation of caspase-3 by H(2)O(2) is only governed by a caspase-8-mediated apoptotic pathway.     

Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.     

Caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain

Caspase activation resulting from cytochrome c release from the mitochondria is an essential component of the mechanism of apoptosis initiated by a range of factors. The activation of Bid by caspase-8 in this pathway promotes further cytochrome c release, thereby completing a positive feedback loop of caspase activation. Although the identity of the caspases necessary for caspase-8 activation in this pathway are known, it is still unclear which protease directly cleaves caspase-8. In order to identify the factor responsible we undertook a biochemical purification of caspase-8 cleaving activity in cytosolic extracts to which cytochrome c had been added. Here we report that caspase-6 is the only soluble protease in cytochrome c activated Jurkat cell extracts that has significant caspase-8 cleaving activity. Furthermore the caspase-6 that we purified was sufficient to induce Bid dependent cytochrome c releasing activity in cell extracts. Inhibition of caspase-6 activity in cells significantly inhibited caspase-8 cleavage and apoptosis, therefore establishing caspase-6 as a major activator of caspase-8 in vivo and confirming that this pathway can have a critical role in promotion of apoptosis. We also show that caspase-6 is inactive until the short prodomain is removed. We suggest that the requirement for two distinct cleavage steps to activate an effector caspase may represent an effective mechanism for restriction of spontaneous caspase activation and aberrant entry into apoptosis.     

Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death through the caspase activation cascade and translocation of cleaved Bid (tBid) by the apical caspase-8 to mitochondria to induce oligomerization of multidomain Bax and Bak. However, the roles of prosurvival Bcl-2 family proteins in TRAIL apoptosis remain elusive. Here we showed that, besides the specific cleavage and activation of Bid by caspase-8 and caspase-3, TRAIL-induced apoptosis in Jurkat T cells required the specific cleavage of Mcl-1 at Asp-127 and Asp-157 by caspase-3, while other prototypic antiapoptotic factors such as Bcl-2 or Bcl-X(L) seemed not to be affected. Mutation at Asp-127 and Asp-157 of Mcl-1 led to cellular resistance to TRAIL-induced apoptosis. In sharp contrast to cycloheximide-induced Mcl-1 dilapidation, TRAIL did not activate proteasomal degradation of Mcl-1 in Jurkat cells. We further established for the first time that the C-terminal domain of Mcl-1 became proapoptotic as a result of caspase-3 cleavage, and its physical interaction and cooperation with tBid, Bak, and voltage-dependent anion-selective channel 1 promoted mitochondrial apoptosis. These results suggested that removal of N-terminal domains of Bid by caspase-8 and Mcl-1 by caspase-3 enabled the maximal mitochondrial perturbation that potentiated TRAIL-induced apoptosis.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.     

Real-time monitoring full length bid interacting with Bax during TNF-α-induced apoptosis

Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-α(tumor necrosis factor α)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-α-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-α treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-α-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.     

Phosphorylation of murine caspase-9 by the protein kinase casein kinase 2 regulates its cleavage by caspase-8

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in tumor necrosis factor (TNF)-alpha-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of Ser(348) on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of Ser(348) abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-alpha-treated caspase-9 knock-out mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, whereas phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9, rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.