Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

Overexpression of cFLIPs inhibits oxaliplatin-mediated apoptosis through enhanced XIAP stability and Akt activation in human renal cancer cells

cFLIP inhibits caspase 8 recruitment and processing at the death-inducing signaling complex (DISC), which is known to inhibits apoptosis mediated by death receptors such as Fas and death receptor 5 (DR5) as well as apoptosis mediated by anticancer therapeutic drugs. We observed that oxaliplatin induced apoptosis, the activation of DEVDase activity, DNA fragmentation, and cleavage of PLC-gamma1 and degradation of XIAP protein in dose-dependent manners, which was prevented by pretreatment with z-VAD or NAC, suggesting that oxaliplatin-induced apoptosis was mediated by caspase- or reactive oxygen species (ROS)-dependent pathways. Furthermore, ectopic expression of cFLIPs potently attenuated oxaliplatin-induced apoptosis, whereas cFLIP(L) had less effect. Interestingly, we found that the protein level of XIAP was sustained in oxaliplatin-treated cFLIPs overexpressing cell, which was caused by the increased XIAP protein stability and that the phospho-Akt level was high compared to vector-transfected cell. The increased XIAP protein stability was lessened by PI3K inhibitor LY294002 treatment in cFLIPs overexpressing cells. Thus, our findings imply that the anti-apoptotic functions of cFLIPs may be attributed to inhibit oxaliplatin-induced apoptosis through the sustained XIAP protein level and Akt activation.     

Fas death receptor signalling: roles of Bid and XIAP

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis.     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

Caspase activation and apoptosis in response to proteasome inhibitors

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. In fact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Mouse mammary gland involution is associated with cytochrome c release and caspase activation

At weaning, milk producing mammary epithelial cells undergo apoptosis and are removed by phagocytosis. Here, we show that mouse mammary gland involution is associated with mitochondrial cytochrome c release and processing of numerous caspases, including caspase-1, -3, -7, -8 and -9. Induction of caspase-3-like activity paralleled cleavage of poly-(ADP--ribose) polymerase. Dexamethasone inhibited processing of caspase-3, -7 and -8 and apoptosis, but had no effect on caspase-1 accumulation and cytochrome c release. In Bcl-2 transgenic animals, cytochrome c release, caspase activation and apoptosis were impaired. Thus, the pro-apoptotic signaling pathway in mammary epithelial cells during involution involves the release of cytochrome c and activation of caspases. It is inhibited by Bcl-2 at the mitochondrial level and by dexamethasone at a post-mitochondrial level.     

PQ1, a quinoline derivative, induces apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9

Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.     

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.     

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.     

Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein.

Bcl-2 and Bcl-x(L) are reported to inhibit CD95-mediated apoptosis in "type II" but not in "type I" cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-x(L) displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-x(L) inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-x(L) proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Mitochondrial Regulation of Cell Death

Although required for life, paradoxically, mitochondria are often essential for initiating apoptotic cell death. Mitochondria regulate caspase activation and cell death through an event termed mitochondrial outer membrane permeabilization (MOMP); this leads to the release of various mitochondrial intermembrane space proteins that activate caspases, resulting in apoptosis. MOMP is often considered a point of no return because it typically leads to cell death, even in the absence of caspase activity. Because of this pivotal role in deciding cell fate, deregulation of MOMP impacts on many diseases and represents a fruitful site for therapeutic intervention. Here we discuss the mechanisms underlying mitochondrial permeabilization and how this key event leads to cell death through caspase-dependent and -independent means. We then proceed to explore how the release of mitochondrial proteins may be regulated following MOMP. Finally, we discuss mechanisms that enable cells sometimes to survive MOMP, allowing them, in essence, to return from the point of no return.In most organisms, mitochondria play an essential role in activating caspase proteases through a pathway termed the mitochondrial or intrinsic pathway of apoptosis. Mitochondria regulate caspase activation by a process called mitochondrial outer membrane permeabilization (MOMP). Selective permeabilization of the mitochondrial outer membrane releases intermembrane space (IMS) proteins that drive robust caspase activity leading to rapid cell death. However, even in the absence of caspase activity, MOMP typically commits a cell to death and is therefore considered a point of no return (Fig. 1). Because of this pivotal role in dictating cell fate, MOMP is highly regulated, mainly through interactions between pro- and antiapoptotic members of the Bcl-2 family. In this article, we begin by discussing how mitochondria may have evolved to become central players in apoptotic cell death. We then provide an overview of current models addressing the mechanics of MOMP, outlining how this crucial event leads to cell death through both caspase-dependent or -independent mechanisms. Finally, we discuss how caspase activity may be regulated post-MOMP and define other processes that allow cells to survive MOMP and, in effect, return from the point of no return.Open in a separate windowFigure 1.Mitochondrial regulation of cell death. Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) can lead to caspase-dependent apoptosis (left) or caspase-independent cell death (right). Following MOMP, soluble proteins are released from the mitochondrial intermembrane space into the cytoplasm. Cytochrome c binds to monomeric Apaf-1 leading to its conformational change and oligomerization. Procaspase-9 is recruited to heptameric Apaf-1 complexes forming the apoptosome. This leads to activation of caspase-9 and, through caspase-9-mediated cleavage, activation of the executioner caspases-3 and -7. Release of Smac and Omi from the mitochondrial intermembrane space facilitates caspase activation by neutralizing the caspase inhibitor XIAP. MOMP can also lead to nonapoptotic cell death through a gradual loss of mitochondrial function and/or release of mitochondrial proteins that kill the cell in a caspase-independent manner.     

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.Subject terms: Cell biology, Molecular biology, Experimental models of disease     

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.     

Mitochondrial pro-apoptotic indices do not precede the transient caspase activation associated with myogenesis

Skeletal muscle differentiation requires activity of the apoptotic protease caspase-3. We attempted to identify the source of caspase activation in differentiating C2C12 skeletal myoblasts. In addition to caspase-3, caspase-2 was transiently activated during differentiation; however, no changes were observed in caspase-8 or -9 activity. Although mitochondrial Bax increased, this was matched by Bcl-2, resulting in no change to the mitochondrial Bax:Bcl-2 ratio early during differentiation. Interestingly, mitochondrial membrane potential increased on a timeline similar to caspase activation and was accompanied by an immediate, temporary reduction in cytosolic Smac and cytochrome c. Since XIAP protein expression dramatically declined during myogenesis, we investigated whether this contributes to caspase-3 activation. Despite reducing caspase-3 activity by up to 57%, differentiation was unaffected in cells overexpressing normal or E3-mutant XIAP. Furthermore, a XIAP mutant which can inhibit caspase-9 but not caspase-3 did not reduce caspase-3 activity or affect differentiation. Administering a chemical caspase-3 inhibitor demonstrated that complete enzyme inhibition was required to impair myogenesis. These results suggest that neither mitochondrial apoptotic signaling nor XIAP degradation is responsible for transient caspase-3 activation during C2C12 differentiation.     

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) do not inhibit caspase-3 and caspase-7 processing in cells exposed to pro-apoptotic inducing stimuli

We recently demonstrated that TLCK and TPCK could act as potent but nonspecific inhibitors of mature caspases [Frydrych and Mlejnek [2008] J Cell Biochem 103:1646-1656]. The question whether TLCK and TPCK inhibit simultaneously caspase activation and/or processing remained, however, open. In this article, we demonstrated that TPCK even enhanced caspase-3 and caspase-7 processing although it substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity in HL-60 cells exposed to various cell death inducing stimuli. Under the same conditions, TLCK had no effect or affected caspase-3 and caspase-7 processing marginally depending on cell treatment used. Importantly, TLCK substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity irrespectively to the treatment used. Interestingly, treatment of cells with toxic concentrations of TPCK alone was accompanied by full caspase-3 and -7 processing even if it induced necrosis. In contrast, treatment of cells with concentrations of TLCK that caused necrosis was accompanied by only partial caspase-3 and caspase-7 processing. Our results clearly indicated that TPCK and TLCK did not inhibit caspase-3 and -7 enzymatic activity by prevention of their activation and/or processing.     

Akt regulates cell survival and apoptosis at a postmitochondrial level

Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.