Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.     

TNF-alpha-induced apoptosis of macrophages following inhibition of NF-kappa B: a central role for disruption of mitochondria

Previously, we established that suppressing the constitutive activation of NF-kappaB in in vitro matured human macrophages resulted in apoptosis initiated by a decrease of the Bcl-2 family member, A1, and the loss of mitochondrial transmembrane potential (Deltapsi(m)). This study was performed to characterize the mechanism of TNF-alpha-induced apoptosis in macrophages following the inhibition of NF-kappaB. The addition of TNF-alpha markedly enhanced the loss of Deltapsi(m) and the induction of apoptotic cell death. Although caspase 8 was activated and contributed to DNA fragmentation, it was not necessary for the TNF-alpha-induced loss of Deltapsi(m). The inhibition of NF-kappaB alone resulted in the release of cytochrome c from the mitochondria, while both cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI were released following the addition of TNF-alpha. Furthermore, c-Jun N-terminal kinase activation, which was sustained following treatment with TNF-alpha when NF-kappaB was inhibited, contributed to DNA fragmentation. These observations demonstrate that cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI may be differentially released from the mitochondria, and that the sustained activation of c-Jun N-terminal kinase modulated the DNA fragmentation independent of the loss of Deltapsi(m).     

Bcl-2 regulates amplification of caspase activation by cytochrome c

Caspases, a family of specific proteases, have central roles in apoptosis [1]. Caspase activation in response to diverse apoptotic stimuli involves the relocalisation of cytochrome c from mitochondria to the cytoplasm where it stimulates the proteolytic processing of caspase precursors. Cytochrome c release is controlled by members of the Bcl-2 family of apoptosis regulators [2] [3]. The anti-apoptotic members Bcl-2 and Bcl-xL may also control caspase activation independently of cytochrome c relocalisation or may inhibit a positive feedback mechanism [4] [5] [6] [7]. Here, we investigate the role of Bcl-2 family proteins in the regulation of caspase activation using a model cell-free system. We found that Bcl-2 and Bcl-xL set a threshold in the amount of cytochrome c required to activate caspases, even in soluble extracts lacking mitochondria. Addition of dATP (which stimulates the procaspase-processing factor Apaf-1 [8] [9]) overcame inhibition of caspase activation by Bcl-2, but did not prevent the control of cytochrome c release from mitochondria by Bcl-2. Cytochrome c release was accelerated by active caspase-3 and this positive feedback was negatively regulated by Bcl-2. These results provide evidence for a mechanism to amplify caspase activation that is suppressed at several distinct steps by Bcl-2, even after cytochrome c is released from mitochondria.     

Nitric oxide as a bioregulator of apoptosis

Nitric oxide (NO), synthesized from l-arginine by NO synthases, is a small, diffusible, highly reactive molecule with dichotomous regulatory roles under physiological and pathological conditions. NO can promote apoptosis (proapoptosis) in some cells, whereas it inhibits apoptosis (antiapoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as iron, thiols, proteins, and reactive oxygen species. Long-lasting production of NO acts as a proapoptotic modulator by activating caspase family proteases through the release of mitochondrial cytochrome c into the cytosol, upregulation of p53 expression, activation of JNK/SAPK, and altering the expression of apoptosis-associated proteins including Bcl-2 family proteins. However, low or physiological concentrations of NO prevent cells from apoptosis induced by trophic factor withdrawal, Fas, TNFalpha, and lipopolysaccharide. The antiapoptotic mechanism can be understood via expression of protective genes such as heat shock proteins, Bcl-2 as well as direct inhibition of the apoptotic caspase family proteases by S-nitrosylation of the cysteine thiol. Our current understanding of the mechanisms by which NO exerts both pro- and antiapoptotic actions is discussed in this review article.     

The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation

Many viruses have evolved strategies to counteract cellular immune responses, including apoptosis. Vaccinia virus, a member of the poxvirus family, encodes an antiapoptotic protein, F1L. F1L localizes to mitochondria and inhibits apoptosis by preventing the release of cytochrome c by an undetermined mechanism (S. T. Wasilenko, T. L. Stewart, A. F. Meyers, and M. Barry, Proc. Natl. Acad. Sci. USA 100:14345-14350, 2003; T. L. Stewart, S. T. Wasilenko, and M. Barry, J. Virol. 79:1084-1098, 2005). Here, we show that in the absence of an apoptotic stimulus, F1L associates with Bak, a proapoptotic member of the Bcl-2 family that plays a pivotal role in the release of cytochrome c. Cells infected with vaccinia virus were resistant to Bak oligomerization and the initial N-terminal exposure of Bak following the induction of apoptosis with staurosporine. A mutant vaccinia virus missing F1L was no longer able to inhibit apoptosis or Bak activation. In addition, the expression of F1L was essential to inhibit tBid-induced cytochrome c release in both wild-type murine embryonic fibroblasts (MEFs) and Bax-deficient MEFs, indicating that F1L could inhibit apoptosis in the presence and absence of Bax. tBid-induced Bak oligomerization and N-terminal exposure of Bak in Bax-deficient MEFs were inhibited during virus infection, as assessed by cross-linking and limited trypsin proteolysis. Infection with the F1L deletion virus no longer provided protection from tBid-induced Bak activation and apoptosis. Additionally, infection of Jurkat cells with the F1L deletion virus resulted in cellular apoptosis, as measured by loss of the inner mitochondrial membrane potential, caspase 3 activation, and cytochrome c release, indicating that the presence of F1L was pivotal for inhibiting vaccinia virus-induced apoptosis. Our data indicate that F1L expression during infection inhibits apoptosis and interferes with the activation of Bak.     

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.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.     

Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation

Serum contains a variety of biomolecules, which play an important role in cell proliferation and survival. We sought to identify the serum factor responsible for mitigating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and to investigate its molecular mechanism. TRAIL induced effective apoptosis without serum, whereas bovine serum decreased apoptosis by suppressing cytochrome c release and caspase activation. Indeed, albumin-bound lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) inhibited TRAIL-induced apoptosis by suppressing caspase activation and cytochrome c release. LPA increased phosphatidylinositol 3-kinase (PI3K)-dependent Akt activation, cellular FLICE-inhibitory protein (cFLIP) expression, and Bad phosphorylation, resulting in inhibition of caspase-8 activation and Bad translocation to mitochondria. The antiapoptotic effect of LPA was abrogated by PI3K inhibitor, transfection with dominant-negative Akt, and specific downregulation of cFLIP expression using siRNA and further increased by siRNA-mediated suppression of Bad expression. Moreover, sera from ovarian cancer patients showed more protective effect against TRAIL-induced apoptosis than those from healthy donors, and this protection was suppressed by PI3K inhibitor. Our results indicate that albumin-bound LPA and S1P prevent TRAIL-induced apoptosis by upregulation of cFLIP expression and in part by Bad phosphorylation, through the activation of PI3K/Akt pathway.     

N‐terminal cleavage of Bax by calpain generates a potent proapoptotic 18‐kDa fragment that promotes Bcl‐2‐independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase‐activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N‐terminus, generating a potent proapoptotic 18‐kDa fragment (Bax/p18). Both the calpain‐mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane‐enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase‐3, cleavage of poly(ADP‐ribose) polymerase, and fragmentation of DNA. Unlike the full‐length Bax, Bax/p18 did not interact with the antiapoptotic Bcl‐2 protein in the mitochondrial fraction of drug‐treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and caspase‐3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase‐3‐mediated apoptosis that was not blocked by overexpression of Bcl‐2 protein. Therefore, Bax/p18 has a cytochrome c–releasing activity that promotes cell death independent of Bcl‐2. Finally, Bcl‐2 overexpression inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution. J. Cell. Biochem. 80:53–72, 2000. © 2000 Wiley‐Liss, Inc.     

Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.     

IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition

The antiapoptotic properties of the inhibitor of apoptosis (IAP) family of proteins have been linked to caspase inhibition. We have previously described an alternative mechanism of XIAP inhibition of apoptosis that depends on the selective activation of JNK1. Here we report that two other members of the IAP family, NAIP and ML-IAP, both activate JNK1. Expression of catalytically inactive JNK1 blocks NAIP and ML-IAP protection against ICE- and TNF-alpha-induced apoptosis, indicating that JNK1 activation is necessary for the antiapoptotic effect of these proteins. The MAP3 kinase, TAK1, appears to be an essential component of this antiapoptotic pathway since IAP-mediated activation of JNK1, as well as protection against TNF-alpha- and ICE-induced apoptosis, is inhibited when catalytically inactive TAK1 is expressed. In addition, XIAP, NAIP, and JNK1 bind to TAK1. Importantly, expression of catalytically inactive TAK1 did not affect XIAP inhibition of caspase activity. These data suggest that XIAP's antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

B-Raf Inhibits Programmed Cell Death Downstream of Cytochrome c Release from Mitochondria by Activating the MEK/Erk Pathway

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.     

Diva/Boo is a negative regulator of cell death in human glioma cells

Diva is a novel proapoptotic member of the Bcl-2 protein family which binds apoptosis activating factor-1 (APAF-1). Diva is identical with Boo which was identified as a novel antiapoptotic Bcl-2 family protein. Here, we report that Diva promotes the cell cycle exit of human glioma cells in response to serum deprivation and inhibits apoptosis of these cells induced by CD95 ligand or chemotherapeutic drugs. In glioma cells, Diva interferes with apoptotic signaling downstream of cytochrome c release, but upstream of caspase activation, consistent with an inhibitory effect on the mitochondrial amplification step involving the apoptosome and APAF-1.     

TUCAN, an antiapoptotic caspase-associated recruitment domain family protein overexpressed in cancer.

Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulated CARD-containing antagonist of caspase nine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c

We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with alpha-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-x(L) and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.