Apoptosis of Mo7e leukemia cells is associated with the cleavage of Bcl-2 into a shortened fragment that is not functional for heterodimerization with Bcl-2 and Bax

Bcl-2 is an integral intracellular membrane protein that can protect cells from apoptosis induced by multiple insults in a variety of cell types. During apoptosis, Bcl-2 was cleaved into a shortened fragment (Bcl-2/Delta34) by a caspase-3-like protease in human Mo7e megakaryocytic leukemia cells deprived of exogenous rhGM-CSF. Results from cell fractionation and immunoblot analyses indicated that both Bcl-2 and Bcl-2/Delta34 were located exclusively on the mitochondria of Mo7e cells. Treatment of isolated mitochondria with recombinant caspase-3 induced the same cleavage of Bcl-2 in vitro and caused the release of cytochrome c from the mitochondria into the supernatant. The antiapoptotic effect of Bcl-2/Delta34 was investigated using an in vitro protein translation approach. Both Bcl-2/Delta34 and Bax proteins generated in wheat germ extract were readily relocated to the mitochondria isolated from control Mo7e cells. Insertion of Bax, but not Bcl-2/Delta34, into mitochondria triggered a rapid release of cytochrome c from the mitochondria. Coimmunoprecipitation studies showed that, unlike Bcl-2, the cleaved Bcl-2 fragment was no longer functional for dimerization with either Bcl-2 or Bax. Taken together, these findings showed that the integrity of Bcl-2 is necessary for its function of heterodimerization with Bax, which appears to be one of the mechanisms of antiapoptotic effect of Bcl-2.     

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.     

Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制

Bcl-2家族蛋白在调控线粒体功能和细胞色素C释放中起重要作用。最近发现Bcl-2分子通过与其他促凋亡分子相互作用调控线粒体外膜通透性,其具体分子机制尚不完全清楚。本课题组采用化学生物学方法,在研究Bax/Bak非依赖的细胞凋亡途径中,发现了一些小分子化合物能够诱导Bim表达量急剧升高,Bim能转位到线粒体上,与Bcl-2相互作用增强,并直接促进Bcl-2构象变化。有意义的是,Bim可以诱导Bcl-2功能发生转换并能够形成大的复合体通道来介导细胞色素C释放。研究结果提示Bcl-2分子可变成促凋亡分子,参与Bax/Bak非依赖的细胞色素C释放和细胞凋亡。     

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.     

The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release

The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp-2 cells with DNAs encoding either the N-terminal (p34) or the C-terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA-GFP and p34-GFP, but not GFP-VacA or GFP-p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34-GFP or VacA-GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP-ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co-transfection of DNA encoding Bcl-2, known to block mitochondria-dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.     

Critical upstream signals of cytochrome C release induced by a novel Bcl-2 inhibitor

Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca(2+). This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca(2+) spike, change in mitochondrial membrane potential (Delta psi(m)) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resulted in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca(2+) uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced Delta psi(m) transition and Bax translocation. This suggests that ROS generation is an event that occurs after the Delta psi(m) transition and Bax translocation. Together these data demonstrate that the Ca(2+) spike, mitochondrial Bcl-2 presensitization, and subsequent Delta psi(m) transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis.     

Failure of Bcl-2 to block cytochrome c redistribution during TRAIL-induced apoptosis

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis and NF-kappaB activation. Here we show that recombinant hu-TRAIL initiates the activation of multiple caspases, the loss of mitochondrial transmembrane potential, the cleavage of BID and the redistribution of mitochondrial cytochrome c. However, whereas Bcl-2 efficiently blocked UV radiation-induced cytochrome c release and consequent apoptosis of CEM cells, it failed to do either in the context of TRAIL treatment. Thus, TRAIL engages a death pathway that is at least partially routed via the mitochondria, but in contrast with other stimuli that engage this pathway, TRAIL-induced cytochrome c release is not regulated by Bcl-2.     

Prosurvival Bcl-2 proteins stabilize pancreatic mitochondria and protect against necrosis in experimental pancreatitis

Acinar cells in pancreatitis die through apoptosis and necrosis, the roles of which are different. The severity of experimental pancreatitis correlates directly with the extent of necrosis and inversely, with apoptosis. Apoptosis is mediated by the release of cytochrome c into the cytosol followed by caspase activation, whereas necrosis is associated with the mitochondrial membrane potential (ΔΨm) loss leading to ATP depletion. Here, we investigate the role of Bcl-2 proteins in apoptosis and necrosis in pancreatitis. We found up-regulation of prosurvival Bcl-2 proteins in pancreas in various experimental models of acute pancreatitis, most pronounced for Bcl-xL. This up-regulation translated into increased levels of Bcl-xL and Bcl-2 in pancreatic mitochondria. Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss and cytochrome c release in isolated mitochondria. Corroborating the results on mitochondria, Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss, ATP depletion and necrosis in pancreatic acinar cells, both untreated and hyperstimulated with CCK-8 (in vitro pancreatitis model). Together Bcl-xL/Bcl-2 inhibitors and CCK induced more necrosis than either treatment alone. Bcl-xL/Bcl-2 inhibitors also stimulated cytochrome c release in acinar cells leading to caspase-3 activation and apoptosis. However, different from their effect on pronecrotic signals, the stimulation by Bcl-xL/Bcl-2 inhibitors of apoptotic responses was less in CCK-treated than control cells. Therefore, Bcl-xL/Bcl-2 inhibitors potentiated CCK-induced necrosis but not apoptosis. Correspondingly, transfection with Bcl-xL siRNA stimulated necrosis but not apoptosis in the in vitro pancreatitis model. Further, in animal models of pancreatitis Bcl-xL up-regulation inversely correlated with necrosis, but not apoptosis. Results indicate that Bcl-xL and Bcl-2 protect acinar cells from necrosis in pancreatitis by stabilizing mitochondria against death signals. We conclude that Bcl-xL/Bcl-2 inhibition would aggravate acute pancreatitis, whereas Bcl-xL/Bcl-2 up-regulation presents a strategy to prevent or attenuate necrosis in pancreatitis.     

In vitro analysis of Bcl-2 proteins in mitochondria and endoplasmic reticulum: similarities in anti-apoptotic functions and differences in regulation

Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.     

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.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Bcl-2 phosphorylation and proteasome-dependent degradation induced by paclitaxel treatment: consequences on sensitivity of isolated mitochondria to Bid

Several studies have suggested that Bcl-2 phosphorylation, which occurs during mitotic arrest induced by paclitaxel, inhibits its antiapoptotic function. In the present study, we demonstrated that the level of phosphorylated Bcl-2 was threefold higher in mitochondria than in the nuclear membrane or endoplasmic reticulum. Our results show, in isolated mitochondria, that phosphorylation of Bcl-2 in mitosis does not modify either its integration into the mitochondrial membrane or the ability to release cytochrome c in response to Bid, a cytochrome c releasing agent. In HeLa cells, in which paclitaxel induces apoptosis, the nonphosphorylated form of Bcl-2 is degraded by a proteasome-dependent degradation pathway, whereas the phosphorylated forms of mitochondrial Bcl-2 appear to be resistant to proteasome-induced degradation. We found that low concentrations of recombinant Bid triggered a greater release of cytochrome c from mitochondria isolated from paclitaxel-treated HeLa cells than from mitochondria isolated from control HeLa cells. Taken together, these results show that Bcl-2 phosphorylation does not inhibit its function. On the contrary, Bcl-2 phosphorylation indirectly regulated its antiapoptotic action via protection against degradation. Indeed, in response to paclitaxel treatment, the level of Bcl-2 expression in mitochondria rather than its phosphorylation state could regulate the sensitivity of mitochondria to cytochrome c releasing agents in vitro.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

Conjugated linoleic acid induces apoptosis of murine mammary tumor cells via Bcl-2 loss

Conjugated linoleic acid (CLA) is a powerful anticancer agent in a number of tumor model systems; however, its precise mechanism of action remains elusive. Here, we report that t10,c12 CLA, a component of synthetic CLA supplements, induced apoptosis and G1 arrest of p53 mutant TM4t murine mammary tumor cells. Furthermore, t10,c12-CLA induced a time- and concentration-dependent cleavage of caspases-3 and -9, and release of cytochrome c from mitochondria to cytosol. Levels of Bcl-2 protein were decreased both in total cellular lysates and in mitochondria after t10,c12-CLA treatment; however, there was no significant change in Bax or Bak. Overexpression of Bcl-2 attenuated apoptosis in response to t10,c12-CLA treatment. These results demonstrate that t10,c12-CLA triggers apoptosis of p53 mutant murine mammary tumor cells through the mitochondrial pathway by targeting Bcl-2.     

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.     

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.     

Natural diterpenoid compound elevates expression of Bim protein, which interacts with antiapoptotic protein Bcl-2, converting it to proapoptotic Bax-like molecule

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.     

Cell death regulation by the Bcl-2 protein family in the mitochondria

An increase in the permeability of the outer mitochondrial membrane is central to apoptotic cell death, since it leads to the release of several apoptogenic factors, such as cytochrome c and Smac/Diablo, into the cytoplasm that activate downstream death programs. During apoptosis, the mitochondria also release AIF and endonuclease G, both of which are translocated to the nucleus and are implicated in apoptotic nuclear changes that occur in a caspase-independent manner. Mitochondrial membrane permeability is directly controlled by the major apoptosis regulator, i.e., the Bcl-2 family of proteins, mainly through regulation of the formation of apoptotic protein-conducting pores in the outer mitochondrial membrane, although the precise molecular mechanisms are still not completely understood. Here, I focus on the mechanisms by which Bcl-2 family members control the permeability of mitochondrial membrane during apoptosis.