Minimal BH3 peptides promote cell death by antagonizing anti-apoptotic proteins

The pro-apoptotic "BH3 domain-only" proteins of the Bcl-2 family (e.g. Bid and Bad) transduce multiple death signals to the mitochondrion. They interact with the anti-apoptotic Bcl-2 family members and induce apoptosis by a mechanism that requires the presence of at least one of the multidomain pro-apoptotic proteins Bax or Bak. Although the BH3 domain of Bid can promote the pro-apoptotic assembly and function of Bax/Bak by itself, other BH3 domains do not function as such. The latter point raises the question of whether, and how, these BH3 domains induce apoptosis. We show here that a peptide comprising the minimal BH3 domain from Bax induces apoptosis but is unable to stimulate the apoptotic activity of microinjected recombinant Bax. This relies on the inability of the peptide to directly induce Bax translocation to mitochondria or a change in its conformation. This peptide nevertheless interferes with Bax/Bcl-xL interactions in vitro and stimulates the apoptotic activity of Bax when combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad stimulates Bax activity only in the presence of Bcl-xL. Thus, BH3 domains do not necessarily activate multidomain pro-apoptotic proteins directly but promote apoptosis by releasing active multidomain pro-apoptotic proteins from their anti-apoptotic counterparts.     

Evidence for a role for notch signaling in the cytokine-dependent survival of activated T cells

Peripheral T cell homeostasis results from a balance between factors promoting survival and those that trigger deletion of Ag-reactive cells. The cytokine IL-2 promotes T cell survival whereas reactive oxygen species (ROS) sensitize T cells to apoptosis. Two pathways of activated T cell apoptosis-one triggered by Fas ligand and the other by cytokine deprivation-depend on ROS, with the latter also regulated by members of the Bcl-2 family. Notch family proteins regulate several cell-fate decisions in metazoans. Ectopic expression of the Notch1 intracellular domain (NICD) in T cells inhibits Fas-induced apoptosis. The underlying mechanism is not known and the role, if any, of Notch in regulating apoptosis triggered by cytokine deprivation or neglect has not been examined. In this study, we use a Notch1/Fc chimera; a blocking Ab to Notch1 and chemical inhibitors of gamma-secretase to investigate the role of Notch signaling in activated T cells of murine origin. We show that perturbing Notch signaling in activated CD4+/CD8+ T cells maintained in IL-2 results in the accumulation of ROS, reduced Akt/protein kinase B activity, and expression of the antiapoptotic protein Bcl-xL, culminating in apoptosis. A broad-spectrum redox scavenger inhibits apoptosis but T cells expressing mutant Fas ligand are sensitive to apoptosis. Activated T cells isolated on the basis of Notch expression (Notch+) are enriched for Bcl-xL expression and demonstrate reduced susceptibility to apoptosis triggered by neglect or oxidative stress. Furthermore, enforced expression of NICD protects activated T cells from apoptosis triggered by cytokine deprivation. Taken together, these data implicate Notch1 signaling in the cytokine-dependent survival of activated T cells.     

c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.     

Anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit

The interleukin-2 receptor (IL-2R) is composed of one affinity-modulating subunit (IL-2Ralpha) and two essential signaling subunits (IL-2Rbeta and gammac). Although most known signaling events are mediated through tyrosine residues located within IL-2Rbeta, no functions have yet been ascribed to gammac tyrosine residues. In this study, we describe a role for gammac tyrosines in anti-apoptotic signal transduction. We have shown previously that a tyrosine-deficient IL-2Rbeta chain paired with wild type gammac stimulated enhancement of bcl-2 mRNA in IL-2-dependent T cells, but it was not determined which region of the IL-2R or which pathway was activated to direct this signaling response. Here we show that up-regulation of Bcl-2 by an IL-2R lacking IL-2Rbeta tyrosine residues leads to increased cell survival after cytokine deprivation; strikingly, this survival signal does not occur in the absence of gammac tyrosine residues. These gammac-dependent signals are revealed only in the absence of IL-2Rbeta tyrosines, indicating that the IL-2R engages at least two distinct signaling pathways to regulate apoptosis and Bcl-2 expression. Mechanistically, the gammac-dependent signal requires activation of Janus kinases 1 and 3 and is sensitive to wortmannin, implicating phosphatidylinositol 3-kinase. Consistent with involvement of phosphatidylinositol 3-kinase, Akt can be activated via tyrosine residues on gammac. Thus, gammac mediates an anti-apoptotic signaling pathway through Akt which cooperates with signals from its partner chain, IL-2Rbeta.     

Glucose-starved Cells Do Not Engage in Prosurvival Autophagy

In response to nutrient shortage or organelle damage, cells undergo macroautophagy. Starvation of glucose, an essential nutrient, is thought to promote autophagy in mammalian cells. We thus aimed to determine the role of autophagy in cell death induced by glucose deprivation. Glucose withdrawal induces cell death that can occur by apoptosis (in Bax, Bak-deficient mouse embryonic fibroblasts or HeLa cells) or by necrosis (in Rh4 rhabdomyosarcoma cells). Inhibition of autophagy by chemical or genetic means by using 3-methyladenine, chloroquine, a dominant negative form of ATG4B or silencing Beclin-1, Atg7, or p62 indicated that macroautophagy does not protect cells undergoing necrosis or apoptosis upon glucose deprivation. Moreover, glucose deprivation did not induce autophagic flux in any of the four cell lines analyzed, even though mTOR was inhibited. Indeed, glucose deprivation inhibited basal autophagic flux. In contrast, the glycolytic inhibitor 2-deoxyglucose induced prosurvival autophagy. Further analyses indicated that in the absence of glucose, autophagic flux induced by other stimuli is inhibited. These data suggest that the role of autophagy in response to nutrient starvation should be reconsidered.     

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.     

Bcl-xL blocks transforming growth factor-beta 1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing Apaf-1-dependent caspase activation in prostate epithelial cells

The mechanism by which transforming growth factor-beta1 (TGF-beta1) induces apoptosis of prostate epithelial cells was studied in the NRP-154 rat prostate epithelial cell line. TGF-beta 1 down-regulates expression of Bcl-xL and poly(ADP-ribosyl)polymerase (PARP), promotes cytochrome c release, up-regulates expression of latent caspase-3, and activates caspases 3 and 9. We tested the role of Bcl-xL in this cascade by stably overexpressing Bcl-xL to prevent loss by TGF-beta 1. Clones overexpressing Bcl-xL are resistant to TGF-beta 1 with respect to induction of apoptosis, cytochrome c release, activation of caspases 9 and 3, and cleavage of PARP; yet they remain sensitive to TGF-beta 1 by cell cycle arrest, induction of both fibronectin and latent caspase-3 expression, and loss of PARP expression. We show that Bcl-xL associates with Apaf-1 in NRP-154 cells; but this association does not inhibit the activation of caspases 9 and 3 by cytochrome c. Together, our data suggest that TGF-beta1 induces apoptosis through loss of Bcl-xL, leading to cytochrome c release and the subsequent activation of caspases 9 and 3. Moreover, our data demonstrate that the antiapoptotic effect of Bcl-xL occurs by inhibition of mitochondrial cytochrome c release and not through antagonizing Apaf-1-dependent processing of caspases 9 and 3.     

The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.     

CD 95-independent mechanisms of IL-2 deprivation-induced apoptosis in activated human lymphocytes

Growth factor deprivation-induced apoptosis plays an important role in several cellular systems. However, knowledge of the molecular mechanisms involved are restricted to a few murine models or tumor cell lines. Therefore, we aimed studying signaling pathways leading to apoptosis in activated human peripheral T cells after IL-2 withdrawal. Lymphoblasts from patients with CD 95 (Fas/APO-1)-deficiency revealed that functional CD95 was not required to induce apoptosis after IL-2 withdrawal. Moreover, apoptosis induction in response to various cytotoxic stimuli was found to be mediated in the absence of functional CD95 but was affirmatorily influenced by IL-2 signaling. Immunoblots showed no downregulation of Bcl-2 or Bcl-xL and no upregulation of Bax, whereas decreased mitochondrial membrane potential was readily measurable 24 h after cytokine deprivation. Tetrapeptide inhibitors showed limited efficacy in preventing apoptosis whereas the caspase inhibitor zVAD-FMK potently blocked induction of apoptosis. Cleavage of different fluorogenic substrates revealed multiple caspase enzyme activities in lymphoblasts, which were not negatively affected by the fas mutation. Starting at 8 h after IL-2 withdrawal, upregulation of active caspase-3 but not of caspase-8 could be detected. Taken together, our data argue for molecular mechanisms of cytokine deprivation-induced apoptosis in activated human lymphocytes independent of CD95.     

Role of phospholipase D2 in anti-apoptotic signaling through increased expressions of Bcl-2 and Bcl-xL

We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells. The transfected cells showed markedly the increases in Bcl-2 and Bcl-xL protein levels, and became resistant to Fas-induced apoptosis, similar to FasR. Treatment of wild-type A20 cells with phosphatidic acid (PA), the metabolic end product of PLD2 derived from phosphatidylcholin, markedly increased levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Moreover, PA-induced expressions of Bcl-2 and Bcl-xL were enhanced by propranolol, an inhibitor of PA phospholydrolase (PAP), whereas completely blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), suggesting that PLA(2) metabolite of PA is responsible for the increases in Bcl-2 and Bcl-xL protein levels. We further confirmed the involvement of arachidonic acid (AA) in PA-induced survival signals by showing that 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), PA without AA, was unable to increase Bcl-2 and Bcl-xL proteins. Moreover, PA notably increased cyclooxygenase (COX)-2 protein expression, and PA-induced expression of both Bcl-2 and Bcl-xL was inhibited by NS-398, a specific inhibitor of COX-2. Taken together, these findings demonstrate that PA generated by PLD2 plays an important role in cell survival during Fas-mediated apoptosis through the increased Bcl-2 and Bcl-xL protein levels which resulted from PLA(2) and AA-COX2 pathway.     

Activation of the phosphatidylinositol 3-kinase/Akt pathway protects against interleukin-3 starvation but not DNA damage-induced apoptosis

Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against gamma-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL.

The Bcl-2 related protein Bad is a promoter of apoptosis and has been shown to dimerize with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of Bad in murine FL5.12 cells demonstrated that the protein not only could abrogate the protective capacity of coexpressed Bcl-XL but could accelerate the apoptotic response to a death signal when it was expressed in the absence of exogenous Bcl-XL. Using deletion analysis, we have identified the minimal domain in the murine Bad protein that can dimerize with Bcl-xL. A 26-amino-acid peptide within this domain, which showed significant homology to the alpha-helical BH3 domains of related apoptotic proteins like Bak and Bax, was found to be necessary and sufficient to bind Bcl-xL. To determine the role of dimerization in regulating the death-promoting activity of Bad and the death-inhibiting activity of Bcl-xL, mutations within the hydrophobic BH3-binding pocket in Bcl-xL that eliminated the ability of Bcl-xL to form a heterodimer with Bad were tested for the ability to promote cell survival in the presence of Bad. Several of these mutants retained the ability to impart protection against cell death regardless of the level of coexpressed Bad protein. These results suggest that BH3-containing proteins like Bad promote cell death by binding to antiapoptotic members of the Bcl-2 family and thus inhibiting their survival promoting functions.     

p53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis.     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.