The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons

DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.     

Ultraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa

To identify the mechanisms of ultraviolet radiation (UVR)-induced cell death, for which the tumor suppressor p53 is essential, we have analyzed mouse embryonic fibroblasts (MEFs) and keratinocytes in mouse skin that have specific apoptotic pathways blocked genetically. Blocking the death receptor pathway provided no protection to MEFs, whereas UVR-induced apoptosis was potently inhibited by Bcl-2 overexpression, implicating the mitochondrial pathway. Indeed, Bcl-2 overexpression boosted cell survival more than p53 loss, revealing a p53-independent pathway controlled by the Bcl-2 family. Analysis of primary MEFs lacking individual members of its BH3-only subfamily identified major initiating roles for the p53 targets Noxa and Puma. In the transformed derivatives, where Puma, unexpectedly, was not induced by UVR, Noxa had the dominant role and Bim a minor role. Furthermore, loss of Noxa suppressed the formation of apoptotic keratinocytes in the skin of UV-irradiated mice. Collectively, these results demonstrate that UVR activates the Bcl-2-regulated apoptotic pathway predominantly through activation of Noxa and, depending on cellular context, Puma.     

Resveratrol induces apoptosis via a Bak-mediated intrinsic pathway in human lung adenocarcinoma cells

Our recent study have shown that resveratrol (RV), a natural plant polyphenol found in red grape skins as well as other food product, induced apoptosis via the downstream factors, caspase-independent AIF and to lesser extent caspase-9, of intrinsic apoptosis pathway in human lung adenocarcinoma (ASTC-a-1) cells. This report is designed to explore the roles of the upstream mediators of the intrinsic pathway, such as Bak/Bax, Bim, Puma and Noxa, during RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1 and A549) cell lines. RV treatment remarkably induced the activation of Bak but not Bax, and silencing Bak but not Bax by shRNA almost completely prevented RV-induced cell death, mitochondrial dysfunction and also largely prevented RV-induced AIF release, demonstrating the preferential engagement of Bak but not Bax during RV-induced apoptosis. In addition, although RV treatment induced a significant degradation of Mcl-1, knockdown of Mcl-1 by shRNA only modestly increased RV-induced Bak activation. Interestingly, silencing Bim but not Puma and Noxa remarkably attenuated RV-induced cell death, loss of mitochondrial membrane potential, and Bak activation, suggesting the important roles of Bim. Collectively, our findings for the first time demonstrate that RV induces apoptosis dominantly via a Bak- but not Bax-mediated AIF-dependent mitochondrial apoptotic signaling pathway in which Bim but not Puma and Noxa may supply the force to trigger Bak activation and subsequent apoptosis in both ASTC-a-1 and A549 cell lines.     

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.     

p53 signalling controls cell cycle arrest and caspase‐independent apoptosis in macrophages infected with pathogenic Leptospira species

Pathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspase‐independent, mitochondrion‐related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoG‐based macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNAdamage indicators) in wild‐type p53‐containing mouse macrophages and p53‐deficient human macrophages. Most leptospire‐infected cells stayed at the G₁ phase, whereas depletion or inhibition of p53 caused a decrease of the G₁‐phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53‐dependent p21^Cip1/WAF1 and pro‐apoptotic Bcl‐2 family proteins (Bax, Noxa and Puma), release of AIF and EndoG from mitochondria, and membrane translocation of Fas occurred during leptospire‐induced macrophage apoptosis. Thus, our study demonstrated that ROS production and DNA damage‐dependent p53‐Bax/Noxa/Puma‐AIF/EndoG signalling mediates the leptospire‐induced cell cycle arrest and caspase‐independent apoptosis of macrophages.     

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.     

JNK- and Akt-mediated Puma expression in the apoptosis of cisplatin-resistant ovarian cancer cells

BH3 (Bcl-2 homology domain 3)-only proteins have an important role in the cisplatin resistance of cells. However, the effect of BH3-only proteins on cisplatin-resistant ovarian cancer cells has not been thoroughly elucidated. Our results from the present study indicate that Puma plays a critical role in the apoptosis of chemo-resistant ovarian cancer cells treated with BetA (betulinic acid). The reduction of Puma expression inhibits Bax activation and apoptosis. However, p53 gene silencing has little effect on Puma activation. Further experiments demonstrated that Akt-mediated FoxO3a (forkhead box O3a) nuclear translocation and the JNK (c-Jun N-terminal kinase)/c-Jun pathway only partially trigger Puma induction and apoptosis, whereas dominant-negative c-Jun expression with FoxO3a reduction completely inhibits Puma expression and cell death. Furthermore, our results suggest that JNK regulates the Akt/FoxO3a signalling pathway. Therefore the dual effect of JNK can efficiently trigger Puma activation and apoptosis in chemoresistant cells. Taken together, our results demonstrate the role of Puma in BetA-induced apoptosis and the molecular mechanisms of Puma expression regulated by BetA during ovarian cancer cell apoptosis. Our findings suggest that the JNK-potentiated Akt/FoxO3a and JNK-mediated c-Jun pathways co-operatively trigger Puma expression, which determines the threshold for overcoming chemoresistance in ovarian cancer cells.     

An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis

The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.     

Decitabine-induced apoptosis is derived by Puma and Noxa induction in chronic myeloid leukemia cell line as well as in PBL and is potentiated by SAHA

Restoration of cellular apoptotic pathways plays a crucial role in cancer therapy strategies. In a broad spectrum of anticancer drugs, epigenetic effectors are in the center of interest mostly because of potential reversibility of their action. Methylation status of the cells is influenced by methyltransferase inhibitor 2-deoxy-5'-azacytidine (decitabine, DAC), but higher concentrations of this agent cause a DNA-damage. In our study, tumor supressor p53-apoptotic pathway was activated in decitabine-induced cell death. Expression of p53-inducible BH3-only apoptotic proteins Puma and Noxa was elevated and large activation of executive caspases was observed. The extent of acetylation in the cell is affected by histonedeacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Combination of SAHA with decitabine brought synergistic effect on apoptosis triggering in CML-T1 cell line, but apoptosis as well as necrosis occurred also in normal peripheral blood lymphocytes. Therefore, promising potential of such combined therapy calls for more detailed investigation of unwanted effects in normal cells.     

Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.