Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death.

The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.

Cell cycle and cell death in disease: past, present and future.

Cell division and cell death are the two predominant physiological processes that regulate tissue homeostasis in the adult organism. The importance of dysregulation of these processes in the pathogenesis of major diseases, such as cancer, myocardial infarction, stroke, atherosclerosis, infection, inflammation and neurodegenerative disorders, is becoming increasingly evident. Hence, attempts to find modulators of the cell cycle and cell death programmes are being made with the hope of creating novel therapeutic approaches to the treatment of these diseases. It is clear that improved understanding of how cells balance life-and-death processes is crucial for this development. In view of this, a Nobel Symposium entitled 'The Cell Cycle and Apoptosis in Disease' was organized in conjunction with the celebration of the 200-year anniversary of the Karolinska Institute in 2010. The symposium focused on the importance of dysregulation of cell cycle/cell death programmes in the pathogenesis of human disease. Three comprehensive reviews based on presentations at this symposium are presented in this issue of the Journal of Internal Medicine. They include a discussion of autophagy in anticancer therapy, the description of a role for type 2 transglutaminase in Huntington's disease and the proposal that 'redox-sensing' mechanisms might act as an orthogonal control in cell cycle and apoptosis signalling.

Mitochondrial targeting of tBid/Bax: a role for the TOM complex?

The release of pro-apoptotic proteins from the mitochondria is a key event in cell death signaling that is regulated by Bcl-2 family proteins. For example, cleavage of the BH3-only protein, Bid, by multiple proteases leads to the formation of truncated Bid that, in turn, promotes the insertion/oligomerization of Bax into the mitochondrial outer membrane, resulting in pore formation and the release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol is targeted to the mitochondria by a yet unknown mechanism. Several proteins of the outer mitochondrial membrane have been proposed to act as receptors for Bax, among them the voltage-dependent anion channel, VDAC, and the mitochondrial protein translocase of the outer membrane, the TOM complex. Alternatively, the unique mitochondrial phospholipid, cardiolipin, has been ascribed a similar function. Here, we review recent work on the mechanisms of activation and the targeting of Bax to the mitochondria and discuss the advantages and limitations of the methods used to study this process.

An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death.

Apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity anchored to the mitochondrial inner membrane, is known to be involved in complex I maintenance. During apoptosis, AIF can be released from mitochondria and translocate to the nucleus, where it participates in chromatin condensation and large-scale DNA fragmentation. The mechanism of AIF release is not fully understood. Here, we show that a prolonged ( approximately 10 min) increase in intracellular Ca(2+) level is a prerequisite step for AIF processing and release during cell death. In contrast, a transient ATP-induced Ca(2+) increase, followed by rapid normalization of the Ca(2+) level, was not sufficient to trigger the proteolysis of AIF. Hence, import of extracellular Ca(2+) into staurosporine-treated cells caused the activation of a calpain, located in the intermembrane space of mitochondria. The activated calpain, in turn, cleaved membrane-bound AIF, and the soluble fragment was released from the mitochondria upon outer membrane permeabilization through Bax/Bak-mediated pores or by the induction of Ca(2+)-dependent mitochondrial permeability transition. Inhibition of calpain, or chelation of Ca(2+), but not the suppression of caspase activity, prevented processing and release of AIF. Combined, these results provide novel insights into the mechanism of AIF release during cell death.

DNA damage induces two distinct modes of cell death in ovarian carcinomas.

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cell's ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3sigma deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53- and Chk2-related proteins.

Functional connection between p53 and caspase-2 is essential for apoptosis induced by DNA damage.

Recent findings have established caspase-2 as an important apical regulator in apoptotic pathways leading from DNA damage to release of mitochondrial cytochrome c and subsequent activation of effector caspases. Yet, the molecular map connecting the embarking stimuli of genotoxic stress with caspase-2 activation remains to be elucidated. Here, we address the question of potential caspase-2 regulators by examining 5-fluorouracil (5-FU)-induced apoptosis in wild-type and p53-deficient human colon carcinoma cells. Apoptosis was observed only in p53(+/+) cells and was preceded by caspase-2 activation. Hence, although no direct interaction between p53 and caspase-2 was observed in the cell system used, our data clearly demonstrate that a functional connection between these two proteins is essential for initiation of the 5-FU-induced apoptotic process. Proposed mediators of caspase-2 activation include PIDDosome complex proteins PIDD and RAIDD. Surprisingly, the presence of a complex encompassing at least RAIDD, PIDD and caspase-2 was verified in both p53(+/+) and p53(-/-) cells, also in the absence of 5-FU treatment. Thus, our results confirm the participation of PIDD and RAIDD in PIDDosome complex formation but question their role as sole mediators of caspase-2 activation. This assumption was further supported by siRNA transfections targeting PIDD or RAIDD. In conclusion, our findings support the hypothesis of p53 as an upstream regulator of caspase activity and provide data concerning caspase-2 processing mechanisms. As suppression of caspase-2 expression in 5-FU-treated cells also affects the level of the p53 protein, possibilities of a reciprocal interaction between these proteins are discussed.

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease.

Apoptosis is a highly regulated process of cell deletion and plays a fundamental role in the maintenance of tissue homeostasis in the adult organism. Numerous studies in recent years have revealed that apoptosis is a constitutive suicide programme expressed in most, if not all cells, and can be triggered by a variety of extrinsic and intrinsic signals. Many human diseases can be attributed directly or indirectly to a derangement of apoptosis, resulting in either cell accumulation, in which cell eradication or cell turnover is impaired, or cell loss, in which the apoptotic programme is inadvertently triggered. In addition, defective macrophage engulfment and degradation of cell corpses may also contribute to a dysregulation of tissue homeostasis. An increased understanding of the signalling pathways that govern the execution of apoptosis and the subsequent clearance of dying cells may thus yield novel targets for therapeutic intervention in a wide range of human maladies.

Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli.

Apoptotic cell death is an essential process in the development of the central nervous system and in the pathogenesis of its degenerative diseases. Efflux of K(+) and Cl(-) ions leads to the shrinkage of the apoptotic cell and facilitates the activation of caspases. Here, we present electrophysiological and immunocytochemical evidences for the activation of a voltage-dependent anion channel (VDAC) in the plasma membrane of neurons undergoing apoptosis. Anti-VDAC antibodies blocked the channel and inhibited the apoptotic process. In nonapoptotic cells, plasma membrane VDAC1 protein can function as a NADH (-ferricyanide) reductase. Opening of VDAC channels in apoptotic cells was associated with an increase in this activity, which was partly blocked by VDAC antibodies. Hence, it appears that there might be a dual role for this protein in the plasma membrane: (1) maintenance of redox homeostasis in normal cells and (2) promotion of anion efflux in apoptotic cells.

Neural stem cells and cell death.

Neural stem cells (NSC) undergo apoptotic cell death as an essential component of neural development. Here, we present the results of our studies on the mechanisms by which NSC undergo cell death in response to neurotoxic insults. As experimental models we used primary culture of adult NSC from the subventricular zone of the rat brain, and the neural stem cell line C17.2 initially derived from developing mouse cerebellum. NSC undergo apoptosis in response to staurosporine (0.25 microM) as well as agents inducing oxidative stress such as 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Exposed cells demonstrate an apoptotic morphology, positive TUNEL staining and phosphatidyl serine exposure as labeled with Annexin V. Using an antibody specific for cytochrome c, we found that cells exposed to staurosporine or DMNQ exhibited diffuse fluorescence throughout the cytosol, implying a release of cytochrome c from the mitochondria. In addition to positive immunoreactivity against the active fragment (p17) of caspase-3, the administration of the pan-caspase inhibitor, zVAD-fmk (40 microM), prevents apoptosis. Both NSC and C17.2 express the Fas receptor, and procaspase-8, but exposure to agonistic Fas mAb (250 ng/ml) fails to induce apoptosis. Pretreatment with cycloheximide or actinomycin D does not influence the cell response to Fas mAb, suggesting that the endogenous inhibitor of caspase-8 FLICE-inhibitory protein (FLIP) is not responsible for the inhibition of the Fas pathway. Thus, it appears that the Fas dependent cell death pathway is not operative in these cells, while the mitochondrial pathway is active and caspase-3 serves as an executioner caspase in the apoptotic machinery. It is known that Fas not only induces apoptosis, but can also deliver growth stimulatory signals through activation of the extracellular-signal regulated kinase (ERK) pathway. The Fas-induced ERK phosphorylation that we detect in C17.2 cells suggests that in NSC Fas may function as a mediator of growth rather than death.

Familial hemophagocytic lymphohistiocytosis: too little cell death can seriously damage your health.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare and fatal disease of early childhood characterized by a non-malignant accumulation of activated T lymphocytes and histiocytes in the reticuloendothelial system. Moreover, immune system derangement, with prominent hypercytokinemia and low or absent cytotoxic T and natural killer (NK) cell activity, is a consistent feature of this autosomal recessive disorder. Recent work has demonstrated that the degree of spontaneous caspase activation in FHL lymphocytes is attenuated in vitro whereas Fas-mediated caspase activation and apoptosis induction remains unmitigated, and FHL can thus be distinguished from the related chronic disorder of immune regulation termed autoimmune lymphoproliferative syndrome or ALPS. However, subsequent studies have identified mutations in the gene encoding perforin, a cytotoxic granule constituent required for apoptotic killing of target cells, in a number of FHL patients. Hence, the underlying defect in FHL may be conceived of as a lack of apoptosis triggering within the immune system, rather than apoptosis resistance per se. These observations represent an important step in our understanding of the pathogenesis of FHL and also serve to emphasize the pivotal role of cellular (perforin-based) cytotoxicity in the regulation of immune homeostasis.

Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli.

Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c-mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.