Death by design: apoptosis, necrosis and autophagy

Apoptosis is the principal mechanism by which cells are physiologically eliminated in metazoan organisms. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response. Autophagy has also been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy in times of stress. Recent advances have helped to define the function of and mechanism for programmed necrosis and the role of autophagy in cell survival and suicide.     

Cell death pathology: the war against cancer

Programmed cell death was a fundamental discovery, awarded with the Nobel price in 2002 to Sulston, Brenner and Horvitz [1]. Since then it has been clear that alteration of apoptotic pathways is a common feature of tumors, enabling cancer cells to survive chemotherapeutic interventions. Thus, apoptosis is an attractive target in cancer therapy, with the aim to revert the cancer-related alterations of the cell death machinery. Here, we overview the fundamental apoptotic pathways and summarize the attempts to target apoptosis to restore cell death in cancer cells with a special focus on the p53-family and autophagy.     

Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells

Apoptotic cell death eventually results in secondary necrotic cell death, whereas caspase-independent primary necrotic cell death has been reported and its mechanism involving RIP1 and RIP3 has been recently elucidated. Dual staining with fluorescent Annexin V and propidium iodide (PI) has been used to discriminate apoptotic and necrotic cell death, in which Annexin V-positive/PI-negative staining is regarded as apoptosis and PI-positive staining as necrosis. Here we demonstrate that primary necrotic cells unexpectedly show Annexin V-positive/PI-negative staining before they become PI-positive, and that primary necrotic and apoptotic Annexin V-positive/PI-negative cells can be discriminated by necrostatin-1, an inhibitor of primary necrosis by inhibition of RIP1.     

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹⁴C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.     

Cell death pathology: cross-talk with autophagy and its clinical implications

Autophagy is a self-digesting mechanism that cells adopt to respond to stressful stimuli. Morphologically, cells dying by autophagy show multiple cytoplasmic double-membraned vacuoles, and, if prolonged, autophagy can lead to cell death, “autophagic cell death”. Thus, autophagy can act both as a temporary protective mechanism during a brief stressful episode and be a mode of cell death in its own right. In this mini-review we focus on recent knowledge concerning the connection between autophagy and programmed cell death, evaluating their possible implications for therapy in pathologies like cancer and neurodegeneration.     

Hsp90α/β associates with the GSK3β/axin1/phospho-β-catenin complex in the human MCF-7 epithelial breast cancer model

Hsp90α/β, the signal transduction chaperone, maintains intracellular communication in normal, stem, and cancer cells. The well characterised association of Hsp90α/β with its client kinases form the framework of multiple signalling networks. GSK3β, a known Hsp90α/β client, mediates β-catenin phosphorylation as part of a cytoplasmic destruction complex which targets phospho-β-catenin to the 26S proteasome. The canonical Wnt/β-catenin pathway promotes stem cell self-renewal as well as oncogenesis. The degree of Hsp90α/β involvement in Wnt/β-catenin signalling needs clarification. Here, we describe the association of Hsp90α/β with GSK3β, β-catenin, phospho-β-catenin and the molecular scaffold, axin1, in the human MCF-7 epithelial breast cancer cell model using selective inhibition of Hsp90α/β, confocal laser scanning microscopy and immunoprecipitation. Our findings suggest that Hsp90α/β modulates the phosphorylation of β-catenin by interaction in common complex with GSK3β/axin1/β-catenin.     

Gadd45�� activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.     

An interaction between human Sec63 and nucleoredoxin may provide the missing link between the SEC63 gene and polycystic liver disease

The formation of multiple cysts in one or several organs is a characteristic of several human inherited diseases. Recent research suggests that problems in planar cell polarity may be the common denominator in polycystic diseases. Mutations in at least two genes are linked to autosomal dominant polycystic liver disease (PCLD), PRKCSH and SEC63. A recent study linked PRKCSH to the signaling- and cytoskeletal adaptor-component β-catenin. In a yeast two hybrid screen we identified the cytosolic protein nucleoredoxin (NRX) as an interaction partner of human Sec63. Since NRX is involved in the Wnt signaling pathways, we characterized this interaction. Thus, Sec63 is linked to the Wnt signaling pathways and this interaction may be the reason why mutations in SEC63 can lead to PCLD.

Structured summary

Sec63physically interacts with NRX by two hybrid(View interaction)NRXbinds to Sec63 by peptide array (View Interaction 1, 2)Sec63binds to NRX by pull down(View interaction)Sec63binds to NRX by peptide array (View Interaction 1, 2, 3)     

Phospholipase C-γ1 is required for the epidermal growth factor receptor-induced squamous cell carcinoma cell mitogenesis

The epidermal growth factor receptor (EGFR) is a key driver in the process of squamous cell carcinoma (SCC) cell mitogenesis. Phospholipase C-γ1 (PLC-γ1) is a downstream target of EGFR signaling, but the role and necessity of PLC-γ1 in EGFR-induced cell mitogenesis remain unclear. In the present study, we report an elevated expression of PLC-γ1 in human SCC biopsies relative to adjacent normal epidermis, and in human SCC cell lines compared to normal human keratinocytes. EGFR-induced SCC cell mitogenesis was blocked by small interfering RNA knockdown of PLC-γ1. However, inhibition of the catalytic activity of phospholipase C had no effect on EGFR-induced SCC cell mitogenesis. In response to the EGFR ligand epidermal growth factor (EGF), PLC-γ1 was translocated not only to the plasma membrane but also to the nucleus. These data suggest that PLC-γ1 is required for EGFR-induced SCC cell mitogenesis and the mitogenic function of PLC-γ1 is independent of its lipase activity.     

Integrin-mediated mechanotransduction processes in TGFbeta-stimulated monolayer-expanded chondrocytes

Previous studies have demonstrated that passage in monolayer detrimentally affects the response of articular chondrocytes to the application of dynamic compression. Transforming growth factor beta (TGFbeta) is known to regulate metabolic processes in articular cartilage and can enhance the re-expression of a chondrocytic phenotype following monolayer expansion. The current study tests the hypothesis that TGFbeta also modulates the response of monolayer-expanded human chondrocytes to the application of dynamic compression, via an integrin-mediated mechanotransduction process. The data presented demonstrate that TGFbeta3 enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release after 48 h of culture when compared to unsupplemented constructs. Dynamic compression also enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release in the presence of TGFbeta3. By contrast, dynamic compression did not alter these parameters in the absence of the growth factor. The addition of the peptide, GRGDSP, which acts as a competitive ligand for the alpha5beta1 integrin, reversed the compression-induced stimulation of 35SO4 incorporation, [3H]thymidine incorporation, and suppression of nitrite release. No effect was observed when the control peptide, GRADSP, was used. The current data clearly demonstrate that the dynamic compression-induced changes observed in cell metabolism for human monolayer-expanded chondrocytes were dependent on the presence of TGFbeta3 and are integrin-mediated.     

B cells promote hepatic inflammation, biliary cyst formation, and salivary gland inflammation in the NOD.c3c4 model of autoimmune cholangitis

There are now several murine models of autoimmune cholangitis that have features both similar and distinct from human PBC. One such model, the NOD.c3c4 mouse, manifests portal cell infiltrates, anti-mitochondrial antibodies but also biliary cysts. The biliary cysts are not a component of PBC and not found in the other murine models. To address the immunopathology in these mice, we generated genetically B cell deficient Igμ^−/− NOD.c3c4 mice and compared the immunopathology of these animals to control B cell sufficient NOD.c3c4 mice. B cell deficient mice demonstrated decreased number of non-B cells in the liver accompanied by reduced numbers of activated natural killer cells. The degree of granuloma formation and bile duct damage were comparable to NOD.c3c4 mice. In contrast, liver inflammation, biliary cyst formation and salivary gland inflammation was significantly attenuated in these B cell deficient mice. In conclusion, B cells play a critical role in promoting liver inflammation and also contribute to cyst formation as well as salivary gland pathology in autoimmune NOD.c3c4 mice, illustrating a critical role of B cells in modulating specific organ pathology and, in particular, in exacerbating both the biliary disease and the sialadenitis.     

Transglutaminase 2 in the balance of cell death and survival

Transglutaminase 2 (TG2), a multifunctional enzyme with Ca(2+)-dependent protein crosslinking activity and GTP-dependent G protein functions, is often upregulated in cells undergoing apoptosis. In cultured cells TG2 may exert both pro- and anti-apoptotic effects depending upon the type of cell, the kind of death stimuli, the intracellular localization of the enzyme and the type of its activities switched on. The majority of data support the notion that transamidation by TG2 can both facilitate and inhibit apoptosis, while the GTP-bound form of the enzyme generally protects cells against death. In vivo studies confirm the Janus face of TG2 in the initiation of the apoptotic program. In addition, they reveal a further role: the prevention of inflammation, tissue injury and autoimmunity once the apoptosis has already been initiated. This function of TG2 is partially achieved by being expressed and activated also in macrophages digesting apoptotic cells and mediating a crosstalk between dying and phagocytic cells.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Neuroprotective cytokines repress PUMA induction in the 1-methyl-4-phenylpyridinium (MPP(+)) model of Parkinson's disease

The hematopoietic cytokines erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) provide neuroprotection in several in vitro and in vivo models of Parkinson’s disease (PD). The molecular mechanism by which Epo and G-CSF signals reduce the neuronal death in PD is not clear. Here, we show that in rat pheochromocytoma PC12 cells, Epo and G-CSF efficiently repressed the 1-methyl-4-phenylpyridinium (MPP⁺)-induced expression of the proapoptotic protein PUMA (p53 up-regulated modulator of apoptosis). Accordingly, Epo and G-CSF treatment reduced the PC12 cell fraction that underwent apoptosis by MPP⁺ treatment and thus improved cell viability. Downregulation of PUMA expression by Epo and G-CSF in MPP⁺-treated PC12 cells seems to be mediated by repression of p53, as the expression of p53 was increased by MPP⁺-treatment and reduced by Epo and G-CSF. Together, these results suggest that the neuroprotective activities of Epo and G-CSF in an experimental model of PD involve the repression of the apoptosis-inducing action of PUMA.     

A receptor-electromigration-based model for cellular electrotactic sensing and migration

Directed cell migration in tissues mediates various physiological processes and is guided by complex cellular factors such as chemoattractant gradients and electric fields. Direct current (DC) electric fields can be generated in physiological settings and the electric field guided migration of various cell types (i.e., electrotaxis) has been demonstrated both in vitro and in vivo. Although several mechanisms have been proposed for electrotaxis, there are so far very few quantitative models. Furthermore, because chemoattractant gradients and electric fields co-exist in tissues, it is important to understand how chemotaxis and electrotaxis interact for mediating cell migration and trafficking. In this study, we developed a mathematical model to investigate the role of electromigration of cell surface chemoattractant receptors in mediating electrochemical sensing and migration of cells. Our results show that electromigration of chemoattractant receptors enables cell electrotactic sensing and migration in the presence of a uniform chemoattractant field. Furthermore, in the physiologically-relevant range of receptor electromigration rates, application of electric fields overcomes chemical guiding signals for directional sensing and migration of cells in co-existing competing electric fields and chemoattractant gradients.     

Catabolism of 4-hydroxy-2-trans-nonenal by THP1 monocytes/macrophages and inactivation of carboxylesterases by this lipid electrophile

Oxidative stress in cells and tissues leads to the formation of an assortment of lipid electrophiles, such as the quantitatively important 4-hydroxy-2-trans-nonenal (HNE). Although this cytotoxic aldehyde is atherogenic the mechanisms involved are unclear. We hypothesize that elevated HNE levels can directly inactivate esterase and lipase activities in macrophages via protein adduction, thus generating a biochemical lesion that accelerates foam cell formation and subsequent atherosclerosis. In the present study we examined the effects of HNE treatment on esterase and lipase activities in human THP1 monocytes/macrophages at various physiological scales (i.e., pure recombinant enzymes, cell lysate, and intact living cells). The hydrolytic activities of bacterial and human carboxylesterase enzymes (pnbCE and CES1, respectively) were inactivated by HNE in vitro in a time- and concentration-dependent manner. In addition, so were the hydrolytic activities of THP1 cell lysates and intact THP1 monocytes and macrophages. A single lysine residue (Lys105) in recombinant CES1 was modified by HNE via a Michael addition reaction, whereas the lone reduced cysteine residue (Cys389) was found unmodified. The lipolytic activity of cell lysates and intact cells was more sensitive to the inhibitory effects of HNE than the esterolytic activity. Moreover, immunoblotting analysis using HNE antibodies confirmed that several cellular proteins were adducted by HNE following treatment of intact THP1 monocytes, albeit at relatively high HNE concentrations (>50 μM). Unexpectedly, in contrast to CES1, the treatment of a recombinant human CES2 with HNE enhanced its enzymatic activity ∼3-fold compared to untreated enzyme. In addition, THP1 monocytes/macrophages can efficiently metabolize HNE, and glutathione conjugation of HNE is responsible for ∼43% of its catabolism. The functional importance of HNE-mediated inactivation of cellular hydrolytic enzymes with respect to atherogenesis remains obscure, although this study has taken a first step toward addressing this important issue by examining the potential of HNE to inhibit this biochemical activity in a human monocyte/macrophage cell line.     

The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h

The tomato AGC protein kinase Adi3 is known to function as a suppressor of PCD and silencing of Adi3 leads to spontaneous cell death on leaves and stems. In an effort to isolate Adi3 interacting proteins, a yeast two-hybrid screen was carried out and identified the autophagy protein Atg8h as an Adi3 interactor. This interaction occurred independent of the kinase activity status of Adi3. Silencing of genes involved in autophagy is known to eliminate the restriction of pathogen-induced PCD to a few cells and leads to run away PCD. Cosilencing Adi3 with several autophagy genes lead to the same run away cell death suggesting Adi3 may be involved in autophagic regulation of PCD.