Use of the monoclonal antibody DAKO-ERbeta (8D5-1) to measure oestrogen receptor beta in breast cancer cells

BACKGROUND: Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. METHODS: Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. RESULTS: Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. CONCLUSIONS: This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry.     

Acinetobacter calcoaceticus–baumannii Complex Strains Induce Caspase-Dependent and Caspase-Independent Death of Human Epithelial Cells

We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.     

Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.     

Ultrastructural effects of low dosage endocrine disrupter chemicals on neural cells of the chicken embryo model.

Previous research suggests that endocrine disrupters (EDCs) like nonylphenol cause apoptosis (both via the intrinsic and extrinsic pathway) and that ROS generation and Ca (2+) play a fundamental role in the process. We have investigated morphological changes induced by 17beta-estradiol, nonylphenol, 17alpha-ethynylestradiol and diethylstilbestrol on the IN OVO neural chick embryo model by using transmission and scanning electron microscopy (TEM and SEM). We found that estrogenic substances such as nonylphenol, diethylstilbestrol (DES) and 17alpha-ethynylestradiol, as well as 17beta-estradiol cause ultrastructural changes to developing neurons, resulting in damage to the plasma, mitochondrial as well as nuclear membranes. Furthermore, both apoptotic blebbing and necrotic (or oncotic) budding was seen in TEM and SEM micrographs. SEM shows that nonylphenol-exposed neurons have irregular cell surfaces with pseudopodia, cell shrinkage and breakages in the plasma membrane--typical of apoptosis. TEM indicated that plasma membranes and double nuclear membranes have structural changes, with apoptotic bodies (blebbing) and disrupted mitochondrial membranes. In 17alpha-ethynylestradiol-exposed neurons, disruption of the plasma membrane with cell swelling and vacuolization was present. No apoptotic bodies or budding were noted here. 17beta-Estradiol induced openings in the plasma membrane, while DES-exposed neurons did not show any morphological changes. Therefore we conclude that EDC damage is morphologically visible and the damage is recognized as apoptosis and oncosis. Estrogenic substances may hence modify hormonal actions thereby leaving the developing nervous system more susceptible to damaging events.     

Apoptosis induced by direct triggering of mitochondrial apoptosis proceeds in the near-absence of some apoptotic markers

Apoptotic cell death is characterized by the activation of the apoptotic signal transduction pathway on one hand and a number of regularly found morphological and biochemical features, such as nuclear condensation and mitochondrial depolarisation. Although much of our knowledge of apoptosis was obtained using noxious stimuli in cell culture, these apoptotic stimuli are likely to have numerous off-target effects that may contribute to or obscure the immediate effects of the apoptotic pathway. We have developed a cellular model where mitochondrial apoptosis is directly triggered by the tetracycline-regulated expression of the pro-apoptotic BH3-only protein Bim_S. We report the comparison of Bim_S-induced apoptosis with the commonly used apoptotic stimuli staurosporine and UV-light. While the release of mitochondrial cytochrome c and Smac/DIABLO, activation of caspases and nuclear morphological changes occurred with very similar kinetics, striking differences were found in other apoptotic assays. In particular, drop in mitochondrial membrane potential, loss of plasma membrane integrity and the appearance of sub-G1 nuclei were strongly reduced in cells dying upon Bim_S-induction, compared to staurosporine- or UV-induced apoptosis. The results thus indicate that the link between the apoptotic pathway and commonly used indicators of apoptosis is less tight than it appears from experiments with cytotoxic stimuli.     

Mitochondrial potassium channels and uncoupling proteins in synaptic plasticity and neuronal cell death

The function of the nervous system relies upon synaptic transmission, a process in which a neurotransmitter released from pre-synaptic terminals of one neuron (in response to membrane depolarization and calcium influx) activates post-synaptic receptors on dendrites of another neuron. Synapses are subjected to repeated bouts of oxidative and metabolic stress as the result of changing ion gradients and ATP usage. Mitochondria play central roles in meeting the demands of synapses for ATP and in regulating calcium homeostasis, and mitochondrial dysfunction can cause dysfunction and degeneration of synapses, and can trigger cell death. We have identified two types of mitochondrial proteins that serve the function of protecting synapses and neurons against dysfunction and death. Mitochondrial ATP-sensitive potassium (MitoKATP) channels modulate inner membrane potential and oxyradical production; mitochondrial potassium fluxes can affect cytochrome c release and caspase activation and may determine whether neurons live or die in experimental models of stroke and Alzheimer's disease. Uncoupling proteins (UCPs) are a family of mitochondrial membrane proteins that uncouple electron transport from ATP production by transporting protons across the inner membrane. Neurons express at least three UCPs including the widely expressed UCP-2 and the neuron-specific UCP-4 and UCP-5 (BMCP-1). We have found that UCP-4 protects neurons against apoptosis by a mechanism involving suppression of oxyradical production and stabilization of cellular calcium homeostasis. The expression of UCP-4 is itself regulated by changes in energy metabolism. In addition to their roles in neuronal cell survival and death, MitoKATP channels and UCPs may play roles in regulating neuronal differentiation during development and synaptic plasticity in the adult.     

Praf2 is a novel Bcl-xL/Bcl-2 interacting protein with the ability to modulate survival of cancer cells

Increased expression of Bcl-xL in cancer has been shown to confer resistance to a broad range of apoptotic stimuli and to modulate a number of other aspects of cellular physiology, including energy metabolism, cell cycle, autophagy, mitochondrial fission/fusion and cellular adhesion. However, only few of these activities have a mechanistic explanation. Here we used Tandem Affinity purification to identify novel Bcl-xL interacting proteins that could explain the pleiotropic effects of Bcl-xL overexpression. Among the several proteins co-purifying with Bcl-xL, we focused on Praf2, a protein with a predicted role in trafficking. The interaction of Praf2 with Bcl-xL was found to be dependent on the transmembrane domain of Bcl-xL. We found that Bcl-2 also interacts with Praf2 and that Bcl-xL and Bcl-2 can interact also with Arl6IP5, an homologue of Praf2. Interestingly, overexpression of Praf2 results in the translocation of Bax to mitochondria and the induction of apoptotic cell death. Praf2 dependent cell death is prevented by the co-transfection of Bcl-xL but not by its transmembrane domain deleted mutant. Accordingly, knock-down of Praf2 increases clonogenicity of U2OS cells following etoposide treatment by reducing cell death. In conclusion a screen for Bcl-xL-interacting membrane proteins let us identify a novel proapoptotic protein whose activity is strongly counteracted exclusively by membrane targeted Bcl-xL.     

Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM)”. The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI− phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.     

Heat stress activates AKT via focal adhesion kinase-mediated pathway in neonatal rat ventricular myocytes

Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-based signaling pathways that are associated with protection from lethal cell injury.     

Towards an understanding of apoptosis detection by SYTO dyes.

BACKGROUND: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis. METHODS: Human follicular lymphoma cell lines were subjected to diverse apoptotic and oncotic stimuli with subsequent multiparametric flow cytometric and fluorescence imaging analysis. SYTO green (SYTO11-16), TMRM, PI, 7AAD, and Hoechst 33342 probes were applied for multivariate analysis of temporal sequence of apoptotic events. Sorting of cells differing in the level of SYTO16 fluorescence and subsequent characterization of obtained subpopulations were also performed. RESULTS: Loss of SYTO16 fluorescence (SYTOlow/PI+ events) has been observed in cells exposed to oncotic stimuli, whereas SYTOhigh/PI+ events did not prevail at any treatment scenario. We tracked similarities and discrepancies between SYTO16 and TMRM probes. Often, SYTO16 and TMRM exhibited the same staining profiles, as loss of their fluorescence was detected in a single cell population. However, both mitochondrial uncoupler FCCP and a small-molecule Bcl-2 inhibitor, HA14-1, appeared to induce distinct staining profiles of SYTO16 and TMRM, with the decrease in TMRM fluorescence preceding the loss of SYTO16 fluorescence. Importantly, in both cases (FCCP and HA14-1) the decrease of SYTO16 fluorescence was blocked by pharmacological inhibition of caspases (with z-VAD-fmk). CONCLUSIONS: The data demonstrate that loss of SYTO16 is caspase-dependent, as is not a mere indicator of Deltapsim dissipation. Commonly observed similarities between SYTO and TMRM may stem from the fast kinetics of apoptotic events once cell death is initiated.     

Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers

Kinetic analysis of dexamethasone-induced apoptosis in the human lymphoblastoid cell line CCRF CEM C7A has revealed a point when cells, morphologically indistinguishable from untreated cells, have irreversibly engaged a program leading to death, measured by a loss of clonogenicity. Since all cells that fail to clone eventually died through apoptosis, measurements of clonogenicity in this system provide an accurate measure of commitment to apoptotic death. Inhibition of caspases by peptide inhibitors blocked proteolysis of endogenous substrates and reduced nuclear condensation yet did not alter either dexamethasone-induced changes in clonogenicity or mitochondrial membrane potential. In contrast to the results with caspase inhibitors, expression of BCL-2 in CCRF CEM C7A cells proved sufficient to block all changes associated with apoptosis, including loss of both clonogenicity and changes in mitochondrial membrane potential. These results demonstrate that commitment to cell death can precede the key biochemical or morphological features of apoptosis by several hours and indicate that separate regulators govern cellular commitment to clonogenic death and the subsequent execution phase characterised as apoptosis.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.     

Mitochondrial Ca(2+) and apoptosis

Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca(2+)) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca(2+) homeostasis and in turn modulate the effectiveness of Ca(2+)-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca(2+) overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.