When nitrosative stress hits the endoplasmic reticulum: Possible implications in oxLDL/oxysterols-induced endothelial dysfunction.

Oxidized LDL (oxLDL) and oxysterols are known to play a crucial role in endothelial dysfunction (ED) by inducing endoplasmic reticulum stress (ERS), inflammation, and apoptosis. However, the precise molecular mechanisms underlying these pathophysiological processes remain incompletely understood. Emerging evidence strongly implicates excessive nitric oxide (NO) production in the progression of various pathological conditions. The accumulation of reactive nitrogen species (RNS) leading to nitrosative stress (NSS) and aberrant protein S-nitrosylation contribute to NO toxicity. Studies have highlighted the involvement of NSS and S-nitrosylation in perturbing ER signaling through the modification of ER sensors and resident isomerases in neurons. This review focuses on the existing evidence that strongly associates NO with ERS and the possible implications in the context of ED induced by oxLDL and oxysterols. The potential effects of perturbed NO synthesis on signaling effectors linking NSS with ERS in endothelial cells are discussed to provide a conceptual framework for further investigations and the development of novel therapeutic strategies targeting ED.

Endothelial cells, endoplasmic reticulum stress and oxysterols.

Oxysterols are bioactive lipids that act as regulators of lipid metabolism, inflammation, cell viability and are involved in several diseases, including atherosclerosis. Mounting evidence linked the atherosclerosis to endothelium dysfunction; in fact, the endothelium regulates the vascular system with roles in processes such as hemostasis, cell cholesterol, hormone trafficking, signal transduction and inflammation. Several papers shed light the ability of oxysterols to induce apoptosis in different cell lines including endothelial cells. Apoptotic endothelial cell and endothelial denudation may constitute a critical step in the transition to plaque erosion and vessel thrombosis, so preventing the endothelial damaged has garnered considerable attention as a novel means of treating atherosclerosis. Endoplasmic reticulum (ER) is the site where the proteins are synthetized and folded and is necessary for most cellular activity; perturbations of ER homeostasis leads to a condition known as endoplasmic reticulum stress. This condition evokes the unfolded protein response (UPR) an adaptive pathway that aims to restore ER homeostasis. Mounting evidence suggests that chronic activation of UPR leads to cell dysfunction and death and recently has been implicated in pathogenesis of endothelial dysfunction. Autophagy is an essential catabolic mechanism that delivers misfolded proteins and damaged organelles to the lysosome for degradation, maintaining basal levels of autophagic activity it is critical for cell survival. Several evidence suggests that persistent ER stress often results in stimulation of autophagic activities, likely as a compensatory mechanism to relieve ER stress and consequently cell death. In this review, we summarize evidence for the effect of oxysterols on endothelial cells, especially focusing on oxysterols-mediated induction of endoplasmic reticulum stress.

Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells.

The pineal gland hormone melatonin has been recently described to downregulate the intrinsic (or damage-induced) pathway of apoptosis in human leukocytes. These properties appear to depend on a specific mitochondrial signaling of melatonin which is associated with a lower generation of reactive oxygen species and a better control of redox-sensitive components such as the antiapoptotic protein Bcl-2. Other elements upstream in this signaling are expected to contribute regulatory roles that remain unexplored. The aim of this study was to investigate whether the extracellular signal-regulated kinase (ERK), which controls the balance between survival and death-promoting genes throughout the MAPK pathway, is involved in the antiapoptotic signaling of melatonin. Human monocytic U937 cells irradiated with UVB light were used as a model of stress-induced apoptosis. In this model we found that pharmacological concentrations of melatonin (1 mM) were able to decrease superoxide anion production, mitochondrial damage, and caspase-dependent apoptosis by improved Bcl-2 levels and decreased Cyt c release in the cytoplasm. Moreover, melatonin increased the phosphorylative activation of ERK 1/2 independently from the presence of UVB stress, and decreased the UVB-mediated activation of the stress kinases p38 MAPK and JNK. The ERK 1/2 inhibitor PD98059, but not the p38 MAPK inhibitor SB203580, abolished to different extents the effects that melatonin had on the UVB-induced ROS generation, mitochondrial dysfunction, and apoptosis. Using these inhibitors, a cross-talk effect between stress and survival-promoting kinases was tentatively identified, and confirmed the hierarchical role of ERK MAPK phosphorylation in the signaling of melatonin. In conclusion, melatonin sustains the activation of the survival-promoting pathway ERK MAPK which is required to antagonize UVB-induced apoptosis of U937 cells. This kinase mediates also the antioxidant and mitochondrial protection effects of this hormonal substance that may find therapeutic applications in inflammatory and immune diseases associated with leukocyte oxidative stress and accelerated apoptosis.

Melatonin reduces early changes in intramitochondrial cardiolipin during apoptosis in U937 cell line.

Cardiolipin (CL) is found exclusively in the inner mitochondrial membrane. CL deficiency leads to an alteration in the stability of mitochondrial membranes, to an increased permeability as well as a decreased respiratory rate, and therefore to mitochondria which are completely dysfunctional. It is known that reactive oxygen species (ROS) cause a decrease and a variation in CL content, concomitantly the formation of the mitochondrial permeability transition pore facilitates the release of cytochrome c (cyt c) into the cytosol. Melatonin (Mel), the secretory product of the pineal gland, is a potent and efficient endogenous radical scavenger. It has been shown to protect, various biomolecules, such as DNA, membrane lipids, and cytosolic proteins from oxidative damage. To evaluate the protective role of Mel, we have studied U937 cells treated with UV-B irradiation. In our model, the administration of 1mM Mel before UV-B irradiation showed a significant protection from apoptotic cell death, in particular, mitochondrial structure and function were preserved through apoptotic pathways when cells were preincubated with 1mM Mel before UV-B exposure. The cardiolipin-sensitive probe 10-nonyl acridine orange (NAO) was used to monitor changes in mitochondrial lipids. Our data suggest that the Mel treatment protects CL from ROS and this suggests a possible link with the reduction of the apoptotic phenomenon.

Effects of hyperthermia in human neuroblastoma cells.

Neuroblastoma is a childhood malignancy derived from the developing sympathetic nervous system (SNS) and often diagnosed during early infancy. To investigate its metastatic properties, also in response to anti-cancer treatment, we have studied hyperthermia (HT) effects on the ultrastructure of SK-N-MC human neuroblastoma tumor cells. Cells undergoing HT showed a significant increase in apoptotic and necrotic events, but also a rearrangement of the cellular shape, appearing as cell detachment and rounding. The most striking effects of HT can be so correlated to primary tumor mass decrease and to a certain impairment of cell adhesion properties and consequently metastatic diffusion potential.

Multiple roles of matrix metalloproteinases during apoptosis.

Structural, molecular and biochemical approaches have contributed to piecing together the puzzle of how matrix metalloproteinases (MMPs) work and contribute to various disease processes. However, MMPs have many unexpected substrates other than components of the extracellular matrix which profoundly influence cell behaviour, survival and death. With the current understanding of diverse/novel roles of matrix metalloproteinases--particularly their direct or indirect relevance for the early steps during programmed cell death--some seemingly contrasting results seem less surprising. To better target MMPs an appreciation of their many extracellular, intracellular and intranuclear functions, often acting in opposing directions with paradoxical roles in cell death, is carefully required.

C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization.

In this study, the differentiation of C2C12 cells, a primary line of murine myoblasts, was investigated by a multiple technical approach. Undifferentiated cells, and those at intermediate and final differentiation times, were studied at the reverted microscope, by conventional and confocal immunofluorescence, and by transmission and scanning electron microscopy. The general monolayer architecture changed during differentiation from fusiform or star-shaped cells to elongated confluent cells, finally originating long, multinucleated myotubes. Sarcomeric actin and myosin are present also in undifferentiated myoblasts, but progressively acquire a structured pattern up to the appearance of sarcomeres and myofibrils at about 5 days after differentiation induction. Myotubes show a particular positivity for actin and myosin, and M-cadherin, an adhesion molecule characteristic, as known, of satellite cells, also seems to be involved in their assembling. Rare apoptotic patterns, as evidenced by the TUNEL technique, appear during myoblast maturation.

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis.

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.

In vitro apoptotic cell death during erythroid differentiation.

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.

Hyperthermia triggers apoptosis and affects cell adhesiveness in human neuroblastoma cells.

Hyperthermia is a known apoptotic inducer and has been recently utilized in combination with chemo-and/or radiotherapy in cancer treatment. In this study we have described its effect on SK-N-MC human neuroblastoma tumor cells, a line which grows as a double adherent and floating population. Considering this particular culture behavior, we also investigated the relationship between hyperthermia and cell adhesiveness by evaluating integrin expression, namely CD11a, which is, as known, closely correlated to cell adhesion properties. By a multiple, ultrastructural and flow cytometrical approach, we have demonstrated that hyperthermia, while triggering apoptosis, also determines a CD11a surface expression decrease in apoptotic and living cells. We thus suggest a further role for this treatment, which, affecting adhesion mechanisms, could down-regulate metastatic diffusion.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds induce apoptotic patterns in cultures of human tumor cell lines.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds were extensively studied as anti-gestational agents and some of these molecules were also described as inhibitors of ornithine decarboxylase. Polyamine depletion has been frequently related to the induction of apoptosis and consequently we investigated DL-111-IT and analogs for this effect in myeloid (HL60), neuroblastic (SK-N-MC) and epithelial (BeWo) human tumor cell lines, by means of electron microscopy and DNA electrophoresis. HL60 and SK-N-MC appeared notably sensitive to apoptosis, whereas BeWo responsiveness was variable and frequently associated with necrosis. Our results indicate that the contragestational effect of DL-111-IT and analogs is associated with apoptotic deletion of chorionic tissue and that these molecules, due to their effect on human tumor cell lines, can be considered as antiblastic lead compounds.

Actin involvement in apoptotic chromatin changes of hemopoietic cells undergoing hyperthermia.

During apoptosis, cell chromatin undergoes characteristic morphological changes, which have been long described in a variety of experimental models but are mostly not yet understood. The aim of the present study was to investigate the mechanisms underlying this phenomenon and the possible role of cytoskeleton, in particular actin. The chosen apoptotic model were HL60 hemopoietic cells undergoing hyperthermia and the starting point was the observation of thin filament bundles in decondensed chromatin of their early apoptotic nuclei. The characterization of these structures was undertaken by cytochemical, fluorescent and immunogold techniques, directed to actin identification. Taken together, our results suggest, in apoptotic cells, a deep actin rearrangement. Moreover, this cytoskeletal component, never present in normal nucleus, appears in the early apoptotic one, where it can be found in polymerized form, promptly recognizable both by conventional and immunogold electron microscopy. We suggest that, similarly to the role played by nuclear matrix in interphase and mitotic nucleus, actin could be directly involved in chromatin rearrangement occurring in apoptosis.

Intracellular detection of Bcl-2 and p53 proteins by flow cytometry: comparison of monoclonal antibodies and sample preparation protocols.

Several techniques have been proposed for flow cytometric evaluation of intracellular antigens. This approach is particularly important for detection at the single cell level of proteins which correlate to tumour progression. Bcl-2 and p53 are two of the most relevant proteins. In the present study we have compared five different cell fixation-permeabilisation protocols and nine fluorochrome-conjugated (FITC or PE) monoclonal antibodies (mAb): four mAb directed against Bcl-2 and five against p53. For detection of Bcl-2 we have analysed three Bcl-2 positive cell lines (K562, Daudi and MCF-7), and peripheral blood samples obtained from nine healthy subjects. To distinguish internal positive (lymphocytes) and negative control cells (granulocytes), it was necessary to perform simultaneous detection of surface and intracellular antigens. For detection of p53 three cell lines, two p53 positive (Raji and CEM) and one p53 negative (HL-60), were analysed. Using these cells we have performed a combined analysis of the efficiency of monoclonal antibodies and sample preparation techniques. In conclusion, clones 124-FITC and Bcl-2/100-PE (Bcl-2), and clones BP53,12-FITC and G59-12-PE (p53) provided the highest specific fluorescence intensity of the respective markers independent of cell preparation protocols. Importantly, our results show that mAb background may depend on the specific fixation/permeabilisation kit and that mAb titration using negative and positive control cells is essential to determine the specificity and the sensitivity of the mAb used.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Disassembly of nuclear bodies during arousal from hibernation: an in vitro study.

In previous studies we demonstrated that during hibernation cell nuclei contain structural constituents usually absent in euthermia. The rapid disappearance of such nuclear bodies upon arousal makes very difficult the in vivo investigation of the disassembly process, which could clarify their functions in nuclear metabolism in the hibernator. In the present study we subjected liver samples taken from hibernating edible dormice ( Glis glis) to different in vitro experimental conditions: at 4 degrees C, to preserve the hypothermic state of the hibernating organism; at 37 degrees C, to simulate the drastic increase in body temperature occurring during arousal; at 37 degrees C, in culture medium containing 10(-5) M delta opioid D-Ala2- D-Leu5 enkephalin, which mimics the activity of the hibernation induction trigger in hibernators. Electron microscopic analysis of hepatocyte nuclei at increasing incubation times revealed the subsequent steps of disassembly of coiled bodies, amorphous bodies and fibro-granular material, the unusual structural constituents accumulating during hibernation in these nuclei. We demonstrated that: (1) a temperature of 37 degrees C induces the disappearance of all nuclear bodies typical of hibernation in a few minutes; (2) both low temperature and hibernation-triggering opioid are able to slow down, although to different extents, the process of disassembly of nuclear bodies; (3) the fibro-granular material rapidly disappears during the early phases of incubation; while (4) coiled bodies and amorphous bodies progressively disassemble as fibrous material. Our results support previous hypotheses based on in vivo observations about a possible role for coiled bodies, amorphous bodies and fibro-granular material as storage/assembly sites of molecules needed for the rapid and massive resumption of transcriptional and post-transcriptional activities upon arousal and suggest a strict correlation between the dynamics and metabolic rate of nuclear bodies.

Behavior of nucleolar proteins during the course of apoptosis in camptothecin-treated HL60 cells.

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders.

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells.

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.