Question: Mitochondria are uninvolved in apoptosis.

Title: Mitochondria: Dynamic Organelles in Disease, Aging, and Development Content: Mitochondria are the primary energy-generating system in most eukaryotic cells. Additionally, they participate in intermediary metabolism, calcium signaling, and apoptosis. Given these well-established functions, it might be expected that mitochondrial dysfunction would give rise to a simple and predictable set of defects in all tissues. However, mitochondrial dysfunction has pleiotropic effects in multicellular organisms. Clearly, much about the basic biology of mitochondria remains to be understood. Here we discuss recent work that suggests that the dynamics (fusion and fission) of these organelles is important in development and disease.

Title: Bioenergetic aspects of apoptosis, necrosis and mitoptosis Content: In this review I summarize interrelations between bioenergetic processes and such programmed death phenomena as cell suicide (apoptosis and necrosis) and mitochondrial suicide (mitoptosis). The following conclusions are made. (I) ATP and rather often mitochondrial hyperpolarization (i.e. an increase in membrane potential, ΔΨ) are required for certain steps of apoptosis and necrosis. (II) Apoptosis, even if it is accompanied by ΔΨ and [ATP] increases at its early stage, finally results in a ΔΨ collapse and ATP decrease. (III) Moderate (about three-fold) lowering of [ATP] for short and long periods of time induces apoptosis and necrosis, respectively. In some types of apoptosis and necrosis, the cell death is mediated by a ΔΨ-dependent overproduction of ROS by the initial (Complex I) and the middle (Complex III) spans of the respiratory chain. ROS initiate mitoptosis which is postulated to rid the intracellular population of mitochondria from those that are ROS overproducing. Massive mitoptosis can result in cell death due to release to cytosol of the cell death proteins normally hidden in the mitochondrial intermembrane space.

Title: At the gates of death. Content: Apoptosis that proceeds via the mitochondrial pathway involves mitochondrial outer membrane permeabilization (MOMP), responsible for the release of cytochrome c and other proteins of the mitochondrial intermembrane space. This essential step is controlled and mediated by proteins of the Bcl-2 family. The proapoptotic proteins Bax and Bak are required for MOMP, while the antiapoptotic Bcl-2 proteins, including Bcl-2, Bcl-xL, Mcl-1, and others, prevent MOMP. Different proapoptotic BH3-only proteins act to interfere with the function of the antiapoptotic Bcl-2 members and/or activate Bax and Bak. Here, we discuss an emerging view, proposed by Certo et al. in this issue of Cancer Cell, on how these interactions result in MOMP and apoptosis.

Title: BAX/BAK-independent mitoptosis during cell death induced by proteasome inhibition? Content: Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds through the intrinsic mitochondrial apoptosis pathway. We identify the cascade of molecular events responsible for cell death induced by a prototypical proteasome inhibitor, MG132, starting with rapid accumulation of BH3-only proteins in the mitochondria, proceeding through mitochondrial membrane permeabilization and subsequent loss of DeltaPsi(m), and leading to irreversible changes of mitochondrial ultrastructure, degradation of mitochondrial network, and detrimental impairment of crucial mitochondrial functions. Our results also establish a rationale for the broader use of proteasome inhibitors to kill apoptosis-resistant tumor cells that lack functional BAX/BAK proteins.

Title: Bax translocation and mitochondrial fragmentation induced by Helicobacter pylori. Content: BACKGROUND AND AIMS Previous in vitro and in vivo studies have revealed an association between Helicobacter pylori infection and apoptosis in gastric epithelial cells. Although involvement of the Bcl-2 family of proteins as well as cytochrome c release has been demonstrated in H pylori induced cell death, the exact role of the mitochondria during this type of programmed cell death has not been fully elucidated. Therefore, we sought to determine whether or not Bax translocation and mitochondrial fragmentation occur on exposure of gastric epithelial cells to H pylori, resulting in cell death. METHODS Experiments were performed with human gastric adenocarcinoma (AGS) cells, AGS cells transfected with the HPV-E6 gene (which inactivates p53 function), AGS-neo cells (transfected with the backbone construct), mouse embryonic fibroblasts (MEFs), and p19(ARF) null (ARF(-/-)) MEFs. Cells were incubated with a cag positive H pylori strain for up to 24 hours, lysed, and cytoplasmic and mitochondrial membrane fractions were analysed by western blot for Bax translocation. RESULTS Bax translocation was detected in AGS, AGS-neo, and normal MEF cells after exposure to H pylori for three hours, but not in ARF(-/-) MEFs cells. Translocation of Bax after H pylori incubation was also detected in AGS-E6 cells (inactive p53 gene) but to a lesser degree than in AGS-neo cells. In parallel studies, the mitochondrial morphology of living cells infected with H pylori was assessed by confocal microscopy. Mitochondrial fragmentation was detectable after 10 hours of H pylori incubation with AGS cells and after seven hours with MEF cells. In wild-type MEFs, mitochondrial fragmentation was significantly increased in comparison with ARF null MEFs (43% v 10.4%, respectively). Furthermore, mitochondrial depolarisation and caspase-3 activity were initiated within four hours in cells incubated with H pylori, and these events were inhibited by forced expression of Bcl-2. CONCLUSIONS These data suggest that during H pylori induced apoptosis, Bax translocates to the mitochondria which subsequently undergo depolarisation and profound fragmentation. Functional ARF and p53 proteins may play an important role in H pylori induced mitochondrial modification.

Title: Examining BCL-2 family function with large unilamellar vesicles. Content: The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis(1). Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)(1). After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues(2). In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)(3-6). Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation(7,8). In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members(7,9). As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization(10). LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)(10). This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)(11). As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Title: Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis. Content: BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis. METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays. RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE. CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

Title: Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes Content: Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Title: A role for mitochondria in NLRP3 inflammasome activation Content: An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger’, including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.

Title: Homocysteine Induces Trophoblast Cell Death with Apoptotic Features1 Content: Abstract Hyperhomocysteinemia has been suggested as a possible risk factor in women suffering from habitual abortions, placental abruption or infarcts, preeclampsia, and/or intrauterine growth retardation. However, little is known about the pathogenic mechanisms underlying the action of homocysteine. The present study investigated the in vitro ability of homocysteine to affect trophoblast gonadotropin secretion and to induce cell death. In primary human trophoblast cells, homocysteine treatment (20 μmol/L) resulted in cellular flattening and enlargement, extension of pseudopodia, and cellular vacuolization. Cellular detachment, apoptosis, and necrosis were favored. With in situ nick end labeling, we investigated DNA degradation, and we used M30 CytoDEATH to selectively stain the cytoplasm of apoptotic cells. Cytochrome c release from mitochondria to the cytosol and DNA cleavage in agarose gel have been investigated. Homocysteine, but not cysteine, induced trophoblast apoptosis and significantly reduced human chorionic gonadotropin secretion. These findings suggest that trophoblast cell death might represent a pathogenic mechanism by which homocysteine may cause pregnancy complications related to placental diseases.

Title: MiD49 and MiD51, new components of the mitochondrial fission machinery. Content: Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.

Title: From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only. Content: Megakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the 2 daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal because accumulation of nonmuscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearance of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible for the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.

Title: The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic, and Ischemic Tissue Damage Content: Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.

Title: Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function Content: Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.

Title: Cell death: the significance of apoptosis. Content: Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis. ” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.

Title: Elimination of paternal mitochondria in mouse embryos occurs through autophagic degradation dependent on PARKIN and MUL1 Content: A defining feature of mitochondria is their maternal mode of inheritance. However, little is understood about the cellular mechanism through which paternal mitochondria, delivered from sperm, are eliminated from early mammalian embryos. Autophagy has been implicated in nematodes, but whether this mechanism is conserved in mammals has been disputed. Here, we show that cultured mouse fibroblasts and pre-implantation embryos use a common pathway for elimination of mitochondria. Both situations utilize mitophagy, in which mitochondria are sequestered by autophagosomes and delivered to lysosomes for degradation. The E3 ubiquitin ligases PARKIN and MUL1 play redundant roles in elimination of paternal mitochondria. The process is associated with depolarization of paternal mitochondria and additionally requires the mitochondrial outer membrane protein FIS1, the autophagy adaptor P62, and PINK1 kinase. Our results indicate that strict maternal transmission of mitochondria relies on mitophagy and uncover a collaboration between MUL1 and PARKIN in this process.

Title: The DNA damage response during mitosis. Content: Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

Title: Mitochondria as sensors and regulators of calcium signalling Content: During the past two decades calcium (Ca2+) accumulation in energized mitochondria has emerged as a biological process of utmost physiological relevance. Mitochondrial Ca2+ uptake was shown to control intracellular Ca2+ signalling, cell metabolism, cell survival and other cell-type specific functions by buffering cytosolic Ca2+ levels and regulating mitochondrial effectors. Recently, the identity of mitochondrial Ca2+ transporters has been revealed, opening new perspectives for investigation and molecular intervention.

Title: tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1 Content: Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of β-oxidation flux by labeled palmitate demonstrates that tBid inhibits β-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on β-oxidation. The unexpected role of tBid in the regulation of lipid β-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

Title: Quercetin attenuates vascular calcification by inhibiting oxidative stress and mitochondrial fission. Content: Vascular calcification is a strong independent predictor of increased cardiovascular morbidity and mortality and has a high prevalence among patients with chronic kidney disease. The present study investigated the effects of quercetin on vascular calcification caused by oxidative stress and abnormal mitochondrial dynamics both in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial potential and ATP production. Disruption of mitochondrial structural integrity was also observed in a rat model of adenine-induced aortic calcification. Increased production of reactive oxygen species, enhanced expression and phosphorylation of Drp1, and excessive mitochondrial fragmentation were also observed in Pi-treated VSMCs. These effects were accompanied by mitochondria-dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol and subsequent activation of caspase-3. Quercetin was shown to block Pi-induced apoptosis and calcification of VSMCs by inhibiting oxidative stress and decreasing mitochondrial fission by inhibiting the expression and phosphorylation of Drp1. Quercetin also significantly ameliorated adenine-induced aortic calcification in rats. In summary, our findings suggest that quercetin attenuates calcification by reducing apoptosis of VSMCs by blocking oxidative stress and inhibiting mitochondrial fission.

Title: A phospholipid transfer function of ER-mitochondria encounter structure revealed in vitro Content: As phospholipids are synthesized mainly in the endoplasmic reticulum (ER) and mitochondrial inner membranes, how cells properly distribute specific phospholipids to diverse cellular membranes is a crucial problem for maintenance of organelle-specific phospholipid compositions. Although the ER-mitochondria encounter structure (ERMES) was proposed to facilitate phospholipid transfer between the ER and mitochondria, such a role of ERMES is still controversial and awaits experimental demonstration. Here we developed a novel in vitro assay system with isolated yeast membrane fractions to monitor phospholipid exchange between the ER and mitochondria. With this system, we found that phospholipid transport between the ER and mitochondria relies on membrane intactness, but not energy sources such as ATP, GTP or the membrane potential across the mitochondrial inner membrane. We further found that lack of the ERMES component impairs the phosphatidylserine transport from the ER to mitochondria, but not the phosphatidylethanolamine transport from mitochondria to the ER. This in vitro assay system thus offers a powerful tool to analyze the non-vesicular phospholipid transport between the ER and mitochondria.

Title: Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies. Content: The research on mitochondrial functions in adipocytes has increasingly evidenced that mitochondria plays an important role in the onset and/or progression of obesity and related pathologies. Mitochondrial function in brown adipose tissue (BAT) has been classically assessed by measuring either the levels/activity of mitochondrial enzymes, or the respiration in isolated mitochondria. Isolation of mitochondria is not advantageous because it demands significant time and amount of tissue and, as tissue homogenates, disrupts biochemical and physical connections of mitochondria within the cell. Here, we described a new and efficient protocol to analyze the mitochondrial respiratory states in BAT biopsies that relies on intracellular triglyceride depletion followed by tissue permeabilization. In addition to minimizing tissue requirements to ∼17 mg wet weight, the proposed protocol enabled analysis of all mitochondrial respiratory states, including phosphorylation (OXPHOS), no-phosphorylation (LEAK), and uncoupled (ETS) states, as well as the use of substrates for complex I, complex II, and cytochrome c; together, these features demonstrated mitochondrial integrity and validated the preparation efficacy. Therefore, the protocol described here increases the possibilities of answering physiological questions related to small BAT regions of human and animal models, which shall help to unravel the mechanisms that regulate mitochondrial function in health and disease.

Title: Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria Content: Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1-Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1-Mdm35 with and without PA. The Ups1-Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes.

Title: Apoptosis in breast cancer: relationship with other pathological parameters. Content: Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. The number of apoptotic cells as a percentage of cells present, or the number of apoptotic cells per square millimetre of neoplastic tissue, is usually described as the apoptotic index (AI). In breast cancer, the AI is not related to tumour size, axillary lymph node metastasis or distant metastasis at diagnosis. It is greater in invasive ductal carcinomas than in other histological types. High AI is also related to high histological grade, high nuclear grade, comedo-type necrosis, lack of tubule formation, and dense infiltration of the tumour by lymphocytes. Sex steroid receptor-negative tumours have greater AIs than the sex steroid receptor-positive ones. Aneuploid breast cancers with high S-phase fractions (SPFs) also have high AI values compared with diploid tumours with low SPFs. p53-Positive breast cancers have high AIs, whereas tumours that are Bcl-2 positive have low AIs. The AI shows a strong positive correlation to all direct or indirect indicators of cell proliferation, such as mitotic index and Ki67 immunolabelling. Univariate survival analyses show that a high AI is linked with unfavourable disease outcome in axillary lymph node-negative and -positive breast cancer, but multivariate analyses indicate that AI is not an independent prognostic factor. In conclusion, a high AI is related to malignant cellular features and indicators of invasiveness and cell proliferation in breast cancer.

Title: Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Content: Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.

Title: Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity Content: Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.

Title: Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics Content: Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s). Melatonin protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting the mitochondrial permeability transition pore (MPTP), and activating uncoupling proteins (UCPs). Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria.

Title: Anaphase catastrophe is a target for cancer therapy. Content: Neoplastic cells are genetically unstable. Strategies that target pathways affecting genome instability can be exploited to disrupt tumor cell growth, potentially with limited consequences to normal cells. Chromosomal instability (CIN) is one type of genome instability characterized by mitotic defects that increase the rate of chromosome mis-segregation. CIN is frequently caused by extra centrosomes that transiently disrupt normal bipolar spindle geometry needed for accurate chromosome segregation. Tumor cells survive with extra centrosomes because of biochemical pathways that cluster centrosomes and promote chromosome segregation on bipolar spindles. Recent work shows that targeted inhibition of these pathways prevents centrosome clustering and forces chromosomes to segregate to multiple daughter cells, an event triggering apoptosis that we refer to as anaphase catastrophe. Anaphase catastrophe specifically kills tumor cells with more than 2 centrosomes. This death program can occur after genetic or pharmacologic inhibition of cyclin dependent kinase 2 (Cdk2) and is augmented by combined treatment with a microtubule inhibitor. This proapoptotic effect occurs despite the presence of ras mutations in cancer cells. Anaphase catastrophe is a previously unrecognized mechanism that can be pharmacologically induced for apoptotic death of cancer cells and is, therefore, appealing to engage for cancer therapy and prevention.

Title: Apoptosis and chemoresistance in transgenic cancer models Content: Multidrug resistance remains an unresolved problem in clinical oncology. Over a decade ago genes encoding cellular efflux pumps were shown to confer resistance to a broad spectrum of biochemically unrelated anticancer drugs even before the compounds reached their intracellular targets. More recently it has become apparent that many drugs induce a common apoptotic program, such that mutations in this program can also produce multidrug resistance. However, a thorough evaluation of the contribution of apoptotic defects to this "postdamage" drug resistant phenotype is technically complicated, and this has led to uncertainty about the overall significance of apoptosis in therapy-induced cell death. For example, correlative analyses using patient specimens are limited by unknown background mutations in the biopsy material, and assays using cancer cell lines can be biased by unphysiological conditions. We sought to circumvent these restrictions by utilizing a tractable transgenic cancer model to examine the impact of apoptosis on treatment outcome. Here we discuss potential caveats of cell culture based assays, highlight features of genetically engineered mice as potential model systems, and describe a tractable transgenic mouse model to study drug responses in a series of primary lymphomas with genetically defined lesions treated at their natural site.

Title: The cell cycle in polyploid megakaryocytes is associated with reduced activity of cyclin B1-dependent cdc2 kinase. Content: The platelet precursor, the megakaryocyte, matures to a polyploid cell as a result of DNA replication in the absence of mitosis (endomitosis). The factors controlling endomitosis are accessible to analysis in our megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocytes of transgenic mice. We aimed to define whether endomitosis consists of a continuous phase of DNA synthesis (S) or of S phases interrupted by gaps. Analysis of the cell cycle in MegT cells revealed that, upon inactivation of large T antigen, the cells shifted from a mitotic cell cycle to an endomitotic cell cycle consisting of S/Gap phases. The level of the G1/S cyclin, cyclin A, as well as of the G1 phase cyclin, cyclin D3, were elevated at the onset of DNA synthesis, either in MegT cells undergoing a mitotic cell cycle or during endomitosis. In contrast, the level of the mitotic cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected during the mitotic cell cycle or during endomitosis; however, cyclin B1-dependent Cdc2 kinase activity was largely abolished in the polyploid cells. Fibroblasts immortalized with the same heat-labile oncogene do not display reduced levels of cyclin B1 upon shifting to high temperature nor do they become polyploid, indicating that reduced levels of cyclin B1 is a property of megakaryocytes and not of the T-antigen mutant. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1.

Title: Stromal-epithelial metabolic coupling in cancer: integrating autophagy and metabolism in the tumor microenvironment. Content: Cancer cells do not exist as pure homogeneous populations in vivo. Instead they are embedded in "cancer cell nests" that are surrounded by stromal cells, especially cancer associated fibroblasts. Thus, it is not unreasonable to suspect that stromal fibroblasts could influence the metabolism of adjacent cancer cells, and visa versa. In accordance with this idea, we have recently proposed that the Warburg effect in cancer cells may be due to culturing cancer cells by themselves, out of their normal stromal context or tumor microenvironment. In fact, when cancer cells are co-cultured with fibroblasts, then cancer cells increase their mitochondrial mass, while fibroblasts lose their mitochondria. An in depth analysis of this phenomenon reveals that aggressive cancer cells are "parasites" that use oxidative stress as a "weapon" to extract nutrients from surrounding stromal cells. Oxidative stress in fibroblasts induces the autophagic destruction of mitochondria, by mitophagy. Then, stromal cells are forced to undergo aerobic glycolysis, and produce energy-rich nutrients (such as lactate and ketones) to "feed" cancer cells. This mechanism would allow cancer cells to seed anywhere, without blood vessels as a food source, as they could simply induce oxidative stress wherever they go, explaining how cancer cells survive during metastasis. We suggest that stromal catabolism, via autophagy and mitophagy, fuels the anabolic growth of tumor cells, promoting tumor progression and metastasis. We have previously termed this new paradigm "The Autophagic Tumor Stroma Model of Cancer Metabolism", or the "Reverse Warburg Effect". We also discuss how glutamine addiction (glutaminolysis) in cancer cells fits well with this new model, by promoting oxidative mitochondrial metabolism in aggressive cancer cells.

Title: Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1. Content: Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Title: Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease Content: Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Title: Transcriptional integration of mitochondrial biogenesis. Content: Gene regulatory factors encoded by the nuclear genome are essential for mitochondrial biogenesis and function. Some of these factors act exclusively within the mitochondria to regulate the control of mitochondrial transcription, translation, and other functions. Others govern the expression of nuclear genes required for mitochondrial metabolism and organelle biogenesis. The peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family of transcriptional coactivators play a major role in transducing and integrating physiological signals governing metabolism, differentiation, and cell growth to the transcriptional machinery controlling mitochondrial functional capacity. Thus, the PGC-1 coactivators serve as a central component of the transcriptional regulatory circuitry that coordinately controls the energy-generating functions of mitochondria in accordance with the metabolic demands imposed by changing physiological conditions, senescence, and disease.

Title: Infection and apoptosis as a combined inflammatory trigger. Content: While inflammatory phagocytosis of microbial pathogens and non-inflammatory phagocytosis of apoptotic cells have each been studied extensively, the consequences of innate immune recognition of host cells undergoing apoptosis as a direct result of infection are unclear. In this situation, the innate immune system is confronted with mixed signals, those from apoptotic cells and those from the infecting pathogen. Nuclear receptor activation has been implicated downstream of apoptotic cell recognition while Toll-like receptors are the prototypical inflammatory receptors engaged during infection. When the two signals combine, a new set of events takes place beginning with transrepression of a subset of inflammatory-response genes and ending with the induction of a T helper-17 adaptive immune response. This response is best suited for clearing the infecting pathogen and repairing the damage that occurred to the host tissue during infection.

Title: Functional reprogramming of polyploidization in megakaryocytes. Content: Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells.

Title: The psychiatric manifestations of mitochondrial disorders: a case and review of the literature. Content: OBJECTIVE Mitochondrial disorders are caused by gene mutations in mitochondrial or nuclear DNA and affect energy-dependent organs such as the brain. Patients with psychiatric illness, particularly those with medical comorbidities, may have primary mitochondrial disorders. To date, this issue has received little attention in the literature, and mitochondrial disorders are likely underdiagnosed in psychiatric patients. DATA SOURCES This article describes a patient who presented with borderline personality disorder and treatment-resistant depression and was ultimately diagnosed with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 3271. We also searched the literature for all case reports of patients with mitochondrial disorders who initially present with prominent psychiatric symptoms by using MEDLINE (from 1948-February 2011), Embase (from 1980-February 2011), PsycINFO (from 1806-February 2011), and the search terms mitochondrial disorder, mitochondria, psychiatry, mental disorders, major depression, anxiety, schizophrenia, and psychosis. STUDY SELECTION Fifty cases of mitochondrial disorders with prominent psychiatric symptomatology were identified. DATA EXTRACTION Information about the psychiatric presentation of the cases was extracted. This information was combined with our case, the most common psychiatric manifestations of mitochondrial disorders were identified, and the important diagnostic and treatment implications for patients with psychiatric illness were reviewed. RESULTS The most common psychiatric presentations in the cases of mitochondrial disorders included mood disorder, cognitive deterioration, psychosis, and anxiety. The most common diagnosis (52% of cases) was a MELAS mutation. Other genetic mitochondrial diagnoses included polymerase gamma mutations, Kearns-Sayre syndrome, mitochondrial DNA deletions, point mutations, twinkle mutations, and novel mutations. CONCLUSIONS Patients with mitochondrial disorders can present with primary psychiatric symptomatology, including mood disorder, cognitive impairment, psychosis, and anxiety. Psychiatrists need to be aware of the clinical features that are indicative of a mitochondrial disorder, investigate patients with suggestive presentations, and be knowledgeable about the treatment implications of the diagnosis.

Title: Human IRGM Regulates Autophagy and Its Cell-Autonomous Immunity Functions Through Mitochondria Content: IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease.

Title: Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons Content: Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.

Title: ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast Content: Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER-mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER-mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER-mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells. DOI:http://dx.doi.org/10.7554/eLife.00422.001.

Title: Non-canonical PI3K-Cdc42-Pak-Mek-Erk Signaling Promotes Immune-Complex-Induced Apoptosis in Human Neutrophils Content: Neutrophils are peripheral blood leukocytes that represent the first line of immune cell defense against bacterial and fungal infections but are also crucial players in the generation of the inflammatory response. Many neutrophil cell surface receptors regulate important cellular processes via activation of agonist-activated PI3Ks. We show here that activation of human neutrophils with insoluble immune complexes drives a previously uncharacterized, PI3K-dependent, non-canonical, pro-apoptotic signaling pathway, FcγR-PI3Kβ/δ-Cdc42-Pak-Mek-Erk. This is a rare demonstration of Ras/Raf-independent activation of Erk and of PI3K-mediated activation of Cdc42. In addition, comparative analysis of immune-complex- and fMLF-induced signaling uncovers key differences in pathways used by human and murine neutrophils. The non-canonical pathway we identify in this study may be important for the resolution of inflammation in chronic inflammatory diseases that rely on immune-complex-driven neutrophil activation.

Title: Metabolic rescue in pluripotent cells from patients with mtDNA disease Content: Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Title: Mitochondrial Dysfunction Leads to Nuclear Genome Instability via an Iron-Sulfur Cluster Defect Content: Mutations and deletions in the mitochondrial genome (mtDNA), as well as instability of the nuclear genome, are involved in multiple human diseases. Here, we report that in Saccharomyces cerevisiae, loss of mtDNA leads to nuclear genome instability, through a process of cell-cycle arrest and selection we define as a cellular crisis. This crisis is not mediated by the absence of respiration, but instead correlates with a reduction in the mitochondrial membrane potential. Analysis of cells undergoing this crisis identified a defect in iron-sulfur cluster (ISC) biogenesis, which requires normal mitochondrial function. We found that downregulation of nonmitochondrial ISC protein biogenesis was sufficient to cause increased genomic instability in cells with intact mitochondrial function. These results suggest mitochondrial dysfunction stimulates nuclear genome instability by inhibiting the production of ISC-containing protein(s), which are required for maintenance of nuclear genome integrity. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

Title: Genetic Drift Can Compromise Mitochondrial Replacement by Nuclear Transfer in Human Oocytes. Content: Replacement of mitochondria through nuclear transfer between oocytes of two different women has emerged recently as a strategy for preventing inheritance of mtDNA diseases. Although experiments in human oocytes have shown effective replacement, the consequences of small amounts of mtDNA carryover have not been studied sufficiently. Using human mitochondrial replacement stem cell lines, we show that, even though the low levels of heteroplasmy introduced into human oocytes by mitochondrial carryover during nuclear transfer often vanish, they can sometimes instead result in mtDNA genotypic drift and reversion to the original genotype. Comparison of cells with identical oocyte-derived nuclear DNA but different mtDNA shows that either mtDNA genotype is compatible with the nucleus and that drift is independent of mitochondrial function. Thus, although functional replacement of the mitochondrial genome is possible, even low levels of heteroplasmy can affect the stability of the mtDNA genotype and compromise the efficacy of mitochondrial replacement.

Title: Direct visualization of the endomitotic cell cycle in living megakaryocytes: differential patterns in low and high ploidy cells. Content: Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (>or=8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Title: Control of Apoptosis by Asymmetric Cell Division Content: Asymmetric cell division and apoptosis (programmed cell death) are two fundamental processes that are important for the development and function of multicellular organisms. We have found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the MIDA1-like protein GlsA of the alga Volvox carteri, as well as the Snail-related proteins Snail, Escargot, and Worniu of Drosophila melanogaster, have previously been implicated in asymmetric cell division. Therefore, C. elegans dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail may be components of a pathway involved in asymmetric cell division that is conserved throughout the plant and animal kingdoms. Furthermore, based on our results, we propose that this pathway directly controls the apoptotic fate in C. elegans, and possibly other animals as well.

Title: Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency. Content: Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.

Title: Glucose Metabolism Inhibits Apoptosis in Neurons and Cancer Cells by Redox Inactivation of Cytochrome c Content: Neurons and cancer cells use glucose extensively, yet the precise advantage of this adaptation remains unclear. These two seemingly disparate cell types also show an increased regulation of the apoptotic pathway, which allows for their long-term survival. Here we show that both neurons and cancer cells strictly inhibit cytochrome c-mediated apoptosis by a mechanism dependent on glucose metabolism. We report that the pro-apoptotic activity of cytochrome c is influenced by its redox state and that increases in reactive oxygen species (ROS) following an apoptotic insult lead to the oxidation and activation of cytochrome c. In healthy neurons and cancer cells, however, cytochrome c is reduced and held inactive by intracellular glutathione (GSH), generated as a result of glucose metabolism by the pentose phosphate pathway. These results uncover a striking similarity in apoptosis regulation between neurons and cancer cells and provide insight into an adaptive advantage offered by the Warburg effect for cancer cell evasion of apoptosis and for long-term neuronal survival.

Title: A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs Content: In mammalian mitochondria, 22 species of tRNAs encoded in mitochondrial DNA play crucial roles in the translation of 13 essential subunits of the respiratory chain complexes involved in oxidative phosphorylation. Following transcription, mitochondrial tRNAs are modified by nuclear-encoded tRNA-modifying enzymes. These modifications are required for the proper functioning of mitochondrial tRNAs (mt tRNAs), and the absence of these modifications can cause pathological consequences. To date, however, the information available about these modifications has been incomplete. To address this issue, we isolated all 22 species of mt tRNAs from bovine liver and comprehensively determined the post-transcriptional modifications in each tRNA by mass spectrometry. Here, we describe the primary structures with post-transcriptional modifications of seven species of mt tRNAs which were previously uncharacterized, and provide revised information regarding base modifications in five other mt tRNAs. In the complete set of bovine mt tRNAs, we found 15 species of modified nucleosides at 118 positions (7.48% of total bases). This result provides insight into the molecular mechanisms underlying the decoding system in mammalian mitochondria and enables prediction of candidate tRNA-modifying enzymes responsible for each modification of mt tRNAs.

Title: Non-receptor tyrosine kinase Etk is involved in the apoptosis of small cell lung cancer cells. Content: Epithelial and endothelial tyrosine kinase (Etk), also known as Bmx (bone marrow X kinase) plays an important role in apoptosis of epithelial cells. The goal of this study was to investigate whether Etk is involved in apoptosis of small cell lung cancer (SCLC) cells and correlated with the expression levels of apoptosis-associated proteins such as Bcl-2, Bcl-X(L) and p53. One hundred and seventy-one cases of lung cancer specimens including seventy-one SCLCs and one hundred NSCLCs were immunostained for Etk, Bcl-2, Bcl-X(L) and p53. Parental SCLC H446 cell line, and its subline (H446-Etk) that overexpresses Etk, were used to study the role of Etk in apoptosis induced by doxorubicin. It was found that high expression of Etk occurs in 74.6% of SCLC cases, but only in 40% of NSCLC cases, and there is marked difference in the expression levels of Bcl-2, Bcl-X(L) and p53 between Etk-positive and Etk-negative SCLC cases. Furthermore, the levels of Bcl-2 and Bcl-X(L) significantly increased in H446-Etk cells than that in H446 cells after doxorubicin treatment, and were positively associated with Etk expression. However, p53 did not correspond with Etk expression although its expression decreased greatly with apoptosis both in H446-Etk and H446 cells. After doxorubicin treatment, the cell viability was significantly higher in H446-Etk cells than in parental H446 cells. Downregulation of Etk by Etk siRNA sensitized H446 cells to doxorubicin. Our results indicate that upregulation of tyrosine kinase Etk may be a new mechanism involved in protection of SCLC cells from apoptosis. Bcl-2 and Bcl-X(L) but not p53 may contribute to doxorubicin-induced apoptosis through Etk pathway.

Title: Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53 Content: The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Title: Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas. Content: Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.

Title: The Inhibition of TDP-43 Mitochondrial Localization Blocks Its Neuronal Toxicity Content: Genetic mutations in TAR DNA-binding protein 43 (TARDBP, also known as TDP-43) cause amyotrophic lateral sclerosis (ALS), and an increase in the presence of TDP-43 (encoded by TARDBP) in the cytoplasm is a prominent histopathological feature of degenerating neurons in various neurodegenerative diseases. However, the molecular mechanisms by which TDP-43 contributes to ALS pathophysiology remain elusive. Here we have found that TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Disease-associated mutations increase TDP-43 mitochondrial localization. In mitochondria, wild-type (WT) and mutant TDP-43 preferentially bind mitochondria-transcribed messenger RNAs (mRNAs) encoding respiratory complex I subunits ND3 and ND6, impair their expression and specifically cause complex I disassembly. The suppression of TDP-43 mitochondrial localization abolishes WT and mutant TDP-43-induced mitochondrial dysfunction and neuronal loss, and improves phenotypes of transgenic mutant TDP-43 mice. Thus, our studies link TDP-43 toxicity directly to mitochondrial bioenergetics and propose the targeting of TDP-43 mitochondrial localization as a promising therapeutic approach for neurodegeneration.

Title: Folate affects apoptosis in human trophoblastic cells. Content: Effects of folate deficiency on the rate of apoptosis in human cytotrophoblastic cells has been investigated. Apoptosis was determined in cytotrophoblastic cells after culture in 1. control medium, 2. folate-free medium and 3. folate-free medium plus 10% fetal calf serum. Apoptosis rates in cells cultured in mediums 2 and 3 were significantly higher than those cultured in the control medium (P<0.02 and P<0.03, respectively). In conclusion, human cytotrophoblastic cells show a significantly increased rate of apoptosis in vitro after culture in a folate-free medium. Possible explanations for the association between folate deficiency and pregnancy complications are suggested.

Title: Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity. Content: Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.

Title: The mitochondrial RNA-binding protein GRSF1 localizes to RNA granules and is required for posttranscriptional mitochondrial gene expression. Content: RNA-binding proteins are at the heart of posttranscriptional gene regulation, coordinating the processing, storage, and handling of cellular RNAs. We show here that GRSF1, previously implicated in the binding and selective translation of influenza mRNAs, is targeted to mitochondria where it forms granules that colocalize with foci of newly synthesized mtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, and the long noncoding RNAs for cytb and ND5, each of which contains multiple consensus binding sequences. RNAi-mediated knockdown of GRSF1 leads to alterations in mitochondrial RNA stability, abnormal loading of mRNAs and lncRNAs on the mitochondrial ribosome, and impaired ribosome assembly. This results in a specific protein synthesis defect and a failure to assemble normal amounts of the oxidative phosphorylation complexes. These data implicate GRSF1 as a key regulator of posttranscriptional mitochondrial gene expression.

Title: Uncoupling Mitochondrial Respiration for Diabesity. Content: Until recently, the mechanism of adaptive thermogenesis was ascribed to the expression of uncoupling protein 1 (UCP1) in brown and beige adipocytes. UCP1 is known to catalyze a proton leak of the inner mitochondrial membrane, resulting in uncoupled oxidative metabolism with no production of adenosine triphosphate and increased energy expenditure. Thus increasing brown and beige adipose tissue with augmented UCP1 expression is a viable target for obesity-related disorders. Recent work demonstrates an UCP1-independent pathway to uncouple mitochondrial respiration. A secreted enzyme, PM20D1, enriched in UCP1+ adipocytes, exhibits catalytic and hydrolytic activity to reversibly form N-acyl amino acids. N-acyl amino acids act as endogenous uncouplers of mitochondrial respiration at physiological concentrations. Administration of PM20D1 or its products, N-acyl amino acids, to diet-induced obese mice improves glucose tolerance by increasing energy expenditure. In short-term studies, treated animals exhibit no toxicity while experiencing 10% weight loss primarily of adipose tissue. Further study of this metabolic pathway may identify novel therapies for diabesity, the disease state associated with diabetes and obesity.

Title: Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes Content: Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes. Expression of genes involved in ER stress was evaluated in insulin-secreting MIN6 cells exposed to elevated lipids, in islets isolated from db/db mice and in pancreas sections of humans with type 2 diabetes. Overproduction of the ER chaperone heat shock 70 kDa protein 5 (HSPA5, previously known as immunoglobulin heavy chain binding protein [BIP]) was performed to assess whether attenuation of ER stress affected lipid-induced apoptosis. We demonstrated that the pro-apoptotic fatty acid palmitate triggers a comprehensive ER stress response in MIN6 cells, which was virtually absent using non-apoptotic fatty acid oleate. Time-dependent increases in mRNA levels for activating transcription factor 4 (Atf4), DNA-damage inducible transcript 3 (Ddit3, previously known as C/EBP homologous protein [Chop]) and DnaJ homologue (HSP40) C3 (Dnajc3, previously known as p58) correlated with increased apoptosis in palmitate- but not in oleate-treated MIN6 cells. Attenuation of ER stress by overproduction of HSPA5 in MIN6 cells significantly protected against lipid-induced apoptosis. In islets of db/db mice, a variety of marker genes of ER stress were also upregulated. Increased processing (activation) of X-box binding protein 1 (Xbp1) mRNA was also observed, confirming the existence of ER stress. Finally, we observed increased islet protein production of HSPA5, DDIT3, DNAJC3 and BCL2-associated X protein in human pancreas sections of type 2 diabetes subjects. Our results provide evidence that ER stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure.

Title: Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. Content: One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.

Title: Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and the bisphosphonate zoledronic acid against prostate cancer cells in vitro. Content: Bisphosphonates are widely used agents for the treatment of malignant bone disease. They inhibit osteoclast-mediated bone resorption and can have direct effects on cancer cells. In this study, we investigated whether the anticancer activity of the third-generation bisphosphonate zoledronic acid (ZOL) could be enhanced by combination with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). We found that ZOL and SAHA cooperated to induce cell death in the prostate cancer cell lines LNCaP and PC-3. The effect was synergistic, as evidenced by combination index isobologram analysis. ZOL and SAHA synergized to induce dissipation of the mitochondrial transmembrane potential, to activate caspase-3, and to trigger DNA fragmentation, showing that the combination of ZOL and SAHA resulted in the initiation of apoptosis. Because ZOL acts by inhibiting the mevalonate pathway, thereby preventing protein prenylation, we explored whether the mevalonate pathway was also the target of the cooperative action of ZOL and SAHA. We found that geranylgeraniol, but not farnesol, significantly reduced ZOL/SAHA-induced cell death, indicating that the synergistic action of the agents was due to the inhibition of geranylgeranylation. Consistently, a direct inhibitor of geranylgeranylation, GGTI-298, synergized with SAHA to induce cell death, whereas an inhibitor of farnesylation, FTI-277, had no effect. In addition, SAHA synergized with mevastatin, an inhibitor of the proximal enzyme in the mevalonate pathway. These in vitro findings provide a rationale for an in vivo exploration into the potential of combining SAHA and ZOL, or other inhibitors of the mevalonate pathway, as an effective strategy for anticancer therapy.

Title: Cyanidin-3-rutinoside, a natural polyphenol antioxidant, selectively kills leukemic cells by induction of oxidative stress. Content: Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including anti-mutagenesis and anticarcinogenesis, which are generally attributed to their antioxidant activities. We studied the effects and the mechanisms of the most common type of anthocyanins, cyanidin-3-rutinoside, in several leukemia and lymphoma cell lines. We found that cyanidin-3-rutinoside extracted and purified from the black raspberry cultivar Jewel induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Paradoxically, this compound induced the accumulation of peroxides, which are involved in the induction of apoptosis in HL-60 cells. In addition, cyanidin-3-rutinoside treatment resulted in reactive oxygen species (ROS)-dependent activation of p38 MAPK and JNK, which contributed to cell death by activating the mitochondrial pathway mediated by Bim. Down-regulation of Bim or overexpression of Bcl-2 or Bcl-x(L) considerably blocked apoptosis. Notably, cyanidin-3-rutinoside treatment did not lead to increased ROS accumulation in normal human peripheral blood mononuclear cells and had no cytotoxic effects on these cells. These results indicate that cyanidin-3-rutinoside has the potential to be used in leukemia therapy with the advantages of being widely available and selective against tumors.

Title: Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl, and the PP2A/B55α,δ phosphatase. Content: Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.

Title: Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos. Content: Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

Title: Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. Content: The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.

Title: Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae. Content: Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

Title: Bcl2 Regulation by the Melanocyte Master Regulator Mitf Modulates Lineage Survival and Melanoma Cell Viability Content: Kit/SCF signaling and Mitf-dependent transcription are both essential for melanocyte development and pigmentation. To identify Mitf-dependent Kit transcriptional targets in primary melanocytes, microarray studies were undertaken. Among identified targets was BCL2, whose germline deletion produces melanocyte loss and which exhibited phenotypic synergy with Mitf in mice. BCL2's regulation by Mitf was verified in melanocytes and melanoma cells and by chromatin immunoprecipitation of the BCL2 promoter. Mitf also regulates BCL2 in osteoclasts, and both Mitf(mi/mi) and Bcl2(-/-) mice exhibit severe osteopetrosis. Disruption of Mitf in melanocytes or melanoma triggered profound apoptosis susceptible to rescue by BCL2 overexpression. Clinically, primary human melanoma expression microarrays revealed tight nearest neighbor linkage for MITF and BCL2. This linkage helps explain the vital roles of both Mitf and Bcl2 in the melanocyte lineage and the well-known treatment resistance of melanoma.

Title: Identification of a novel p53 functional domain that is necessary for mediating apoptosis. Content: The ability of p53 to induce apoptosis requires its sequence-specific DNA binding activity; however, the transactivation-deficient p53(Gln22-Ser23) can still induce apoptosis. Previously, we have shown that the region between residues 23 and 97 in p53 is necessary for such activity. In an effort to more precisely map a domain necessary for apoptosis within the N terminus, we found that deletion of the N-terminal 23 amino acids compromises, but does not abolish, p53 induction of apoptosis. Surprisingly, p53(Delta1-42), which lacks the N-terminal 42 amino acids and the previously defined activation domain, retains the ability to induce apoptosis to an even higher level than wild-type p53. A more extensive deletion, which eliminates the N-terminal 63 amino acids, renders p53 completely inert in mediating apoptosis. In addition, we found that both p53(Delta1-42) and p53(Gln22-Ser23) can activate a subset of cellular p53 targets. Furthermore, we showed that residues 53 and 54 are critical for the apoptotic and transcriptional activities of both p53(Delta1-42) and p53(Gln22-Ser23). Taken together, these data suggest that within residues 43-63 lie an apoptotic domain as well as another transcriptional activation domain. We therefore postulate that the apoptotic activity in p53(Gln22-Ser23) and p53(Delta1-42) is still transcription-dependent.

Title: Antiapoptotic BCL-2 is required for maintenance of a model leukemia. Content: Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).

Title: Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils. Content: The recently identified Fas antigen (Ag) is a cell surface molecule that can mediate apoptosis. The cytoplasmic product of proto-oncogene bcl-2 has been shown to prolong the cellular survival by inhibiting apoptosis. To elucidate the physiologic significance of expression of both molecules, we examined the expression of Fas Ag and bcl-2 on blood leukocyte populations and evaluated their sensitivity to the cytolytic action of anti-Fas antibody. Although Fas Ag was expressed on a fraction of lymphocytes, both neutrophils and monocytes expressed Fas Ag constitutively. In contrast, there was marked difference among these leukocytes regarding bcl-2 expression. Lymphocytes expressed bcl-2 intensely, but monocytes showed weaker bcl-2 expression, and neutrophils were essentially absent for bcl-2 expression. Seemingly reflecting this lack of bcl-2-expression, neutrophils more easily underwent apoptotic cell death in vitro as compared with monocytes and lymphocytes. We showed that anti-Fas antibody affectively accelerated apoptotic cell death in neutrophils. However, the apoptosis-inducing effect of anti-Fas antibody was minimal on monocytes, and lymphocytes were resistant to this antibody. These results suggest that anti-Fas-mediated cell death may, in part, be determined by bcl-2 expression status in Fas+ lymphoid and hematopoietic cells.

Title: Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint Content: Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The “destruction box” (D-box) of cyclin A is 10–20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.

Title: Mechanisms of Renal Cell Apoptosis Induced by Cyclosporine A: A Systematic Review of in vitro Studies Content: Background:Chronic cyclosporine A (CsA) nephrotoxicity (CCN) is a major cause of chronic renal dysfunction and has no effective clinical interventions yet. Objective: To reveal the mechanisms of renal cell apoptosis in CCN, we analyzed all in vitro studies of such mechanisms. Methods: We collected all in vitro studies about the mechanisms of renal cell apoptosis induced by CsA in Medline (1966 to July 2010), Embase (1980 to July 2010) and ISI (1986 to July 2010), evaluated their quality according to in vitro standards and extracted data following the PICOS principles and synthesized the data. Results: First,CsA could upregulate Fas and Fas-L expression, increase FADD and apoptosis enzymes (caspase-2, -3, -4, -7, -8, -9 and -10) and downregulate the Bcl-2 and Bcl-xL. Second, CsA could induce oxidative stress and damage the antioxidant defense system. Third, CsA could increase the expression of HERP, GRP78 and CHOP. Fourth, CsA could induce renal cell apoptosis and increase their iNOS and p53 expression in cultured cells. Conclusions: At least four pathways are involved in renal cell apoptosis induced by CsA in different cell species. Caspases might be their final common pathway in vitro. They might all provide potential points for interventions, but these need to be confirmed in vivo.

Title: Phosphorylation of ATM by Cdk5 mediates DNA damage signaling and regulates neuronal death Content: The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) has a central role in coordinating DNA damage responses, including cell-cycle checkpoint control, DNA repair and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. However, the mechanism by which DNA damage activates ATM is poorly understood. Here we show that Cdk5 (cyclin-dependent kinase 5), activated by DNA damage, directly phosphorylates ATM at Ser 794 in post-mitotic neurons. Phosphorylation at Ser 794 precedes, and is required for, ATM autophosphorylation at Ser 1981, and activates ATM kinase activity. The Cdk5-ATM signal regulates phosphorylation and function of the ATM targets p53 and H2AX. Interruption of the Cdk5-ATM pathway attenuates DNA-damage-induced neuronal cell cycle re-entry and expression of the p53 targets PUMA and Bax, protecting neurons from death. Thus, activation of Cdk5 by DNA damage serves as a critical signal to initiate the ATM response and regulate ATM-dependent cellular processes.

Title: Membrane topology of the 60-kDa Oxa1p homologue from Escherichia coli. Content: We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule.

Title: IAP regulation of metastasis. Content: Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-kappaB, which in turn leads to increased fibronectin gene expression, signaling by beta1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer.

Title: Latent membrane protein 1 of Epstein–Barr virus coordinately regulates proliferation with control of apoptosis Content: Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein–Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBV's induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1's signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1's signals are cell cycle arrested and survive quiescently. LMP1's activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1's induction and maintenance of proliferation.

Title: Doxycycline induces apoptosis in PANC-1 pancreatic cancer cells. Content: BACKGROUND Tetracyclines such as doxycycline are reported to possess cytotoxic activity against mammalian tumor cells, but the mechanism of their effects on cell proliferation remains unclear. MATERIALS AND METHODS The antitumor effect of doxycycline was investigated in human pancreatic cancer cell line, PANC-1. We also investigated the effect of doxycycline on expression of a potent proangiogenic factor, interleukin (IL)-8. RESULTS In excess of 20 microg/ml, cytotoxic effects of doxycycline were accompanied by G(1)-S cell cycle arrest and DNA fragmentation in PANC-1 cells. Doxycycline consistently activated transcription of p53, p21 and Fas/FasL-cascade-related genes, while reducing the expression of Bcl-xL and Mcl-1. Doxycycline (5 microg/ml) below the cytotoxic level suppressed endogenous and paclitaxel-induced IL-8 expression. In the mouse xenograft model, doxycycline treatment was shown to suppress tumor growth by 80%. CONCLUSION These data suggest that doxycycline exerts its antitumor effect by activating proapoptotic genes, inhibiting IL-8 expression, and suppressing antiapoptotic genes.

Title: Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin. Content: Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.

Title: Noxa controls Mule-dependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction. Content: The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.

Title: Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Content: Programmed cell death (PCD), referring to apoptosis, autophagy and programmed necrosis, is proposed to be death of a cell in any pathological format, when mediated by an intracellular program. These three forms of PCD may jointly decide the fate of cells of malignant neoplasms; apoptosis and programmed necrosis invariably contribute to cell death, whereas autophagy can play either pro-survival or pro-death roles. Recent bulk of accumulating evidence has contributed to a wealth of knowledge facilitating better understanding of cancer initiation and progression with the three distinctive types of cell death. To be able to decipher PCD signalling pathways may aid development of new targeted anti-cancer therapeutic strategies. Thus in this review, we present a brief outline of apoptosis, autophagy and programmed necrosis pathways and apoptosis-related microRNA regulation, in cancer. Taken together, understanding PCD and the complex interplay between apoptosis, autophagy and programmed necrosis may ultimately allow scientists and clinicians to harness the three types of PCD for discovery of further novel drug targets, in the future cancer treatment.

Title: Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone. Content: Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 nM rotenone increased mitochondrial superoxide (O2*-) levels and potentiated glutamate-induced cytoplasmic Ca2+ deregulation, the first irreversible stage of necrotic cell death. However, the potent cell-permeant O2*- trap manganese tetrakis (N-ethylpyridinium-2yl) porphyrin failed to prevent the effects of the inhibitor. The bioenergetic consequences of rotenone addition were quantified by monitoring cell respiration. Glutamate activation of NMDA receptors used the full respiratory capacity of the in situ mitochondria, and >80% of the glutamate-stimulated respiration was attributable to increased cellular ATP demand. Rotenone at 20 nM inhibited basal and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated cell respiration and caused respiratory failure in the presence of glutamate. ATP synthase inhibition by oligomycin was also toxic in the presence of glutamate. We conclude that the cell vulnerability in the rotenone model of partial complex I deficiency under these specific conditions is primarily determined by spare respiratory capacity rather than oxidative stress.

Title: Key role of Ubc5 and lysine-63 polyubiquitination in viral activation of IRF3. Content: The mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, and CARDIF) is essential for innate immune response against RNA viruses. MAVS transduces signals from the cytosolic RIG-I-like receptors, which bind to viral RNAs. But how MAVS activates downstream transcription factors such as IRF3 to induce type-I interferons is not well understood. We have established a cell-free system in which mitochondria derived from virus-infected cells activate IRF3 in the cytosol. Fractionation of the cytosol led to the identification of Ubc5 as a ubiquitin-conjugating enzyme (E2) required for IRF3 activation. Using an inducible RNAi strategy, we demonstrate that catalytically active Ubc5 is required for IRF3 activation by viral infection. The activation of IRF3 also requires two ubiquitin-binding domains of NEMO. Furthermore, we show that replacement of endogenous ubiquitin with its K63R mutant abolishes viral activation of IRF3, demonstrating that K63 polyubiquitination plays a key role in IRF3 activation.

Title: Circulating mitochondrial DNA in serum of patients with granulomatosis with polyangiitis. Content: Neutrophil is a key cell in pathophysiology of granulomatosis with polyangiitis. Recently, neutrophil extracellular traps were described in this disease. Mitochondrial DNA is also released during traps formation. We measured circulating cell-free mitochondrial and genomic DNA in serum of patients with granulomatosis with polyangiitis. Subjects with the disease (14 active and 11 in remission stage) and 10 healthy controls were enrolled. Quantitative real-time polymerase chain reaction (PCR) was used to measure 79 base pairs (bp) and 230 bp mtDNA fragments. Alu repeats were quantified to evaluate abundance of nuclear DNA in serum at the presence of plasmid control. Both fragments of mtDNA (79 bp and 230 bp) and genomic DNA were elevated significantly in granulomatosis with polyangiitis compared to controls. Only the shorter 79 bp mtDNA correlated with active stage of granulomatosis with polyangiitis and clinical symptoms. A mechanism of extracellular release of mitochondrial DNA accompanies the active stage of the disease. Circulating mtDNA is extremely high in untreated patients. This suggests that biomarker properties of mtDNA are useful for monitoring of treatment.

Title: Metabolic regulation by the mitochondrial phosphatase PTPMT1 is required for hematopoietic stem cell differentiation. Content: The regulation and coordination of mitochondrial metabolism with hematopoietic stem cell (HSC) self-renewal and differentiation is not fully understood. Here we report that depletion of PTPMT1, a PTEN-like mitochondrial phosphatase, in inducible or hematopoietic-cell-specific knockout mice resulted in hematopoietic failure due to changes in the cell cycle and a block in the differentiation of HSCs. Surprisingly, the HSC pool was increased by ∼40-fold in PTPMT1 knockout mice. Reintroduction of wild-type PTPMT1, but not catalytically deficient PTPMT1 or truncated PTPMT1 lacking mitochondrial localization, restored differentiation capabilities of PTPMT1 knockout HSCs. Further analyses demonstrated that PTPMT1 deficiency altered mitochondrial metabolism and that phosphatidylinositol phosphate substrates of PTPMT1 directly enhanced fatty-acid-induced activation of mitochondrial uncoupling protein 2. Intriguingly, depletion of PTPMT1 from myeloid, T lymphoid, or B lymphoid progenitors did not cause any defects in lineage-specific knockout mice. This study establishes a crucial role of PTPMT1 in the metabolic regulation of HSC function.

Title: Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival Content: Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand- or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.

Title: The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities Content: According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.

Title: Efficient Mitochondrial Genome Editing by CRISPR/Cas9 Content: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

Title: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells Content: The aim of this study was to investigate the mechanism through which Sphingosine kinase-1 (SPHK1) exerts its anti-apoptosis activity in glioma cancer cells. We here report that dysregulation of SPHK1 alters the sensitivity of glioma to apoptosis both in vitro and in vivo. Further mechanistic study examined the expression of Bcl-2 family members, including Bcl-2, Mcl-1, Bax and Bim, in SPHK1-overexpressing glioma cells and revealed that only pro-apoptotic Bim was downregulated by SPHK1. Moreover, the transcriptional level of Bim was also altered by SPHK1 in glioma cells. We next confirmed the correlation between SPHK1 and Bim expression in primary glioma specimens. Importantly, increasing SPHK1 expression in glioma cells markedly elevated Akt activity and phosphorylated inactivation of FOXO3a, which led to downregulation of Bim. A pharmacological approach showed that these effects of SPHK1 were dependent on phosphatidylinositol 3-kinase (PI3K). Furthermore, effects of SPHK1 on Akt/FOXO3a/Bim pathway could be reversed by SPHK1 specific RNA interference or SPHK1 inhibitor. Collectively, our results indicate that regulation of the Akt/FOXO3a/Bim pathway may be a novel mechanism by which SPHK1 protects glioma cells from apoptosis, thereby involved in glioma tumorigenesis.

Title: PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening. Content: The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation.

Title: Genomic stability and tumour suppression by the APC/C cofactor Cdh1 Content: The anaphase promoting complex or cyclosome (APC/C) is a ubiquitin protein ligase that, together with Cdc20 or Cdh1, targets cell-cycle proteins for degradation. APC/C–Cdh1 specifically promotes protein degradation in late mitosis and G1. Mutant embryos lacking Cdh1 die at E9.5–E10.5 due to defects in the endoreduplication of trophoblast cells and placental malfunction. This lethality is prevented when Cdh1 is expressed in the placenta. Cdh1-deficient cells proliferate inefficiently and accumulate numeric and structural chromosomal aberrations, indicating that Cdh1 contributes to the maintenance of genomic stability. Cdh1 heterozygous animals show increased susceptibility to spontaneous tumours, suggesting that Cdh1 functions as a haploinsufficient tumour suppressor. These heterozygous mice also show several defects in behaviour associated with increased proliferation of stem cells in the nervous system. These results indicate that Cdh1 is required for preventing unscheduled proliferation of specific progenitor cells and protecting mammalian cells from genomic instability.

Title: Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion. Content: Sustained signaling from the T cell receptor (TCR) and costimulatory molecules is thought necessary for generating high numbers of effector T cells. Here, we show that Survivin is controlled in peripheral T cells by OX40 cosignaling via sustained PI3k and PKB activation. Survivin is induced by OX40 independent of mitotic progression in late G1, and blocking Survivin suppresses S-phase transition and division of T cells and leads to apoptosis. Moreover, Survivin expression alone is sufficient to restore proliferation and to antagonize apoptosis in costimulation-deficient T cells and can rescue T cell expansion in vivo. Survivin allows effector T cells to accumulate in large numbers, but Bcl-2 family proteins are required for T cell survival after the phase of active division. Thus, sustained Survivin expression from costimulatory signaling maintains T cell division over time and regulates the extent of clonal expansion.

Title: Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria Content: Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.

Title: The flavonoid component isorhamnetin in vitro inhibits proliferation and induces apoptosis in Eca-109 cells. Content: Isorhamnetin is one member of flavonoid components which has been used in the treatment of heart disease. Recently the in vitro anti-cancer effect of isorhamnetin on human esophageal squamous carcinoma cell line Eca-109 was investigated in our lab. When Eca-109 cells were in vitro exposed to the graded doses of isorhamnetin (0-80 microg/ml) for 48 h, respectively, isorhamnetin exhibited cytostatic effect on the treated cells, with an IC(50) of 40+/-0.08 microg/ml as estimated by MTT assay. Inhibition on proliferation by isorhamnetin was detected by trypan blue exclusion assay, clone formation test, immunocytochemical assay of PCNA and (3)H-thymidine uptake analysis. Cell cycle distribution was measured by FCM. It was found that the viability of Eca-109 cells was significantly hampered by isorhamnetin. Compared with the negative control group, the treated group which was exposed to isorhamnetin had increased population in G(0)/G(1) phase from 74.6 to 84 while had a significant reduction in G(2)/M phase from 11.9 to 5.8. In addition to its cytostatic effect, isorhamnetin also showed stimulatory effect on apoptosis. Typical apoptotic morphology such as condensation and fragmentation of nuclei and blebbing membrane of the apoptotic cells could be observed through transmission electron microscope. Moreover, the sharp increase in apoptosis rate between the control and treated group were detected by FCM from 6.3 to 16.3. To explore the possible molecular mechanisms that underlie the growth inhibition and apoptosis stimulatory effects of isorhamnetin, the expressions of six proliferation- and death-related genes were detected by FCM. Expressions of bcl-2, c-myc and H-ras were downregulated whereas Bax, c-fos and p53 were upregulated. However, the in vivo experiments were required to further confirm the anti-cancer effects of isorhamnetin. In conclusion, isorhamnetin appears to be a potent drug against esophageal cancer due to its in vitro potential to not only inhibit proliferation but also induce apoptosis of Eca-109 cells.

Title: Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency III-infected cells. Content: Epstein-Barr virus (EBV) latency III infection converts B lymphocytes into lymphoblastoid cell lines (LCLs) by expressing EBV nuclear and membrane proteins, EBNAs, and latent membrane proteins (LMPs), which regulate transcription through Notch and tumor necrosis factor receptor pathways. The role of NF-kappa B in LMP1 and overall EBV latency III transcriptional effects was investigated by treating LCLs with BAY11-7082 (BAY11). BAY11 rapidly and irreversibly inhibited NF-kappa B, decreased mitochondrial membrane potential, induced apoptosis, and altered LCL gene expression. BAY11 effects were similar to those of an NF-kappa B inhibitor, Delta N-I kappa B alpha, in effecting decreased JNK1 expression and in microarray analyses. More than 80% of array elements that decreased with Delta N-I kappa B alpha expression decreased with BAY11 treatment. Newly identified NF-kappa B-induced, LMP1-induced, and EBV-induced genes included pleckstrin, Jun-B, c-FLIP, CIP4, and I kappa B epsilon. Of 776 significantly changed array elements, 134 were fourfold upregulated in EBV latency III, and 74 were fourfold upregulated with LMP1 expression alone, whereas only 28 were more than fourfold downregulated by EBV latency III. EBV latency III-regulated gene products mediate cell migration (EBI2, CCR7, RGS1, RANTES, MIP1 alpha, MIP1 beta, CXCR5, and RGS13), antigen presentation (major histocompatibility complex proteins and JAW1), mitogen-activated protein kinase pathway (DUSP5 and p62Dok), and interferon (IFN) signaling (IFN-gamma R alpha, IRF-4, and STAT1). Comparison of EBV latency III LCL gene expression to immunoglobulin M (IgM)-stimulated B cells, germinal-center B cells, and germinal-center-derived lymphomas clustered LCLs with IgM-stimulated B cells separately from germinal-center cells or germinal-center lymphoma cells. Expression of IRF-2, AIM1, ASK1, SNF2L2, and components of IFN signaling pathways further distinguished EBV latency III-infected B cells from IgM-stimulated or germinal-center B cells.

Title: IAP antagonists induce anti-tumor immunity in multiple myeloma Content: The cellular inhibitors of apoptosis (cIAP) 1 and 2 are amplified in about 3% of cancers and have been identified in multiple malignancies as being potential therapeutic targets as a result of their role in the evasion of apoptosis. Consequently, small-molecule IAP antagonists, such as LCL161, have entered clinical trials for their ability to induce tumor necrosis factor (TNF)-mediated apoptosis of cancer cells. However, cIAP1 and cIAP2 are recurrently homozygously deleted in multiple myeloma (MM), resulting in constitutive activation of the noncanonical nuclear factor (NF)-κB pathway. To our surprise, we observed robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and in patients with relapsed-refractory MM, where the addition of cyclophosphamide resulted in a median progression-free-survival of 10 months. This effect was not a result of direct induction of tumor cell death, but rather of upregulation of tumor-cell-autonomous type I interferon (IFN) signaling and a strong inflammatory response that resulted in the activation of macrophages and dendritic cells, leading to phagocytosis of tumor cells. Treatment of a MM mouse model with LCL161 established long-term anti-tumor protection and induced regression in a fraction of the mice. Notably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all of the treated mice.

Title: Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia Content: For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Title: Unravelling mechanisms of p53-mediated tumour suppression Content: p53 is a crucial tumour suppressor that responds to diverse stress signals by orchestrating specific cellular responses, including transient cell cycle arrest, cellular senescence and apoptosis, which are all processes associated with tumour suppression. However, recent studies have challenged the relative importance of these canonical cellular responses for p53-mediated tumour suppression and have highlighted roles for p53 in modulating other cellular processes, including metabolism, stem cell maintenance, invasion and metastasis, as well as communication within the tumour microenvironment. In this Opinion article, we discuss the roles of classical p53 functions, as well as emerging p53-regulated processes, in tumour suppression.

Title: Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Content: Type 1 and type 2 diabetes are characterized by progressive beta-cell failure. Apoptosis is probably the main form of beta-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced beta-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1beta, nuclear factor (NF)-kappaB, and Fas. We review herein the similarities and differences between the mechanisms of beta-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. IL-1beta and/or TNF-alpha plus IFN-gamma induce beta-cell apoptosis via the activation of beta-cell gene networks under the control of the transcription factors NF-kappaB and STAT-1. NF-kappaB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of beta-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes beta-cell dysfunction and may induce beta-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially "glucose hypersensitization" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1beta, NF-kappaB, or inducible nitric oxide synthase in rat or human beta-cells in vitro or in vivo in Psammomys obesus. FFAs may cause beta-cell apoptosis via ER stress, which is NF-kappaB and NO independent. Thus, cytokines and nutrients trigger beta-cell death by fundamentally different mechanisms, namely an NF-kappaB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-kappaB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of beta-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent beta-cell death in type 1 and type 2 diabetes.

Title: Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2. Content: Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro. Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents. Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

Title: Molecular mechanisms of (-)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line. Content: Dietary polyphenols have been associated with reduced risk of chronic diseases, but the precise molecular mechanisms of protection remain unclear. This work was aimed at studying the effect of (-)-epicatechin (EC) and chlorogenic acid (CGA) on the regulation of apoptotic and survival/proliferation pathways in a human hepatoma cell line (HepG2). EC or CGA treatment for 18 h had a slight effect on cell viability and decreased reactive oxygen species formation, and EC alone promoted cell proliferation, whereas CGA increased glutathione levels. Phenols neither induced the caspase cascade for apoptosis nor affected expression levels of Bcl-xL or Bax. A sustained activation of the major survival signals AKT/PI-3-kinase and ERK was shown in EC-treated cells, rather than in CGA-exposed cells. These data suggest that EC and CGA have no effect on apoptosis and enhance the intrinsic cellular tolerance against oxidative insults either by activating survival/proliferation pathways or by increasing antioxidant potential in HepG2.

Title: BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax Content: BCL-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma1–3. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths4–13. An emerging family of Bcl-2 -related proteins share two highly conserved regions14–20 referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-214. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitu-tion of Gly 145 in BHl domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in inter-leukin-3 deprivation, γ-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homo-dimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.