NO signalling in cytokinin-induced programmed cell death

Cell death can be induced by cytokinin 6-benzylaminopurine (BA) at high dosage in suspension-cultured Arabidopsis cells. Herein, we provide evidence that BA induces nitric oxide (NO) synthesis in a dose-dependent manner. A reduction in cell death can be observed when the cytokinin is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or the nitric oxide synthase (NOS) inhibitors: 2-aminoethyl-isothiourea (AET) and N^G.-monomethyl- l -arginine ( l -NMMA), which suggests that NO is produced via a NOS and is a signalling component of this form of programmed cell death. In BA-treated cells, mitochondrial functionality is altered via inhibition of respiration. This inhibition can be prevented by addition of either cPTIO or AET implying that NO acts at the mitochondrial level.     

Palmitate-induced NO production has a dual action to reduce cell death through NO and accentuate cell death through peroxynitrite formation

The objective of this study was to determine the role of palmitate-induced stimulation of nitric oxide synthase (NOS) on palmitate-induced cell death, specifically distinguishing the effects of the subtype NOS2 from NOS3, defining the effect of NO on mitochondria death pathways, and determining whether palmitate induces peroxynitrite formation which may impact cardiomyocyte cell survival. Cardiomyocytes from embryonic chick hearts were treated with palmitate 300-500 microM. Cell death was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The ability of palmitate to induce NO production and its consequences were tested by using the NOS inhibitor 7-nitroindazole (7-N) and the peroxynitrite scavenger (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride) (FeTPPS). The effect of palmitate on the mitochondria was assessed by Western blotting for cytochrome c release into the cytosol, and assessment of mitochondrial transmembrane potential (DeltaPsi(m)) by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide staining and immunocytochemistry. The NOS inhibitor 7-N, which is selective for NOS2 and not for NOS3, significantly (p<0.05) increased palmitate-induced cell death. In contrast, 7-N did not alter cell death produced by the combination of potassium cyanide and deoxyglucose, which, respectively, inhibit glycolysis and oxidative phosphorylation. The mitochondrial actions of palmitate, specifically palmitate-induced translocation of mitochondrial cytochrome c to cytosol and loss of mitochondrial transmembrane potential, were not altered by pretreatment with 7-N. FeTPPS, which isomerizes peroxynitrite to nitrate and thereby reduces the toxic effects of peroxynitrite, produced a significant reduction in palmitate-induced cell death. In summary, these data suggest that palmitate stimulates NO production, which has a dual action to protect against cell death or to induce cell death. Palmitate-induced cell death is mediated, in part, through NO generation, which leads to peroxynitrite formation. The protective effect of NO is operative through stimulation of NOS2 but not NOS3. The actions of NO on palmitate-induced cell death are independent of mitochondrial cell death pathways.     

Yeast programmed cell death: an intricate puzzle

Yeasts as eukaryotic microorganisms with simple, well known and tractable genetics, have long been powerful model systems for studying complex biological phenomena such as the cell cycle or vesicle fusion. Until recently, yeast has been assumed as a cellular 'clean room' to study the interactions and the mechanisms of action of mammalian apoptotic regulators. However, the finding of an endogenous programmed cell death (PCD) process in yeast with an apoptotic phenotype has turned yeast into an 'unclean' but even more powerful model for apoptosis research. Yeast cells appear to possess an endogenous apoptotic machinery including its own regulators and pathway(s). Such machinery may not exactly recapitulate that of mammalian systems but it represents a simple and valuable model which will assist in the future understanding of the complex connections between apoptotic and non-apoptotic mammalian PCD pathways. Following this line of thought and in order to validate and make the most of this promising cell death model, researchers must undoubtedly address the following issues: what are the crucial yeast PCD regulators? How do they play together? What are the cell death pathways shared by yeast and mammalian PCD? Solving these questions is currently the most pressing challenge for yeast cell death researchers.     

Serine protease inhibitor 2A inhibits caspase-independent cell death

The release of cysteine cathepsins from the lysosome into the cytoplasm can trigger programs of cell death (PCD) that do not require caspase executioner proteases but instead are mediated by toxic reactive oxygen species (ROS). Here, we show that a cytoplasmic inhibitor of papain-like cathepsins - Serine protease inhibitor 2A (Spi2A) - is required for the protection of cells from caspase-independent PCD triggered by tumor necrosis factor-alpha. In the absence of caspase activity, Spi2A suppressed PCD by inhibiting cathepsin B after it was released into the cytoplasm. Spi2A also directly protected against ROS-mediated PCD, which is consistent with a role in suppressing caspase-independent pathways of PCD. We conclude that inhibition of lysosomal executioner proteases by Spi2A is a physiological mechanism by which cells are protected from caspase-independent programmed cell death.     

Oxygen deprivation induced cell death: an update

Mammalian cells have multiple responses to low or zero oxygen concentrations. In the complete absence of oxygen, cells undergo cell death through apoptosis, and not necrosis. Apoptotic signaling during oxygen deprivation occurs through the release of cytochrome c and apaf-1 mediated caspase-9 activation. The upstream regulators of cytochrome c release are the Bcl-2 family members. Pro-apoptotic Bcl-2 family members such as bax or bak are clearly required to initiate cytochrome c/apaf-1/caspase-9 mediated cell death during oxygen deprivation. Here we review what is currently known oxygen deprivation induced cell death and speculate about initiating mechanisms resulting in the activation of pro-apoptotic Bcl-2 family members.     

Membrane lipids and cell death: an overview

In this article we overview major aspects of membrane lipids in the complex area of cell death, comprising apoptosis and various forms of programmed cell death. We have focused here on glycerophospholipids, the major components of cellular membranes. In particular, we present a detailed appraisal of mitochondrial lipids that attract increasing interest in the field of cell death, while the knowledge of their re-modelling and traffic remains limited. It is hoped that this review will stimulate further studies by lipid experts to fully elucidate various aspects of membrane lipid homeostasis that are discussed here. These studies will undoubtedly reveal new and important connections with the established players of cell death and their action in promoting or blocking membrane alteration of mitochondria and other organelles. We conclude that the new dynamic era of cell death research will pave the way for a better understanding of the 'chemistry of apoptosis'.     

Induction of mammalian cell death by a plant Bax inhibitor

Arabidopsis thaliana AtBI-1 is an orthologue of mammalian Bax inhibitor-1 capable of suppressing Bax-induced cell death in yeast as well as mammalian cells. Here we investigated whether or not AtBI-1 suppresses Bax-induced cell death using human fibrosarcoma HT1080 cells. Surprisingly, AtBI-1 did not block Bax-induced cell death, but it triggered apoptotic cell death in mammalian cells. The proapoptotic effect of AtBI-1 was blocked by the X-linked caspase inhibitor XIAP, suggesting that the cell death caused by AtBI-1 is similar to that caused by Bax.     

Neurotrophin receptor-interacting mage homologue is an inducible inhibitor of apoptosis protein-interacting protein that augments cell death

The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and pathways to fulfil their anti-apoptotic role. In the search for novel IAP-interacting proteins we identified the neurotrophin receptor-interacting MAGE homologue (NRAGE) as being able to bind to the avian IAP homologue ITA. This interaction requires the RING domain of ITA. NRAGE additionally coimmunoprecipitates with XIAP. When overexpressed in 32D cells NRAGE augments interleukin-3 withdrawal induced apoptosis, possibly through binding endogenous XIAP. Moreover, NRAGE is able to overcome the anti-apoptotic effect of Bcl-2.     

Ferroptosis: an iron-dependent form of nonapoptotic cell death

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.     

Possible usefulness of apocynin, an NADPH oxidase inhibitor, for nitrate tolerance: prevention of NO donor-induced endothelial cell abnormalities

The long-term benefits of nitroglycerin therapy are limited by tolerance development. Understanding the precise nature of mechanisms underlying nitroglycerin-induced endothelial cell dysfunction may provide new strategies to prevent tolerance development. In this line, we tested interventions to prevent endothelial dysfunction in the setting of nitrate tolerance. When bovine aortic endothelial cells (BAECs) were continuously treated with nitric oxide (NO) donors, including nitroglycerin, over 2-3 days, basal production of nitrite and nitrate (NO(x)) was diminished. The diminished basal NO(x) levels were mitigated by intermittent treatment allowing an 8-h daily nitrate-free interval during the 2- to 3-day treatment period. Addition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin restored the basal levels of NO(x) that were decreased by continuous nitroglycerin treatment of BAECs. Apocynin caused significant improvement of increased mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in BAECs given nitroglycerin continuously over the treatment period. Apocynin also reduced endothelial production of reactive oxygen species (ROS) after continuous nitroglycerin treatment. These results showed an essential similarity to the effects of a nitrate-free interval. Application of the NOS inhibitor N(omega)-nitro- l-arginine methyl ester caused a recovery effect on basal NO(x) and eNOS expression but was without effect on ROS levels in continuously NO donor-treated BAECs. In conclusion, the present study characterized abnormal features and functions of endothelial cells following continuous NO donor application. We suggest that inhibition of NADPH oxidase, by preventing NO donor-induced endothelial dysfunction, may represent a potential therapeutic strategy that confers protection from nitrate tolerance development.     

Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death

Viral protein R (Vpr), one of the human immunodeficiency virus type 1 (HIV-1) accessory proteins, contributes to multiple cytopathic effects, G2 cell cycle arrest and apoptosis. The mechanisms of Vpr have been intensely studied because it is believed that they underlie HIV-1 pathogenesis. We here report a cell-based small molecule screen on Vpr induced cell death in the context of HIV-1 infection. From the screen of 504 bioactive compounds, we identified damnacanthal (Dam), a component of noni [corrected] as an inhibitor of Vpr induced cell death. Our studies illustrate a novel efficient platform for drug discovery and development in anti-HIV therapy which should also be applicable to other viruses.     

Interdigital cell death function and regulation: new insights on an old programmed cell death model

Interdigital cell death (ICD) is the oldest and best-studied model of programmed cell death (PCD) in vertebrates. The classical view of ICD function is the separation of digits by promotion of tissue regression. However, in addition, ICD can contribute to digit individualization by restricting interdigital tissue growth. Depending on the species, the relative contribution of either regression or growth-restricting functions of ICD to limb morphogenesis may differ. Under normal conditions, most cells appear to die by apoptosis during ICD. Accordingly, components of the apoptotic machinery are found in the interdigits, though their role in the initiation and execution of cell death is yet to be defined. Fgf8 has been identified as a survival factor for the distal mesenchymal cells of the limb such that ICD can initiate following specific downregulation of Fgf8 expression in the ectoderm overlying the interdigital tissue. On the other hand, Bmps may promote cell death directly by acting on the interdigital tissue, or indirectly by downregulating Fgf8 expression in the ectoderm. In addition, retinoic acid can activate ICD directly or through a Bmp-mediated mechanism. Interactions at different levels between these factors establish the spatiotemporal patterning of ICD activation. Defining the regulatory network behind ICD activation will greatly advance our understanding of the mechanisms controlling PCD in general.     

Identification of PKCzetaII: an endogenous inhibitor of cell polarity

A new member of the atypical protein kinase C (aPKC) family, designated PKCzetaII, is identified in this study. The gene contains no introns and is 98% homologous with the cDNA encoding PKCzeta. The PKCzetaII coding region is frame-shifted with respect to the PKCzeta open reading frame, resulting in expression of an aPKC regulatory domain without associated kinase activity. PKCzetaII mRNA is detected in various mouse tissues and an immunoreactive 45 kDa protein is present in epithelial cell cultures. PKCzetaII is shown to interact with the Par6 protein and functions in the development of cell polarity. HC11 epithelial cells express PKCzetaII and are maintained in a nondifferentiated state characterised by the absence of tight junctions and cell overgrowth. HC11 cells harbouring a PKCzetaII-specific RNAi, recruit ZO-1 and other tight junction markers to cell-cell boundaries and adopt a monolayer phenotype in the presence of growth factors. The data demonstrate a regulatory role for PKCzetaII in the maintenance of cell transformation and the development of cell polarity.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Superoxide anion is a natural inhibitor of FAS-mediated cell death.

The cell surface receptor Fas is a major trigger of apoptosis. However, expression of the Fas receptor in many tumor cell types does not correlate with sensitivity to Fas-mediated cell death. Because a prooxidant state is a common feature of tumor cells, we examined the role of intracellular reactive oxygen intermediates in the regulation of Fas-mediated cytotoxicity. Our results show that an oxidative stress induced by increasing the intracellular superoxide anion (O2-) concentration can abrogate Fas-mediated apoptosis in cells which are constitutively sensitive to Fas. Conversely, an O2- concentration decrease is observed to sensitize cells which are naturally resistant to Fas signals. These observations suggest that intracellular O2- may play a key role in regulating cell sensitivity to a potentially lethal signal and provide tumor cells with a natural, inducible mechanism of resistance to Fas-mediated apoptosis.