Apoptosis: Programmed cell death in health and disease

Apoptosis is a normal physiological cell death process of eliminating unwanted cells from living organisms during embryonic and adult development. Apoptotic cells are characterised by fragmentation of nuclear DNA and formation of apoptotic bodies. Genetic analysis revealed the involvement of many death and survival genes in apoptosis which are regulated by extracellular factors. There are multiple inducers and inhibitors of apoptosis which interact with target cell specific surface receptors and transduce the signal by second messengers to programme cell death. The regulation of apoptosis is elusive, but defective regulation leads to aetiology of various ailments. Understanding the molecular mechanism of apoptosis including death genes, death signals, surface receptors and signal pathways will provide new insights in developing strategies to regulate the cell survival/death. The current knowledge on the molecular events of apoptotic cell death and their significance in health and disease is reviewed.     

Cloning and sequencing of a cDNA encoding the rat Bcl-2 protein

A rat cDNA encoding the Bcl-2 protein was cloned and sequenced. The primary amino-acid sequence deduced from the nucleotide sequence reveals a 236-aa protein having extensive homology with the mouse (95%), human (87%) and chicken (71%) Bcl-2 proteins.     

Morphological and Biochemical Changes During Programmed Cell Death of Rat Cerebellar Granule Cells

Cultured cerebellar granule cells deprived of depolarizing concentrations of KCl and serum die by programmed cell death. Recently, it was shown that serum removal by itself can lead to oxidative stress and DNA fragmentation in these cells. We have modified the protocol which initiates cell death in such a way that only the effect of KCl withdrawal-induced cell death was observed. We have performed a series of experiments to correlate the structural and biochemical changes in this process of cell death. Significant morphological alterations occur in cell bodies and neurites during a 48-hour period of KCl removal. Cell viability dropped to 53%, 34% or 10% of control levels, respectively, as a result of 1-, 2-, or 3-day KCl removal. A series of experiments was conducted to determine the change of total protein level, protein synthesis rate, RNA synthesis rate, and mitochondrial activity during the first 48 hours of KCl removal. These studies not only provide a picture correlating the morphological and biochemical changes in the process of programmed cell death, but also serve as a reference for future studies of this complex phenomenon.     

Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.     

Mfn2 modulates the UPR and mitochondrial function via repression of PERK

Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2‐deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP‐1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2‐ablated cells in response to ER stress. XBP‐1 loss‐of‐function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2‐ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss‐of‐function reveals that PERK is a key regulator of mitochondrial morphology and function.     

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.     

Growth and senescence of Medicago truncatula cultured cells are associated with characteristic mitochondrial morphology

Here mitochondrial morphology and dynamics were investigated in Medicago truncatula cell-suspension cultures during growth and senescence. Cell biology techniques were used to measure cell growth and death in culture. Mitochondrial morphology was investigated in vivo using a membrane potential sensor probe coupled with confocal microscopy. Expression of a senescence-associated gene (MtSAG) was evaluated in different cell-growth phases. Mitochondria appeared as numerous, punctuate organelles in cells at the beginning of the subculture cycle, while interconnected networks were observed in actively growing cells. In senescent cells, giant mitochondria were associated with dying cells. The release of cytochrome c from mitochondria was detected in different growth phases of cultured cells. Studies on plant cell cultures allowed us to identify physiological and molecular markers of senescence and cell death, and to associate distinct mitochondrial morphology with cells under different physiological conditions.     

Coumarin A/AA induces apoptosis‐like cell death in HeLa cells mediated by the release of apoptosis‐inducing factor

It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral‐blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis‐inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase‐3 was not detected. The overall results indicate that coumarin A/AA induces a caspase‐independent apoptotic‐like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:263–272, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20288     

Apoptosis in CHO cell batch cultures: examination by flow cytometry

Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G₁/G₀ and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.     

一氧化氮对植物细胞基因表达和代谢产物合成的影响机制

一氧化氮(NO)作为一种气体信号分子,在植物体内具有多种生理功能,许多研究逐步揭示了NO在植物发育、新陈代谢和疾病响应等方面的分子机制。内源和外源NO都可以使植物组织或悬浮细胞基因表达发生变化,高通量的基因表达的研究,如转录分析等,为信号网络通路分析提供了有力的证据。我们简要综述了NO的合成途径,并讨论了次生代谢产物及细胞程序性死亡过程中依赖NO调控的相应基因及蛋白的变化,揭示了NO是一种重要的植物信号分子,有较高的研究价值。     

Human eosinophils: Apoptosis versus survival in the mediation of inflammation

Eosinophil-derived mediators are thought to make a major contribution to the inflammation underlying a number of allergic diseases, most notably asthma. The toxic potential of eosinophils at tissue sites of inflammation might be limited if they were cleared by the process of programmed cell death or apoptosis. In this review we have examined the relationship between the signal transduction pathways important in controlling cytokine-induced prolonged survival and the mechanisms responsible for the induction and control of apoptosis in the eosinophil. A greater understanding of these processes might result in the development of novel therapeutic agents which would promote the safe and rapid removal by apoptosis of this important pro-inflammatory cell.     

Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53

Murine preimplantation embryos exposed to hyperglycemia experience decreased glucose transport, and overexpression of the proapoptotic protein BAX, leading to increased apoptosis. These changes may account for the increased rates of miscarriages and malformations seen in women with diabetes mellitus. To test whether p53 expression is necessary for hyperglycemia-induced apoptosis, p53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52 mM D- or L-glucose. Apoptosis was detected using terminal dUTP nick end labeling (TUNEL) assays. In vivo studies were performed in the same manner using blastocysts recovered from streptozotocin-induced diabetic mothers. Both in vitro and in vivo studies showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX, immunofluorescent confocal microscopy and immunoprecipitation/ immunoblotting were performed on blastocysts cultured in high vs. control glucose conditions. Blastocysts from p53+/+ mice exhibited increased BAX staining vs. p53+/- and -/- embryos. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose transport was measured in individual blastocysts from p53+/+ and +/- diabetic vs. nondiabetic mice. Embryos from diabetic p53+/- mice exhibit a 44% decrease in glucose transport, similar to the 38% decrease seen in embryos from diabetic p53+/+ mice. Taken together, these results strongly indicate that p53 plays a role in hyperglycemia-induced apoptosis, upstream of BAX overexpression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo.     

胆汁酸对代谢性疾病的调控

胆汁酸作为一种信号分子通过激活肝、肠道和外周组织中的胆汁酸受体影响体内葡萄糖和脂质的代谢平衡,对于调节肥胖、2型糖尿病和非酒精性脂肪肝等代谢性疾病具有非常重要的意义。胆汁酸与相应核受体,如法尼醇X受体(farnesoid X receptor, FXR)和Takeda G蛋白偶联受体5 (Takeda G protein-coupled receptor 5,TGR5)的相互作用影响了这些代谢性疾病。FXR主要通过影响胆汁酸的合成及转运对非酒精性脂肪肝发挥作用,TGR5则是间接增加褐色脂肪组织中的生热作用,改善肥胖和2型糖尿病。这些调控机制的研究是非常必要的。本文综述了胆汁酸代谢及其对代谢性疾病调控的分子机制的研究进展,以期为科研工作者提供一定的参考。     

Apoptosis of cells lacking mitochondrial DNA

The mitochondrial genome of animals encodes a few subcomponents of the respiratory chain complexes I, III and IV, whereas nuclear DNA encodes the overwhelming majority, both in quantitative and qualitative terms, of mitochondrial proteins. Complete depletion of mitochondrial DNA (mtDNA) can be achieved by culturing cells in the presence of inhibitors of mtDNA replication or mitochondrial protein synthesis, giving rise to mutant cells (ϱ∘ cells) which carry morphological near-to-intact mitochondria with respiratory defects. Such cells can be used to study the impact of mitochondrial respiration on apoptosis. ϱ∘ cells do not undergo cell death in response to determined stimuli, yet they conserve their potential to undergo full-blown apoptosis in many experimental systems. This indicates that mtDNA and associated functions (in particular mitochondrial respiration) are irrelevant to apoptosis execution. However, the finding that mtDNA-deficient mitochondria can undergo apoptosis does not argue against the involvement of mitochondria in the apoptotic process, since mitochondria from ϱ∘ cells conserve most of their functions including those involved in the execution of the death programme: permeability transition and release of one or several intermembrane proteins causing nuclear apoptosis. Supported by ARC, ANRS, CNRS, FRM, Fondation de France, INSERM, NATO, Ligue contre le Cancer Ministère de la Recherche et de l'Industrie (France), and Sidaction (to GK). SAS receives a fellowship from the Spanish Government (Ministerio de Ciencia y Educación).     

Programmed cell death in plants: distinguishing between different modes

Programmed cell death (PCD) in plants is a crucial componentof development and defence mechanisms. In animals, differenttypes of cell death (apoptosis, autophagy, and necrosis) havebeen distinguished morphologically and discussed in these morphologicalterms. PCD is largely used to describe the processes of apoptosisand autophagy (although some use PCD and apoptosis interchangeably)while necrosis is generally described as a chaotic and uncontrolledmode of death. In plants, the term PCD is widely used to describemost instances of death observed. At present, there is a vastarray of plant cell culture models and developmental systemsbeing studied by different research groups and it is clear fromwhat is described in this mass of literature that, as with animals,there does not appear to be just one type of PCD in plants.It is fundamentally important to be able to distinguish betweendifferent types of cell death for several reasons. For example,it is clear that, in cell culture systems, the window of timein which ‘PCD’ is studied by different groups varieshugely and this can have profound effects on the interpretationof data and complicates attempts to compare different researcher'sdata. In addition, different types of PCD will probably havedifferent regulators and modes of death. For this reason, inplant cell cultures an apoptotic-like PCD (AL-PCD) has beenidentified that is fairly rapid and results in a distinct corpsemorphology which is visible 4–6 h after release of cytochromec and other apoptogenic proteins. This type of morphology, distinctfrom autophagy and from necrosis, has also been observed inexamples of plant development. In this review, our model systemand how it is used to distinguish specifically between AL-PCDand necrosis will be discussed. The different types of PCD observedin plants will also be discussed and the importance of distinguishingbetween different forms of cell death will be highlighted. Key words: Apoptosis, apoptosis-like programmed cell death (AL-PCD), Arabidopsis, autophagy, mitochondria, necrosis, programmed cell death (PCD) Received 5 June 2007; Revised 13 September 2007 Accepted 20 September 2007     

Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.     

The mitochondrial pathway in yeast apoptosis

Mitochondria are not only important for the energetic status of the cell, but are also the fatal organelles deciding about cellular life and death. Complex mitochondrial features decisive for cell death execution in mammals are present and functional in yeast: AIF and cytochrome c release to the cytosol, mitochondrial fragmentation as well as mitochondrial hyperpolarisation followed by an oxidative burst, and breakdown of mitochondrial membrane potential. The easy accessibility of mitochondrial manipulations such as repression of respiration by growing yeast on glucose or deletion of mitochondrial DNA (rho⁰) on the one hand and the unique ability of yeast cells to grow on non-fermentable carbon sources by switching on mitochondrial respiration on the other hand have made yeast an excellent tool to delineate the necessity for mitochondria in cell death execution. Yeast research indicates that the connection between mitochondria and apoptosis is intricate, as abrogation of mitochondrial function can be either deleterious or beneficial for the cell depending on the specific context of the death scenario. Surprisingly, mitochondrion dependent yeast apoptosis currently helps to understand the aetiology (or the complex biology) of lethal cytoskeletal alterations, ageing and neurodegeneration. For example, mutation of mitochondrial superoxide dismutase or CDC48/VCP mutations, both implicated in several neurodegenerative disorders, are associated with mitochondrial impairment and apoptosis in yeast.     

Molecular mechanisms of programmed cell death

Programmed cell death is currently under active investigation. A recent meeting focused on the molecular machinery of programmed cell death and on its role in the pathogenesis of human diseases.