DNA nick end labeling: a reliable marker for apoptosis?

1995 ◽  
Vol 53 ◽  
pp. 962-963
Author(s):  
Anne F. Bushnell ◽  
Sarah Webster ◽  
Lynn S. Perlmutter
Keyword(s):  
Cell Death ◽  
Cultured Cells ◽  
Apoptotic Cell ◽  
Morphological Characteristics ◽  
Detection Methods ◽  
Tissue Preparation ◽  
Preparation Methods ◽  
Dna Laddering ◽  
Disease States ◽  
Reliable Marker

Apoptosis, or programmed cell death, is an important mechanism in development and in diverse disease states. The morphological characteristics of apoptosis were first identified using the electron microscope. Since then, DNA laddering on agarose gels was found to correlate well with apoptotic cell death in cultured cells of dissimilar origins. Recently numerous DNA nick end labeling methods have been developed in an attempt to visualize, at the light microscopic level, the apoptotic cells responsible for DNA laddering.The present studies were designed to compare various tissue processing techniques and staining methods to assess the occurrence of apoptosis in post mortem tissue from Alzheimer's diseased (AD) and control human brains by DNA nick end labeling methods. Three tissue preparation methods and two commercial DNA nick end labeling kits were evaluated: the Apoptag kit from Oncor and the Biotin-21 dUTP 3' end labeling kit from Clontech. The detection methods of the two kits differed in that the Oncor kit used digoxigenin dUTP and anti-digoxigenin-peroxidase and the Clontech used biotinylated dUTP and avidinperoxidase. Both used 3-3' diaminobenzidine (DAB) for final color development.

Redistribution of phosphatidylethanolamine and phosphatidylserine precedes reperfusion-induced apoptosis

1998 ◽  
Vol 274 (1) ◽  
pp. H242-H248 ◽  
Author(s):  
Nilanjana Maulik ◽  
Valerian E. Kagan ◽  
Vladimir A. Tyurin ◽  
Dipak K. Das
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Genomic Dna ◽  
High Performance ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Apoptotic Cell Death ◽  
Dna Laddering ◽  
Outer Leaflet ◽  
Induced Apoptosis

Although cardiomyocyte death and infarction associated with ischemia-reperfusion are traditionally believed to be induced via necrosis, recent studies implicated apoptotic cell death in ischemic reperfused tissue. To examine whether myocardial ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15 and 30 min of ischemia as well as 15 min of ischemia followed by 30, 90, or 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from cardiomyocytes to 1.8% agarose gel electrophoresis and photographed under ultraviolet illumination. In addition, high-performance thin-layer chromatography (HPTLC) of aminophospholipids labeled with 2,4,6-trinitrobenzenesulfonate was performed to evaluate phospholipid topography in cardiomyocytes. The results of our study revealed apoptotic cells only in the 90- and 120-min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscope. None of the ischemic hearts showed any evidence of apoptosis. These results corroborated with the findings of DNA fragmentation that showed increased ladders of DNA bands in the 120-min reperfused hearts, representing integer multiples of the internucleosomal DNA length (∼180 bp). Two-dimensional HPTLC of the phospholipids obtained from the cardiomyocytes and transbilayer organization of the phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the myocytes indicated translocation of both PE and PS from the inner leaflet to the outer leaflet of the membrane as early as after 20 min of ischemia. These results demonstrate that the redistribution of PS and PE precedes the apototic cell death and DNA fragmentation associated with the reperfusion of ischemic myocardium, suggesting that ischemia may trigger the signal for apoptosis although it becomes evident during reperfusion.

scholarly journals Cytotoxic T lymphocyte–induced killing in the absence of granzymes A and B is unique and distinct from both apoptosis and perforin-dependent lysis

2006 ◽  
Vol 173 (1) ◽  
pp. 133-144 ◽  
Author(s):  
Nigel J. Waterhouse ◽  
Vivien R. Sutton ◽  
Karin A Sedelies ◽  
Annette Ciccone ◽  
Misty Jenkins ◽  
...  
Keyword(s):  
Cell Death ◽  
T Lymphocyte ◽  
Target Cell ◽  
Cytotoxic T Lymphocyte ◽  
Apoptotic Cell ◽  
Morphological Characteristics ◽  
Granule Exocytosis ◽  
Molecular Events ◽  
Membrane Protrusions ◽  
Cell Gene

Cytotoxic T lymphocyte (CTL)–induced death triggered by the granule exocytosis pathway involves the perforin-dependent delivery of granzymes to the target cell. Gene targeting has shown that perforin is essential for this process; however, CTL deficient in the key granzymes A and B maintain the ability to kill their targets by granule exocytosis. It is not clear how granzyme AB−/− CTLs kill their targets, although it has been proposed that this occurs through perforin-induced lysis. We found that purified granzyme B or CTLs from wild-type mice induced classic apoptotic cell death. Perforin-induced lysis was far more rapid and involved the formation of large plasma membrane protrusions. Cell death induced by granzyme AB−/− CTLs shared similar kinetics and morphological characteristics to apoptosis but followed a distinct series of molecular events. Therefore, CTLs from granzyme AB−/− mice induce target cell death by a unique mechanism that is distinct from both perforin lysis and apoptosis.

scholarly journals Chikungunya virus–induced autophagy delays caspase-dependent cell death

2012 ◽  
Vol 209 (5) ◽  
pp. 1029-1047 ◽  
Author(s):  
Pierre-Emmanuel Joubert ◽  
Scott W. Werneke ◽  
Claire de la Calle ◽  
Florence Guivel-Benhassine ◽  
Alessandra Giodini ◽  
...  
Keyword(s):  
Cell Death ◽  
Chikungunya Virus ◽  
Cultured Cells ◽  
Apoptotic Cell ◽  
Kinetic Studies ◽  
Mtor Inhibition ◽  
Viral Propagation ◽  
Dependent Cell ◽  
Induced Apoptosis

Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α–XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16LHM mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease.

scholarly journals Induction of Apoptotic Cell Death Leads to the Development of Bacterial Rot Caused by Pseudomonas cichorii

2006 ◽  
Vol 19 (2) ◽  
pp. 112-122 ◽  
Author(s):  
Akinori Kiba ◽  
Yasutaka Sangawa ◽  
Kouhei Ohnishi ◽  
Nan Yao ◽  
Pyoyun Park ◽  
...  
Keyword(s):  
Cell Death ◽  
De Novo ◽  
Apoptotic Cell ◽  
Leaf Tissue ◽  
Apoptotic Cell Death ◽  
Pseudomonas Cichorii ◽  
Disease Symptoms ◽  
Dna Laddering ◽  
Bacterial Rot ◽  
De Novo Protein Synthesis

Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P.cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii.

scholarly journals Oxidized unsaturated fatty acids induce apoptotic cell death in cultured cells

2019 ◽  
Author(s):  
Katsuya Iuchi ◽  
Mika Ema ◽  
Moe Suzuki ◽  
Chikako Yokoyama ◽  
Hisashi Hisatomi
Keyword(s):  
Fatty Acids ◽  
Cell Death ◽  
Unsaturated Fatty Acids ◽  
Cultured Cells ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death

scholarly journals Ceramide phosphoethanolamine synthase SMSr is a target of caspase-6 during apoptotic cell death

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Birol Cabukusta ◽  
Niclas T. Nettebrock ◽  
Matthijs Kol ◽  
Angelika Hilderink ◽  
Fikadu G. Tafesse ◽  
...  
Keyword(s):  
Cell Death ◽  
Cultured Cells ◽  
Apoptotic Cell ◽  
Expression System ◽  
Apoptotic Cell Death ◽  
Negative Regulator ◽  
Specific Substrate ◽  
Sam Domain ◽  
Effector Caspase ◽  
Caspase 6

Ceramides are essential precursors of sphingolipids with a dual role as mediators of apoptotic cell death. Previous work revealed that the ER-resident ceramide phosphoethanolamine (CPE) synthase SMSr/SAMD8 is a suppressor of ceramide-mediated apoptosis in cultured cells. Anti-apoptotic activity of SMSr requires a catalytically active enzyme but also relies on the enzyme’s N-terminal sterile α-motif or SAM domain. Here, we demonstrate that SMSr itself is a target of the apoptotic machinery. Treatment of cells with staurosporine or the death receptor ligand FasL triggers caspase-mediated cleavage of SMSr at a conserved aspartate located downstream of the enzyme’s SAM domain and upstream of its first membrane span. Taking advantage of reconstitution experiments with SMSr produced in a cell-free expression system, specific caspase-inhibitors and gene silencing approaches, we show that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Huntington’s and Alzheimer’s diseases. Our findings underscore a role of SMSr as negative regulator of ceramide-induced cell death and, in view of a prominent expression of the enzyme in brain, raise questions regarding its potential involvement in neurodegenerative disorders.

Participation of reactive oxygen species in PGF2alpha-induced apoptosis in rat luteal cells

Reproduction ◽  
2000 ◽  
pp. 239-245 ◽  
Author(s):  
M Tanaka ◽  
T Miyazaki ◽  
S Tanigaki ◽  
K Kasai ◽  
K Minegishi ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Apoptotic Cell ◽  
Morphological Characteristics ◽  
Reactive Oxygen ◽  
Radical Scavenger ◽  
Oxygen Species ◽  
Luteal Cells ◽  
Prostaglandin F ◽  
Induced Apoptosis

Prostaglandin F(2alpha) (PGF(2alpha)) is implicated in the process of luteal regression in many species. Treatment of rat luteal tissue with PGF(2alpha) increases the generation of reactive oxygen species. Since reactive oxygen species have been implicated in apoptosis, the present study was undertaken to determine whether reactive oxygen species play a role in the PGF(2alpha)-induced apoptosis of rat luteal cells. Rat luteal cells were loaded with 6-carboxy-2, 7'-dichlorodihydro-fluorescein (CDCFH) diacetate, di (acetomethyl ester), which can be oxidized by reactive oxygen species to yield CDCF, a fluorescent molecule, and the cells were treated with different doses of PGF(2alpha). Incubation with 100 micromol PGF(2alpha) l(-1) induced an increase in CDCF fluorescence (P < 0. 05). Treatment of cells with PGF(2alpha) for 48 h in serum-free medium induced a dose-dependent increase in cell death, and these cells exhibited the morphological characteristics typical of apoptosis, including condensed or fragmented nuclei and fragmentation of internucleosomal DNA. Pretreatment of these cells with ascorbic acid, N,N'-dimethylthiourea, or superoxide dismutase, which acts as an antioxidant or a radical scavenger, prevented the PGF(2alpha)-induced apoptosis. These results demonstrate that PGF(2alpha) produces reactive oxygen species and induces apoptosis in rat luteal cells, indicating that the reactive oxygen species may induce apoptotic cell death during luteolysis.

scholarly journals Nitric Oxide and Reactive Oxygen Species Do Not Elicit Hypersensitive Cell Death but Induce Apoptosis in the Adjacent Cells During the Defense Response of Oat

2004 ◽  
Vol 17 (3) ◽  
pp. 245-253 ◽  
Author(s):  
Yasuomi Tada ◽  
Tomoyo Mori ◽  
Takeshi Shinogi ◽  
Nan Yao ◽  
Satsuki Takahashi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Cell Death ◽  
Defense Response ◽  
Apoptotic Cell ◽  
Reactive Oxygen ◽  
Apoptotic Cell Death ◽  
Oxygen Species ◽  
Hypersensitive Cell Death ◽  
Dna Laddering

Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2 probe DCF demonstrated that NO and H2O2 were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2 accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2 by interacting with O2 - in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2 - but not H2O2 are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.

scholarly journals Apoptosis: Dual Role in Aetiology and Cure

2019 ◽  
pp. 5
Author(s):  
Aye Aye Wynn ◽  
Ohnmar Myint ◽  
Nang Khin Mya
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Apoptotic Cell ◽  
Transplant Rejection ◽  
Detection Methods ◽  
Host Cell Apoptosis ◽  
Different Types ◽  
Apoptosis Detection ◽  
Infected Cells ◽  
Parenchymal Cells

Apoptosis is a programmed cell death which occurs following a variety of stimuli. Physiologically the process is important for morphogenesis of organs and homeostasis of different types of cells. Apoptotic cell death is responsible for a variety of pathologic states such as elimination of cell death in mutated cells, infected cells, tumour cells and transplant rejection well as the pathological atrophy. In this review, there is discussion about the control of apoptosis, detection methods of apoptosis, its association with infectious and non-communicable diseases. Intracellular microorganisms survive through inhibition of host cell apoptosis as well as they destroy the parenchymal cells causing impaired functions. It plays important role in tumourigenesis. There are possible therapeutic roles of drugs that modify apoptosis in human diseases.

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