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.

scholarly journals Accelerated Apoptotic DNA Laddering Protocol

BioTechniques ◽  
2002 ◽  
Vol 33 (4) ◽  
pp. 734-736 ◽  
Author(s):  
Michael C. Yeung
Keyword(s):  
Dna Laddering

Stem cell factor protects germ cells from apoptosis in vitro

2000 ◽  
Vol 113 (1) ◽  
pp. 161-168 ◽  
Author(s):  
W. Yan ◽  
J. Suominen ◽  
J. Toppari
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Primordial Germ Cells ◽  
Survival Function ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Dna Laddering ◽  
Survival Effect

Stem cell factor (SCF) plays an important role in migration, adhesion, proliferation, and survival of primordial germ cells and spermatogonia during testicular development. However, the function of SCF in the adult testis is poorly described. We have previously shown that, in the presence of SCF, there were more type A spermatogonia incorporating thymidine at stage XII of rat seminiferous tubules cultured in vitro than in the absence of SCF, implying that the increased DNA synthesis might result from enhanced survival of spermatogonia. To explore the potential pro-survival function of SCF during spermatogenesis, the seminiferous tubules from stage XII were cultured in the presence or absence of SCF (100 ng/ml) for 8, 24, 48, and 72 hours, respectively, and apoptosis was analyzed by DNA laddering and in situ 3′-end labeling (ISEL) staining. Surprisingly, not only spermatogonia, but also spermatocytes and spermatids, were protected from apoptosis in the presence of SCF. Apoptosis took place much later and was less severe in the SCF-treated tubules than in the controls. Based on previous studies showing that FSH prevents germ cells from undergoing apoptosis in vitro, and that SCF level is increased dramatically in response to FSH stimulation, we also tested if the pro-survival effect of FSH is mediated through SCF by using a function-blocking monoclonal antibody, ACK-2, to block SCF/c-kit interaction. After 24 hours of blockade, the protective effect of FSH was partially abolished, as manifested by DNA laddering and ISEL analyses. The present study demonstrates that SCF acts as an important survival factor for germ cells in the adult rat testis and FSH pro-survival effect on germ cells is mediated partially through the SCF/c-kit pathway.

scholarly journals Programmed Cell Death in Human Ovary Is a Function of Follicle and Corpus Luteum Status*

1997 ◽  
Vol 82 (9) ◽  
pp. 3148-3155
Author(s):  
Wei Yuan ◽  
Linda C. Giudice
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Corpus Luteum ◽  
Dna Fragmentation ◽  
Low Molecular Weight ◽  
Corpora Lutea ◽  
Positive Staining ◽  
Human Ovary ◽  
Dna Laddering

Abstract Although extensive investigation on follicular apoptosis (programmed cell death) has been conducted in the infraprimate ovary, there is little information regarding apoptosis and its relationship to follicular status in the human. In this study, apoptosis was investigated in 116 human ovarian follicles (primordial to dominant) and 5 corpora lutea from a total of 27 premenopausal women. Follicles and corpora lutea were evaluated for the presence of DNA fragmentation, characteristic of apoptosis, by two methods: in situ hybridization using 3′ end-labeling of DNA with digoxigenin-labeled nucleotides and subsequent digoxigenin antibody and peroxidase staining, and/or biochemical analysis of low molecular weight DNA laddering. Follicle functional status was evaluated by determining follicle sizes and follicular fluid androgen/estrogen (A/E) ratios. No apoptosis was observed in 67 primordial, primary, or secondary follicles. Positive staining for DNA fragmentation was found in a few granulosa cells in 0.1- to 2-mm follicles, whereas abundant staining in granulosa was detected in 2.1- to 9.9-mm follicles. In contrast, no DNA fragmentation was detected in dominant follicles (10–16 mm). The frequency of apoptosis in follicles was calculated to be 37% in 0.1- to 2-mm follicles, 50% in 2.1- to 5-mm follicles, and 27% in 5.1- to 9.9-mm follicles. Abundant low molecular weight DNA laddering was only found in androgen-dominant follicles and not in estrogen-dominant follicles. Positive staining for DNA fragmentation and low molecular weight DNA laddering were observed in degenerating but not healthy-appearing corpora lutea. In the former, DNA fragmentation was found primarily in large luteal cells. These data suggest that follicular atresia in human ovary results from normal programmed cell death and primarily occurs in the granulosa cell layers of the early antral and <10-mm antral follicles primarily. Furthermore, because apoptosis occurs as early as the 200-mm stage, follicle selection may begin as early as the initial formation of the antrum. The results also suggest that degeneration of the corpus luteum occurs by apoptotic mechanisms.

Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats

1998 ◽  
Vol 274 (5) ◽  
pp. L714-L720 ◽  
Author(s):  
Sue Buckley ◽  
Lora Barsky ◽  
Barbara Driscoll ◽  
Kenneth Weinberg ◽  
Kathryn D. Anderson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Epithelial Cells ◽  
Cellular Response ◽  
Ex Vivo ◽  
Keratinocyte Growth Factor ◽  
Alveolar Epithelial Cells ◽  
Alveolar Epithelial ◽  
Dna Laddering

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

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 Mannose Induces an Endonuclease Responsible for DNA Laddering in Plant Cells

1999 ◽  
Vol 121 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Joshua C. Stein ◽  
Geneviève Hansen
Keyword(s):  
Plant Cells ◽  
Dna Laddering

Mitochondrial-Mediated Apoptosis by the Sesquiterpene Oil α-Bisabolol and Its Synergism with Imatinib and Nilotinib In BCR-ABL+ Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4456-4456
Author(s):  
Massimiliano Bonifacio ◽  
Antonella Rigo ◽  
Elisabetta Cavalieri ◽  
Emanuele Guardalben ◽  
Christian Bergamini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Single Agent ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Ros Production ◽  
Dna Laddering

Abstract Abstract 4456 Background. The plant-derived agent α-bisabolol is a small oily sesquiterpene alcohol that has been demonstrated to be cytotoxic against human malignant non-hematological and leukemic cells (Bonifacio M et al, Blood, 2009 ASH annual meeting abstracts;114:4800). Here we tested its activity against BCR-ABL+ cell lines and primary cells from patients, alone or in combination with the Tyrosine-Kinase Inhibitors (TKIs) Imatinib and Nilotinib. Also, the mechanism of α-bisabolol cytotoxicity in BCR-ABL+ cells was assessed. Methods. We used the BCR-ABL+ K562, LAMA-84 and CML-T1 cell lines and primary leukemic cells from 14 patients with BCR-ABL+ Acute Lymphoblastic Leukemia at diagnosis. First, the citotoxicity of single-agent α-bisabolol was determined by MTT. Then, mitochondrial membrane potential of treated cells was evaluated by the JC-1 dye in flow cytometry and fluorescence microscopy. Permeabilized leukemic cells were assayed for oxygen consumption by measuring mitochondrial state 3 and uncoupled respiration. Reactive oxygen species (ROS) production in α-bisabolol treated cells were quantified in flow cytometry by oxidation of CM-H2DCFDA, measuring the fluorescence intensity of the DCF products. Apoptosis was studied by the poly(ADP-ribose) polymerase (PARP) cleavage and internucleosomal DNA laddering analysis. Finally, the combination effects between α-bisabolol and Imatinib or Nilotinib (kindly provided by Novartis) were analyzed according to the median-effect method of Chou and Talalay using the CalcuSyn software. Results. α-bisabolol reduced the viability of BCR-ABL+ cells in a dose-dependent manner. The mean IC50 values of α-bisabolol were 46±11 μ M for primary leukemic cells and ranged from 62 to 115 μ M in the cell lines. JC-1 staining of BCR-ABL+ primary leukemic cells treated with 40 μ M α-bisabolol for 3 to 5 hours demonstrated a dissipation of the mitochondrial transmembrane potential (ΔΨm), thus indicating the start of the apoptotic process. Moreover, NADH-supported state 3 respiration in α-bisabolol treated leukemic cells was significantly decreased in comparison with untreated leukemic controls (140.0±70.5 vs 280.7±11.9 pmol O2/min/106 cells; p<.05). Finally, PARP cleavage and DNA laddering followed α-bisabolol exposure of leukemic BCR-ABL+ blasts. The apoptosis induction was accompanied by ROS production. When tested in combination at constant ratio with Imatinib or Nilotinib, α-bisabolol showed overall slight to strong synergistic effects, without evidence for antagonism across a range of doses (Table 1). In 3 patients with mutation of BCR-ABL (T315I, E255V and Y253H, respectively) we observed full activity of α-bisabolol as single agent and confirmed the synergism between α-bisabolol and Imatinib. Conclusion. This study indicates that α-bisabolol is an effective pro-apoptotic agent for human acute BCR-ABL+ leukemia cells via induction of mitochondrial membrane damage. The combination of α-bisabolol with Imatinib or Nilotinib allows a dose reduction up to 90% of each drug to obtain the same cytotoxic effect, so indicating a clear synergism. α-bisabolol may be a potential candidate for the treatment of BCR-ABL+ leukemias and the effective dose of TKIs could be reduced in a combined treatment with α-bisabolol. Disclosures: No relevant conflicts of interest to declare.

Altered expression of prostaglandin synthases in NSCLC: Therapeutic strategies and targets for intervention

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 18083-18083
Author(s):  
M. Cathcart ◽  
K. Gatley ◽  
E. Kay ◽  
G. P. Pidgeon ◽  
K. J. O’ Byrne
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cell Survival ◽  
Lower Grade ◽  
Altered Expression ◽  
Over Expression ◽  
Chemotherapy Drugs ◽  
Dna Laddering ◽  
Induced Apoptosis ◽  
Brdu Assay

18083 Background: Prostacyclin Synthase (PGIS) and Thromboxane synthase (TXS) metabolize PGH(2) into prostacyclin and thromboxane respectively. PGIS over-expression inhibits cancer growth in a mouse model, while over-expression of TXS caused opposite effects. TXS is expressed in a variety of tumours, associated with poor prognosis and increased metastasis. The aim of this study was to examine the expression of PGIS and TXS in NSCLC, the effect of targeted TXS inhibition, and the mechanisms regulating these effects. Methods: A panel of resected human lung tumours were stained for PGIS and TXS expression by IHC and by western analysis. A 170- core tissue microarray was stained for TXS and intensity correlated with tumour grade. Cell survival was examined by BrdU assay in A-549 (adenocarcinoma) and SKMES-1 (squamous cell carcinoma) cells following 24h selective TXS inhibition with Ozagrel (500nM) alone, or in combination with chemotherapy. Multi-parameter apoptosis signalling was examined after TXS inhibition using In Cell Analyser and confirmed by DNA laddering and cell death ELISA. PCR arrays were used to examine genes involved in tumourigenesis following TXS inhibition. Results: PGIS was absent in lung cancer sections. TXS was over-expressed in lung cancer relative to matched normal, with significantly increased expression in lower grade tumours. PGIS was down-regulated or absent, while TXS expression was up-regulated in tumours v’s normal tissue. Ozagrel significantly reduced cell survival and induced apoptosis, determined by both DNA laddering and Cell Death ELISA. Multi-parameter apoptosis analysis revealed enlarged nuclei, decreased f-actin staining and decreased mitochondrial mass potential, while PCR arrays confirmed upregulation of the pro-apoptotic gene BAX following TXS inhibition. Ozagrel in combination with Doxorubicin (10nM) showed greatest efficacy compared to a number of other chemotherapy drugs. Conclusions: Expression of PGIS and TXS are altered in NSCLC. Overexpression of TXS may regulate tumour survival as its inhibition induces apoptosis, potentially through upregulation of pro- apoptotic proteins. Targeting TXS, alone or in combination with chemotherapy is a potential therapeutic strategy for the treatment of NSCLC. No significant financial relationships to disclose.

Sign in / Sign up

Export Citation Format

Share Document