Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 269-279 ◽  
Author(s):  
Dušan Fabian ◽  
Alexandra Bukovská ◽  
Štefan Juhás ◽  
Juraj Koppel
Keyword(s):  
Cell Death ◽  
Cytosine Methylation ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Blastocyst Stage ◽  
Nuclear Condensation ◽  
Cell Stage ◽  
Genome Methylation ◽  
Dna Cytosine Methylation

SummaryThe present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (α-amanitin, actinomycin D, TNF-α). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death

2009 ◽  
Vol 37 (2) ◽  
pp. 209-218 ◽  
Author(s):  
Mathieu Vinken ◽  
Elke Decrock ◽  
Elke De Vuyst ◽  
Luc Leybaert ◽  
Tamara Vanhaecke ◽  
...  
Keyword(s):  
Cell Death ◽  
In Vitro Model ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cell Death ◽  
Biochemical Characterisation ◽  
Vitro Model

This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.

scholarly journals Target-induced death by apoptosis in human lymphokine-activated natural killer cells

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2411-2418 ◽  
Author(s):  
K Taga ◽  
A Yamauchi ◽  
K Kabashima ◽  
ET Bloom ◽  
J Muller ◽  
...  
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Dna Cleavage ◽  
Nk Cell ◽  
Apoptotic Cell ◽  
Interleukin 2 ◽  
Nuclear Condensation

Activated human natural killer (NK) cells undergo rapid apoptotic cell death after ligand binding to the Fc receptor (CD16). We examined whether human NK cells die after engagement in cytolytic functions. Peripheral blood NK cells, with and without prior activation in vitro with interleukin-2 (IL-2), were tested for the occurrence of cell death after incubation with K562, the prototype NK-sensitive target cell. A proportion (15.2%) of NK cells that were stimulated for 3 days with IL- 2 and then incubated for 4 hours with K562 cells showed rapid cell death, but NK cells not stimulated with IL-2 did not. This cell death was found to involve nuclear condensation and fragmentation and DNA cleavage, all of which are characteristic of apoptosis. These data indicate that a proportion of activated human NK cells undergo apoptosis as they engage in target cell lysis. Target-induced NK cell death may represent an important mechanism for regulation of inflammatory processes involving NK cells.

scholarly journals Effect of Increased Urea Levels on Mouse Preimplantation Embryos Developed in Vivo and in Vitro

2012 ◽  
Vol 56 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Ján Bystriansky ◽  
Ján Burkuš ◽  
Štefan Juhás ◽  
Dušan Fabian ◽  
Juraj Koppel
Keyword(s):  
Apoptotic Cell ◽  
Nitrogen Concentration ◽  
High Plasma ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Plasma Urea ◽  
High Concentrations

Abstract High plasma urea nitrogen concentration has been proposed as an important factor contributing to the decline in reproductive parameters of domestic animals. The aim of this study was to evaluate the effect of urea on the development of preimplantation embryos in a mouse model. During in vivo tests, acute renal failure (ARF) accompanied by hyper-uraemia was induced by intramuscular administration of glycerol (50%) into hind limbs of fertilised dams. During in vitro tests, embryos collected from healthy dams were cultured in a medium with the addition of various concentrations of urea from the 4-cell stage to the blastocyst stage. Stereomicroscopic evaluation and fluorescence staining of embryos obtained from dams with ARF showed that high blood urea is connected with an increase in the number blastocysts containing at least one apoptotic cell and in the incidences of dead cells per blastocyst, but it did not affect their ability to reach the blastocyst stage. In vitro tests showed that culture of embryos with urea at concentration of 10 mM negatively affected the quality of obtained blastocysts. Blastocysts showed significantly lower numbers of cells and increased incidence of dead cells. An increase in apoptosis incidence was observed even in blastocysts obtained from cultures with 5 mM urea. Urea at concentrations 50 mM and higher negatively affected the ability of embryos to reach the blastocyst stage and the highest used concentrations (from 500 mM) caused overall developmental arrest of embryos at the 4- or 5- cell stage. These results show that elevated levels of urea may cause changes in the microenvironment of developing preimplantation embryos, which can negatively affect their quality. Embryo growth remains un-affected up to very high concentrations of urea.

scholarly journals Annona cherimola Seed Extract Activates Extrinsic and Intrinsic Apoptotic Pathways in Leukemic Cells

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 506 ◽  
Author(s):  
Tony Haykal ◽  
Peter Nasr ◽  
Mohammad H. Hodroj ◽  
Robin I. Taleb ◽  
Rita Sarkis ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Seed Extract ◽  
Leukemic Cells ◽  
Western Blots ◽  
Toxic Activity ◽  
Annona Cherimola

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways.

115 APOPTOSIS EVENT OF PREIMPLANTATION DEVELOPMENT STAGES IN PORCINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 157
Author(s):  
S. M. Hong ◽  
S. H. Jeong ◽  
S. H. Hyun
Keyword(s):  
Cell Death ◽  
Actinomycin D ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Development Stages

Little is known about apoptosis events in porcine preimplantation embryos. In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro-fertilized (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22 h postinsemination were treated at different concentrations of actinomycin D (0, 5, 50 and 500 ng mL–1 in NCSU medium). Four groups were incubated at 37°C in 5% CO2, 5%O2 for 8 h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2 days and BL development rate at 7 days after in vitro culture (IVC). A significantly less rate of cleavage was found in the 500 ng mL–1 group compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 15.4% v. 48.6%, 40%, 32%). In the results of BL formation rate, there was a significantly less BL formation rate in 500 ng mL–1 compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 0% v. 10%, 8.8%, 9%). In experiment 2, to evaluate apoptotic cells at different stage (2-cell, 4-cell, 8-cell and BL stage) of all groups, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng mL–1 actinomycin D). A high number of the BL derived control group contained at least one apoptotic cell. Actinomycin D treated BL responded to the presence of apoptotic inductor by a significant decrease in the average number of blastomeres and a significant increase in the incidence of apoptotic cell death. In the 500 ng mL–1 group, the incidence of apoptosis increased at the 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is a useful tool for the apoptosis study of porcine preimplantation embryos. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

163 SUCROSE ADDITION AT EARLY CULTURE STAGE IMPROVES DEVELOPMENT OF PRE-IMPLANTATION PORCINE NT AND IVF EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
I.-S. Hwang ◽  
J.-S. Seo ◽  
H.-S. Park ◽  
S.-W. Kim ◽  
D.-H. Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Culture Stage ◽  
Membrane Changes ◽  
Analysis System

Apoptosis is a form of cell death leading to fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes, and cell death without lysis or damage to neighboring cells. It might contribute to the low developmental rate of in vitro-produced (IVP) embryos, but apoptosis in porcine embryos is still unclear. This study investigated the effect of sucrose in the culture medium on the development of porcine NT and IVP embryos. Oocytes were aspirated from the follicles in ovaries collected from a local abattoir, and then matured in TCM-199 for 40-44 h. Fresh semen was diluted and equilibrated at 16�C. A final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus and used as donor cells. Embryos were cultured in PZM-3 supplemented with 0.05 M sucrose for 2 days, and then cultured in the PZM-3 without sucrose for 4 days at 38.5�C under 5% CO2 in air. Apoptotic cell death was analyzed by using a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. All data were subjected to a generalized linear model procedure (PROC-GLM) of the statistical analysis system (SAS; SAS Institute, Inc., Cary, NC, USA). NT and IVF embryos cultured in the medium supplemented with sucrose showed a significantly higher blastocyst formation rate than those cultured with no addition (28.8 vs. 20% and 32.3 vs. 17.9%; P < 0.05, respectively). For apoptosis, both NT and IVF embryos cultured in the medium with sucrose showed significantly lower frequency of apoptosis compared to embryos cultured in the medium without sucrose (3.4 vs. 6.3% and 0.6 vs. 1.8%; P < 0.05, respectively). Finally, the number of nuclei in NT blastocysts cultured in the medium with sucrose was higher than that of NT blastocysts cultured without sucrose (30.8 vs. 25.5; P < 0.05, respectively). However, the number of nuclei in the IVF blastocysts was not significantly different between groups. These results indicate that sucrose addition may increase the development of porcine NT and IVF embryos to the blastocyst stage and decrease the rate of apoptotic cells.

scholarly journals Aprepitant Promotes Caspase-Dependent Apoptotic Cell Death and G2/M Arrest through PI3K/Akt/NF-κB Axis in Cancer Stem-Like Esophageal Squamous Cell Carcinoma Spheres

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Hossein Javid ◽  
Amir R. Afshari ◽  
Farnaz Zahedi Avval ◽  
Jahanbakhsh Asadi ◽  
Seyed Isaac Hashemy
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Single Agent ◽  
Annexin V ◽  
Apoptotic Cell Death ◽  
Anticancer Activities ◽  
M Cell ◽  
Apoptotic Pathway ◽  
Neurokinin 1

The antagonists of the neurokinin-1 receptor (NK1R) are known for their anti-inflammatory, anxiolytic, antiemetic, and anticancer activities. Aprepitant, a nonpeptide NK1R antagonist, is used in nausea and vomiting, the most common side effects of cancer chemotherapy in patients. It has been established that NK1R activation by substance P (SP), which links cancer promotion and progression to a neurokinin-mediated environment, became one mechanism that corresponds to the mitogenesis of tumor cells. Therefore, this study is aimed at explaining and evaluating the anticancer impacts of aprepitant on esophageal squamous cancer cell (ESCC) spheres by using in vitro experiments, such as resazurin, ROS, annexin-V binding, RT-PCR, and Western blot analysis. As a result, we showed that aprepitant had strong antiproliferative and cytotoxic effects on ESCC cell spheres. Also, aprepitant caused significant G2-M cell cycle arrest depending on concentration increase. Further, exposure of cells to this agent resulted in caspase -8/-9-dependent apoptotic pathway activation by modifying the expression of genes involved in apoptosis. Besides, treatment of the cells by aprepitant abrogates of the PI3K/Akt pathway, as shown by reducing the level of Akt, induces apoptotic cell death. In summary, pharmacological inhibition of NK1R with aprepitant seems to have a significant chance of treating ESCC as a single agent or in conjunction with other chemotherapeutic drugs.

scholarly journals Multiorganelle Localization of Metallated Phthalocyanine Photosensitizer in Colorectal Cancer Cells (DLD-1 and CaCo-2) Enhances Efficacy of Photodynamic Therapy

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Palesa Rose Sekhejane ◽  
Nicolette Nadene Houreld ◽  
Heidi Abrahamse
Keyword(s):  
Colorectal Cancer ◽  
Cell Death ◽  
Photodynamic Therapy ◽  
Cancer Cells ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Annexin V ◽  
Zinc Phthalocyanine ◽  
Cell Death Pathway

Colorectal cancer is the third most commonly diagnosed cancer. Amongst treatments that have been explored, photodynamic therapy (PDT) is a treatment that is of interest as it poses ideal advantages such as affinity for cancer cells. This study aimed to determine the correlation between the localization site of a sulfonated zinc phthalocyanine (ZnPcSmix) photosensitizer (PS) and its associated cell death pathwayin vitroin colorectal cancer cell lines (DLD-1 and CaCo-2). Visible morphological changes were observed in PDT treated cells after 24 h. Reactive oxygen species (ROS) were detected and visualized 1 h after PDT.ZnPcSmixwas predominantly localized in lysosomes and partially in the mitochondria. FITC Annexin V staining showed a significant decrease in the percentage of viable DLD-1 and CaCo-2 cells 24 h after PDT, with an increase in apoptotic cell population. Moreover, there was a significant increase in both cathepsin D and cytochrome C at 1 and 24 h. In conclusion,ZnPcSmixshowed the ability of inducing apoptotic cell death features in PDT treated cells.

scholarly journals KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sachiko Iwai ◽  
Hanako O. Ikeda ◽  
Hisashi Mera ◽  
Kohei Nishitani ◽  
Motoo Saito ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Apoptotic Cell ◽  
Intraperitoneal Administration ◽  
Atp Depletion ◽  
Knee Joints ◽  
Cellular Protection ◽  
Monosodium Iodoacetate

AbstractCurrently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

scholarly journals AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 73-80 ◽  
Author(s):  
JongYeob Choi ◽  
MinWha Jo ◽  
EunYoung Lee ◽  
DooSeok Choi
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Apoptotic Cell ◽  
Follicular Development ◽  
Negative Regulator ◽  
Metabolic Clearance ◽  
Akt Inhibitor ◽  
Western Blots

In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition,in-vitroFSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death.

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