scholarly journals Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D

1999 ◽  
Vol 112 (13) ◽  
pp. 2223-2232
Author(s):  
K. Rothbarth ◽  
E. Spiess ◽  
B. Juodka ◽  
U. Yavuzer ◽  
P. Nehls ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Binding ◽  
Bystander Effect ◽  
Fluorescent Protein ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Apoptotic Cell Death ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Suspension Cells ◽  
Transfected Cells

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12–24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.

Bufalin Induces Apoptotic Cell Death in Human Nasopharyngeal Carcinoma Cells through Mitochondrial ROS and TRAIL Pathways

2019 ◽  
Vol 47 (01) ◽  
pp. 237-257 ◽  
Author(s):  
En-Yun Su ◽  
Yung-Lin Chu ◽  
Fu-Shin Chueh ◽  
Yi-Shih Ma ◽  
Shu-Fen Peng ◽  
...  
Keyword(s):  
Cell Death ◽  
Nasopharyngeal Carcinoma ◽  
Er Stress ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Chromatin Condensation ◽  
Apoptotic Cell Death ◽  
Dapi Staining ◽  
Human Nasopharyngeal Carcinoma ◽  
Associated Proteins

The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G2/M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca[Formula: see text] production and decreased the levels of [Formula: see text]. Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1[Formula: see text], IRE-1[Formula: see text], caspase-4, ATF-6[Formula: see text], Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.

scholarly journals In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2234-2243 ◽  
Author(s):  
Giorgio Zauli ◽  
Marco Vitale ◽  
Elisabetta Falcieri ◽  
Davide Gibellini ◽  
Alessandra Bassini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Apoptotic Cell ◽  
High Surface ◽  
Suspension Cultures ◽  
Apoptotic Cell Death ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Terminal Phase ◽  
Serum Free

Abstract To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.

Failure of gelsolin overexpression to regulate lymphocyte apoptosis

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3483-3488 ◽  
Author(s):  
S. Celeste Posey ◽  
Maria Paola Martelli ◽  
Toshifumi Azuma ◽  
David J. Kwiatkowski ◽  
Barbara E. Bierer
Keyword(s):  
Cell Death ◽  
Growth Factor ◽  
Actin Filaments ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Apoptotic Cell Death ◽  
Model Systems ◽  
Dependent Manner ◽  
Amino Terminal

Abstract The actin regulatory protein gelsolin cleaves actin filaments in a calcium- and polyphosphoinositide-dependent manner. Gelsolin has recently been described as a novel substrate of the cysteinyl protease caspase-3, an effector protease activated during apoptosis. Cleavage by caspase-3 generates an amino-terminal fragment of gelsolin that can sever actin filaments independently of calcium regulation. The disruption of the actin cytoskeleton by cleaved gelsolin is hypothesized to mediate many of the downstream morphological changes associated with apoptosis. In contrast, overexpression of full-length gelsolin has also been reported to inhibit apoptotic cell death upstream of the activation of caspase-3, suggesting that gelsolin may also act prior to commitment to cell death. The authors previously observed that actin stabilization by the cell permeant agent jasplakinolide enhanced cell death upon interleukin (IL)-2 or IL-3 withdrawal from growth-factor–dependent lymphocyte cell lines, and hypothesized that actin polymerization could alter the activity of gelsolin, thus enhancing apoptosis. Here the authors show that constitutive overexpression of gelsolin did not, however, inhibit or dramatically enhance apoptotic cell death upon growth-factor withdrawal, nor did it modify sensitivity to jasplakinolide. In contrast to previous reports, overexpression of gelsolin in Jurkat T cells did not prevent or delay apoptosis induced by Fas ligation or ceramide treatment. Overexpressed gelsolin protein was cleaved during apoptosis, as seen previously in this and other cell types. In these model systems, therefore, the level of gelsolin expression was not a rate-limiting determinant in commitment to or time to the morphological changes of apoptosis.

Functional Validation of DownRegulated MicroRNAs in HeLa Cells Treated with Polyalthia longifolia Leaf Extract Using Different Microscopic Approaches: A Morphological Alteration-Based Validation

2019 ◽  
Vol 25 (05) ◽  
pp. 1263-1272 ◽  
Author(s):  
Shanmugapriya ◽  
Soundararajan Vijayarathna ◽  
Sreenivasan Sasidharan
Keyword(s):  
Cell Death ◽  
Electron Microscope ◽  
Hela Cells ◽  
Leaf Extract ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Apoptotic Cell Death ◽  
Morphological Alteration ◽  
Polyalthia Longifolia

AbstractSeveral microscopy methods have been developed to assess the morphological changes in cells in the investigations of the mode of cell death in response to a stimulus. Our recent finding on the treatment of the IC50 concentration (26.67 μg/mL) of Polyalthia longifolia leaf extract indicated the induction of apoptotic cell death via the regulation of miRNA in HeLa cells. Hence, the current study was conducted to validate the function of these downregulated microRNAs in P. longifolia-treated HeLa cells using microscopic approaches. These include scanning electron microscope (SEM), transmission electron microscope (TEM), and acridine orange/propidium iodide (AO/PI)-based fluorescent microscopy techniques by observing the morphological alterations to cells after transfection with mimic miRNA. Interestingly, the morphological changes observed in this study demonstrated the apoptotic hallmarks, for instance, cell blebbing, cell shrinkage, cytoplasmic and nuclear condensation, vacuolization, cytoplasmic extrusion, and the formation of apoptotic bodies, which proved the role of dysregulated miRNAs in apoptotic HeLa cell death after treatment with the P. longifolia leaf extract. Conclusively, the current study proved the crucial role of downregulated miR-484 and miR-221-5p in the induction of apoptotic cell death in P. longifolia-treated HeLa cells using three approaches—SEM, TEM, and AO/PI-based fluorescent microscope.

Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron

1998 ◽  
Vol 275 (3) ◽  
pp. H1046-H1053 ◽  
Author(s):  
Daniel W. Goldman ◽  
Richard J. Breyer ◽  
David Yeh ◽  
Beth A. Brockner-Ryan ◽  
Abdu I. Alayash
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Cell Death ◽  
Dna Fragmentation ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Blood Substitutes ◽  
Apoptotic Cell Death ◽  
Redox Activity ◽  
Chemically Modified

We tested the hypothesis that chemical modifications used to produce stable, oxygen-carrying, Hb-based blood substitutes can induce cytotoxicity in endothelial cells in culture because of altered redox activity. We examined the interaction of hydrogen peroxide with nonmodified hemoglobin (HbA0) and two chemically modified hemoglobins, α-cross-linked hemoglobin (α-DBBF) and its polymerized form (poly-α-DBBF). Hydrogen peroxide-induced cell death (as assessed by lactate dehydrogenase release) in bovine aortic endothelial cells (BAEC) was completely inhibited by all three hemoglobin preparations, consistent with their known pseudoperoxidase activity [hemoglobin consumes peroxide as it cycles between ferric (Fe3+) and ferryl (Fe4+) hemes]. However, reaction of the modified hemoglobins, but not HbA0, with hydrogen peroxide induced apoptotic cell death (as assessed by morphological changes and DNA fragmentation) that correlated with the formation of a long-lived ferrylhemoglobin. A preparation of ferryl-α-DBBF free of residual peroxide rapidly induced morphological changes and DNA fragmentation in BAEC, indicative of apoptotic cell death. Redox cycling of chemically modified hemoglobins by peroxide yielded a persistent ferryl iron that was cytotoxic to endothelial cells.

scholarly journals Green-Synthesized Silver Nanoparticles Induced Apoptotic Cell Death in MCF-7 Breast Cancer Cells by Generating Reactive Oxygen Species and Activating Caspase 3 and 9 Enzyme Activities

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Ikram Ullah ◽  
Ali Talha Khalil ◽  
Muhammad Ali ◽  
Javed Iqbal ◽  
Waqar Ali ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Silver Nanoparticles ◽  
Cell Death ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Reactive Oxygen ◽  
Apoptotic Cell Death ◽  
Oxygen Species ◽  
Cell Viability Assay ◽  
Mcf 7

Silver nanoparticles are among the most significant diagnostic and therapeutic agents in the field of nanomedicines. In the current study, the green chemistry approach was made to optimize a cost-effective synthesis protocol for silver nanoparticles from the aqueous extract of the important anticancer plant Fagonia indica. We investigated the anticancer potential and possible involvement of AgNPs in apoptosis. The biosynthesized AgNPs are stable (zeta potential, -16.3 mV) and spherical with a crystal size range from 10 to 60 nm. The MTT cell viability assay shows concentration-dependent inhibition of the growth of Michigan Cancer Foundation-7 (MCF-7) cells (IC50, 12.35 μg/mL). In addition, the fluorescent microscopic analysis shows activation of caspases 3 and 9 by AgNPs that cause morphological changes (AO/EB assay) in the cell membrane and cause nuclear condensation (DAPI assay) that eventually lead to apoptotic cell death (Annexin V/PI assay). It was also observed that AgNPs generate reactive oxygen species (ROS) that modulate oxidative stress in MCF-7 cells. This is the first study that reports the synthesis of a silver nanoparticle mediated by Fagonia indica extract and evaluation of the cellular and molecular mechanism of apoptosis.

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