Apoptosis and Necrosis after Reversible Focal Ischemia: An in Situ DNA Fragmentation Analysis

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain ( Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385–388). We now show that a significant number of striatal and cortical neurons exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

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Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells.

Acta Biochimica Polonica ◽

10.18388/abp.2006_3324 ◽

2006 ◽

Vol 53 (3) ◽

pp. 531-538 ◽

Cited By ~ 20

Author(s):

Adriana Magalska ◽

Agnieszka Brzezinska ◽

Anna Bielak-Zmijewska ◽

Katarzyna Piwocka ◽

Grazyna Mosieniak ◽

...

Keyword(s):

Cell Death ◽

Cell Viability ◽

Dna Fragmentation ◽

Caspase 3 ◽

Apoptotic Cell ◽

Morphological Changes ◽

Chromatin Condensation ◽

Human Lymphocytes ◽

Dna Degradation ◽

Cd8 Cells

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.

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The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

Eukaryotic Cell ◽

10.1128/ec.00138-13 ◽

2013 ◽

Vol 12 (9) ◽

pp. 1225-1234 ◽

Cited By ~ 29

Author(s):

F. Shirazi ◽

D. P. Kontoyiannis

Keyword(s):

Cell Death ◽

Dna Fragmentation ◽

Rhizopus Oryzae ◽

Apoptotic Cell ◽

Chromatin Condensation ◽

Membrane Integrity ◽

Ros Generation ◽

Content Type ◽

Intracellular Ca ◽

Calcineurin Pathway

ABSTRACT The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales . We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca 2+ , phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 μg/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 μg/ml) for Rhizopus oryzae , Cunninghamella bertholletiae , and Mucor circinelloides . Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca 2+ and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies.

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Mitochondria-independent morphological and biochemical apoptotic alterations promoted by the anti-tumor agent bleomycin in Saccharomyces cerevisiae

Biochemistry and Cell Biology ◽

10.1139/o06-147 ◽

2007 ◽

Vol 85 (1) ◽

pp. 49-55 ◽

Cited By ~ 14

Author(s):

Mustapha Aouida ◽

Halima Mekid ◽

Omrane Belhadj ◽

Lluis M. Mir ◽

Omar Tounekti

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Potent Inhibitor ◽

Apoptotic Cell ◽

Morphological Changes ◽

Chromatin Condensation ◽

Model System ◽

Dna Breaks ◽

Double Stranded Dna ◽

Tumor Agent

Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded DNA breaks in a catalytic way. Using Saccharomyces cerevisiae as a model system, we show that when a high amount of bleomycin molecules is internalized (100 µmol/L), morphological changes identical to those usually associated with apoptosis, i.e., a sub-G1 region peak, chromatin condensation, and very rapid DNA fragmentation into oligonucleosomal-sized fragments, are observed. The known bleomycin inhibitors cobalt and EDTA were able to prevent bleomycin nucleasic activity and thus apoptotic cell death. However, both oligomycin, a potent inhibitor of the mitochondial F0F1-ATPase, and antimycin, a drug affecting mitochondria respiration, were unable to prevent the bleomycin-induced apoptotic-like cell death. These results suggest that high bleomycin concentrations induce an apoptosis-like mitochondria-independent cell death in yeast.

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Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Journal of Biological Chemistry ◽

10.1074/jbc.m112.411371 ◽

2013 ◽

Vol 288 (13) ◽

pp. 9200-9215 ◽

Cited By ~ 23

Author(s):

Victoria Iglesias-Guimarais ◽

Estel Gil-Guiñon ◽

María Sánchez-Osuna ◽

Elisenda Casanelles ◽

Mercè García-Belinchón ◽

...

Keyword(s):

Cell Death ◽

Dna Fragmentation ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Single Strand ◽

Dna Breaks

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Apoptotic cell death at tooth eruption in rat molar observed by in situ labeling of nuclear DNA fragmentation.

Japanese Journal of Oral Biology ◽

10.2330/joralbiosci1965.37.488 ◽

1995 ◽

Vol 37 (6) ◽

pp. 488-492 ◽

Cited By ~ 4

Author(s):

Yoshihiro Abiko ◽

Hidetoshi Kanno ◽

Jiro Arai ◽

Michiko Nishimura ◽

Masato Saitoh ◽

...

Keyword(s):

Cell Death ◽

Dna Fragmentation ◽

Nuclear Dna ◽

Apoptotic Cell ◽

Tooth Eruption ◽

Apoptotic Cell Death ◽

Nuclear Dna Fragmentation ◽

Rat Molar

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Transcriptional CDK inhibitors, CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation

Cell Death and Disease ◽

10.1038/s41419-021-04050-7 ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Viktorija Juric ◽

Lance Hudson ◽

Joanna Fay ◽

Cathy E. Richards ◽

Hanne Jahns ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Cell Cycle Arrest ◽

Cortical Neurons ◽

Apoptotic Cell ◽

Cdk Inhibitors ◽

Induce Cell Death ◽

Viability Loss ◽

Cycle Arrest ◽

Recurrent Gbm

AbstractActivation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

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Two steroidal saponins from Camassia cusickii induce L1210 cell death through the apoptotic mechanism

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y01-068 ◽

2001 ◽

Vol 79 (11) ◽

pp. 953-958 ◽

Cited By ~ 3

Author(s):

Ellyawati Candra ◽

Kimihiro Matsunaga ◽

Hironori Fujiwara ◽

Yoshihiro Mimaki ◽

Yutaka Sashida ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Apoptotic Cell ◽

Chromatin Condensation ◽

Flow Cytometric Analysis ◽

Apoptotic Cell Death ◽

Morphological Observation ◽

Steroidal Saponin ◽

Steroidal Saponins ◽

L1210 Cells

Two steroidal saponins, tigogenin hexasaccharide-1 (TGHS-1, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside) and tigogenin hexasaccharide-2 (TGHS-2, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (β-D-glucopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside), were isolated from the fresh bulbs of Camassia cusickii. In murine leukemic L1210 cells, both compounds showed cytotoxicity with an EC50 value of 0.06 µM. The morphological observation revealed that TGHS-1 and TGHS-2 induced shrinkage in cell soma and chromatin condensation, suggesting apoptotic cell death. The cell death was confirmed to be apoptosis by Annexin V binding to phosphatidylserine in the cell membrane and excluding propidium iodide. A typical apoptotic DNA ladder and the cleavage of caspase-3 were observed after treatment with TGHS-1 and TGHS-2. In the presence of both the compounds, cells with sub-G1 DNA content were detected by flow cytometric analysis, indicating that TGHS-1 and TGHS-2 (each EC50 value of 0.1 µM) are the most powerful apoptotic saponins known. These results suggest that TGHS-1 and TGHS-2 induce apoptotic cell death through caspase-3 activation.Key words: steroidal saponin, tigogenin hexasaccharide, apoptosis, DNA fragmentation, murine leukemic L1210 cells.

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A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.1.7678025 ◽

1993 ◽

Vol 41 (1) ◽

pp. 7-12 ◽

Cited By ~ 542

Author(s):

J H Wijsman ◽

R R Jonker ◽

R Keijzer ◽

C J van de Velde ◽

C J Cornelisse ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Staining Method ◽

Morphological Characteristics ◽

Image Cytometry ◽

Apoptotic Cells ◽

Dna Breaks ◽

Paraffin Sections ◽

Tissue Sections

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

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Survivin in relation to Bcl-2, Bax and in situ apoptotic cell death in anaplastic thyroid carcinoma

Archives of Biological Sciences ◽

10.2298/abs1104955s ◽

2011 ◽

Vol 63 (4) ◽

pp. 955-963

Author(s):

Sonja Selemetjev ◽

Dubravka Cvejic ◽

Svetlana Savin ◽

I. Paunovic ◽

S. Tatic

Keyword(s):

Cell Death ◽

Thyroid Carcinoma ◽

Apoptotic Cell ◽

Anaplastic Thyroid Carcinoma ◽

Apoptotic Cell Death ◽

Staining Score ◽

Human Malignancy ◽

Apoptotic Pathways ◽

Individual Scores

Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive human malignancy. It is known that disturbances in apoptotic pathways have a great impact on tumor progression and aggressiveness. In this study the apoptosisrelated molecules Bcl-2 (antiapoptotic), Bax (proapoptotic) and survivin (an inhibitor of apoptosis) were analyzed immunohistochemically in thirty archival cases of ATC. In situ apoptotic cell death was analyzed by the TUNEL method. Mean Bcl-2 staining score (calculated from individual scores from 0-3) was low compared to those for Bax and survivin (p<0.05). High expression of survivin was associated with high Bax expression, and was significantly segregated from high Bcl-2 expressing cases (p<0.05). Despite high Bax expression, apoptotic cell death was low in the investigated carcinomas. In addition, the mean apoptotic index in high survivin expressing carcinomas was significantly lower than in low survivin expressing carcinomas (p<0.05). It could be concluded that down-regulation of Bcl-2 is counterbalanced by up-regulation of survivin, which may overcome the effects of high Bax expression, and, at least partly, explain the low apoptosis rate and high biological aggressiveness of ATC.

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