scholarly journals Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Majtnerova Pavlina ◽  
Capek Jan ◽  
Petira Filip ◽  
Handl Jiri ◽  
Rousar Tomas
Keyword(s):  
Cell Death ◽  
Low Cost ◽  
Hoechst 33258 ◽  
Dna Ladder ◽  
Nuclear Condensation ◽  
Intact Cells ◽  
Fast Processing ◽  
Nuclear Changes ◽  
Parp 1 ◽  
Hepatoma Hepg2

AbstractAt present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6–48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

scholarly journals Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells

2021 ◽  
Author(s):  
Pavlina Majtnerova ◽  
Jan Capek ◽  
Filip Petira ◽  
Jiri Handl ◽  
Tomáš Roušar
Keyword(s):  
Cell Death ◽  
Low Cost ◽  
Caspase Activity ◽  
Hoechst 33258 ◽  
Dna Ladder ◽  
Nuclear Condensation ◽  
Intact Cells ◽  
Fast Processing ◽  
Nuclear Changes ◽  
Cells Cultured

Abstract At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods has used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in intact cells. We used HepG2 and HK‑2 cells cultured in 96-well plates which were treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Then, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting apoptosis (i.e. TUNEL, DNA ladder, caspase activity). We found that the developed assay provides results of same sensitivity as TUNEL assay but the advantages of the spectrofluorometric assay are fast processing, low-cost and high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis

1997 ◽  
Vol 110 (7) ◽  
pp. 819-828 ◽  
Author(s):  
A. Simm ◽  
G. Bertsch ◽  
H. Frank ◽  
U. Zimmermann ◽  
J. Hoppe
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Structural Changes ◽  
Energy State ◽  
Energy Charge ◽  
High Energy ◽  
Serum Starvation ◽  
Nuclear Condensation ◽  
Mitochondrial Potential ◽  
Intact Cells

AKR-2B cells disintegrate after serum removal. After a delay of approximately 90 minutes, cell death began and reached after six hours a plateau of 40–50% remaining living cells. We used time-lapse video microscopy to monitor dynamic structural changes and to measure the time span of individual cells to die. The first change was the rapid appearance of membrane blebs. Membrane vesicles were rapidly extruded and reintegrated by the cell. This highly dynamic process of an affected cell stopped after 80+/−20 minutes with its death. Conductivity measurements showed that at that time the membrane was electrically permeable. By using fluorescence double staining with propidium iodide and Hoechst 33258, we show that membrane leakage leading to disintegration is accompanied, and for some cells preceded, by nuclear condensation. The energy state of the intact cells was monitored by measuring the intracellular ATP content which remained high (6 mM) throughout the entire time of investigation. Mitochondrial potential was determined by rhodamine 123 fluorescence in parallel to the measurement of membrane permeability via uptake of propidium iodide and lead to the detection of a cell population that exhibits a high mitochondrial potential and an uptake of propidium iodide indicating a membrane disruption of cells which still have a high energy charge. It is shown by electron microscopy that mitochondria were swollen and damaged in parallel to nuclear condensation. There was no DNA fragmentation as shown by two independent methods. Addition of the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone immediately after serum starvation lead to an almost complete survival of the cells up to 6 hours. A pronounced protection was still observed after 24 hours, suggesting an involvement of this type of protease in the onset of cell death after serum removal. Apparently, serum withdrawal activates a succession of initial events that are similar to those defined as ‘apoptosis’, i.e. nuclear condensation and membrane blebbing. These steps are, however, accompanied or rapidly followed by cell lysis and disruption of mitochondria, both of which are characteristic of necrosis.

Cell death induced by granzyme C

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3093-3101 ◽  
Author(s):  
Hillary Johnson ◽  
Luca Scorrano ◽  
Stanley J. Korsmeyer ◽  
Timothy J. Ley
Keyword(s):  
Cell Death ◽  
Granzyme B ◽  
The Other ◽  
Cytotoxic Lymphocytes ◽  
Nuclear Condensation ◽  
Intact Cells ◽  
Isolated Mitochondria ◽  
Molar Basis ◽  
Dna Nicking ◽  
Kinetics Of

Abstract Although the functions of granzymes A and B have been defined, the functions of the other highly expressed granzymes (Gzms) of murine cytotoxic lymphocytes (C, D, and F) have not yet been evaluated. In this report, we describe the ability of murine GzmC (which is most closely related to human granzyme H) to cause cell death. The induction of death requires its protease activity and is characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. The kinetics of these events are similar to those caused by granzyme B, and its potency (defined on a molar basis) is also equivalent. The induction of death did not involve the activation of caspases, the cleavage of BID, or the activation of the CAD nuclease. However, granzyme C did cause rapid mitochondrial swelling and depolarization in intact cells or in isolated mitochondria, and this mitochondrial damage was not prevented by cyclosporin A pretreatment. These results suggest that granzyme C rapidly induces target cell death by attacking nuclear and mitochondrial targets and that these targets are distinct from those used by granzyme B to cause classical apoptosis.

scholarly journals Characterization of Host Cell Death Induced by Chlamydia trachomatis

2006 ◽  
Vol 74 (11) ◽  
pp. 6057-6066 ◽  
Author(s):  
Songmin Ying ◽  
Silke F. Fischer ◽  
Matthew Pettengill ◽  
Debye Conte ◽  
Stefan A. Paschen ◽  
...  
Keyword(s):  
Cell Death ◽  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Chlamydial Infection ◽  
Slight Reduction ◽  
Nuclear Condensation ◽  
Apoptotic Pathway ◽  
Host Cell Death ◽  
Nuclear Changes ◽  
Infected Cells

ABSTRACT Chlamydia are obligate intracellular bacteria that modulate apoptosis of the host cell. Strikingly, chlamydial infection has been reported both to inhibit and to induce apoptosis. Although the ability to inhibit apoptosis has been corroborated by the identification of cellular targets, confirmation of cell death induction has been complicated by a mixture of apoptotic features and atypical cell death during infection, as well as by differences in the experimental techniques used to measure cell death. Here we use a panel of well-established approaches in the study of apoptosis to define the form of cell death induced by Chlamydia trachomatis infection. Infected cells displayed apoptotic features such as nuclear condensation and fragmentation, as well as positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining. Fragmentation of genomic DNA occurred, but was atypical. Clear evidence against the activation of effector caspases was found. Nuclear changes were measured in fibroblasts lacking one or both of the effectors of mitochondrial apoptosis, Bax and Bak. A slight reduction in nuclear changes was observed in Bax-deficient cells and in Bax/Bak double-deficient cells. Most surprisingly, this reduction was almost complete in Bak-deficient cells. Finally, dying infected cells were efficiently taken up by professional phagocytes, suggesting that Chlamydia-induced host-cell death could play a role in the immune response. In conclusion, chlamydial infection can induce cell death. Although Chlamydia-induced cell death has certain morphological features of apoptosis, it does not result from activation of the apoptotic pathway.

scholarly journals Discovery of pyrano[2,3-d]pyrimidine-2,4-dione derivatives as novel PARP-1 inhibitors: design, synthesis and antitumor activity

RSC Advances ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 4454-4464
Author(s):  
Nour E. A. Abd El-sattar ◽  
Eman H. K. Badawy ◽  
Eman Z. Elrazaz ◽  
Nasser S. M. Ismail
Keyword(s):  
Cell Death ◽  
Dna Repair ◽  
Antitumor Activity ◽  
Cancer Cell ◽  
Cytotoxic Agents ◽  
Repair Mechanism ◽  
Design Synthesis ◽  
Parp 1

PARP-1 are involved in DNA repair damage and so PARP-1 inhibitors have been used as potentiators in combination with DNA damaging cytotoxic agents to compromise the cancer cell DNA repair mechanism, resulting in genomic dysfunction and cell death.

scholarly journals Poly (ADP-Ribose) Polymerase 1 Protein Expression in Normal Pancreas and Pancreatic Adenocarcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-4
Author(s):  
Roberto Castiglione ◽  
Aldo E. Calogero ◽  
Enzo Vicari ◽  
Giovanna Calabrini ◽  
Anna Cosentino ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Pancreatic Cancer ◽  
Cell Death ◽  
Pancreatic Adenocarcinoma ◽  
Cancer Tissue ◽  
Normal Pancreas ◽  
Normal Prostate ◽  
Cytoplasmic Staining ◽  
Cancer Of The Pancreas ◽  
Parp 1

Pancreatic cancer is a most frequent cancer in Europe, and the majority of cases of cancer of the pancreas are diagnosed above the age of 65. Radical surgery is the first curative treatment of pancreatic cancer, and alternative or combined therapeutic options, in particular, consist of adjuvant or neoadjuvant chemotherapy, with or without radiotherapy. Many factors, including diet and genetics, have been implicated in the development of cancer of the pancreas. Poly (ADP-ribose) polymerase 1 (PARP-1) protein is required for translocation of the apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. It is involved in programmed cell death processes. Different PARP-1 gene expression proteins have been observed in various tumors such as lung, ovarian, endometrial, skin, and glioblastoma. We evaluated the expression of PARP-1 protein in pancreatic adenocarcinoma and normal pancreas tissues by immunohistochemistry. Protein PARP-1 in the nucleus was found in all samples (normal pancreas and pancreatic adenocarcinoma tissues). No cytoplasmic staining was observed in any sample. PARP-1-positive cells resulted higher in the normal pancreas compared with the pancreas with adenocarcinoma. PARP-1 overexpression in prostate cancer tissue compared with normal prostate suggests a greater activity of PARP-1 in these tumors. These findings suggest that PARP-1 expression in prostate cancer is an attempt to trigger apoptosis in this type of tumor, similarl to that reported in other cancers. This finding suggests that PARP-1-mediated cell death pathways are inhibited in this cancer.

scholarly journals DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lama Tarayrah-Ibraheim ◽  
Elital Chass Maurice ◽  
Guy Hadary ◽  
Sharon Ben-Hur ◽  
Alina Kolpakova ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Germ Cells ◽  
Nuclear Translocation ◽  
Primordial Germ Cells ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Dnase Ii ◽  
Cell Death Pathway ◽  
Parp 1

AbstractDuring Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.

Poly(ADP-ribose) polymerase-1 gene ablation protects mice from ischemic renal injury

2005 ◽  
Vol 288 (2) ◽  
pp. F387-F398 ◽  
Author(s):  
Jianfeng Zheng ◽  
Kishor Devalaraja-Narashimha ◽  
Kurinji Singaravelu ◽  
Babu J. Padanilam
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Mouse Model ◽  
Renal Injury ◽  
Atp Depletion ◽  
Inflammatory Cascade ◽  
Renal Functions ◽  
Atp Levels ◽  
Ischemic Renal Injury ◽  
Parp 1

Increased generation of reactive oxygen species (ROS) and the subsequent DNA damage and excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in the pathogenesis of ischemic injury. We previously demonstrated that pharmacological inhibition of PARP protects against ischemic renal injury (IRI) in rats (Martin DR, Lewington AJ, Hammerman MR, and Padanilam BJ. Am J Physiol Regul Integr Comp Physiol 279: R1834–R1840, 2000). To further define the role of PARP-1 in IRI, we tested whether genetic ablation of PARP-1 attenuates tissue injury after renal ischemia. Twenty-four hours after reperfusion following 37 min of bilateral renal pedicle occlusion, the effects of the injury on renal functions in PARP−/− and PARP+/+ mice were assessed by determining glomerular filtration rate (GFR) and the plasma levels of creatinine. The levels of plasma creatinine were decreased and GFR was augmented in PARP−/− mice. Morphological evaluation of the kidney tissues showed that the extent of damage due to the injury in PARP−/− mice was less compared with their wild-type counterparts. The levels of ROS and DNA damage were comparable in the injured kidneys of PARP+/+ and PARP−/− mice. PARP activity was induced in ischemic kidneys of PARP+/+ mice at 6–24 h postinjury. At 6, 12, and 24 h after injury, ATP levels in the PARP+/+ mice kidney declined to 28, 26, and 43%, respectively, whereas it was preserved close to normal levels in PARP−/− mice. The inflammatory cascade was attenuated in PARP−/− mice as evidenced by decreased neutrophil infiltration and attenuated expression of inflammatory molecules such as TNF-α, IL-1β, and intercellular adhesion molecule-1. At 12 h postinjury, no apoptotic cell death was observed in PARP−/− mice kidneys. However, by 24 h postinjury, a comparable number of cells underwent apoptosis in both PARP−/− and PARP+/+ mice kidneys. Thus activation of PARP post-IRI contributes to cell death most likely by ATP depletion and augmentation of the inflammatory cascade in the mouse model. PARP ablation preserved ATP levels, renal functions, and attenuated inflammatory response in the setting of IRI in the mouse model. PARP inhibition may have clinical efficacy in preventing the progression of acute renal failure complications.

scholarly journals Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study

2004 ◽  
Vol 382 (3) ◽  
pp. 877-884 ◽  
Author(s):  
Bruno GUIGAS ◽  
Dominique DETAILLE ◽  
Christiane CHAUVIN ◽  
Cécile BATANDIER ◽  
Frédéric De OLIVEIRA ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Cytochrome C Release ◽  
Intact Cells ◽  
Human Carcinoma ◽  
Butyl Hydroperoxide ◽  
Mitochondrial Permeability ◽  
New Findings

Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition.

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