Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

Wolbers, F.; Buijtenhuijs, P.; Haanen, C.; Vermes, I.

doi:10.1023/b:appt.0000025816.16399.7a

Cited by 53 publications

(44 citation statements)

References 16 publications

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“…In endothelial cells, this tends to happen soon after the induction of apoptosis (56). Fluorescence microscopy was performed immediately after staining.…”

Section: Methodsmentioning

confidence: 99%

Plasmodium falciparumMalaria: Reduction of Endothelial Cell Apoptosis In Vitro

Hemmer

Lehr²,

Westphal

et al. 2005

Infect Immun

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Organ failure in Plasmodium falciparum malaria is associated with neutrophil activation and endothelial damage. This study investigates whether neutrophil-induced endothelial damage involves apoptosis and whether it can be prevented by neutralization of neutrophil secretory products. Endothelial cells from human umbilical veins were coincubated with neutrophils from healthy donors and with sera from eight patients with P. falciparum malaria, three patients with P. vivax malaria, and three healthy controls. Endothelial apoptosis was demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V staining. The rate of apoptosis of cells was markedly increased after incubation with patient serum compared to that with control serum. Apoptosis was most pronounced after incubation with sera from two patients with fatal cases of P. falciparum malaria, followed by sera of survivors with severe P. falciparum malaria and, finally, by sera of patients with mild P. falciparum and P. vivax malaria. Ascorbic acid, tocopherol, and ulinastatin reduced the apoptosis rate, but gabexate mesilate and pentoxifylline did not. Furthermore, in fatal P. falciparum malaria, apoptotic endothelial cells were identified in renal and pulmonary tissue by TUNEL staining. These findings show that apoptosis caused by neutrophil secretory products plays a major role in endothelial cell damage in malaria. The antioxidants ascorbic acid and tocopherol and the protease inhibitor ulinastatin can reduce malaria-associated endothelial apoptosis in vitro.

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“…In endothelial cells, this tends to happen soon after the induction of apoptosis (56). Fluorescence microscopy was performed immediately after staining.…”

Section: Methodsmentioning

confidence: 99%

Plasmodium falciparumMalaria: Reduction of Endothelial Cell Apoptosis In Vitro

Hemmer

Lehr²,

Westphal

et al. 2005

Infect Immun

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“…By contrast, nearly all of the HS-treated cells ( Figure 1J) displayed a loss of plasma membrane integrity (99 Ϯ 1% PI ϩ ) with light scatter changes and uptake of PI ϩ , typical of necrosis. 31 By 48 hours, both etoposide-and rotenone-treated cells were 70 Ϯ 4% and 72 Ϯ 1% PI ϩ , respectively (Supplemental Figure 2, see http://ajp. amjpathol.org), suggesting late apoptosis/secondary necrosis.…”

Section: Chemically Induced Hypoxia-mediated Apoptosismentioning

confidence: 99%

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

Orozco

Jorgez

Horne

et al. 2008

The American Journal of Pathology

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Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA ؉ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. (Am J

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“…According to product manuals, these membrane-permeable and nontoxic inhibitors enter the cell and bind covalently to the reactive cysteine residue that resides on the large subunit of the active caspase heterodimer. This system undoubtedly represents a useful research tool to monitor apoptosis progression (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). It was also used to study the intracellular localization of caspases and their changes during the apoptosis (15).…”

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confidence: 99%

Labeling of apoptotic JURL‐MK1 cells by fluorescent caspase‐3 inhibitor FAM‐DEVD‐fmk occurs mainly at site(s) different from caspase‐3 active site

2007

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Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as an alternative tool for the detection of caspase activation in whole cells. They should label the active site of the corresponding caspase through a covalent attachment to the reactive cysteine residue. One of the FLICAs, FAM-DEVD-fmk, was used to monitor apoptosis progression in leukemic JURL-MK1 cells by means of flow cytometry. The effects of unlabeled caspase inhibitors z-DEVD-fmk and z-VAD-fmk on FLICA staining were analyzed to evaluate the contribution of caspase-bound FLICA to the fluorescent signal. Covalent binding of inhibitors to caspase-3 subunit was revealed by Western blotting. Although the unlabeled inhibitors irreversibly bind to caspase-3, completely inhibit its activity, and prevent FLICA binding to caspase-3 even at concentrations lower than 5 lM, they have no effect on FLICA staining of apoptotic cells. Fluorescent signal of FLICA is characteristic for apoptotic cells but originates mainly from yet unspecified site(s) that differ from the caspase active site. This finding puts in doubt the specificity of staining by various FLICAs with regard to individual caspases and shows the need for an extreme care in the interpretation of data obtained using these labels. ' 2007International Society for Analytical Cytology Key termsFLICA; apoptosis; JURL-MK1; Imatinib; z-DEVD-fmk; z-VAD-fmk; flow cytometry; Western blotting CASPASES are widely considered to be the key mediators of apoptosis. This enzyme family participates in a series of reactions that are triggered in response to proapoptotic signals and result in the cleavage of protein substrates and finally in the controlled disassembly of the cell. Caspases are synthesized as inactive precursors (procaspases) that undergo proteolytic maturation upon apoptotic stimulation.Detection of caspase activation provides a useful assay for analyzing one of the earliest known biochemical events associated with apoptosis. The cleavage of procaspases into the active fragments can be observed on immunoblots. Anticaspase antibodies which specifically recognize the active enzyme forms are also available for use in immunoblotting as well as in flow cytometry. Alternatively, the enzyme activity rather than its presence can be monitored by means of fluorogenic substrates generating strong fluorescence signal upon cleavage by caspases. Another approach to study activation of caspases in situ utilizes fluorescently labeled irreversible caspase inhibitors (1,2). Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as affinity labels of the enzyme active center and consist of a short peptide, a fluoromethylketone (fmk) moiety, and a fluorescent tag, usually carboxyfluorescein (FAM) or fluorescein isothiocyanate (FITC). The amino acid composition of the peptide reflects the substrate

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Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

Abstract: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.

Cited by 53 publications

References 16 publications

Plasmodium falciparumMalaria: Reduction of Endothelial Cell Apoptosis In Vitro

Plasmodium falciparumMalaria: Reduction of Endothelial Cell Apoptosis In Vitro

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

Labeling of apoptotic JURL‐MK1 cells by fluorescent caspase‐3 inhibitor FAM‐DEVD‐fmk occurs mainly at site(s) different from caspase‐3 active site

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