PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 15502242

Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance spectroscopy and annexin-conjugated iron.

James P Fabisiak·Grigory G Borisenko·Valerian E Kagan
View original source ↗Export metadata ↓

Abstract

We present here the application of a novel assay that measures the absolute amount of phosphatidylserine (PS) externalized on the surface of cells. Although the assay is based on the same annexin binding principle as the fluorescent flow cytometry assay, we use paramagnetic iron as the ultimate reporter molecule, establishing a linear relationship between signal amplitude and amount of PS on the cell surface, allowing a quantitative assay of PS externalization over a wide dynamic range. The application of this technique, alone and in concert with the PS oxidation method presented in the previous chapter, will greatly aid in studying the mechanistic connection between lipid peroxidation and translocation events during apoptosis.

Explore related subjects

AnnexinsApoptosisCell MembraneElectron Spin Resonance SpectroscopyHL-60 CellsHumansIronJurkat CellsLipid PeroxidationPhosphatidylserinesJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

James P Fabisiak, Grigory G Borisenko, Valerian E Kagan. 2005. Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance spectroscopy and annexin-conjugated iron.. https://doi.org/10.1385/1-59259-840-4%3A457

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins.

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up-regulation of survivin, c-myc, and CD1 protein expression, and documented comparable colony formation by annexin(+) trisomy 8(-) CD34(+) and annexin(-) CD34 cells. There were low levels of DNA degradation in annexin(+) trisomy 8 CD34 cells, which were comparable with annexin(-) CD34 cells. Trisomy 8 cells were resistant to apoptosis induced by gamma irradiation. Knock-down of survivin by siRNA resulted in preferential loss of trisomy 8 cells. These results suggest that trisomy 8 cells undergo incomplete apoptosis and are nonetheless capable of colony formation and growth.

Annexins↗
PubMed2006

Annexins as intracellular calcium sensors.

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.

Annexins↗
PubMed2006

Different effects of two cyclic chalcone analogues on cell cycle of Jurkat T cells.

It has been previously shown that the cyclic chalcone analogues E-2-(4'-methoxybenzylidene)- (2) and E-2-(4'-methylbenzylidene)-1-benzusuberone (3) inhibited proliferation of various murine and human tumor cells. In order to gain new insights into the cytotoxic mechanism of the two compounds detection of apoptosis and necrosis of Jurkat T cells exposed to 2 and 3 were performed by flow cytometry using the Annexin V-FITC and propidium iodide double staining method. Analysis of the DNA histograms at 8, 24 and 48 h exposure times showed that near equitoxic doses of 2 and 3 had different effects on the cell cycle of the exposed cells. The immediate (8h) effect of 2 was a remarkable decrease of cells in the G(0)/G(1) phase and increase in the G(2)/M phase. This effect could also be seen in the histogram of cells at the 24h time point. On the contrary, such an effect of 3 could not be observed. At the 24 and 48 h time points accumulation of sub-G(0) (apoptotic and necrotic) and hyperdiploid cells could be detected after both treatments. Incubation of 2 and 3 with reduced glutathione under cell-free conditions indicated spontaneous conjugation (non-redox) reaction at pH 7.4 and pH 9.0. Analyzing the mechanism of action the total thiol content of the cells exposed to compounds 2 and 3 was determined. Compound 2 showed to reduce the total cellular thiol level both under nutrient-free and nutrient-supplemented conditions. Under the latter conditions an increase of the total thiol level of the cells exposed to 3 for 4h could be observed. The different effect of the two compounds on the cellular thiol status might contribute to the different tumor cytotoxicity of the cyclic chalcone analogues 2 and 3. Investigation of antioxidant capacity of the compounds by monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose indicated that both compounds displayed hydroxyl radical scavenger activity. The experiments provide further details of dual--cytotoxic and cytoprotective (chemopreventive)--effects of the compounds.

Annexins↗