Limitations of the single-cell gel electrophoresis assay to monitor apoptosis in U937 and HepG2 cells exposed to 7beta-hydroxycholesterol

Biochemical Pharmacology
Y C O'CallaghanN M O'Brien

Abstract

The single-cell gel electrophoresis (comet) assay is a method which allows the detection of DNA strand breaks in individual cells. It has been suggested that the single cell gel electrophoresis assay, as an index of DNA fragmentation during cell death, may be applied to monitor apoptosis. The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to discriminate between the mode of cell death in two cell lines (U937, a human monocytic blood cell line and HepG2, a human hepatocarcinoma cell line) which were treated with 30 microM 7beta-hydroxycholesterol (7betaOHC) over a 48 hr period. The single cell gel electrophoresis assay was compared with more established methods for the determination of apoptosis such as morphological examination, flow cytometry and DNA laddering. The percentage of maximally damaged nuclei as measured by the single cell gel electrophoresis assay was found to be similar at 48 hr in both U937 and HepG2 cells when treated with 7betaOHC. However, morphological examination, flow cytometry and DNA laddering techniques showed that 7betaOHC induced apoptosis in U937 cells but not in HepG2 cells. Thus, although the alkaline ...Continue Reading

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Citations

Dec 12, 2012·Drug Development and Industrial Pharmacy·Kevin P O'Donnell, Robert O Williams
Dec 11, 2003·The Journal of Veterinary Medical Science·Seiichi WadaNobuhiko Ito
Mar 3, 2004·Journal of Biochemical and Molecular Toxicology·Lisa RyanNora M O'Brien
Jun 1, 2005·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Peggy L Olive, Ralph E Durand

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis