Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis.
The single cell gel electrophoresis assay, or Comet assay, is a powerful tool for measurement of DNA strands breaks, oxidative damage, and alkali labile sites, and the assay was recently modified to detect DNA cross-links. It has also been proposed as a measure of apoptosis since apoptotic cells are suspected to result in total migration of the DNA from the nucleus into the tail. Cells with this appearance are called ghost cells, clouds, hedgehogs, or NDCN (nondetectable cell nuclei). The aim of this study was to determine if ghost cells can be used to measure apoptosis in the standard alkaline comet assay. To answer this question, we made use of two cell lines: CTLL-2 cells that can enter apoptosis upon addition of apoptosis stimuli or IL-2 deprivation, and CTLL-2 bcl2 cells that are protected from apoptosis due to the overexpression of the apoptosis inhibitor gene bcl2. The two cell lines were treated with cytotoxins (nongenotoxic apoptosis inducers, nongenotoxic necrotic agents) or genotoxins. They were also subjected to growth factor withdrawal, which induced apoptosis in the CTLL-2 cell line. The level of apoptosis was measured by the Annexin V-FITC method in parallel with performing the Comet assay. The results obtained in the two cell lines suggest that apoptotic or necrotic death does not correlate well with the detection of ghost cells, presumably because these cells are lost upon electrophoresis. A variant of the alkaline Comet assay that was performed without electrophoresis (halo method) was able to efficiently detect cells undergoing apoptosis, but it was unable to clearly distinguish between apoptosis and genotoxic damage.
Meintières
Sophie
S
Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, Lille, France.
Nesslany
Fabrice
F
Pallardy
Marc
M
Marzin
Daniel
D
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D020552:Comet Assay; D003902:Detergents / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL
D003902:Detergents; D007376:Interleukin-2