Online citations, reference lists, and bibliographies.
← Back to Search

Sizing Highly Fragmented DNA In Individual Apoptotic Cells Using The Comet Assay And A DNA Crosslinking Agent.

P. L. Olive, J. Banáth
Published 1995 · Biology, Medicine

Cite This
Download PDF
Analyze on Scholarcy
TK6 human B lymphoblast cells exposed to ionizing radiation undergo apoptosis in a time and dose-dependent manner. The resulting highly fragmented DNA is easily detected using the comet assay, a sensitive microscopic gel electrophoresis method capable of measuring DNA strand breaks in individual cells. The degree of DNA fragmentation may be indicative of different stages in the fragmentation process, responses to different agents, and/or cell type-dependent differences. In an effort to determine the number of breaks present in each apoptotic cell, we first applied a DNA-crosslinking agent, mechlorethamine, to TK6 cells containing a known number of DNA double-strand breaks produced by X rays. As the concentration of mechlorethamine increased, crosslinked DNA was less able to migrate during gel electrophoresis. Exposure of TK6 cells to 5 microM mechlorethamine prior to irradiation with 20 Gy was sufficient to "hide" the presence of these breaks by preventing DNA from migrating during electrophoresis. However, in apoptotic TK6 cells, it was necessary to apply a dose of mechlorethamine several times higher in order to produce a similar degree of inhibition of DNA migration. Calibrations using either the alkaline or neutral comet assays indicate that the average DNA fragment size in apoptotic TK6 cells is about 50 kb. Even in cells containing only 10-20% of the original amount of DNA, the remaining fragments still averaged about 50 kb, indicating that fragmentation to much smaller sizes occurs in some parts of the genome before others. When Chinese hamster V79 cells were exposed to hyperthermia (45 degrees C for 20 min), necrosis was induced over a period of several days. The size of DNA fragments in these cells was considerably larger (200-400 kb) and heterogeneity in appearance of comets was larger than observed for TK6 cells. This crosslinking method may be useful in discriminating cells dying by apoptosis from cells damaged or dying by other mechanisms.

This paper is referenced by
Genotoxic potential and in vitro tumour-promoting potential of 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, two radiolytic products of fatty acids.
K. Yamakage (2014)
A unique program for cell death in xylem fibers of Populus stem.
C. Courtois-Moreau (2009)
Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cells
Mei Wu (2008)
Urinary levels of insecticide metabolites and DNA damage in human sperm.
J. Meeker (2004)
Impact of the comet assay in radiobiology.
P. L. Olive (2009)
Enhanced uptake of antisense oligonucleotides using cationic liposomes and the apoptotic effect of idarubicin in K-562 cell line.
L. Vellón (2002)
A comprehensive study of the toxicity of natural multi-contaminated sediments: New insights brought by the use of a combined approach using the medaka embryo-larval assay and physico-chemical analyses.
Iris Barjhoux (2017)
Evaluation of the Use of DNA Adduct Dosimetry to Optimize the Timing of High Dose Therapy for Breast Cancer
R. Everson (2000)
Assessment of chemotherapy-induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay.
N. Kopjar (2002)
Inflammatory and genotoxic responses during 30-day welding-fume exposure period.
I. Yu (2004)
The radiomimetic enediyne, 20'-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance.
T. Beerman (2014)
Assessment of DNA damage induced by high-LET ions in human lymphocytes using the comet assay.
I. Testard (2000)
Comments on: "DNA strand breaks" by Diem et al. [Mutat. Res. 583 (2005) 178-183] and Ivancsits et al. [Mutat. Res. 583 (2005) 184-188].
Vijayalaxmi (2006)
Heat effects on the repair of DNA double-strand breaks in CHO cells.
J. Dahm-Daphi (1997)
Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations.
A. Hartmann (2004)
The Comet Assay
G. Speit (2005)
Northern lights assay: a versatile method for comprehensive detection of DNA damage
Bjarki Agust Gudmundsson (2018)
Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt-tungsten carbide.
F. Van Goethem (1997)
The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair.
G. Speit (2012)
Efeitos tóxicos e genotóxicos do herbicida Roundup Transorb®em Guppy (Poecilia reticulata) submetido a tratamento agudo
J. D. S. Filho (2011)
Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis
S. Yasuhara (2003)
Detection of DNA‐crosslinking agents with the alkaline comet assay
S. Pfuhler (1996)
Molecular Approaches for Detecting DNA Damage
P. L. Olive (1998)
The DNA of annexin V-binding apoptotic cells is highly fragmented.
Z. Bacso (2000)
Genotoxicity of (+)- and (-)-usnic acid in mice.
I. Prokopiev (2019)
Assessment by Ames test and comet assay of toxicity potential of polymer used to develop field-capable rapid-detection device to analyze environmental samples
A. Hebert (2014)
Milena Kirilova (2005)
Effect of Light Irradiation and Sex Hormones on Jurkat T Cells: 17β-Estradiol but Not Testosterone Enhances UVA-Induced Cytotoxicity in Jurkat Lymphocytes
H. Cohly (2005)
Glutathione transferase omega 1-1 (GSTO1-1) plays an anti-apoptotic role in cell resistance to cisplatin toxicity.
S. Piaggi (2010)
Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles
Z. Magdolénová (2015)
Modification of gamma radiation induced response of peritoneal macrophages and splenocytes by Hippophae rhamnoides (RH‐3) in mice
H. Prakash (2005)
Cytotoxic Property of Surfactant–Cobalt(III) Complexes on a Human Breast Cancer Cell Line
R. Kumar (2011)
See more
Semantic Scholar Logo Some data provided by SemanticScholar