Detection of radiation-induced apoptosis using the comet assay.
The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis.
Wada
Seiichi
S
Biotechnology Lab., JAERI-Takasaki, Watanuki-machi 1233, Takasoki, Gunma 370-1292, Japan.
Khoa
Tran Van
TV
Kobayashi
Yasuhiko
Y
Funayama
Tomoo
T
Yamamoto
Kazuo
K
Natsuhori
Masahiro
M
Ito
Nobuhiko
N
D000818:Animals; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects*; D002478:Cells, Cultured; D020552:Comet Assay / Q000379:methods*; D005720:Gamma Rays; D020287:In Situ Nick-End Labeling / Q000379:methods; D008856:Microscopy, Fluorescence