抄録
We have modified the method of in situ nick translation
(ISNT) for analysis of the 3-dimensional (3-D)
progression of DNA cleavage during the course of
apoptosis. HL-60 cells treated with all-trans retinoic
acid (RA) were used. DNA strand breaks are visualized
as the incorporation of biotin-16-dUTP by
DNA polymerase I, detected with streptavidin-FITC.
To analyze the spatial progression of apoptotic
change by ISNT, two experimental conditions were
stipulated: first, that the nick translation reaction
takes place at an equal rate throughout the nucleus;
and second, that the procedure for fixing DNA
preserves the structure in vivo. To fulfill these
prerequisites, cells were fixed with 4% paraformaldehyde,
permeabilized by NP-40 and digested
by Proteinase K, followed by the second fixation.
After these treatments, ISNT was performed and the
fluorescent signal was analyzed either by conventional
fluorescence microscopy or by confocal laser
scanning microscopy. The first change detected by
ISNT in the process of apoptosis was the homogeneous
labeling of the interior of the nucleus, with
the concurrent occurrence of several highly labeled
distinct foci. These foci appear to increase in size and
intensity as apoptosis proceeds. Finally the foci change
into discretely delineated spherical regions, becoming
the canonical apoptotic bodies. Although temporal,
these sequential morphological changes during the
early stages of apoptosis are a novel observation,
establishing the usefulness of this method.
