Spatial Progression of DNA Strand Breaks in Apoptotic HL-60 Cells

• lkeda-Nishizawa Yoko

  Department of Life Science, Tokyo Institute of Technology The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo

• Yazaki Yoshio

  The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo

• Hirai Hisamaru

  The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo

• lshikawa Fuyuki

  Department of Life Science, Tokyo Institute of Technology

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.

収録刊行物

• bioimages

  bioimages 4 (2), 63-72, 1996

  日本バイオイメージング学会

キーワード

• in situ nick translation
• cell fixation
• three dimensional nuclear structure
• confocal laser microscopy

詳細情報

• CRID: 1390852271223986560
• NII論文ID: 10002037706
• NII書誌ID: AA11084187
• DOI: 10.11169/bioimages.4.63
• ISSN: 09192719
• 本文言語コード: en
• データソース種別:
  • JaLC
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可