Apoptotic condensations in M-phase cells.
Apoptosis is a morphologically distinctive form of programmed cell death/cell suicide in which genomic DNA degradation/fragmentation and variegated dense chromatin aggregates are characteristic hallmarks that have never been demonstrated in mitotic cells. Perceptions of mutual exclusivity between apoptosis and mitosis imply that M-phase cells cannot be apoptotic. However, in the present study we show apoptotic morphologies in M-phase cells after an acute oxidative stress and endonuclease digestion.



Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by flow cytometric cell-by-cell evaluation of (a) propidium iodide intercalative binding to DNA and (b) terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmented DNA. Oxidative stress was imposed by a 30-min prepulse with 200 microM vanadyl(4), which produces hydroxyl free radicals (OH*), the most reactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated monochlorobimane-glutathione adduct fluorescence for glutathione content, the main reducing agent of a cell, and methylene blue redox metachromasia, which is a deep color when oxidized and colorless when reduced. Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic morphologies were visualized by staining with Giemsa and neutral red dyes and by DNA-propidium iodide binding to chromatin. Direct endonuclease induction of apoptotic morphologies in permeabilized M-phase cells was produced by 1 hr incubation (37 degrees C) with 16 units/ml of micrococcal nuclease.



The genomic DNA of proliferative cells, namely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepulsing and by micrococcal nuclease digestion, concomitantly with DNA fragmentation shown by TUNEL. Cytological profiles showed GSH depletion and M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase cells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to "pulverized" aggregates of a mitotic catastrophe, buddings, and "apoptotic bodies," were found in prophase, metaphase, anaphase, and telophase mitotic cells. The observed separation of condensed chromatin aggregates from the main chromosome mass in prophase and metaphase cells could explain micronuclei, linking it with apoptosis. Direct endonuclease digestion readily produced apoptotic morphologies in interphase and in M-phase cells.



Apoptotic morphologies in M-phase cells can be induced indirectly via oxidative stress or directly via endonuclease activity, which has long been established as a pervading hallmark of apoptosis.
Sit
K H
KH
Department of Anatomy, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore.
Yin
L
L
Paramanantham
R
R
D017209:Apoptosis; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004720:Endonucleases / Q000494:pharmacology; D005978:Glutathione / Q000032:analysis; D006801:Humans; D008099:Liver / Q000166:cytology; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000502:physiology*; D018384:Oxidative Stress / Q000502:physiology*; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D004247:DNA; D004720:Endonucleases; D005978:Glutathione