APOPTOTIC CONDENSATIONS IN M-PHASE CELLS

Background: Apoptosis is a morphologically distinctive form of program med cell death/cell suicide in which genomic DNA degradation/fragmenta tion and variegated dense chromatin aggregates are characteristic hall marks 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 apop totic morphologies in M-phase cells after an acute oxidative stress an d endonuclease digestion. Methods: Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by how cytometric cell-by-cell evaluation of (a) propidiu m iodide intercalative binding to DNA and (b) terminal deoxynucleotidy l transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmen ted DNA. Oxidative stress was imposed by a 30-min prepulse with 200 mu M vanadyl(4), which produces hydroxyl free radicals (OH), the most r eactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated m onochlorobimane-glutathione adduct fluorescence for glutathione conten t, the main reducing agent of a cell, and methylene blue redox metachr omasia, which is a deep color when oxidized and colorless when reduced . Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic mo rphologies were visualized by staining with Giemsa and neutral red dye s and by DNA-propidium iodide binding to chromatin. Direct endonucleas e induction of apoptotic morphologies in permeabilized M-phase cells w as produced by 1 hr incubation (37 degrees C) with 16 units/ml of micr ococcal nuclease. Results: The genomic DNA of proliferative cells, nam ely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepul sing and by micrococcal nuclease digestion, concomitantly with DNA fra gmentation shown by TUNEL. Cytological profiles showed GSH depletion a nd M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase c ells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to ''pulverized'' aggregates o f a mitotic catastrophe, buddings, and ''apoptotic bodies,'' were foun d in prophase, metaphase, anaphase, and telophase mitotic cells. The o bserved separation of condensed chromatin aggregates from the main chr omosome mass in prophase and metaphase cells could explain micronuclei , linking it with apoptosis. Direct endonuclease digestion readily pro duced apoptotic morphologies in interphase and in M-phase cells. Concl usion: Apoptotic morphologies in M-phase cells can be induced indirect ly via oxidative stress or directly via endonuclease activity, which h as long been established as a pervading hallmark of apoptosis. (C) 199 7 Wiley-Liss, Inc.