Apoptotic condensations in m-phase cells

Abstract

Background: 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. Methods 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 μM 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°C) with 16 units/ml of micrococcal nuclease. Results 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. Conclusion 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. Anat. Rec. 248:149–158, 1997. © 1997 Wiley‐Liss, Inc.

Method of Specific Detection of Apoptosis Using Formamide-induced DNA Denaturation Assay

“…A question concerning the specificity of the TUNEL method for detecting apoptosis has been raised because it detects not only apoptotic cells but also necrotic and mitotic cells (Grasl-Kraupp et al 1995;Sit et al 1997;Wolvekamp et al 1998;Otsuki 2000Otsuki ,2004, and it recognizes only free 39-OH DNA ends. In necrotic cells, however, free 39-OH DNA ends are also generated.…”

“…The TUNEL method is generally applied more often for the detection of apoptosis than TEM because the TUNEL method does not require special equipment or a complex sample preparation as in TEM. Recent studies using the TUNEL method have cast doubt on its specificity of detecting apoptosis because the TUNEL method detects not only apoptotic cells but also necrotic and mitotic cells (Grasl-Kraupp et al 1995;Sit et al 1997;Wolvekamp et al 1998;Otsuki 2000Otsuki ,2004. The TUNEL method by TEM (TUNEL/TEM) was developed for retrospective studies using archival epoxyresin-embedded materials and enables the morphological discrimination of apoptotic cells among many cell types including necrotic or mitotic cells based on ultrastructural characteristics (Inoki et al 1997;Hayashi et al 1998;Otsuki 2000Otsuki ,2004.…”

Transforming growth factor beta mediates hepatocyte apoptosis through Smad3 generation of reactive oxygen species

“…Hepatocytes were visualized under green UV light using an IX-70 Olympus microscope (Olympus, Tokyo, Japan), and the number of condensed nuclei indicative of apoptotic hepatocytes was quantified in 5 high powered fields (hpf) at 400× magnification [17].…”

Primary cirrhotic hepatocytes resist TGF$beta;-induced apoptosis through a ROS-dependent mechanism

“…Hepatocytes were fixed and permeabilized in 3:1 methanol/acetic acid for 10 min at 4 8C and stained with 2 mg/ml propidium iodide (PI) following 0, 24, 36, and 48 h of treatment with TGFb, TNFa, or UV-C. Hepatocytes were visualized under green UV light using an IX-70 Olympus microscope (Olympus, Tokyo, Japan), and the number of condensed nuclei indicative of apoptotic hepatocytes was quantified in five high powered fields (hpf) at 400X magnification [30].…”