Background Fragmentation of genomic DNA in apoptosis/programmed cell death (PCD) is a characteristic hallmark in which both 2N and 4N DNA from G1, S, and G2/M cell cycle phases were seen degraded to the sub‐2N A0 level in PCD such as from serum deprivation, glucocorticoid treatment, and γ‐radiation. However M‐phase (mitotic) cells are said to perish only via non‐programmed or necrotic cell death unless they were allowed to complete cytokinesis and re‐enter interphase. The morphological criteria of PCD refer only to interphase cells with intact nuclear membranes, none seems applied to mitotic cells. We show here autophagic sequestration of mitotic chromosomes in a typical PCD response where G1, S, and G2/M DNA were replaced by a sub‐2N A0 peak, suggesting that mitotic cells may yet have the option of PCD or suicide. Autophagy is absent in necrosis. Methods Mitotic human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts initiated by a burst of hydroxyl free radicals (OH*) generated from vanadium internalized by an NH4Cl prepulse containing vanadyl(4) ions. Total (free and bound) vanadium uptake was quantitated by elemental spectral analysis of single cells using a) Particle‐Induced X‐ray Emission (PIXE) profiling, and b) Scanning Transmission Ion Microscopy (STIM) in the nuclear microscope. The Coulter EPICS PROFILE II flow cytometer was used for a) the cell cycle analysis using propidium iodide‐DNA binding, b) intracellular pH (pHi) evaluation in the acidification‐and‐recovery cycle, using ratiometric 2′,7′‐bis(2‐carboxyethyl)‐5(and‐6)‐carboxyfluorescein (BCECF) fluorescence quantitation. Transmission electron microscopy examined the morphological changes. Vanadyl(4)‐generated hydroxyl free radicals (OH*) were evaluated by measuring OH*‐benzoic adduct fluorescence at 304/413 nm using the SPEX Fluoromax photon counting spectrofluorometer. Results Nuclear microscopy showed that a 30 min acidification prepulse containing 4 mM vanadyl(4) ions, V(4), had increased the total (free and bound) vanadium concentration of human Chang liver cells from normal ultratrace levels to 56,922 ppm of dry wt (1.1174 Eq per kg dry wt). After the prepulse, cells realkalinized in DMEM growth medium, recovering to the physiological pHi level in 30 min. At the physiological pH 7 level, V(4) generated a burst of OH* free radicals in the order of 15,000 folds above the prepulse (pH 4.5) level. In these conditions, spectrofluorometric evaluation showed loss of DNA intercalation with propidium iodide (PI‐DNA binding) indicating DNA degradation. Cell‐by‐cell evaluation of the PI‐DNA binding by flow cytometry showed abolition of G1, S, and G2/M phases and their replacement by a sub‐2N A0 peak of fragmented DNA, emulating serum deprivation PCD in these cells. Immediately upon initiating an OH* burst ultrastructural profiles showed mitotic chromosomes (M‐phase chromatin) being surrounded by rough endoplasmic reticulum (RER) and small vesicles, indicating their sequestration in autophagosomes. Autophag...

Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pHi upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pHi upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death.

Very little is known about the modulation of vanadium accumulation in cells, although this ultratrace element has long been seen as an essential nutrient in lower life forms, but not necessarily in humans where factors modulating cellular uptake of vanadium seem unclear. Using nuclear microscopy, which is capable of the direct evaluation of free and bound (total) elemental concentrations of single cells we show here that an NH4Cl acidification prepulse causes distinctive accumulation of vanadium (free and bound) in human Chang liver cells, concentrating particularly in the nucleus. Vanadium loaded with acidification but leaked away with realkalinization, suggests proton-dependent loading. Vanadyl(4), the oxidative state of intracellular vanadium ions, is known to be a potent source of hydroxyl free radicals (OH). The high oxidative state of nuclei after induction of vanadyl(4) loading was shown by the redox indicator methylene blue, suggesting direct oxidative damage to nuclear DNA. Flow cytometric evaluation of cell cycle phase-specific DNA composition showed degradation of both 2N and 4N DNA phases in G1, S and G2/M cell cycle profiles to a solitary IN DNA peak, in a dose-dependent manner, effective from micromolar vanadyl(4) levels. This trend was reproduced with microccocal nuclease digestion in a time response, supporting the notion of DNA fragmentation effects. Several other approaches confirmed fragmentation occurring in virtually all cells after 4mM V(4) loading. Ultrastructural profiles showed various stages of autophagic autodigestion and well defined plasma membrane outlines, consistent with programmed cell death but not with necrotic cell death. Direct intranuclear oxidative damage seemed associated with the induction of mass suicide in these human Chang liver cells following vanadium loading and nuclear sequestration.

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.

Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects, such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo administration of Zn2+ in C57/6J mice induces weight loss and massive hair loss where the normal course hair becomes replaced by fine vello hair, simulating the side effects from cancer chemotherapy where oxidative free radical damage is implicated in association with DNA fragmentation and programmed cell death (PCD). Here, in vitro flow cytometric studies on human Chang liver showed Zn2+ causing cell condensation with DNA fragmentation that occurred in a dose-dependent manner, an effect replicated by micrococcal nuclease digestion. Specific terminal deoxynucleotidyl transferase-(TdT) mediated labeling of 3'-OH ends of DNA nicks corroborated the flow cytometric profiles of propidium iodide-DNA binding where degradation of both 2 and 4 N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an established hallmark of PCD. We further showed that Zn2+ could enhance the generation of hydroxyl free radicals (OH.) by the transition metal vanadium. Glutathione, the cell's main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.