Adding Zn2+ induces DNA fragmentation and cell condensation in cultured human Chang liver cells.

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.

Apoptotic condensations in M-phase cells.

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 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. 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.

Hydroxyl free radicals generated by vanadyl[IV] induce cell blebbing in mitotic human Chang liver cells.

Vanadium has recently been reported to induce interphase and M-phase (mitotic) programmed cell death via the generation of hydroxyl free radicals (OH*). In this paper, the effects of antioxidants on: (a) vanadyl[IV]-generated OH* free radical levels; and (b) cellular glutathione in vanadyl [IV]-treated Chang liver cells were evaluated. The surface morphology of vanadyl-treated mitotic cells was studied by confocal and scanning microscopy. The free radical scavengers zinc chloride, glucose and thiourea reduced the levels of vanadyl-induced OH* free radicals and partially prevented the depletion of cellular glutathione. Concurrent with OH* free radical production, vanadyl-treated telophase cells exhibited excessive cell blebbing and cell shrinkage. The morphological features demonstrated in vanadyl-induced mitotic programmed cell death as a consequence of oxidative stress is novel.

Flow cytometric evaluation of the DNA profile and cell cycle of zinc supplemented human Chang liver cells.

Zinc, an essential trace element, is important for normal cell growth. Growing children, especially at puberty, require increased zinc (2.8 mg/day for males and 2.65 mg/day for females). The DNA profile and cell cycle of human Chang liver cells grown in 0-900 mumol/L zinc chloride supplemented serum-free media for 24 h were analyzed using a Coulter flow cytometer. There was no significant difference in the G1, S and G2/M phases between zinc treated cells and control cultures except at 90 and 900 mumol/L zinc chloride. At these two higher dosages, fragmentation of genomic DNA into sub-2N DNA (sub-G1 DNA), generally considered a hallmark of programmed cell death (PCD), was noted. Results of the present study seem to suggest that growth regulation by zinc during growth spurts such as at puberty, could also be influenced by other factors besides its direct effect on DNA synthesis. In addition, high dosages of zinc could be cytotoxic.

ATP induces large channel endocytosis with concomitant increase in cell density.

Large channel endocytosis is considered to be the characteristic of specialized endocytic cells like macrophages and phagocytes while small pit endocytosis involving clathrin protein coatings are the membrane recycling macromolecular pathways for most eukaryotic cells. We show here that extracellular ATP induced cells to internalize their plasma membrane by large channel endocytosis. In the process of plasma membrane internalization, flat protracted cells round up and become easily detachable from the substrate. Scanning transmission ion microscopy (STIM) revealed an increase in cell density as the ATP treated cells assumed a rounded morphology. The increase in cell density could be attributed to endocytic internalization of cell membrane and debris.

Induction of vanadium accumulation and nuclear sequestration causing cell suicide in human Chang liver cells.

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.

Acidification and recovery results in nuclear accumulation of supravital dyes during interphase.

Recent studies using real time imaging demonstrated relative nuclear insulation for ion-size particles. We show here that acidification and recovery converted the insulated interphase nuclei of KB carcinoma and nontumorigenic Chang cells into intense nuclear accumulating states marked by sequestration of the exogenous supravital dyes neutral red, methylene blue, and brilliant cresyl blue. The phenomenon was not affected by Na(+)-free and HCO3(-)-free conditions nor by the presence of cationic and anionic antiport regulators of cytosolic pH. Cytological, microspectrophotometric, and flow cytometric evaluation of whole cell populations showed that the nuclear influx was abolished by omitting the pH recovery response, and by modulating the recovery response. The abolition of nuclear influx in the presence of the P-ATPase and Fzero-ATPase inhibitors, vanadyl(IV) ions and oligomycin, respectively, suggest that H(+)-translocating ATPase pumps are involved in regulating cytosolic acidification in Na(+)-free and HCO3-conditions vanadyl(IV) inhibited nuclear uptake of supravital dyes in a dose dependent manner. Nuclear uptake of dyes, however, was not affected by up to 1 mM of genistein even though tyrosine-specific phosphorylation and DNA synthesis were abolished. Upgradient nuclear influx involving proton pump is novel. KB cancer cells and nontumorigenic Chang cells had differential dye accumulations induced by acidification and recovery.

Acidification-and-recovery induces nuclear accumulation of neutral red and DNA into human KB oral carcinoma cells.

In cultures of oral cancers, gene transfer has been achieved by delivery systems which introduce DNA into cells but not specifically into the nucleus. We observed that acidification and recovery converted the nuclear-insulated interphase KB carcinoma cells into intense nuclear-accumulating states. Nuclear sequestration of the vital dye neutral red and T7 oligonucleotide was demonstrated qualitatively, and quantitatively by image analysis of single cells. pGem beta gal plasmids of 6.8 kb were introduced into KB cells by similar acidification and recovery pulses. Successful integration into the host KB genome was shown by expression of the lacZ gene of the plasmids. Enhanced nucleo-cytoplasmic transport demonstrated here in KB oral epidermoid carcinoma cells could potentially facilitate gene therapy in oral cancers.

Sequestration of mitotic (M-phase) chromosomes in autophagosomes: mitotic programmed cell death in human Chang liver cells induced by an OH* burst from vanadyl(4).

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 Ao level in PCD such as from serum deprivation, glucocorticoid treatment, and gamma-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 Ao 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 4mM vanadyl(4) ions, V(4), had increased the total (free and bound) vanadium concentration of human Chang liver cells from normal ultratrace levels to 56,992 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 Ao 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. Autophagy was also seen to be a prominent feature in serum deprivation PCD. CONCLUSION: Sequestration of mitotic chromosomes by autophagosomes in a typical PCD response showed a well-defined morphological pathway for direct degradation of M-phase chromatin without first completing cytokinesis. Mitotic cells could commit suicide via autophagy directed at its own chromatin. Autophagic sequestration of chromatin in PCD is novel.

Reduced surface area in apoptotic rounding of human Chang liver cells from serum deprivation.

BACKGROUND: The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and "condensation of the cytoplasm" in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding. METHODS: Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied 1) the cell cycle from propidium iodide-DNA bindings, 2) uptake of neutral red (NR) dye, a viable cell marker, and 3) cytosolic pH (pHi) modulations from 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH4Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy. RESULTS: Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary Ao (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H+]i/[H+]o gradient promoting proton extrusion. The pHi upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pHi upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells. CONCLUSIONS: 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.

Azide- and vanadate-sensitive M-phase alkalinity and cytosolic acidification of Chang liver cells.

Flow cytometric cell-by-cell evaluation of NH4Cl acidification of human Chang cells showed that at steady state, 3% of the cells remained alkalinized (> pHi 7) over an extended period (up to 80 min) despite the absence of extracellular Na+ and HCO3-. In fluorescence microscopy, the acidification-resistant cells were characteristically rounded M-phase cells. Both mean cytosolic pH and M-phase alkalinity were however sensitive to (a) azide and oligomycin, inhibitors of F-ATPase (ATP synthase), and to (b) vanadium ions, the phosphate analogue of P-ATPase (ATP-hydrolyzing), in dose-dependent and time-dependent manners. Dead cell indices were constant at approximately 10%. Thiocyanate chaotrophic anions, which cleave the V-ATPase structure, had no effect. Since ATP synthesizing F-ATPase (ATP synthase) is coupled to ATP-hydrolyzing P-ATPase as 'master-&-slave', azide- and oligomycin-sensitivity corroborated with vanadate-sensitivity in suggesting energized proton pumping modulating (a) M-phase alkalinity and (b) cytosolic pH, against acidification.