K.H. Sit

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.

Antiport-mediated cell retraction: viable rounding and distinctive endocytosis

Cells that are detached through Na+/H+ antiport-mediated cell retraction (the flat-to-round or FTR change) are as viable as trypsinized cells despite unusually high trypan blue dye inclusion. Even high molecular weight dextran and carbon particles are easily loaded following the FTR change. ECM of confluent cultures of human keloid fibroblasts and Chang liver epitheloid cells stained directly by fluorescein-conjugated monoclonal anti-fibronectin is dramatically reduced or removed upon cell rounding. At the ultrastructural level, distinctive channels and vacuoles filled with extracellular material are seen, resolved at later stages as peripheral granular patches which too disappear when the rounded cells are reflattened in round-to-flat (RTF) change. Antiport-mediated endocytosis (AME) could explain the drastic reduction in cell area concomitant with cell rounding and detachment. AME provides a new way of loading cells with impermeant macromolecules.

Necrosis has orderly DNA fragmentations

Necrosis from freezing manifested an orderly pattern of DNA fragmentations, including the apoptosis signature of 200 bp ladder, in three different cell populations, despite pancaspase suppression by zVAD-fmk. Immediately on thawing, all three populations had 100% dead cell indices and 2.2, 1.6, and 1.1 megabase fragmentations, which marked the point of death. Kilobase and 200 bp DNA ladder fragmentations manifested later together with overt necrotic morphologies. CpG oligodeoxynucleotides (ODNs) complementary to highly conserved GCn(x)GC motifs inhibited the megabase fragmentations and retarded their electrophoretic mobility (gel shift), indicating ODN-DNA binding, which is known to confer site-specific resistance to cleavage. Cleavage specificity was confirmed using EDTA-CpG ODN conjugates to direct free-radical-producing transitional element, vanadyl(4), to the binding sites to reproduce the megabase fragmentations in normal cells. Specific orderly fragmentations in necrosis suggested a necrosis-apoptosis convergence after death has been committed.

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 1N 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 4 mM 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.

Induced interphase cell retraction: Its reversal and EGF potentiation

Human epidermoid A431 carcinoma cells retract into globular shapes upon activation with epidermal growth factor (EGF) but not normal cells. We find that normal anchor-dependent cultured cells will retract from attachment on incubation in saline with pH upshifting. There is significant potentiation of that phenomenon by EGF prepulsing as seen in (a) dose- and time-response experiments quantitated by measuring profile area and perimeter of cells (b) 22Na+-uptake studies and (c) pHi measurements. EGF action by itself produces quantitatively significant retractions, although only to a very small degree. Blocking Na+/H+ exchanges using (a) trifluoperazine (150 microM), (b) amiloride (2 mM) and (c) Na+-omission could block the cell retractions. Retracted cells can be reversed in low [Na+]o during which rapid 22Na+-efflux is observed. A dose-response ouabain (1 mM) sensitive relationship exists between [Na+]o and retardation of the reversal phenomenon. Ouabain however does not prevent reversal in low [Na+]o, nor even in combination with high [Na+]o given the presence of Ca2+. Retraction into spherical shapes would maximize volume-holding for a given surface area, however they are quantitatively more bulgy than cells rounded by trypsinization.

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.

An efficient and simple method of non-enzymatic detachment of monolayer cultures into single cell suspension

Activating the Na+/H+ antiporter causes a distinctive and rapid endocytosis which internalizes the plasma membrane leading to cell retraction and detachment as rounded cells with much reduced surface area. Antiport activation is by a combination of two individually effective motivations, viz. a) steep [Na+] and [H+] gradients across the plasma membrane and b) allosteric activation by second messengers initiated with simple inorganic sulphate. The dissociated cells are as viable as those released by trypsinization. Thus it provides an effective enzyme-free alternative to trypsin digestion in cell dissociation.

UDP-glucuronyltransferase activity toward harmol in human liver and human fetal liver cells in culture

This paper presents a fast HPLC assay for measuring UDP-glucuronyltransferase (UDPGT) activity in extracts of adult human liver and human fetal liver cells in culture. Harmol glucuronide formed was quantitated directly without prior hydrolysis after a simple step of selective extraction of harmol. The method is applicable to crude liver homogenates as well as to partially fractionated preparations. It is several fold more sensitive than the conventional detection of harmol glucuronide by TLC, making it possible to distinguish the low and high affinity forms of UDPGT of adult human liver and to detect the low affinity form in fetal cells. The possible participation of both forms of GT in adults under different conditions and the apparent lack of the high affinity form in the fetal liver is discussed.

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 4N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an estabilished hallmark of PCD. We further showed that Zn2+ could enhance the generation of hydroxyl free radicals (OH•) by the transition metal vanadium. Glutathione, the cells main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.

Effect of genistein, a tyrosine-specific protein kinase inhibitor, on cell rounding by pH upshifting

SummaryCell rounding is generally regarded as a cytoskeletal change exemplified by the mitotic state of monolayer cell cultures in which mitosis-specific phosphorylation was recently cited as the molecular mechanism. Mechanistically, there appears a convergence with earlier suggestions of tyrosyl phosphorylation of cytoskeletal elements in growth-factor-induced rounding inasmuch as protein tyrosine kinase plays a major regulatory role in cell cycle progression. On the other hand, despite the permissive association between cell activation and intracellular alkalinization, inducing rounding via pH upshifts in the presence of even high concentrations (135µg/ml) of a tyrosine specific protein kinase inhibitor, genistein, did not suppress the rounding response, although a distinct difference in phosphotyrosine level was demonstrated by monoclonal antiphosphotyrosine antibodies.