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

Abstract

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.

Implications of Therapy-Induced Selective Autophagy on Tumor Metabolism and Survival

“…Emerging evidence suggests that the selective digestion of portions of the nucleus by autophagy plays a central role in upholding nuclear integrity when structural damage occurs [12]. While nucleophagy in mammalian cells has recently been reported [12, 95–97], this process has primarily been described in yeast [94]. In most yeast models, nutrient deprivation is the stressor of choice used to induce nucleophagy [98, 99].…”

“…In contrast to PMN seen in yeast, mammalian cells undergoing nucleophagy are able to form large autophagosomes characteristic of macroautophagy [12, 95–97]. These large double-membraned macroautophagosomes are observed to colocalize with LC3 and have been seen to envelop large portions of structurally deformed nuclei as well as small nuclear blebs [12].…”

Transient G2M arrest and subsequent release of apoptotic and mitotic cells in vanadyl(4)-prepulsed human Chang liver cells

“…When oxidized, methylene blue becomes metachromatic and appears purple. Protocol was as previously described (Sit et al, 1996b).…”

“…By itself, MCB is non-fluorescent. Protocol was as previously described (Sit et al, 1996b, Kane et al, 1993.…”

“…The principle involved was templete-independent addition of dUTP-digoxigenin to 3'OH ends of the fragmented DNA catalyzed by exogenous terminal deoxynucleotidyl transferase (TdT), which was followed by binding with fluorescein-labelled antidigoxigenin antibody for detection. Procedure was previously described (Sit et al, 1996b).…”