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).

Abstract

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. 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. 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. 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.