SEQUESTRATION OF MITOTIC (M-PHASE) CHROMOSOMES IN AUTOPHAGOSOMES - MITOTIC PROGRAMMED CELL-DEATH IN HUMAN CHANG LIVER-CELLS INDUCED BY AN OH-ASTERISK 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 f rom G(1), S, and G(2)/M cell cycle phases were seen degraded to the su b-2N A(0) level in PCD such as from serum deprivation, glucocorticoid treatment, and gamma-radiation. However M-phase (mitotic) cells are sa id to perish only via non-programmed or necrotic cell death unless the y were allowed to complete cytokinesis and re-enter interphase. The mo rphological 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 respo nse where G(1), S, and G(2)/M DNA were replaced by a sub-SN A(o) peak, suggesting that mitotic cells may yet have the option of PCD or suici de. Autophagy is absent in necrosis. Methods: Mitotic human Chang live r cells in normal monolayer culture were compared with apoptotic count erparts 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 eleme ntal spectral analysis of single cells using a) Particle-Induced X-ray Emission (PIXE) profiling, and b) Scanning Transmission Ion Microscop y (STIM) in the nuclear microscope. The Coulter EPICS PROFILE II flow cytometer was used for a) the cell cycle analysis using propidium iodi de-DNA binding, b) intracellular pH (pH(i)) evaluation in the acidific ation-and-recovery cycle, using ratiometric 7'-bis(2-carboxyethyl)-5(a nd-6)-carboxyfluorescein (BCECF) fluorescence quantitation. Transmissi on electron microscopy examined the morphological changes. Vanadyl(4-g enerated hydroxyl free radicals (OH) were evaluated by measuring OH*- benzoic adduct fluorescence at 304/413 nm using the SPEX Fluoromax pho ton counting spectrofluorometer. Results: Nuclear microscopy showed th at 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 o f dry wt (1.1174 Eq per kg dry wt). After the prepulse, cells realkali nized in DMEM growth medium, recovering to the physiological pH(i) lev el 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 sho wed loss of DNA intercalation with propidium iodide (PI-DNA binding) i ndicating DNA degradation. Cell-by-cell evaluation of the PI-DNA bindi ng by flow cytometry showed aboliton of G(1), S, and G(2)/M phases and their replacement by a sub-SN A(o) peak of fragmented DNA, emulating serum deprivation PCD in these cells. Immediately upon initiating an O H burst ultrastructural profiles showed mitotic chromosomes (M-phase chromatin) being surrounded by rough endoplasmic reticulum (RER) and s mall vesicles, indicating their sequestration in autophagosomes. Autop hagy 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 fo r direct degradation of M-phase chromatin without first completing cyt okinesis. Mitotic cells could commit suicide via autophagy directed at its own chromatin. Autophagic sequestration of chromatin in PCD is no vel. (C) 1996 Wiley-Liss, Inc.