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)

Sit, K.H.; Paramanantham, R.; Bay, Boon‐Huat; Chan, Hardy Sze On; Wong, Kim Ping; Thong, Patricia S. P.; Watt, Fiona M.

doi:10.1002/(sici)1097-0185(199605)245:1<1::aid-ar1>3.0.co;2-2

Cited by 40 publications

(19 citation statements)

References 37 publications

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“…For example, in addition to genetic alterations, cancer cells usually have tremendous changes in their epigenetic landscape (Sharma et al 2010). Given that autophagosomes can sequester chromosomes (Sit et al 1996) and autophagic breakdown of the nucleus is well documented in yeast (Kvam and Goldfarb 2007), it might be possible for autophagy to regulate epigenetic regulatory factors such as histone methyltransferases and deacetylases in the nucleus of mammalian cells. Could this be an additional role for autophagy in cancer?…”

Section: Discussionmentioning

confidence: 99%

Autophagy and Cancer

Mah

Ryan

2011

Cold Spring Harbor Perspectives in Biology

147

109

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(Macro)autophagy is a cellular membrane trafficking process that serves to deliver cytoplasmic constituents to lysosomes for degradation. At basal levels, it is critical for maintaining cytoplasmic as well as genomic integrity and is therefore key to maintaining cellular homeostasis. Autophagy is also highly adaptable and can be modified to digest specific cargoes to bring about selective effects in response to numerous forms of intracellular and extracellular stress. It is not a surprise, therefore, that autophagy has a fundamental role in cancer and that perturbations in autophagy can contribute to malignant disease. We review here the roles of autophagy in various aspects of tumor suppression including the response of cells to nutrient and hypoxic stress, the control of programmed cell death, and the connection to tumor-associated immune responses.

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Section: Discussionmentioning

confidence: 99%

Autophagy and Cancer

Mah

Ryan

2011

Cold Spring Harbor Perspectives in Biology

147

109

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“…V(4)-prepulsed cells with a 2 h recovery in normal DMEM had pH i =6.9 and mean GSH-MCB=108.7 units thereby indicating that GSH was already depleted to basal limits immediately upon realkalinization. GSH depletion coincided with (a) the triggering of an OH* burst from alkalinization of V(4) (Sit et al, 1996a), and (b) the demonstration of high oxidative reactivity in the nuclei (see Figure 4B). GSH depletion would enhance the oxidative stress, and contribute to the oxidative genomic damage (Kane et al, 1993;Dawson and Dawson, 1996).…”

Section: Intranuclear Oxidative Damagementioning

confidence: 80%

“…Methylene blue is colourless in the reduced state. The OH* burst (Sit et al, 1996a) coincided with depletion of glutathione (GSH), the main reducing agent of the cell ( Figure 4C and D), thereby enhancing the oxidative stress. Flow cytometric cellby-cell evaluation of GSH content (measuring GSH-Monochlorobimane (GSH-MCB) adduct fluorescence) corroborated the cytological demonstration of GSH decline with realkalinization after a V(4) prepulse ( Figure 5A).…”

Section: Intranuclear Oxidative Damagementioning

confidence: 96%

“…Hydrogen peroxide and superoxides were sources of hydroxyl free radicals (OH*) generated via the transition metal-ion driven Fenton and Haber-Weiss reactions (Halliwell and Gutteridge, 1984). An OH* burst was triggered with realkalinization in normal growth medium after the V(4) prepulse (Sit et al, 1996a). OH* could react with most cellular biomolecules of which DNA is a known target molecule (Farber et al, 1990).…”

Section: Vanadyl(4) Internalizationmentioning

confidence: 99%

“…Cells were given a 30 min prepulse (378C) in the V(4) buffer, before reincubation in normal DMEM growth medium containing 10% foetal bovine serum. Protocol was as previously described (Sit et al, 1996a).…”

Section: Vanadyl(4) Prepulsementioning

confidence: 99%

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

Sit

Chen

1997

Cell Death Differ

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The relationship between cell cycling and apoptosis/ programmed cell death has been perceived as either checkpoint arrests or mitotic aberration where common pathways between mitosis and apoptosis seem suggested. We show here evidence implicating both perceptions of cell cycle involvement. The process was initiated by hydroxyl free radicals (OH*) generated intracellularly from internalized vanadyl(4). Intranuclear sequestration of vanadyl(4) was verified by nuclear microscopy. Resultant high oxidative reactivity in the nucleus was shown by the redox indicator methylene blue, suggesting direct oxidative damage to genomic DNA. Oxidative stress was further enhanced by depletion of glutathione which is the main cellular reducing agent. Genomic degradation and fragmentation was confirmed by flow cytometric evaluation of terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH end-labelling (TUNEL) of DNA nicks, and cell cycle DNA profiling demonstrating sub-G1 (sub-2N) accumulation. With DNA degradation, there was a G2M transient with hyperdiploid right-shifting, consistent with G2 arrest. G2 arrest was subsequently 'released' with abolition of G2M and all other cell cycle phases except for a solitary sub-G1 (apoptotic) peak. The cytological profile of this 'release' phenomenon was initially marked by the appearance of clusters of mitotic and apoptotic cells. At later stages, the cell population was composed exclusively of nuclear ghosts, apoptotic cells, mitotic cells, and mitotic cells with both chromosomes and apoptotic condensations. Concurrent and conjoint expression of cell death and cell division as the exclusive process of an entire cell population refuted the notion of mutual exclusivity between life and death. Zn 2+ , an endonuclease inhibitor, abolished all observed cytological and DNA profile changes.

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Apoptotic condensations in m-phase cells

Sit¹,

Yin²,

Paramanantham³

1997

Anat. Rec.

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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 μM 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°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. Anat. Rec. 248:149–158, 1997. © 1997 Wiley‐Liss, Inc.

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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)

Cited by 40 publications

References 37 publications

Autophagy and Cancer

Autophagy and Cancer

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

Apoptotic condensations in m-phase cells

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