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Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

Bottom Line: A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering.We find that all depend directly or indirectly on caspase-3 activation.CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Dynamics Unit, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

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In TNF-α induced apoptosis, H2AX S139 phosphorylation is a caspase-3 dependent event but does not influence HMGB1 immobilization.(A) Western Blot of MCF-7 cells stably transfected with empty vector (MCF-7/pv) MCF-7 cells transfected with caspase-3 (MCF-7/c3). Cell were either proliferating (L) or apoptotic (A), pretreated with z-DEVD-fmk (A/z-D) or not. The absence of caspase-3 or its inhibition abolish H2AX phosphorylation. (B) Western Blot of proliferating (L) and apoptotic (A) HeLa cells, treated with 100 ng/ml TSA just prior to apoptosis (A/TSA) or not. In TSA treated cells H2AX is phosphorylated, but HMGB1 is mobile; thus, the two events do not correlate. (C) FRAP experiments performed in HeLa cells (living, apoptotic, and apoptotic treated with TSA) transiently expressing HMGB1-GFP. Each line corresponds to the fluorescence recovery measured in a different cell.
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pone-0006234-g004: In TNF-α induced apoptosis, H2AX S139 phosphorylation is a caspase-3 dependent event but does not influence HMGB1 immobilization.(A) Western Blot of MCF-7 cells stably transfected with empty vector (MCF-7/pv) MCF-7 cells transfected with caspase-3 (MCF-7/c3). Cell were either proliferating (L) or apoptotic (A), pretreated with z-DEVD-fmk (A/z-D) or not. The absence of caspase-3 or its inhibition abolish H2AX phosphorylation. (B) Western Blot of proliferating (L) and apoptotic (A) HeLa cells, treated with 100 ng/ml TSA just prior to apoptosis (A/TSA) or not. In TSA treated cells H2AX is phosphorylated, but HMGB1 is mobile; thus, the two events do not correlate. (C) FRAP experiments performed in HeLa cells (living, apoptotic, and apoptotic treated with TSA) transiently expressing HMGB1-GFP. Each line corresponds to the fluorescence recovery measured in a different cell.

Mentions: Given these contrasting data, we investigated whether in TNF-α induced apoptosis H2AX phosphorylation is dependent on caspase-3 activation. We used proliferating and apoptotic MCF-7/pv and MCF-7/c3 cells, pre-treated or not with the caspase-3 inhibitor z-DEVD-fmk. In apoptotic MCF-7/pv cells there is no detectable level of H2AX S139 phosphorylation, while in apoptotic MCF-7 cells reconstituted with caspase-3 (MCF-7/c3) there is a strong induction of this histone modification. Moreover, H2AX S139 phosphorylation is completely abolished by the treatment with z-DEVD-fmk prior to apoptosis induction (Figure 4A). Thus, in TNF-α induced apoptosis H2AX S139 phosphorylation is a caspase-3 dependent event.


Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

In TNF-α induced apoptosis, H2AX S139 phosphorylation is a caspase-3 dependent event but does not influence HMGB1 immobilization.(A) Western Blot of MCF-7 cells stably transfected with empty vector (MCF-7/pv) MCF-7 cells transfected with caspase-3 (MCF-7/c3). Cell were either proliferating (L) or apoptotic (A), pretreated with z-DEVD-fmk (A/z-D) or not. The absence of caspase-3 or its inhibition abolish H2AX phosphorylation. (B) Western Blot of proliferating (L) and apoptotic (A) HeLa cells, treated with 100 ng/ml TSA just prior to apoptosis (A/TSA) or not. In TSA treated cells H2AX is phosphorylated, but HMGB1 is mobile; thus, the two events do not correlate. (C) FRAP experiments performed in HeLa cells (living, apoptotic, and apoptotic treated with TSA) transiently expressing HMGB1-GFP. Each line corresponds to the fluorescence recovery measured in a different cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713420&req=5

pone-0006234-g004: In TNF-α induced apoptosis, H2AX S139 phosphorylation is a caspase-3 dependent event but does not influence HMGB1 immobilization.(A) Western Blot of MCF-7 cells stably transfected with empty vector (MCF-7/pv) MCF-7 cells transfected with caspase-3 (MCF-7/c3). Cell were either proliferating (L) or apoptotic (A), pretreated with z-DEVD-fmk (A/z-D) or not. The absence of caspase-3 or its inhibition abolish H2AX phosphorylation. (B) Western Blot of proliferating (L) and apoptotic (A) HeLa cells, treated with 100 ng/ml TSA just prior to apoptosis (A/TSA) or not. In TSA treated cells H2AX is phosphorylated, but HMGB1 is mobile; thus, the two events do not correlate. (C) FRAP experiments performed in HeLa cells (living, apoptotic, and apoptotic treated with TSA) transiently expressing HMGB1-GFP. Each line corresponds to the fluorescence recovery measured in a different cell.
Mentions: Given these contrasting data, we investigated whether in TNF-α induced apoptosis H2AX phosphorylation is dependent on caspase-3 activation. We used proliferating and apoptotic MCF-7/pv and MCF-7/c3 cells, pre-treated or not with the caspase-3 inhibitor z-DEVD-fmk. In apoptotic MCF-7/pv cells there is no detectable level of H2AX S139 phosphorylation, while in apoptotic MCF-7 cells reconstituted with caspase-3 (MCF-7/c3) there is a strong induction of this histone modification. Moreover, H2AX S139 phosphorylation is completely abolished by the treatment with z-DEVD-fmk prior to apoptosis induction (Figure 4A). Thus, in TNF-α induced apoptosis H2AX S139 phosphorylation is a caspase-3 dependent event.

Bottom Line: A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering.We find that all depend directly or indirectly on caspase-3 activation.CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Dynamics Unit, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

Show MeSH
Related in: MedlinePlus