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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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The lack of caspase-3 expression in MCF-7 cells abolishes H2BS14 phosphorylation and preserves HMGB1 mobility in the nucleus of apoptotic cells.(A) Proliferating (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells were probed by Western blotting. MCF-7 cells, which lack caspase-3, do not cleave Mst1 during apoptosis and do not phosphorylate histone H2B. Mst1 cleavage and H2B phosphorylation are re-established after caspse 3 re-expression. (B) FRAP experiments were performed in proliferating and apoptotic MCF-7/pv and MCF-7/c3 cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 16). The asterisk indicates that the difference in the fraction of mobile HMGB1-GFP between apoptotic MCF-7/pv and MCF-7/c3 cells is highly significant (P<0.001, unpaired t-test).
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pone-0006234-g003: The lack of caspase-3 expression in MCF-7 cells abolishes H2BS14 phosphorylation and preserves HMGB1 mobility in the nucleus of apoptotic cells.(A) Proliferating (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells were probed by Western blotting. MCF-7 cells, which lack caspase-3, do not cleave Mst1 during apoptosis and do not phosphorylate histone H2B. Mst1 cleavage and H2B phosphorylation are re-established after caspse 3 re-expression. (B) FRAP experiments were performed in proliferating and apoptotic MCF-7/pv and MCF-7/c3 cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 16). The asterisk indicates that the difference in the fraction of mobile HMGB1-GFP between apoptotic MCF-7/pv and MCF-7/c3 cells is highly significant (P<0.001, unpaired t-test).

Mentions: In 3T3 fibroblasts, the caspase inhibitor z-DEVD-fmk abrogated HMGB1 immobilization in dying cells (data not shown). We then used MCF-7 cells, which lack functional caspase-3 [19], reconstituted with a plasmid expressing caspase-3 (MCF-7/c3) or with the pBabe/puro empty vector (MCF-7/pv) as a control [20]. We first showed that Mst1 cleavage and H2B S14 phosphorylation occurred only in MCF-7/c3 cells and not in MCF-7/pv cells (Figure 3A). However, both MCF-7/c3 and MCF-7/pv cells died after stimulation with TNF-α and CHX, either by apoptosis (MCF-7/c3 cells) or a different caspase-3–independent mode of death that produces extensive morphological changes including swelling and detachment from the solid support (MCF-7/pv cells) (data not shown). We then investigated the mobility of HMGB1 in dying cells treated with TNF-α and CHX: only 20% of HMGB1-GFP remains mobile in MCF-7/c3 cells, while essentially all HMGB1 remains mobile in MCF-7/pv cells (Figure 3B).


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

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

The lack of caspase-3 expression in MCF-7 cells abolishes H2BS14 phosphorylation and preserves HMGB1 mobility in the nucleus of apoptotic cells.(A) Proliferating (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells were probed by Western blotting. MCF-7 cells, which lack caspase-3, do not cleave Mst1 during apoptosis and do not phosphorylate histone H2B. Mst1 cleavage and H2B phosphorylation are re-established after caspse 3 re-expression. (B) FRAP experiments were performed in proliferating and apoptotic MCF-7/pv and MCF-7/c3 cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 16). The asterisk indicates that the difference in the fraction of mobile HMGB1-GFP between apoptotic MCF-7/pv and MCF-7/c3 cells is highly significant (P<0.001, unpaired t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713420&req=5

pone-0006234-g003: The lack of caspase-3 expression in MCF-7 cells abolishes H2BS14 phosphorylation and preserves HMGB1 mobility in the nucleus of apoptotic cells.(A) Proliferating (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells were probed by Western blotting. MCF-7 cells, which lack caspase-3, do not cleave Mst1 during apoptosis and do not phosphorylate histone H2B. Mst1 cleavage and H2B phosphorylation are re-established after caspse 3 re-expression. (B) FRAP experiments were performed in proliferating and apoptotic MCF-7/pv and MCF-7/c3 cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 16). The asterisk indicates that the difference in the fraction of mobile HMGB1-GFP between apoptotic MCF-7/pv and MCF-7/c3 cells is highly significant (P<0.001, unpaired t-test).
Mentions: In 3T3 fibroblasts, the caspase inhibitor z-DEVD-fmk abrogated HMGB1 immobilization in dying cells (data not shown). We then used MCF-7 cells, which lack functional caspase-3 [19], reconstituted with a plasmid expressing caspase-3 (MCF-7/c3) or with the pBabe/puro empty vector (MCF-7/pv) as a control [20]. We first showed that Mst1 cleavage and H2B S14 phosphorylation occurred only in MCF-7/c3 cells and not in MCF-7/pv cells (Figure 3A). However, both MCF-7/c3 and MCF-7/pv cells died after stimulation with TNF-α and CHX, either by apoptosis (MCF-7/c3 cells) or a different caspase-3–independent mode of death that produces extensive morphological changes including swelling and detachment from the solid support (MCF-7/pv cells) (data not shown). We then investigated the mobility of HMGB1 in dying cells treated with TNF-α and CHX: only 20% of HMGB1-GFP remains mobile in MCF-7/c3 cells, while essentially all HMGB1 remains mobile in MCF-7/pv cells (Figure 3B).

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