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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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CAD immobilization during apoptosis is not affected in HMGB1shRNA cells.(A) HeLa cells were stably transfected with HMGB1shRNA or with the empty vector (evshRNA) as control. Western blotting shows that HMGB1 expression is abolished in HMGB1shRNA cells. (B) Western blots of living (L) and apoptotic (A) evshRNA and HMGB1shRNA cells show that caspase-3 and caspase-6 are cleaved in both cell lines; H2BS14 and H2AX phosphorylation are clearly induced in both cell lines. (C) FRAP experiments to measure CAD mobility were performed in living and apoptotic evshRNA and HMGB1shRNA cells transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).
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pone-0006234-g006: CAD immobilization during apoptosis is not affected in HMGB1shRNA cells.(A) HeLa cells were stably transfected with HMGB1shRNA or with the empty vector (evshRNA) as control. Western blotting shows that HMGB1 expression is abolished in HMGB1shRNA cells. (B) Western blots of living (L) and apoptotic (A) evshRNA and HMGB1shRNA cells show that caspase-3 and caspase-6 are cleaved in both cell lines; H2BS14 and H2AX phosphorylation are clearly induced in both cell lines. (C) FRAP experiments to measure CAD mobility were performed in living and apoptotic evshRNA and HMGB1shRNA cells transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).

Mentions: Both CAD and HMGB1 are immobilized in apoptotic cells, and might influence each other. To test directly this hypothesis, we generated HeLa cells stably transfected with a vector encoding HMGB1 shRNA (HMGB1shRNA), and with the empty vector as a control (evshRNA). We confirmed by Western Blotting that HMGB1 expression is abolished in cells stably expressing HMGB1 shRNA (Figure 6A). However, HMGB1 depletion does not impair the induction of apoptosis, as demonstrated by procaspase-cleavage and histone post-translational modifications (Figure 6B), or DNA laddering (Figure S1).


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

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

CAD immobilization during apoptosis is not affected in HMGB1shRNA cells.(A) HeLa cells were stably transfected with HMGB1shRNA or with the empty vector (evshRNA) as control. Western blotting shows that HMGB1 expression is abolished in HMGB1shRNA cells. (B) Western blots of living (L) and apoptotic (A) evshRNA and HMGB1shRNA cells show that caspase-3 and caspase-6 are cleaved in both cell lines; H2BS14 and H2AX phosphorylation are clearly induced in both cell lines. (C) FRAP experiments to measure CAD mobility were performed in living and apoptotic evshRNA and HMGB1shRNA cells transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713420&req=5

pone-0006234-g006: CAD immobilization during apoptosis is not affected in HMGB1shRNA cells.(A) HeLa cells were stably transfected with HMGB1shRNA or with the empty vector (evshRNA) as control. Western blotting shows that HMGB1 expression is abolished in HMGB1shRNA cells. (B) Western blots of living (L) and apoptotic (A) evshRNA and HMGB1shRNA cells show that caspase-3 and caspase-6 are cleaved in both cell lines; H2BS14 and H2AX phosphorylation are clearly induced in both cell lines. (C) FRAP experiments to measure CAD mobility were performed in living and apoptotic evshRNA and HMGB1shRNA cells transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).
Mentions: Both CAD and HMGB1 are immobilized in apoptotic cells, and might influence each other. To test directly this hypothesis, we generated HeLa cells stably transfected with a vector encoding HMGB1 shRNA (HMGB1shRNA), and with the empty vector as a control (evshRNA). We confirmed by Western Blotting that HMGB1 expression is abolished in cells stably expressing HMGB1 shRNA (Figure 6A). However, HMGB1 depletion does not impair the induction of apoptosis, as demonstrated by procaspase-cleavage and histone post-translational modifications (Figure 6B), or DNA laddering (Figure S1).

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