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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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Neither HMGB1 nor CAD immobilization onto apoptotic chromatin depend on caspase-6 expression.(A) Western blot of living (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells. Caspase-7, but not caspase-6, is activated in MCF-7/pv forced to undergo apoptosis using TNF-α and CHX. Both caspases are activated in apoptotic MCF-7/c3 cells. (B) HeLa cells were stably transfected with Casp6shRNA or with the empty vector (evshRNA). Western blots show that caspase-6 expression is severely reduced in casp6shRNA HeLa cells. (C) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 20). (D) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).
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pone-0006234-g007: Neither HMGB1 nor CAD immobilization onto apoptotic chromatin depend on caspase-6 expression.(A) Western blot of living (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells. Caspase-7, but not caspase-6, is activated in MCF-7/pv forced to undergo apoptosis using TNF-α and CHX. Both caspases are activated in apoptotic MCF-7/c3 cells. (B) HeLa cells were stably transfected with Casp6shRNA or with the empty vector (evshRNA). Western blots show that caspase-6 expression is severely reduced in casp6shRNA HeLa cells. (C) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 20). (D) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).

Mentions: Besides caspase-3, in mammals there are two other effector caspases, caspase-6 and 7. Both proteins have some substrates in common with caspase-3, and both are involved in the regulation of nuclear changes during apoptosis, including nuclear lamina breakdown [9], [23]. To investigate the role of the different caspases in the immobilization of HMGB1 and CAD, we first determined the level of activation of effector caspases in MCF-7/pv and MCF-7/c3 treated with TNF-α and CHX. Procaspase-7 is processed both in apoptotic MCF-7/pv and MCF-7/c3 cells (Figure 7A). Therefore, caspase-7 cannot be responsible for the immobilization of HMGB1 and CAD in the nucleus of apoptotic cells. Indeed, procaspase-7 appeared to be processed to a greater extent in cells expressing caspase-3, suggesting that its cleavage may in part depend on caspase-3 activation, probably indirectly.


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

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

Neither HMGB1 nor CAD immobilization onto apoptotic chromatin depend on caspase-6 expression.(A) Western blot of living (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells. Caspase-7, but not caspase-6, is activated in MCF-7/pv forced to undergo apoptosis using TNF-α and CHX. Both caspases are activated in apoptotic MCF-7/c3 cells. (B) HeLa cells were stably transfected with Casp6shRNA or with the empty vector (evshRNA). Western blots show that caspase-6 expression is severely reduced in casp6shRNA HeLa cells. (C) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 20). (D) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently 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-g007: Neither HMGB1 nor CAD immobilization onto apoptotic chromatin depend on caspase-6 expression.(A) Western blot of living (L) and apoptotic (A) MCF-7/pv and MCF-7/c3 cells. Caspase-7, but not caspase-6, is activated in MCF-7/pv forced to undergo apoptosis using TNF-α and CHX. Both caspases are activated in apoptotic MCF-7/c3 cells. (B) HeLa cells were stably transfected with Casp6shRNA or with the empty vector (evshRNA). Western blots show that caspase-6 expression is severely reduced in casp6shRNA HeLa cells. (C) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with HMGB1-GFP. The results are expressed as the mean +/− standard deviation (n = 20). (D) FRAP experiments were performed in living and apoptotic evshRNA and casp6shRNA HeLa cells transiently transfected with CAD-GFP. The results are expressed as the mean +/− standard deviation (n = 20).
Mentions: Besides caspase-3, in mammals there are two other effector caspases, caspase-6 and 7. Both proteins have some substrates in common with caspase-3, and both are involved in the regulation of nuclear changes during apoptosis, including nuclear lamina breakdown [9], [23]. To investigate the role of the different caspases in the immobilization of HMGB1 and CAD, we first determined the level of activation of effector caspases in MCF-7/pv and MCF-7/c3 treated with TNF-α and CHX. Procaspase-7 is processed both in apoptotic MCF-7/pv and MCF-7/c3 cells (Figure 7A). Therefore, caspase-7 cannot be responsible for the immobilization of HMGB1 and CAD in the nucleus of apoptotic cells. Indeed, procaspase-7 appeared to be processed to a greater extent in cells expressing caspase-3, suggesting that its cleavage may in part depend on caspase-3 activation, probably indirectly.

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