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Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells.

Lechardeur D, Xu M, Lukacs GL - J. Cell Biol. (2004)

Bottom Line: We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD.The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells.Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells.

View Article: PubMed Central - PubMed

Affiliation: Hospital for Sick Children Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.

ABSTRACT
Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3-dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment.

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Biogenesis and stability of CAD and ICAD in dividing and apoptotic cells. (A) The stability of endogenous ICAD and CAD in HeLa cells during STS-induced apoptosis. Cellular proteins (40 μg) were separated by SDS-PAGE and probed by immunoblotting with anti-CAD and anti-ICAD antibodies. (B) Internucleosomal DNA fragmentation after 2.5 h apoptosis (lane 3). The accumulation of 200-bp nucleosomal DNA fragments is characteristic of late stage of apoptosis. Soluble DNA fragments were absent in control HeLa cells (lane 2). 1-kb DNA ladder; lane 1. (C and D) HeLa cells were transiently transfected with the HA-CAD-GFP and ICAD-myc vectors or carrier DNA. (C) Cell lysates from normal (lane 2) and STS treated HeLa cells (lane 3) were immunoprecipitated with an anti-GFP antibody. Precipitates (top panel) and 10% of the lysates (bottom panels) were immunoblotted. (D) The expression and stability of ICAD-myc and CAD-GFP were determined as described in A. (E) Indirect immunofluorescence of HeLa cells expressing CAD-GFP/ICAD-myc fixed before (−perm) or after (+perm) permeabilization as in Fig. 1 A. (F) Number of cells retaining nuclear CAD-GFP and GFP-NLS under permeabilization. After in vivo permeabilization as in A, cells were fixed and chromatin was counter-stained with DAPI. Approximately 500 nuclei were scored for their fluorescence for each condition. The number of cells expressing CAD-GFP was expressed as the percentage of nuclei remaining fluorescent upon permeabilization of cells. (G) DNase activity of CAD-GFP. Transiently expressed HA-CAD-GFP/ICAD myc was immunoisolated from HeLa cell lysate with an anti-HA antibody on protein G–Sepharose. Purified HA-CAD-GFP/ICAD-myc complex was incubated with plasmid DNA alone (lane 3, −casp) or in the presence of recombinant caspase-3 (lane 4, + casp). Nontransfected cells were used as control (lanes 5 and 6).
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fig2: Biogenesis and stability of CAD and ICAD in dividing and apoptotic cells. (A) The stability of endogenous ICAD and CAD in HeLa cells during STS-induced apoptosis. Cellular proteins (40 μg) were separated by SDS-PAGE and probed by immunoblotting with anti-CAD and anti-ICAD antibodies. (B) Internucleosomal DNA fragmentation after 2.5 h apoptosis (lane 3). The accumulation of 200-bp nucleosomal DNA fragments is characteristic of late stage of apoptosis. Soluble DNA fragments were absent in control HeLa cells (lane 2). 1-kb DNA ladder; lane 1. (C and D) HeLa cells were transiently transfected with the HA-CAD-GFP and ICAD-myc vectors or carrier DNA. (C) Cell lysates from normal (lane 2) and STS treated HeLa cells (lane 3) were immunoprecipitated with an anti-GFP antibody. Precipitates (top panel) and 10% of the lysates (bottom panels) were immunoblotted. (D) The expression and stability of ICAD-myc and CAD-GFP were determined as described in A. (E) Indirect immunofluorescence of HeLa cells expressing CAD-GFP/ICAD-myc fixed before (−perm) or after (+perm) permeabilization as in Fig. 1 A. (F) Number of cells retaining nuclear CAD-GFP and GFP-NLS under permeabilization. After in vivo permeabilization as in A, cells were fixed and chromatin was counter-stained with DAPI. Approximately 500 nuclei were scored for their fluorescence for each condition. The number of cells expressing CAD-GFP was expressed as the percentage of nuclei remaining fluorescent upon permeabilization of cells. (G) DNase activity of CAD-GFP. Transiently expressed HA-CAD-GFP/ICAD myc was immunoisolated from HeLa cell lysate with an anti-HA antibody on protein G–Sepharose. Purified HA-CAD-GFP/ICAD-myc complex was incubated with plasmid DNA alone (lane 3, −casp) or in the presence of recombinant caspase-3 (lane 4, + casp). Nontransfected cells were used as control (lanes 5 and 6).

Mentions: Staurosporine (STS), a nonspecific inhibitor of PKC and an effective apoptosis-inducing agent, activates caspase-3 and triggers the proteolysis of ICAD (Samuelsson et al., 2002). A significant fraction of both endogenous and heterologous ICAD was degraded after 2 h of STS treatment (Fig. 2 A; Lechardeur et al., 2000). As a corollary, substantial DNA fragmentation occurred, as indicated by the appearance of the oligonucleosomal DNA ladder in apoptotic cells (Fig. 2 B). STS-induced apoptosis delayed the release of HA-CAD from permeabilized cells as compared with control (Fig. 1 A). Meanwhile, the extractability of neither GFP-NLS (Fig. 1 B, right panels) nor the cleaved endogenous nor the exogenous ICAD was altered (Fig. 1 A, right, +perm and unpublished data).


Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells.

Lechardeur D, Xu M, Lukacs GL - J. Cell Biol. (2004)

Biogenesis and stability of CAD and ICAD in dividing and apoptotic cells. (A) The stability of endogenous ICAD and CAD in HeLa cells during STS-induced apoptosis. Cellular proteins (40 μg) were separated by SDS-PAGE and probed by immunoblotting with anti-CAD and anti-ICAD antibodies. (B) Internucleosomal DNA fragmentation after 2.5 h apoptosis (lane 3). The accumulation of 200-bp nucleosomal DNA fragments is characteristic of late stage of apoptosis. Soluble DNA fragments were absent in control HeLa cells (lane 2). 1-kb DNA ladder; lane 1. (C and D) HeLa cells were transiently transfected with the HA-CAD-GFP and ICAD-myc vectors or carrier DNA. (C) Cell lysates from normal (lane 2) and STS treated HeLa cells (lane 3) were immunoprecipitated with an anti-GFP antibody. Precipitates (top panel) and 10% of the lysates (bottom panels) were immunoblotted. (D) The expression and stability of ICAD-myc and CAD-GFP were determined as described in A. (E) Indirect immunofluorescence of HeLa cells expressing CAD-GFP/ICAD-myc fixed before (−perm) or after (+perm) permeabilization as in Fig. 1 A. (F) Number of cells retaining nuclear CAD-GFP and GFP-NLS under permeabilization. After in vivo permeabilization as in A, cells were fixed and chromatin was counter-stained with DAPI. Approximately 500 nuclei were scored for their fluorescence for each condition. The number of cells expressing CAD-GFP was expressed as the percentage of nuclei remaining fluorescent upon permeabilization of cells. (G) DNase activity of CAD-GFP. Transiently expressed HA-CAD-GFP/ICAD myc was immunoisolated from HeLa cell lysate with an anti-HA antibody on protein G–Sepharose. Purified HA-CAD-GFP/ICAD-myc complex was incubated with plasmid DNA alone (lane 3, −casp) or in the presence of recombinant caspase-3 (lane 4, + casp). Nontransfected cells were used as control (lanes 5 and 6).
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fig2: Biogenesis and stability of CAD and ICAD in dividing and apoptotic cells. (A) The stability of endogenous ICAD and CAD in HeLa cells during STS-induced apoptosis. Cellular proteins (40 μg) were separated by SDS-PAGE and probed by immunoblotting with anti-CAD and anti-ICAD antibodies. (B) Internucleosomal DNA fragmentation after 2.5 h apoptosis (lane 3). The accumulation of 200-bp nucleosomal DNA fragments is characteristic of late stage of apoptosis. Soluble DNA fragments were absent in control HeLa cells (lane 2). 1-kb DNA ladder; lane 1. (C and D) HeLa cells were transiently transfected with the HA-CAD-GFP and ICAD-myc vectors or carrier DNA. (C) Cell lysates from normal (lane 2) and STS treated HeLa cells (lane 3) were immunoprecipitated with an anti-GFP antibody. Precipitates (top panel) and 10% of the lysates (bottom panels) were immunoblotted. (D) The expression and stability of ICAD-myc and CAD-GFP were determined as described in A. (E) Indirect immunofluorescence of HeLa cells expressing CAD-GFP/ICAD-myc fixed before (−perm) or after (+perm) permeabilization as in Fig. 1 A. (F) Number of cells retaining nuclear CAD-GFP and GFP-NLS under permeabilization. After in vivo permeabilization as in A, cells were fixed and chromatin was counter-stained with DAPI. Approximately 500 nuclei were scored for their fluorescence for each condition. The number of cells expressing CAD-GFP was expressed as the percentage of nuclei remaining fluorescent upon permeabilization of cells. (G) DNase activity of CAD-GFP. Transiently expressed HA-CAD-GFP/ICAD myc was immunoisolated from HeLa cell lysate with an anti-HA antibody on protein G–Sepharose. Purified HA-CAD-GFP/ICAD-myc complex was incubated with plasmid DNA alone (lane 3, −casp) or in the presence of recombinant caspase-3 (lane 4, + casp). Nontransfected cells were used as control (lanes 5 and 6).
Mentions: Staurosporine (STS), a nonspecific inhibitor of PKC and an effective apoptosis-inducing agent, activates caspase-3 and triggers the proteolysis of ICAD (Samuelsson et al., 2002). A significant fraction of both endogenous and heterologous ICAD was degraded after 2 h of STS treatment (Fig. 2 A; Lechardeur et al., 2000). As a corollary, substantial DNA fragmentation occurred, as indicated by the appearance of the oligonucleosomal DNA ladder in apoptotic cells (Fig. 2 B). STS-induced apoptosis delayed the release of HA-CAD from permeabilized cells as compared with control (Fig. 1 A). Meanwhile, the extractability of neither GFP-NLS (Fig. 1 B, right panels) nor the cleaved endogenous nor the exogenous ICAD was altered (Fig. 1 A, right, +perm and unpublished data).

Bottom Line: We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD.The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells.Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells.

View Article: PubMed Central - PubMed

Affiliation: Hospital for Sick Children Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.

ABSTRACT
Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3-dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment.

Show MeSH
Related in: MedlinePlus