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Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils.

Altznauer F, Martinelli S, Yousefi S, Thürig C, Schmid I, Conway EM, Schöni MH, Vogt P, Mueller C, Fey MF, Zangemeister-Wittke U, Simon HU - J. Exp. Med. (2004)

Bottom Line: Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo.Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle-independent manner.Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland.

ABSTRACT
Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle-dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle-independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

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Survivin-antisense treatment abolishes the inhibitory effect of survival cytokines on caspase-3 activation in mature neutrophils. (A) Immunoblotting. Neutrophils cultured in the presence of GM-CSF for 10 h maintained greater amounts of the caspase-3 proform and had decreased levels of the 17-kD fragment compared with untreated or oligonucleotide-treated cells. Survivin-antisense treatment (as), but not mismatch control oligonucleotides (ms), increased caspase-3 processing in the presence of GM-CSF in a dose-dependent manner. The same results were obtained in two additional experiments. (B) Caspase-3 activity assay. Increased enzymatic activity was detectable in neutrophils undergoing spontaneous apoptosis compared with GM-CSF– or G-CSF–treated cells. Survivin-as but not ms increased caspase-3–like enzymatic activity in the presence of GM-CSF or G-CSF in a dose-dependent manner. Results of two independent experiments are shown (circles, experiment 1; triangles, experiment 2).
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fig8: Survivin-antisense treatment abolishes the inhibitory effect of survival cytokines on caspase-3 activation in mature neutrophils. (A) Immunoblotting. Neutrophils cultured in the presence of GM-CSF for 10 h maintained greater amounts of the caspase-3 proform and had decreased levels of the 17-kD fragment compared with untreated or oligonucleotide-treated cells. Survivin-antisense treatment (as), but not mismatch control oligonucleotides (ms), increased caspase-3 processing in the presence of GM-CSF in a dose-dependent manner. The same results were obtained in two additional experiments. (B) Caspase-3 activity assay. Increased enzymatic activity was detectable in neutrophils undergoing spontaneous apoptosis compared with GM-CSF– or G-CSF–treated cells. Survivin-as but not ms increased caspase-3–like enzymatic activity in the presence of GM-CSF or G-CSF in a dose-dependent manner. Results of two independent experiments are shown (circles, experiment 1; triangles, experiment 2).

Mentions: We also monitored caspase-3 activation as a consequence of survival factor and/or survivin antisense treatment. Freshly isolated neutrophils contained procaspase-3, but no active 17-kD fragment (Fig. 8 A). Culturing of cells for 10 h resulted in decreased amounts of procaspase-3 and in the appearance of the 17-kD form. Antisense oligonucleotide treatment had no additional effects. GM-CSF delayed the proteolysis of procaspase-3. Similar effects were seen using G-CSF (reference 33 and unpublished data). This effect of the survival factors on procaspase-3 processing was completely abrogated by optimal concentrations of antisense but not mismatch oligonucleotides. Furthermore, caspase-3–like DEVDase activity was suppressed by G-CSF and GM-CSF in 10-h cultures, and this was also abolished by antisense but not mismatch oligonucleotides (Fig. 8 B). Together, to counteract G-CSF–mediated protection of procaspase-3 and inhibition of caspase-3 activity, 5.0 μM antisense oligonucleotides were required, whereas 2.5 μM were sufficient to abolish the GM-CSF effect. These data correlated well with the levels of survivin protein expression (Fig. 7 B) and the induction of apoptosis (Fig. 7 C).


Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils.

Altznauer F, Martinelli S, Yousefi S, Thürig C, Schmid I, Conway EM, Schöni MH, Vogt P, Mueller C, Fey MF, Zangemeister-Wittke U, Simon HU - J. Exp. Med. (2004)

Survivin-antisense treatment abolishes the inhibitory effect of survival cytokines on caspase-3 activation in mature neutrophils. (A) Immunoblotting. Neutrophils cultured in the presence of GM-CSF for 10 h maintained greater amounts of the caspase-3 proform and had decreased levels of the 17-kD fragment compared with untreated or oligonucleotide-treated cells. Survivin-antisense treatment (as), but not mismatch control oligonucleotides (ms), increased caspase-3 processing in the presence of GM-CSF in a dose-dependent manner. The same results were obtained in two additional experiments. (B) Caspase-3 activity assay. Increased enzymatic activity was detectable in neutrophils undergoing spontaneous apoptosis compared with GM-CSF– or G-CSF–treated cells. Survivin-as but not ms increased caspase-3–like enzymatic activity in the presence of GM-CSF or G-CSF in a dose-dependent manner. Results of two independent experiments are shown (circles, experiment 1; triangles, experiment 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211817&req=5

fig8: Survivin-antisense treatment abolishes the inhibitory effect of survival cytokines on caspase-3 activation in mature neutrophils. (A) Immunoblotting. Neutrophils cultured in the presence of GM-CSF for 10 h maintained greater amounts of the caspase-3 proform and had decreased levels of the 17-kD fragment compared with untreated or oligonucleotide-treated cells. Survivin-antisense treatment (as), but not mismatch control oligonucleotides (ms), increased caspase-3 processing in the presence of GM-CSF in a dose-dependent manner. The same results were obtained in two additional experiments. (B) Caspase-3 activity assay. Increased enzymatic activity was detectable in neutrophils undergoing spontaneous apoptosis compared with GM-CSF– or G-CSF–treated cells. Survivin-as but not ms increased caspase-3–like enzymatic activity in the presence of GM-CSF or G-CSF in a dose-dependent manner. Results of two independent experiments are shown (circles, experiment 1; triangles, experiment 2).
Mentions: We also monitored caspase-3 activation as a consequence of survival factor and/or survivin antisense treatment. Freshly isolated neutrophils contained procaspase-3, but no active 17-kD fragment (Fig. 8 A). Culturing of cells for 10 h resulted in decreased amounts of procaspase-3 and in the appearance of the 17-kD form. Antisense oligonucleotide treatment had no additional effects. GM-CSF delayed the proteolysis of procaspase-3. Similar effects were seen using G-CSF (reference 33 and unpublished data). This effect of the survival factors on procaspase-3 processing was completely abrogated by optimal concentrations of antisense but not mismatch oligonucleotides. Furthermore, caspase-3–like DEVDase activity was suppressed by G-CSF and GM-CSF in 10-h cultures, and this was also abolished by antisense but not mismatch oligonucleotides (Fig. 8 B). Together, to counteract G-CSF–mediated protection of procaspase-3 and inhibition of caspase-3 activity, 5.0 μM antisense oligonucleotides were required, whereas 2.5 μM were sufficient to abolish the GM-CSF effect. These data correlated well with the levels of survivin protein expression (Fig. 7 B) and the induction of apoptosis (Fig. 7 C).

Bottom Line: Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo.Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle-independent manner.Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland.

ABSTRACT
Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle-dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle-independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

Show MeSH
Related in: MedlinePlus