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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 levels are markedly increased in mature neutrophils exposed to survival factors in vitro and in neutrophils from patients with CF. (A) Immunoblotting. Levels of survivin in neutrophils were increased by culturing normal control neutrophils in the presence of GM-CSF or G-CSF for 12 h. Moreover, freshly purified neutrophils from CF patients (n = 4) showed strongly increased survivin protein levels compared with control blood neutrophils. 20 ng purified recombinant survivin served as a positive control. The filter was reprobed with an anti–β-actin mAb to ensure equal loading of the gel. (B) Real-time PCR. The lung cancer cell line A549 served as a standard (100%). Neutrophils from patients with CF contained increased amounts of survivin mRNA compared with normal neutrophils (left). The increase in survivin mRNA was mimicked by addition of GM-CSF or G-CSF to normal control neutrophils in vitro (right). Values are means ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.001. (C) Proliferation assay. Freshly purified blood neutrophils from normal donors or CF patients did not demonstrate evidence for cell proliferation. The same was true for G-CSF– or GM-CSF–stimulated normal mature neutrophils. In contrast, immature neutrophils demonstrated significant proliferation, which was inducible by both GM-CSF and G-CSF. Pan–T cell–stimulated PBMCs were used as positive controls. Results of four independent experiments are shown for each cell population.
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fig4: Survivin levels are markedly increased in mature neutrophils exposed to survival factors in vitro and in neutrophils from patients with CF. (A) Immunoblotting. Levels of survivin in neutrophils were increased by culturing normal control neutrophils in the presence of GM-CSF or G-CSF for 12 h. Moreover, freshly purified neutrophils from CF patients (n = 4) showed strongly increased survivin protein levels compared with control blood neutrophils. 20 ng purified recombinant survivin served as a positive control. The filter was reprobed with an anti–β-actin mAb to ensure equal loading of the gel. (B) Real-time PCR. The lung cancer cell line A549 served as a standard (100%). Neutrophils from patients with CF contained increased amounts of survivin mRNA compared with normal neutrophils (left). The increase in survivin mRNA was mimicked by addition of GM-CSF or G-CSF to normal control neutrophils in vitro (right). Values are means ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.001. (C) Proliferation assay. Freshly purified blood neutrophils from normal donors or CF patients did not demonstrate evidence for cell proliferation. The same was true for G-CSF– or GM-CSF–stimulated normal mature neutrophils. In contrast, immature neutrophils demonstrated significant proliferation, which was inducible by both GM-CSF and G-CSF. Pan–T cell–stimulated PBMCs were used as positive controls. Results of four independent experiments are shown for each cell population.

Mentions: During maturation, neutrophils are exposed to high levels of cytokines in the bone marrow. Some of these cytokines, such as GM-CSF and G-CSF, are also elevated in bacterial infectious diseases (19). Interestingly, mature neutrophils derived from the blood of patients suffering from CF exhibited significantly higher levels of survivin compared with normal blood neutrophils as assessed by immunoblotting. Moreover, GM-CSF and G-CSF increased survivin expression of normal neutrophils upon in vitro stimulation for 12 h (Fig. 4 A). Elevated survivin protein levels correlated with increased mRNA expression as assessed by real-time PCR analysis. The mean mRNA levels in peripheral blood neutrophils from patients with CF were increased ∼10-fold compared with control cells. Moreover, GM-CSF and G-CSF dramatically increased survivin mRNA expression in normal mature neutrophils within 4 h (Fig. 4 B). Both cytokine-stimulated normal neutrophils and CF neutrophils did not demonstrate any evidence for cell proliferation as assessed by [3H]thymidine incorporation assays (Fig. 4 C) and DNA analysis (not depicted). In contrast with mature neutrophils, immature neutrophils demonstrated significant baseline proliferation and proliferation was inducible by both GM-CSF and G-CSF.


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 levels are markedly increased in mature neutrophils exposed to survival factors in vitro and in neutrophils from patients with CF. (A) Immunoblotting. Levels of survivin in neutrophils were increased by culturing normal control neutrophils in the presence of GM-CSF or G-CSF for 12 h. Moreover, freshly purified neutrophils from CF patients (n = 4) showed strongly increased survivin protein levels compared with control blood neutrophils. 20 ng purified recombinant survivin served as a positive control. The filter was reprobed with an anti–β-actin mAb to ensure equal loading of the gel. (B) Real-time PCR. The lung cancer cell line A549 served as a standard (100%). Neutrophils from patients with CF contained increased amounts of survivin mRNA compared with normal neutrophils (left). The increase in survivin mRNA was mimicked by addition of GM-CSF or G-CSF to normal control neutrophils in vitro (right). Values are means ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.001. (C) Proliferation assay. Freshly purified blood neutrophils from normal donors or CF patients did not demonstrate evidence for cell proliferation. The same was true for G-CSF– or GM-CSF–stimulated normal mature neutrophils. In contrast, immature neutrophils demonstrated significant proliferation, which was inducible by both GM-CSF and G-CSF. Pan–T cell–stimulated PBMCs were used as positive controls. Results of four independent experiments are shown for each cell population.
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fig4: Survivin levels are markedly increased in mature neutrophils exposed to survival factors in vitro and in neutrophils from patients with CF. (A) Immunoblotting. Levels of survivin in neutrophils were increased by culturing normal control neutrophils in the presence of GM-CSF or G-CSF for 12 h. Moreover, freshly purified neutrophils from CF patients (n = 4) showed strongly increased survivin protein levels compared with control blood neutrophils. 20 ng purified recombinant survivin served as a positive control. The filter was reprobed with an anti–β-actin mAb to ensure equal loading of the gel. (B) Real-time PCR. The lung cancer cell line A549 served as a standard (100%). Neutrophils from patients with CF contained increased amounts of survivin mRNA compared with normal neutrophils (left). The increase in survivin mRNA was mimicked by addition of GM-CSF or G-CSF to normal control neutrophils in vitro (right). Values are means ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.001. (C) Proliferation assay. Freshly purified blood neutrophils from normal donors or CF patients did not demonstrate evidence for cell proliferation. The same was true for G-CSF– or GM-CSF–stimulated normal mature neutrophils. In contrast, immature neutrophils demonstrated significant proliferation, which was inducible by both GM-CSF and G-CSF. Pan–T cell–stimulated PBMCs were used as positive controls. Results of four independent experiments are shown for each cell population.
Mentions: During maturation, neutrophils are exposed to high levels of cytokines in the bone marrow. Some of these cytokines, such as GM-CSF and G-CSF, are also elevated in bacterial infectious diseases (19). Interestingly, mature neutrophils derived from the blood of patients suffering from CF exhibited significantly higher levels of survivin compared with normal blood neutrophils as assessed by immunoblotting. Moreover, GM-CSF and G-CSF increased survivin expression of normal neutrophils upon in vitro stimulation for 12 h (Fig. 4 A). Elevated survivin protein levels correlated with increased mRNA expression as assessed by real-time PCR analysis. The mean mRNA levels in peripheral blood neutrophils from patients with CF were increased ∼10-fold compared with control cells. Moreover, GM-CSF and G-CSF dramatically increased survivin mRNA expression in normal mature neutrophils within 4 h (Fig. 4 B). Both cytokine-stimulated normal neutrophils and CF neutrophils did not demonstrate any evidence for cell proliferation as assessed by [3H]thymidine incorporation assays (Fig. 4 C) and DNA analysis (not depicted). In contrast with mature neutrophils, immature neutrophils demonstrated significant baseline proliferation and proliferation was inducible by both GM-CSF and G-CSF.

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