Limits...
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.

Show MeSH

Related in: MedlinePlus

Delayed spontaneous apoptosis in immature bone marrow neutrophils compared with mature blood neutrophils. (A) Viability assay in the absence of survival and death factors. Each value represents mean ± SEM calculated from 3–16 (immature cells) and 4–22 (mature cells) independent experiments, respectively. (B) Phosphatidylserine redistribution (left) and DNA fragmentation (right) assays. Mature cells (top) were analyzed after 20-h cultures and immature cells (bottom) were analyzed after 72-h cultures. The results are representative of three independent experiments performed in both cell populations. (C) Caspase-3 is rapidly processed in mature, but not in immature, neutrophils. In mature cells, a 20-kD fragment is usually seen in neutrophils cultured for 4 h. The enzymatically active 17-kD fragment is regularly detected in 8-h neutrophil cultures. In immature cells, small amounts of the 17-kD fragment are frequently seen, suggesting baseline caspase-3 activity in these cells in the absence of apoptosis. Filters were reprobed with an anti-GAPDH or anti–β-actin mAb to ensure equal loading of the gels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211817&req=5

fig2: Delayed spontaneous apoptosis in immature bone marrow neutrophils compared with mature blood neutrophils. (A) Viability assay in the absence of survival and death factors. Each value represents mean ± SEM calculated from 3–16 (immature cells) and 4–22 (mature cells) independent experiments, respectively. (B) Phosphatidylserine redistribution (left) and DNA fragmentation (right) assays. Mature cells (top) were analyzed after 20-h cultures and immature cells (bottom) were analyzed after 72-h cultures. The results are representative of three independent experiments performed in both cell populations. (C) Caspase-3 is rapidly processed in mature, but not in immature, neutrophils. In mature cells, a 20-kD fragment is usually seen in neutrophils cultured for 4 h. The enzymatically active 17-kD fragment is regularly detected in 8-h neutrophil cultures. In immature cells, small amounts of the 17-kD fragment are frequently seen, suggesting baseline caspase-3 activity in these cells in the absence of apoptosis. Filters were reprobed with an anti-GAPDH or anti–β-actin mAb to ensure equal loading of the gels.

Mentions: Purification of immature neutrophils from morphologically normal human bone marrow aspirates yielded a pure population of CD34− CD36−CD7−, but also MPO positive cells (Fig. 1, >97%). However, the cell population contained a mix of neutrophils representing different maturation stages (Table I) . When culturing these mostly immature cells, we observed that they did not rapidly undergo cell death (Fig. 2 A) and apoptosis (Fig. 2 B) as seen in mature neutrophils. Because caspase-3 has been described as a key effector caspase in neutrophil apoptosis (27–29), we measured caspase-3 cleavage by immunoblotting. Both freshly purified immature and mature neutrophils contained significant amounts of procaspase-3 (Fig. 2 C). However, the active 17-kD fragment was not detectable in the immature population. Culturing the mature cells was associated with a partial cleavage of procaspase-3 and in the appearance of the p17 fragment usually after 4–8 h of culture. At later time points, cleavage of procaspase-3 continued until the molecule was no longer detectable. In contrast, culturing of immature cells up to 72 h did not result in cleavage of procaspase-3 (Fig. 2 C). Together, these data suggest that immature neutrophils have much lower caspase-3 activity compared with mature neutrophils, resulting in prolonged in vitro survival.


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)

Delayed spontaneous apoptosis in immature bone marrow neutrophils compared with mature blood neutrophils. (A) Viability assay in the absence of survival and death factors. Each value represents mean ± SEM calculated from 3–16 (immature cells) and 4–22 (mature cells) independent experiments, respectively. (B) Phosphatidylserine redistribution (left) and DNA fragmentation (right) assays. Mature cells (top) were analyzed after 20-h cultures and immature cells (bottom) were analyzed after 72-h cultures. The results are representative of three independent experiments performed in both cell populations. (C) Caspase-3 is rapidly processed in mature, but not in immature, neutrophils. In mature cells, a 20-kD fragment is usually seen in neutrophils cultured for 4 h. The enzymatically active 17-kD fragment is regularly detected in 8-h neutrophil cultures. In immature cells, small amounts of the 17-kD fragment are frequently seen, suggesting baseline caspase-3 activity in these cells in the absence of apoptosis. Filters were reprobed with an anti-GAPDH or anti–β-actin mAb to ensure equal loading of the gels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211817&req=5

fig2: Delayed spontaneous apoptosis in immature bone marrow neutrophils compared with mature blood neutrophils. (A) Viability assay in the absence of survival and death factors. Each value represents mean ± SEM calculated from 3–16 (immature cells) and 4–22 (mature cells) independent experiments, respectively. (B) Phosphatidylserine redistribution (left) and DNA fragmentation (right) assays. Mature cells (top) were analyzed after 20-h cultures and immature cells (bottom) were analyzed after 72-h cultures. The results are representative of three independent experiments performed in both cell populations. (C) Caspase-3 is rapidly processed in mature, but not in immature, neutrophils. In mature cells, a 20-kD fragment is usually seen in neutrophils cultured for 4 h. The enzymatically active 17-kD fragment is regularly detected in 8-h neutrophil cultures. In immature cells, small amounts of the 17-kD fragment are frequently seen, suggesting baseline caspase-3 activity in these cells in the absence of apoptosis. Filters were reprobed with an anti-GAPDH or anti–β-actin mAb to ensure equal loading of the gels.
Mentions: Purification of immature neutrophils from morphologically normal human bone marrow aspirates yielded a pure population of CD34− CD36−CD7−, but also MPO positive cells (Fig. 1, >97%). However, the cell population contained a mix of neutrophils representing different maturation stages (Table I) . When culturing these mostly immature cells, we observed that they did not rapidly undergo cell death (Fig. 2 A) and apoptosis (Fig. 2 B) as seen in mature neutrophils. Because caspase-3 has been described as a key effector caspase in neutrophil apoptosis (27–29), we measured caspase-3 cleavage by immunoblotting. Both freshly purified immature and mature neutrophils contained significant amounts of procaspase-3 (Fig. 2 C). However, the active 17-kD fragment was not detectable in the immature population. Culturing the mature cells was associated with a partial cleavage of procaspase-3 and in the appearance of the p17 fragment usually after 4–8 h of culture. At later time points, cleavage of procaspase-3 continued until the molecule was no longer detectable. In contrast, culturing of immature cells up to 72 h did not result in cleavage of procaspase-3 (Fig. 2 C). Together, these data suggest that immature neutrophils have much lower caspase-3 activity compared with mature neutrophils, resulting in prolonged in vitro survival.

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