Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils

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Abstract

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associated with cancer, but recent reports suggest that this molecule is also expressed in several immature and mature hematopoietic cells. Here, we provide evidence that treatment of immature neutrophils with anticancer drugs reduced endogenous survivin levels causing apoptosis. The anticancer drugs did not directly target survivin, instead they blocked the activity of phosphatidylinositol-3-OH kinase, which regulated survivin expression and apoptosis in these cells. Strikingly, and in contrast to other cells, this pathway did not involve the serine/threonine kinase c-akt/PKB. Moreover, in combination with anticancer drug therapy, rapamycin did not induce increased myelosuppression in an experimental lymphoma mouse model. These data suggest that drugs that block either c-akt/PKB or signaling molecules located distal to c-akt/PKB may preferentially induce apoptosis of cancer cells as they exhibit no cytotoxicity for immature neutrophils.

Introduction

Most anticancer drugs produce severe myelosuppression that limits their clinical usefulness. Consequently, patients develop neutropenia associated with frequent episodes of fever (Dale, 2003; Straka et al., 2004). Several mechanisms may contribute to the myelotoxicity associated with anticancer drug treatment. DNA damage might be one important event, resulting in the activation of a p53-mediated proapoptotic pathway (Wang and El-Deiry, 2004). However, as cytotoxicity induced by anticancer drugs does not always correlate with the p53 status (Herr et al., 2000), additional death pathways might exist. Such pathways appear to be induced by the cellular stress response (Rich et al., 2000). Multiple stress-inducible molecules, such as Jun kinase, mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-κB and ceramide, have been shown to regulate apoptosis pathways (Debatin, 2004).

Another signaling pathway that regulates apoptosis involves phosphatidylinositol-3-OH kinase (PI3K) and the serine–threonine kinase c-akt/PKB (Franke et al., 2003). The PI3K–c-akt/PKB pathway is positively regulated by Ras and negatively by phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Some Ras mutations in cancer activate PI3K (McIlroy et al., 1997; Suzuki et al., 1997). Moreover, decreased expression and/or phosphatase activity of PTEN also enhance PI3K in hematopoietic tumors (Sakai et al., 1998; Dahia et al., 1999). PI3K activity has been linked to a variety of non-hematopoietic tumors (Chang et al., 2003), and several mutations in the PI3K gene were found in human cancers that likely increase kinase activity (Samuels et al., 2004). Furthermore, c-akt/PKB, a kinase acting distal to PI3K, may participate in malignant transformation (Nicholson and Anderson, 2002). Therefore, inhibition of the PI3K–c-akt/PKB pathway has been considered as a major strategy to treat cancer (Vivanco and Sawyers, 2002; Chang et al., 2003).

Survivin is a member of the inhibitor of apoptosis (IAP) family and it was reported to be expressed in fetal tissues as well as in transformed cell lines and malignancies, but not in normal adult tissues (Ambrosini et al., 1997). This suggested that reactivation of the survivin gene occurs frequently during tumorigenesis. Moreover, survivin expression in cancer cells correlated with drug resistance and clinical outcome (Zangemeister-Wittke and Simon, 2004). Recently, several studies reported that survivin is also expressed in normal hematopoietic cells. In particular, adult hematopoietic stem cells (Fukuda et al., 2002, 2004) and immature neutrophils (Altznauer et al., 2004) express survivin at high levels.

Here, we show that anticancer drugs reduce survivin in normal immature bone marrow neutrophils, a mechanism that likely contributes to treatment-associated myelosuppression. This effect of the anticancer drugs is mediated by reducing PI3K activity. Strikingly, and in contrast to cancer cells, the regulation of the PI3K–survivin pathway did not involve c-akt/PKB in immature neutrophils. Even following full maturation of these cells, this antiapoptotic pathway remained c-akt/PKB-independent, suggesting that PI3K does not always act via c-akt/PKB, at least in the neutrophil lineage. These data provide the rationale for reducing survivin levels in cancer cells by using inhibitors that block signaling molecules at the level of or distal to c-akt/PKB, which do not exhibit proapoptotic effects in neutrophil precursors.

Results

Survivin blocks Apaf-1 apoptosome formation

We used cell-free extracts from HL-60 cells (which express survivin, data not shown) and explored whether addition of exogenous cytochrome c/dATP triggers apoptosome formation (Murphy et al., 2003) in these extracts. Addition of 5 μg/ml cytochrome c was sufficient to demonstrate proteolytic cleavage of caspase-3. Cleavage occurred rapidly and was detectable within 15 min (Figure 1a). To demonstrate a potential antiapoptotic function of survivin in these cells, we applied suboptimal caspase activation conditions. For instance, if we added 1 μg/ml cytochrome c only, no detectable caspase-3 cleavage was seen in cell-free HL-60 extracts. Following immunodepletion of survivin, however, caspase-3 cleavage products were clearly detectable within 1 h, suggesting that even low concentrations of cytochrome c are able to trigger assembly of the Apaf-1 apoptosome under survivin-deficient conditions (Figure 1b, upper panel). We also analyzed caspase-9 cleavage in this system and observed more cleavage upon immunological neutralization of survivin (Figure 1b, lower panel). These data suggest that survivin blocks apoptosome formation in HL-60 cells and support the view that survivin exhibits an antiapoptotic function in granulocytes (Altznauer et al., 2004).

Figure 1
figure1

Survivin exhibits antiapoptotic properties and the modulation of survivin expression by anticancer drug treatment in immature neutrophils and some cancer cell lines. (a) Cell-free HL-60 extracts were incubated in the presence of the indicated amounts of cytochrome c/dATP at 37°C for 1 h, followed by caspase-3 immunoblotting (upper panel). Kinetics of 25 μg/ml cytochrome c/dATP-mediated caspase-3 cleavage in HL-60 cell-free extracts. Cell-free reactions were assembled at 37°C for the indicated times (lower panel). The data are representative of three independent experiments. (b) HL-60 cell-free extract was immunodepleted of survivin and then stimulated with 1 μg/ml cytochrome c/dATP at 37°C for 1 h. At this low concentration, only the survivin immunodepleted but not control extracts demonstrated detectable caspase-3 cleavage (upper panel). The filters were re-probed with anti-caspase-9 Ab. Note that depletion of survivin resulted in accelerated caspase-9 cleavage (lower panel). (c) Immunoblotting. Survivin was detectable in all cells. Whereas both cisplatin (50 μ M) and etoposide (50 μ M) reduced survivin levels in immature neutrophils, HL-60 cells and Jurkat cells, no change of survivin expression was seen in lymphocytic Raji cells. Cells were cultured for 48 h. The filters were reprobed with an anti-GAPDH mAb to ensure equal loading of the gels. Same results were observed when the anticancer drugs were used at 25 μ M. (d) DNA fragmentation assay. Cells were cultured for 48 h with the indicated concentrations of cisplatin and etoposide. Each value represents mean±s.e.m. of three independent experiments.

Anticancer drugs differentially modify survivin levels in apoptosis-sensitive and -resistant cells

Survivin expression has been described in both immature neutrophils (Altznauer et al., 2004) and cancer cells (Ambrosini et al., 1997). Treatment of these cells with the anticancer drugs cisplatin and etoposide allowed the distinction of two types of cells. Whereas in immature neutrophils, Jurkat and HL-60 cells, a reduction of survivin protein levels was observed, it was surprising to see that the expression of survivin did not change in the B-cell lymphoma cell line Raji (Figure 1c). Interestingly, cells with reduced survivin levels were apoptosis sensitive, whereas cells with unchanged survivin expression appeared to be apoptosis resistant following anticancer drug treatment (Figure 1d).

Both survivin expression and apoptosis are regulated by PI3K, which is targeted by anticancer drugs, in immature neutrophils

As survivin appeared to have a cytoprotective role in immature neutrophils and its downregulation by anticancer drugs could play a role in chemotherapy-associated myelosuppression, we wanted to understand how these drugs reduce survivin levels in these cells. To this end, we incubated immature neutrophils with different defined signaling inhibitors. Specifically, LY294002, a PI3K inhibitor, SB203580, a selective inhibitor of p38 MAPK, PD98059, which blocks p42/44 MAPKs and SN50, a NF-κB inhibitor, were used. Immunoblotting showed a selective reduction of survivin levels associated with PI3K inhibition, whereas inhibition of NF-κB or MAPK pathways had no effect (Figure 2a, upper panel). The effect of PI3K inhibition was both concentration- and time-dependent, as well as somehow specific as Mcl-1 levels were not affected under the same conditions (Figure 2a, lower two panels). Moreover, reduced survivin protein expression following pharmacological inhibition of PI3K was the consequence of decreased transcription of the survivin gene (Figure 2b).

Figure 2
figure2

Survivin expression and apoptosis are regulated by PI3K in immature neutrophils. (a) Immunoblotting. Immature neutrophils were cultured in the presence of different pharmacological inhibitors for the indicated times. Survivin levels were reduced by LY294002 (25 μ M), but not by SB203580 (25 μ M), PD98059 (25 μ M) or SN50 (50 μg/ml) (upper panel). The effect of LY294002 was concentration-dependent, specific (Mcl-1 levels were not affected) and time-dependent (middle and lower panels). Data are representative of at least three independent experiments in each panel. (b) Reverse transcriptase–PCR. HL-60 cells express survivin mRNA. A 25 μ M portion of LY294002 reduced survivin mRNA expression in cells cultured for 24 h and further declined after 48 h.

PI3K inhibition also caused cell death in immature neutrophils in a concentration-dependent manner (Figure 3a, left panel). The type of death was apoptosis, as determined by phosphatidylserine redistribution (Figure 3a, right panel), DNA fragmentation and morphological analysis (data not shown). To determine whether anticancer drugs target PI3K, we analyzed phosphorylation of its downstream target c-akt/PKB (Burgering and Coffer, 1995; Franke et al., 1995), in HL-60 cells, Jurkat cells and immature neutrophils. Both cisplatin and etoposide reduced phosphophylation of c-akt/PKB at either Ser473 or Thr308 or at both phosphorylation sites in all three cell populations (Figure 3b). We also directly measured PI3K activity in immature neutrophils and HL-60 cells, and observed a dramatic inhibition upon short-term anticancer drug treatment (8 and 12 h, respectively) (Figure 3c). Taken together, anticancer drug-induced apoptosis might be at least partially owing to inhibition of PI3K and subsequent reduced survivin levels in neutrophil precursors.

Figure 3
figure3

Pharmacological inhibition of PI3K reduces viability and anticancer drugs inhibit PI3K activity in immature neutrophils. (a) Viability and phosphatidylserine redistribution assays. Immature neutrophils were cultured in the presence of different pharmacological inhibitors for 48 h. Cell death and apoptosis, were induced by LY294002 (at 25 μ M), but not by SB203580 or PD98059. Each value represents mean±s.e.m. of three independent experiments. (b) c-akt/PKB phosphorylation. HL-60 cells, Jurkat cells and immature neutrophils were cultured for 24 h in the presence or absence of cisplatin (25 μ M) or etoposide (25 μ M). Both anticancer drugs reduced c-akt/PKB phosphorylation at Ser473 or Thr308 or both phosphorylation sites in HL-60 cells, Jurkat cells and immature neutrophils. The data are representative of three independent experiments for each cell population. (c) Effects of cisplatin (25 μ M) or etoposide (25 μ M) on PI3K activity in immature neutrophils (8-h cultures) and HL-60 cells (12-h cultures). As a positive control, cells were cultured in the presence of 25 μ M LY294002.

Both survivin expression and apoptosis are not regulated by c-akt/PKB in immature and mature neutrophils

To investigate whether survivin expression is controlled by c-akt/PKB, we exposed immature neutrophils with the specific c-akt/PKB inhibitor SH-6. This inhibitor is a propidium iodide (PI) analog and has been shown to reduce c-akt/PKB phosphorylation at Ser473 without affecting activation of the upstream kinase, PDK-1 (Kozikowski et al., 2003). We observed reduced expression of survivin in Jurkat and HL-60 cells following treatment with SH-6. In contrast, c-akt/PKB inhibition did not affect survivin levels in immature neutrophils (Figure 4a). This suggests that survivin expression is regulated by c-akt/PKB in Jurkat and HeLa cells, but not in immature neutrophils.

Figure 4
figure4

Survivin expression and apoptosis are not regulated by c-akt/PKB in immature neutrophils. (a) Immunoblotting. Immature neutrophils, Jurkat and HL-60 cells were cultured in the presence of LY294002 (25 μ M) or SH-6 (25 μ M) for 48 h. SH-6 reduced survivin expression in Jurkat and HL-60 cells but not in immature neutrophils. (b) Immunoblotting. Cells were cultured as described in (a). SH-6 blocked phosphorylation at both Ser473 and Thr308 in all cell types. Note that LY294002 did not reduce phosphorylation at Thr308 in Jurkat and HL-60 cells. (c) Viability assay. Cells were cultured as described in (a). SH-6 did not induce cell death in immature neutrophils. In contrast, SH-6 exhibited a strong cytotoxic effect on Jurkat and HL-60 cells. Each value represents mean±s.e.m. of three independent experiments. Same results were observed using the DNA fragmentation assay (data not shown).

As c-akt/PKB activation is reflected by its phosphorylation at Ser473 and Thr308 (Scheid and Woodgett, 2003), we controlled pharmacological c-akt/PKB inhibition by SH-6 by immunoblotting. SH-6 reduced phosphorylation of both Ser473 and Thr308 to almost undetectable levels in immature neutrophils, Jurkat cells and HL-60 cells (Figure 4b), suggesting that SH-6 inactivated c-akt/PKB in all cell populations, including immature neutrophils. Moreover, LY294002 reduced Ser473 phosphorylation in all three cell populations, in accordance with previous reports indicating that PI3K activity is required to achieve Ser473 phosphorylation of c-akt/PKB (Andjelkovic et al., 1999; Scheid et al., 2002). In contrast, Thr308 was either not (Jurkat) or only partially (HL-60) affected by pharmacological inhibition of PI3K.

Inhibition of c-akt/PKB had no effect on survivin levels in immature neutrophils, also it did not induce cell death in these cells (Figure 4c). In contrast, c-akt/PKB inhibition resulted in strongly reduced cell viability in both Jurkat and HL-60 cells. Staurosporine was used as a positive control in these cellular systems. Therefore, in contrast to Jurkat cells, HL-60 cells and other cellular systems (Franke et al., 2003), c-akt/PKB does not appear to exhibit an antiapoptotic function in immature neutrophils.

We also investigated the effects of blocking PI3K and c-akt/PKB in mature neutrophils. In contrast to immature neutrophils, constitutive phosphorylation of c-akt/PKB was almost undetectable in mature neutrophils, suggesting that terminal differentiation is associated with lowering c-akt/PKB activity. However, stimulation of mature neutrophils with granulocyte–macrophage colony-stimulating factor (GM-CSF) resulted in c-akt/PKB phosphorylation at both Ser473 and Thr308. This inducible phosphorylation of c-akt/PKB was blocked by both LY294002 and SH-6 (Figure 5a), demonstrating the pharmacological efficacy of these inhibitors. Although both inhibitors blocked c-akt/PKB activity, only LY294002 but not SH-6 blocked the anti-death effect of GM-CSF (Figure 5b), suggesting that the PI3K-mediated antiapoptosis pathway does not involve c-akt/PKB in mature neutrophils.

Figure 5
figure5

Pharmacological inhibition of c-akt/PKB has no effect on cytokine-mediated survival of mature neutrophils. (a) Immunoblotting. Mature neutrophils were preincubated with either LY294002 (25 μ M) or SH-6 (25 μ M) for 30 min. Stimulation with GM-CSF (50 ng/ml) was performed over a time period of 3 min. GM-CSF induced phosphorylation of c-akt/PKB at both Ser473 and Thr308 in these cells. Phosphorylation was completely blocked by both LY294002 and SH-6. (b) Viability assay. Mature neutrophils were cultured in the presence of either LY294002 (25 μ M) or SH-6 (25 μ M) in conjunction with GM-CSF (50 ng/ml) for 48 h. SH-6, in contrast to LY294002, did not prevent GM-CSF-mediated increased neutrophil survival.

Anticancer drugs exhibit greater cytotoxic effects in association with inhibition of PI3K but not c-akt/PKB in immature neutrophils

We next examined whether pharmacological inhibition of PI3K and c-akt/PKB would enhance the cytotoxic effects of anticancer drugs in immature neutrophils. Apoptosis was measured by DNA fragmentation. We observed that inhibition of PI3K with a suboptimal concentration of LY294002 (10 μ M) augmented both cisplatin- and etoposide-induced apoptosis (Figure 6, upper panel). However, blocking of c-akt/PKB by using an optimal concentration of SH-6 did not enhance the anticancer drug-mediated cytotoxic effect on immature neutrophils (Figure 6, lower panel). In contrast, SH-6 clearly accelerated drug-induced apoptosis in HL-60 cells. These data support the view that inhibition of c-akt/PKB does not affect cell survival of immature neutrophils, even under chemotherapy-mediated stress conditions.

Figure 6
figure6

Pharmacological inhibition of PI3K but not c-akt/PKB enhances the proapoptotic effect of anticancer drugs in immature neutrophils. Immature neutrophils (open symbols) were cultured in the presence or absence of LY294002 (10 μ M) (upper panels) or SH-6 (25 μ M) (lower panels) in conjunction with cisplatin (50 μ M) or etoposide (50 μ M), for 72 h. Apoptosis was measured by DNA fragmentation assay. Results of multiple independent experiments are shown. HL-60 cells were used for comparison (closed symbols). Note that HL-60 cells were cultured with etoposide (10 h) or cisplatin (7 h), much shorter than the immature neutrophils. LY294002 but not SH-6 enhanced the proapoptotic effect of anticancer drugs in immature neutrophils. Same results were observed using the viability assay (data not shown).

Rapamycin does not reduce neutrophil numbers in combination with anticancer therapy in vivo

Pharmacologically, c-akt/PKB inhibitors are currently not available. An important branch of c-akt/PKB-mediated cancer cell survival involves the serine/threonine kinase mTOR (McCormick, 2004). We therefore tested the hypothesis that additional inhibition of the c-akt/PKB-mediated signaling pathway does not induce additional myelosuppression during anticancer drug treatment in vivo using rapamycin, which blocks mTOR activity. We have previously shown that rapamycin induced in combination with anticancer therapy massive apoptosis in lymphoma cells, which were drug resistant in the absence of rapamycin (Wendel et al., 2004). In this model, doxorubicin reduced neutrophil numbers in blood. The combination of doxorubicin and rapamycin, however, did not cause a further reduction of neutrophil numbers. In fact, in two independent experiments, we observed even less doxorubicin-mediated cytotoxicity in the presence of rapamycin (Figure 7). These data confirm our in vitro observations and suggest that the c-akt/PKB–mTOR pathway does not exhibit an antiapoptotic function in immature neutrophils under in vivo conditions.

Figure 7
figure7

Combined doxorubicin/rapamycin treatment does not cause additional myelosuppression compared to doxorubicin alone under in vivo conditions. Mice were treated as described in Materials and methods. After 5 days, differential blood counts were analyzed. Results of two independent experiments are shown. Same data were observed after 10-day treatment.

Discussion

Survivin has been shown to regulate chromosome alignment and segregation during mitosis (Vong et al., 2005). Interestingly, survivin also plays an essential role in cytokine-mediated antiapoptosis in terminally differentiated neutrophils, which are unable to divide (Altznauer et al., 2004). In this report, we demonstrate that anticancer drugs reduce survivin levels via inhibition of PI3K activity in immature neutrophils, resulting in apoptosis, a mechanism that may contribute, at least partially, to myelosuppression often seen in patients receiving chemotherapy.

Survivin expression has previously been reported to be regulated by the PI3K–c-akt/PKB pathway in endothelial cells (Daly et al., 2004) as well as in leukemia (Carter et al., 2001) and prostate cancer cells (Fornaro et al., 2003). Besides survivin, however, c-akt/PKB has additional cellular targets that could promote survival. For instance, c-akt/PKB protects cells from apoptosis by phosphorylating and inactivating several key apoptotic molecules such as Bad, pro-caspase-9 and the forkhead transcription factor 1, which results in decreased transcription of proapoptotic genes (Chang et al., 2003). Moreover, c-akt/PKB was reported to induce the expression of antiapoptotic genes such as IAPs, Bcl-xL and c-FLIP (Mitsiades et al., 2002; Hersey and Zhang, 2003; Daly et al., 2004).

Although we cannot exclude that these mechanisms also support survival in immature neutrophils, we show that survivin levels decline upon pharmacological inhibition of PI3K and confirm that survivin participates in the control of caspase activation and exhibits an antiapoptotic function in granulocytic cells (Altznauer et al., 2004). Strikingly, however, inhibition of c-akt/PKB neither reduced survivin levels nor induced apoptosis in immature neutrophils.

One way to explain these findings is that survivin expression is under the control of different PI3K isoforms in different cells (Vanhaesebroeck and Waterfield, 1999). On the other hand, different downstream signaling molecules have been reported to be activated by the same PI3K isoform. For instance, class IA PI3Ks not only activate c-akt/PKB, but also the small GTP-binding proteins CDC42 and RAC1 as well as the serum and glucocorticoid-inducible kinases (SGKs) (Vivanco and Sawyers, 2002). The SGKs have attracted attention because of their high homology to c-akt/PKB and similar functional effects on survival signaling pathways (Brunet et al., 2001). However, PI3K activation of SGKs differs from c-akt/PKB, as SGKs do not contain a pleckstrin homology (PH) domain (Vivanco and Sawyers, 2002), which is required to recruit c-akt/PKB.

Blocking of PI3K but not c-akt/PKB abolished GM-CSF-mediated survival in mature neutrophils, suggesting that neutrophils do not develop a c-akt/PKB-dependent antiapoptotic pathway during terminal differentiation. That cytokine-mediated survival of mature neutrophils can be pharmacologically blocked by PI3K inhibitors has previously been reported. Several mechanisms explaining PI3K-mediated neutrophil survival have recently been proposed including phosphorylation of Bad (Cowburn et al., 2002) and Bax (Gardai et al., 2004). In both reports, neutrophils were exposed to LY294002 but not to a c-akt/PKB inhibitor, and c-akt/PKB-mediated phosphorylation of Bax was demonstrated in PLB-985 cells but not in neutrophils (Gardai et al., 2004). Therefore, although a role for c-akt/PKB in antiapoptotic signaling has been suggested in neutrophils (Gardai et al., 2004), it has not been demonstrated, at least in primary neutrophils. Moreover, interferon-β has been shown to induce PI3K-dependent neutrophil survival in the absence of c-akt/PKB activation (Scheel-Toellner et al., 2002), supporting our concept that c-akt/PKB may not be a regulator of apoptosis in these cells.

Independent of the exact molecular mechanisms, the newly identified PI3K/survivin pathway, which does not involve c-akt/PKB and its downstream target mTOR, points to a novel therapeutic approach in which the apoptotic program can be preferentially activated in cancer cells but not in immature neutrophils. c-akt/PKB and mTOR have previously been considered as potential new anticancer drug targets. For instance, combined treatment with doxorubicin and rapamycin reversed c-akt/PKB-mediated drug resistance in lymphomas in an experimental in vivo model (Wendel et al., 2004) and the mTOR inhibitor RAD001 enhanced cisplatin-induced apoptosis in wild-type p53-containing cancer cells (Beuvink et al., 2005).

Interestingly, drug resistance of cancers has often been associated with high survivin levels (Ambrosini et al., 1997). The data provided in this report suggest that anticancer drugs may reduce survivin levels in immature neutrophils by inhibition of PI3K. In contrast, the same drugs do not reduce survivin levels in some cancer cells, a mechanism that potentially contributes to drug resistance (Belyanskaya et al., 2005). The mechanisms by which DNA damage differentially influences PI3K activity in different cells remain to be investigated. Nevertheless, our study suggests that reducing survivin levels by pharmacological inhibition of the c-akt/PKB-mediated signaling pathway contributes to the reversed drug resistance against conventional chemotherapy. The same treatment strategy, however, does not seem to induce additional apoptosis in immature neutrophils compared to anticancer treatment alone. Therefore, no additional myelosuppressive effects should be expected by such a combined therapy. Preliminary in vivo data provided in this study support this concept.

Materials and methods

Cell isolations

Immature neutrophils were isolated from bone marrow aspirates with normal cellular morphology and distribution as described previously (Altznauer et al., 2004; Martinelli et al., 2004). Briefly, after centrifugation on a two-step discontinuous Percoll density gradient, cells were negatively isolated using anti-CD7 and anti-CD36 monoclonal antibodies (mAbs) (BD Biosciences, Basel, Switzerland) to eliminate contaminating lymphoid and erythroid precursors. The resulting cell population contained >97% cells of the neutrophil lineage as determined by myeloperoxidase staining and analysis of lineage-associated surface proteins as well as with Diff-Quik (Medion, GmbH, Düdingen, Switzerland) and light microscopy. The distribution of the different maturation stages within the immature neutrophil populations was determined after each isolation (Altznauer et al., 2004; Martinelli et al., 2004). In average, we counted 30% myeloblasts, 21% promyelocytes, 26% myelocytes, 20% metamyelocytes and band cells, and 2% mature bone marrow neutrophils.

Mature peripheral blood neutrophils and peripheral blood mononuclear cells (PBMC) were purified from healthy normal individuals by Ficoll–Hypaque centrifugation (Altznauer et al., 2004; Martinelli et al., 2004). The resulting cell populations contained less than 5% contaminating cells. Eosinophil contamination was always less than 2%. Written informed consent was obtained from all patients and control individuals who donated bone marrow aspirates and blood, respectively. The study was approved by the ethics committee of the Canton Bern.

Cell cultures

Human immature and mature neutrophils were cultured at a concentration of 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS). Jurkat and Raji cells were cultured in the same medium. HL-60 cells were cultured in Iscove's medium containing 10% FCS. Cells were treated with GM-CSF (Novartis Pharma GmbH, Nürnberg, Germany), G-CSF (Aventis Pharma AG, Zurich, Switzerland), SH-6 (Alexis Corp., Lausen, Switzerland), LY294002, SB203580, PD98059, SN50 (all from Calbiochem, distributed by Juro Supply GmbH, Lucerne, Switzerland) and etoposide, cisplatin (both Bristol-Myers Squibb AG, Baar, Switzerland) at the indicated concentrations for the indicated times.

Cell death and apoptosis assays

Cell death was assessed at the indicated times by exclusion/uptake of 1 μ M ethidium bromide and flow cytometric analysis (FACS Calibur™) (Baumann et al., 2003; Altznauer et al., 2004; Bruno et al., 2005). To detect apoptosis, redistribution of phosphatidylserine in the absence of PI uptake was measured by flow cytometry. Apoptosis was also assessed by oligonucleosomal DNA fragmentation (Baumann et al., 2003; Bruno et al., 2005). Briefly, cells were resuspended in hypotonic fluorochrome solution containing 50 μg/ml PI, 0.1% sodium citrate and 0.1% (vol/vol) Triton X-100, incubated at 4°C for 6 h, and analyzed by flow cytometry.

Immunoblotting

Cells (1–2.5 × 106) were exposed to stimuli as indicated, washed with phosphate-buffered saline (PBS) and lysed with modified RIPA buffer (0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mM Tris, 150 mM NaCl, 1 mM ethyleneglycol tetraacetate (EGTA), 1 mM NaF) supplemented with a protease inhibitor-cocktail (Sigma, Buchs, Switzerland) and phenylmethylsulfonyl fluoride (PMSF) for 15 min on ice. After a 15-min centrifugation step to remove insoluble particles, equal amounts of the cell lysates were loaded on NuPage-Gels (Invitrogen Corp., Groningen, The Netherlands). In other experiments, total cell lysates (phosphorylation experiments) and cell-free extracts (see below) were used. Separated proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Immobilion-P, Millipore, Bedford, MA, USA). The filters were incubated overnight with anti-survivin (1/1000) Ab (R&D Systems, Abingdon, UK; 1/1000), anti-caspase-9 Ab (Cell Signaling, distributed by New England Biolabs, Frankfurt/Main, Germany; 1/1000) and anti-caspase-3 Ab (BD Biosciences; 1/1000) at 4 °C in Tris-buffered saline (TBS)/0.1% Tween 20/5% non-fat dry milk. For loading controls, stripped filters were incubated with anti-β-actin (Sigma; 1/10 000) or anti-glyceraldehyde-3-phosphate (GAPDH) (Chemicon International Inc., distributed by Juro, Lucerne, Switzerland; 1/2000) mAbs. Filters were washed in TBS/0.1% Tween 20 for 30 min and incubated with an appropriate horseradish peroxidase-conjugated secondary Ab (Amersham Pharmacia Biotech, Dübendorf, Switzerland) in TBS/0.1% Tween 20/5% non-fat dry milk for 1 h. Filters were developed by an enhanced chemiluminescence (ECL) technique (ECL-Kit, Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Polymerase chain reaction

HL-60 cells were cultured in the presence or absence of 25 μ M LY294002 for the indicated time periods and RNA extracted (TRIzol solution, Invitrogen). RNA was reverse transcribed with oligo(dT) 15 primer (Promega, distributed by Catalys AG, Wallisellen, Switzerland) and Superscript Reverse Transcriptase (Invitrogen). Primers for survivin cDNA amplification were as follows: 5′-IndexTermGAC CAC CGC ATC TCT ACA TTC AAG-3′ and 5′-IndexTermAAG GAA AGC GCA ACC GGA C-3′. Primers for GAPDH were as described previously (Conus et al., 2005). Survivin (217 bp) and GAPDH (418 bp) PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining.

Preparation of cell-free extracts

Cell-free extracts of HL-60 cells were prepared as described previously for mature neutrophils (Murphy et al., 2003). Ice-cold buffer (30 μl) (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, 1 mM EGTA, 1 mM dithiothreitol, 100 μ M PMSF, 10 μg/ml leupeptin, 2 μg/ml aprotinin) was added to 10 × 106 cells, which were allowed to swell under these hypotonic conditions for 30 min on ice. Cells were then disrupted by passing them 10–15 times through a 26-gauge needle. Disruption of cells was controlled by examination of a small aliquot of the suspension under a light microscope. Crude extracts were then centrifuged at 12 000 g for 30 min at 4°C to remove nuclei, unbroken cells and other debris. Supernatants were frozen in aliquots at −80°C until analysis.

Immunodepletion of cell-free extracts

Survivin was immunodepleted from extract as described (Deveraux et al., 1998). Briefly, slurry protein A/G agarose beads (Santa Cruz Biotechnology, distributed by LabForce, Nunningen, Switzerland; 40 μl) were coated with 20 μg polyclonal anti-survivin Ab in PBS, pH 7.3, at 4°C for 4 h. Purified rabbit Ab was used as control (mock). Ab-coated beads were washed twice with PBS, which was completely removed before adding the cell-free extracts. For antibody depletion, 50–100 μl aliquots of cell-free extracts were immunodepleted overnight at 4°C under constant rotation. Beads were removed from the extracts by centrifugation. Depleted extracts were frozen in aliquots at −80°C until analysis.

Cell-free reactions

Cell-free reactions were set up in 50–100 μl volumes. Total protein concentrations were brought to 7 mg/ml and kept equal in each series of experiments. Caspase activation was initiated by addition of the indicated concentrations of cytochrome c and 1 mM dATP (both from Sigma) at 37°C. At the indicated time periods, 15-μl aliquots were removed and frozen at −80°C until SDS–polyacrylamide gel electrophoresis/immunoblot analysis.

Analysis of c-akt/PKB phosphorylation

Cells were exposed to stimuli as indicated, and washed with ice-cold PBS. A 0.5 ml portion of a solution containing 20% (w/v) trichloroacetic acid (TCA), 40 mM NaF and 10 mM Na2HPO4 was added and the cells were kept on ice for 20 min. The precipitates were collected by centrifugation and washed with 0.5 ml of 5% TCA and subsequently with 0.5% TCA. The pellets were solubilized by boiling for 10 min in sample buffer (Invitrogen), followed by separation of proteins on a 4–12% NuPage-Gel. Separated proteins were electrotransferred onto PVDF membranes. The filters were incubated overnight with Abs specific for c-akt/PKB phosphorylated on serine 473 and threonine 308 as well as with Abs reacting with c-akt/PKB independent of its state of phosphorylation (Cell Signaling) at 4°C in TBS/0.1% Tween 20/5% non-fat dry milk. Incubation with secondary Abs and development by ECL was performed as described above.

Measurement of PI3K activity

Immature neutrophils and HL-60 cell were treated with the indicated concentrations of etoposide and cisplatin, respectively, for the indicated times. For controls, cells were cultured in the presence or absence of 25 μ M LY294002. PI3K activity was measured by enzyme-linked immunosorbent assay (Echelon Biosciences Inc., Salt Lake City, UT, USA), according to the manufacturer's instructions and as published previously (Uruno et al., 2006).

In vivo treatment study

Mice were treated as described previously (Wendel et al., 2004). Briefly, we used 3-month-old female C57BL/6 mice, which were treated with rapamycin (4 mg/kg intraperitoneally for 3 days), doxorubicin (10 mg/kg intraperitoneally on day 1) or both drugs given in combination. Blood leukocyte and neutrophil numbers were counted on day 5.

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Acknowledgements

This work was supported by grants from the Swiss National Science Foundation (Grant No. 310000-107526), Bernische Krebsliga (Bern) and Stiftung zur Krebsbekämpfung (Zurich).

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Correspondence to H-U Simon.

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Martinelli, S., Kostylina, G., Niggli, V. et al. Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils. Oncogene 25, 6915–6923 (2006) doi:10.1038/sj.onc.1209692

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Keywords

  • apoptosis
  • cancer
  • chemotherapy
  • myelosuppression
  • neutrophils
  • survivin

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