Address correspondence to Hans-Uwe Simon, Dept. of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. Phone: 41-31-632-3281; Fax: 41-31-632-4992; email: hus{at}pki.unibe.ch
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Abstract |
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Key Words: antisense cancer cytokines differentiation infection
Abbreviations used in this paper: CF, cystic fibrosis; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IAP, inhibitor of apoptosis protein; MPO, myeloperoxidase; Smac, second mitochondrial activator of caspase.
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Introduction |
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Independent of its mode of action, survivin has garnered great interest due to its differential pattern of tissue expression. Although fetal tissues abundantly express survivin mRNA and protein, it has been reported to be absent from most normal adult tissues. Interestingly, the majority of human tumors express survivin protein at high levels, suggesting that reactivation of the survivin gene occurs frequently during carcinogenesis and malignant progression (10, 11). If indeed survivin inactivates Smac (7), its overexpression in tumor cells might result in resistance to a broad range of apoptotic stimuli, including chemo- and radiation therapy. Based on these considerations, survivin has been suggested as an attractive target for cancer therapy (12).
In this paper, we demonstrate that survivin is highly expressed in immature neutrophils, whereas in mature neutrophils, survivin expression is absent or only marginal. Strikingly, neutrophil hematopoietic growth factors reactivated the survivin gene in these terminally differentiated cells, which are unable to resume cell cycle progression at G2-M, and high levels of survivin were expressed in mature neutrophils under inflammatory conditions in vivo. Using genetically modified mouse and human neutrophils, we demonstrate that survivin has an exclusive antiapoptotic function in these cells. Moreover, our data question the value of survivin as a specific target for cancer therapy. Instead, survivin may be a new therapeutic target for the development of antiinflammatory drugs.
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Materials and Methods |
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The 20-mer first generation antisense oligonucleotide 4003 targeting the survivin mRNA has been described previously (13). The second generation 2'-O-(2-methoxy)-ethyl-modified (2'-MOE) gapmer version of 4003 (as) and its three-base mismatch sequence control (ms) were synthesized using an automated DNA synthesizer as described previously (model 394B; Applied Biosystems; reference 14). Sequences were as follows: antisense survivin, 5'-cscscsasgsCsCsTsTsCsCsAsGsCsTscscststsg-3'; and mismatch survivin, 5'-cscstsasgsCsCsTsTsCsCsAsGsGsTscscstsasg-3'. Small letters refer to 2'-O-(2-methoxy)-ethyl-modified nucleotides, capital letters to DNA, and "s" refers to phosphorothioate linkages.
Cells.
Bone marrow aspirates were obtained from cancer patients at staging or restaging and analyzed by an experienced hematologist. Only samples with normal cellular morphology and distribution were considered in this work. The patients did not receive any chemotherapeutic or immunosuppressive treatment at the time of investigation. To induce sedimentation of the erythrocytes, an equal volume of 2% dextran T-500 (Amersham Biosciences) in 0.9% NaCl was added to the bone marrow aspirates. The bone marrow leukocytes were layered on top of a two-step discontinuous Percoll density gradient and centrifuged at 1,000 g at 4°C for 20 min through which a mixed population of immature neutrophils was obtained (15). Because this population of cells also contained erythroid and lymphoid precursors, we developed a new simple technique to improve the purity of the immature neutrophil population based on negative selection. The cells were incubated with a combination of anti-CD7 and anti-CD36 mAbs and subsequently with secondary Ab microbeads. The labeled cells were depleted by passing them through a magnetic cell separation system (Miltenyi Biotec) with LS+/VS+ column in the field of a permanent magnet. The resulting cell population contained >97% cells of the neutrophil lineage as determined by MPO staining (16), analysis of lineage-associated surface proteins (17), as well as by staining with Diff-Quik (Medion GmbH) and light microscopy.
Peripheral blood neutrophils were purified from healthy normal individuals or patients suffering from cystic fibrosis (CF) and associated Pseudomonas aeruginosa infection by Ficoll-Hypaque centrifugation (18, 19). The resulting cell populations contained >95% neutrophils. Written consent was obtained from all patients and control individuals who donated bone marrow aspirates and blood, respectively. The study was approved by the local ethics committee.
Peripheral blood neutrophils from heterozygous survivin+/ and normal survivin+/+ mice (20) were isolated by centrifugation over a Histopaque 1119 and Histopaque 1077 gradient (Sigma-Aldrich) using a standard protocol (21) 24 h after i.p. administration of 30 µg LPS. The purity of the resulting mouse neutrophil population was between 82 and 85%. Blood samples of two mice were pooled to obtain sufficient numbers of neutrophils to set up one ex vivo viability experiment.
Characterization of Purified Bone Marrow Neutrophils.
After purification, cells were stained with saturated concentrations of fluorescence-labeled anti-CD7, anti-CD11b, anti-CD14, anti-CD15, anti-CD16, anti-CD34, anti-CD36, and appropriate control mAbs according to standard protocols for flow cytometric analysis (FACS CaliburTM; Becton Dickinson). Based on the expression of lineage-associated surface proteins and light scattering properties, bone marrow neutrophils were assigned to six distinct maturational stages (17). The distribution of the different maturation stages was quite similar among the 17 normal bone marrow populations used in this work (see Table I). The percentage of segmented mature neutrophils was usually <10%. The purified mixed neutrophilic population was scarcely contaminated (<3%) with CD34+ progenitor cells, CD7+ lymphoid cells, CD36+ erythroid cells, and CD14+ monocytes.
Cell Cultures.
Human immature and mature neutrophils were cultured at 106/ml in complete culture medium (RPMI 1640 containing 10% FCS) and, where indicated, treated with 50 ng/ml GM-CSF, 25 ng/ml G-CSF, or 500 ng/ml IL-3. Survivin antisense oligonucleotides (as) or survivin mismatch control oligonucleotides (ms) were used at the indicated concentrations. When applied in combination with cytokines, the oligonucleotides were given 2 h before addition of cytokines.
Determination of Cell Death and Apoptosis.
Cell death was assessed at the indicated times by uptake of 1 µM ethidium bromide and flow cytometric analysis (FACS CaliburTM; references 21, 22). To determine whether cell death was due to apoptosis, redistribution of phosphatidylserine in the absence of propidium iodide uptake was measured by flow cytometry (22). Neutrophil apoptosis was also assessed by oligonucleosomal DNA fragmentation (23, 24).
Immunoblotting.
Gel electrophoresis and immunoblotting were performed as described previously (24). In brief, after electrotransfer of the separated proteins, the filters were incubated overnight with antisurvivin (1/1,000), anti-IAP-2 (1/666), anti-NAIP (1/1,000), anti-XIAP (1/250), antiIAP-1 (1/333), antiMcl-1 (1/1,000), or anticaspase-3 (1/1,000) Abs at 4°C in TBS/0.1% Tween 20/3% nonfat dry milk. The final concentrations of the primary Abs ranged between 1 and 3 µg/ml. 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting. For loading controls, stripped filters were incubated with antiß-actin (1/10,000) or anti-GAPDH (1/2,000) mAbs. Filters were washed in TBS/0.1% Tween 20 for 30 min and incubated with the appropriate HRP-conjugated secondary Ab (Amersham Biosciences) in TBS/0.1% Tween 20/5% nonfat dry milk for 1 h. Filters were developed by an ECL technique (ECL-Kit; Amersham Biosciences) according to the manufacturer's instructions.
Densitometry Analysis.
In some experiments, protein expression levels were analyzed by densitometry (24). OD of survivin, XIAP, and Mcl-1 bands divided by the OD of the corresponding ß-actin band are expressed as percentage of OD (protein of interest)/OD (ß-actin) of freshly isolated neutrophils that was defined as 100%.
Real-Time PCR.
107 neutrophils were washed with PBS, and total cellular RNA was isolated according to the RNeasy protocol (QIAGEN), which includes DNase digestion. RNA was reverse transcribed with the first strand cDNA synthesis kit (Amersham Biosciences) by using random hexanucleotides according to the manufacturer's instructions. Specific real-time monitoring of PCR amplification of survivin cDNA was performed with a 1/100 dilution of neutrophil cDNA using the S2 primers and probe as described previously (13). Quantification of survivin mRNA levels was performed using ribosomal RNA as internal standard for all samples. For comparison, survivin expression in A549 lung cancer cells was quantified and taken as a 100% reference value.
Enzymatic Caspase Assay.
2.5 x 106 neutrophils were cultured in the presence or absence of 50 ng/ml GM-CSF, 25 ng/ml G-CSF, antisense, or mismatch survivin oligonucleotides at the indicated concentrations for 10 h, washed with cold PBS, and subsequently lysed in 50 µl of cell lysis buffer (50 mM Hepes, pH 7.4/0.1% Chaps/5 mM DTT/0.1 mM EDTA) using a Teflon glass homogenizer (VWR International) on ice for 10 min. After a 10-min centrifugation step at 10,000 g at 4°C, an aliquot of the supernatant was saved for caspase-3 immunoblotting, and caspase-3like activity was measured in 10 µl supernatants as enzymatic conversion of the colorimetric substrate Ac-DEVD-pNA at 405 nm according to the manufacturer's instructions (QuantiZyme caspase-3 cellular activity assay kit; BIOMOL Research Laboratories, Inc.).
Confocal Laser Scanning Microscopy.
106 cells/ml cytospins were made from freshly purified cells on noncoated slides. Cells were fixed in 4% paraformaldehyde at room temperature for 10 min and washed three times in PBS, pH 7.4. Permeabilization of cells was performed with 0.05% saponin in buffer A (3% BSA in PBS) at room temperature for 5 min and with acetone at 20°C for 15 min. To prevent nonspecific binding, slides were incubated in blocking buffer (25% human immunoglobulins, 25% normal goat serum, 25% normal donkey serum, and 25% BSA) at room temperature for 1 h. Indirect immunostainings of CD15, survivin, and caspase-3 were performed at room temperature for 1 h by using the following primary Abs: anti-CD15 mAb (1/20; diluted in buffer A), rabbit antisurvivin polyclonal Ab (1/50), and anticaspase-3 mAb (1/50). Mouse and rabbit control antibodies, respectively, were used at the same concentrations in each experiment.
Immunofluorescent stainings were also performed on 5-µm-thick paraformaldehyde-fixed paraffin-embedded tissue sections from CF, appendicitis, and ulcerative colitis patients. Slides were dried at 52°C for 2 h and deparaffinized using NeoClear Solution (Merck), ethanol (100, 90, 80, 60, and 40%), and water at room temperature. After microwave treatment in TE-buffer (10 mM Tris, pH 8.0, and 1 mM EDTA), slides were washed in water, blocked, and stained with primary Ab as aforementioned.
After incubation with primary Ab, cells and tissues, respectively, were incubated with appropriate TRITC- and FITC-conjugated secondary Abs (1/100) in the dark at room temperature for 1 h. The antifading agent Mowiol (Calbiochem) was added. Slides were covered by coverslips and analyzed by confocal laser scanning microscopy (LSM models 410 and 510; Carl Zeiss MicroImaging, Inc.) equipped with Ar and HeNe lasers.
Immunohistochemistry.
Aside from immunofluorescence analysis, survivin expression on tissue neutrophils was also analyzed by regular immunohistochemistry on paraffin-embedded tissue sections from appendicitis patients according to previously established protocols with slight modifications (25, 26). In brief, sections were deparaffinized, and a subsequent antigen retrieval treatment was performed. Staining with antisurvivin (1/50) was performed using EnVision+System peroxidase kit (AEC; DakoCytomation). A rabbit control Ab was used at the same concentration as a negative control. Binding of the primary Ab was detected by a goat antirabbit Ab that was available in the kit. Sections were slightly counterstained with hematoxylin, mounted, and examined under a microscope (Axiovert model 35; Carl Zeiss MicroImaging, Inc.) at original magnifications (400 and 1,000).
Proliferation Assays.
Proliferation of mature and immature neutrophils (106/ml) was analyzed in the presence and absence of GM-CSF and G-CSF, respectively. PBMCs were used as controls. PBMCs were stimulated with 10 µg/ml PHA and 5 µg/ml anti-CD3 mAb, respectively. Total culture times were 48 h (mature neutrophils and PBMCs) and 72 h (immature neutrophils). Pulsing the cells with 1 µCi/ml (methyl-[3H]thymidine; Hartmann Analytic) was performed for 16 h and its incorporation was measured by using a liquid scintillation counter (Wallac ADL).
Statistical Analysis.
An analysis of variance test and Student's t test were used to compare mean levels. P < 0.05 was considered statistically significant. Mean levels are presented together with SEM. In the antisense oligonucleotide experiments, antisense and mismatch survivin-treated cells were compared at the indicated concentrations.
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Results |
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To confirm the data obtained by lowering survivin protein expression using antisense oligonucleotides, we purified blood neutrophils from survivin+/ mice (20). These mice express 50% of normal survivin levels and demonstrated evidence for increased caspase activation in hepatocytes. Purified neutrophils from survivin+/ mice did not exhibit an accelerated cell death in vitro when compared with normal neutrophils. However, the IL-3mediated antideath effect, observed in normal neutrophils, was significantly reduced in the genetically modified mice (Fig. 7 D).
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-3like 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-CSFmediated 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).
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Discussion |
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Survivin is a member of the IAP family, which seems to constitute a final false-safe mechanism to prevent intracellular damage due to premature caspase activation. For instance, it has been reported that XIAP, another member of the IAP family (1), prevents death receptormediated apoptosis by inhibition of caspase-3 (35). In these cells, it appears that Bid-mediated release of Smac from mitochondria is required to neutralize the IAP function to activate caspase-3 (36). Further support for the potency of IAPs to prevent premature apoptosis is provided by the observation that in some cells, mitochondria can be activated to release cytochrome c, but do not induce apoptosis (37). Thus, mitochondrial and caspase activation do not necessarily represent a "point of no return" in response to a death stimulus.
XIAP appears to be the most potent inhibitor of active caspases compared with other members of the IAP family (38). The expression of XIAP (31), as well as of IAP-1 (39) and IAP-2 (39), has been reported previously in mature neutrophils. In this work, we obtained evidence for the expression of XIAP, IAP-1, IAP-2, and survivin in these cells. XIAP and survivin were found to be inducible by G-CSF. Because XIAP expression was not always increased in immature compared with mature neutrophils, we focused our investigations in this work on the cytoprotective role of survivin.
Survivin expression seems to be a matter of active regulation in neutrophils during differentiation and inflammation. Reduced expression in mature compared with immature neutrophils was associated with caspase-3 activation and spontaneous apoptosis. Moreover, antisense-mediated inhibition of survivin expression prevented the antiapoptotic effect of GM-CSF and G-CSF in human neutrophils. A similar antiapoptosis block was observed in neutrophils from survivin+/ mice. Therefore, although survival cytokines may also increase the ratio between anti- and proapoptotic Bcl-2 family members (32, 40), their ability to increase survivin levels seems to be crucial for delayed neutrophil apoptosis.
A role of survivin in the regulation of apoptosis has previously been suggested in other cell types, particularly in tumor cells (12). The protective effect of survivin was reported to depend on its localization at the mitotic spindle and hence to be cell cycledependent (35). Moreover, survivin expression was seen at the G2-M boundary where its inhibition was accompanied by cell cycle defects. In addition, the embryonic lethal phenotype of survivin/ mice implicated a key role for survivin in mitosis (40). The concept that survivin acts as an IAP has also been challenged by other papers in which a role in cytokinesis was suggested (4143). Here, we demonstrate that survivin expression is not necessarily linked to the cell cycle, and that it indeed acts as an IAP without the requirement of spindle association. The notion that survivin expression is not limited to the G2-M phase is supported by earlier findings in CD34+ cells, which express survivin in all phases of the cell cycle (34, 44). Because the expression of survivin is highly regulated by survival factors and subject to rapid changes, it appears that survivin plays a key role in modulating the apoptotic threshold in neutrophils according to the biological requirements in a given physiologic or pathologic situation.
Another important finding of this work is that high survivin levels are not restricted to embryonic tissues and tumors, and particularly that inflammatory diseases are also associated with increased survivin levels. In conjunction with other papers describing its expression in hematopoietic progenitor cells (34, 44, 45) and T cells (46), this is of paramount importance and questions the value of survivin as a therapeutic target in cancer (12), due to the profound myelo- and immunosuppressive effects that might be expected from this treatment.
In summary, this paper demonstrates the importance of survivin in the regulation of neutrophil apoptosis and confirms its role as an antiapoptotic protein. Elevated survivin levels are present in neutrophils during normal differentiation in the bone marrow and represent a physiologic response in mature terminally differentiated cells under inflammatory conditions. Therefore, survivin might represent a novel target for antiinflammatory therapy. Moreover, and in contrast with previous papers, we demonstrate that survivin expression and its function as an IAP does not require cell cycle progression and mitotic spindle formation. Further analyses are needed to clarify whether additional signals exist that regulate survivin expression in primary cells and to elucidate the molecular mechanisms underlying the regulation of survivin expression in various biological systems.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation (grant nos. 31-58916.99 and 31-68449.02); the Krebsliga Schweiz (grant no. 1063-09-2000); the Bernische Krebsliga; the Kurt and Senta Herrmann Foundation; and the Edoardo R., Giovanni, Giuseppe and Chiarina Sassella Foundation. E.M. Conway was supported in part by the Fonds voor Wetenschappelijk Onderzoek (grant no. G.0382.02).
Submitted: 25 November 2003
Accepted: 5 April 2004
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References |
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