Nuclear localization of Survivin renders HeLa tumor cells more sensitive to apoptosis by induction of p53 and Bax

Cancer Letters 250 (2007) 177–193 www.elsevier.com/locate/canlet

Nuclear localization of Survivin renders HeLa tumor cells more sensitive to apoptosis by induction of p53 and Bax Achim Temme a,*, Jose A. Rodriguez b, Sandy Hendruschk a, Serap Gu¨nes a, Bernd Weigle c, Knut Scha¨kel a, Marc Schmitz a, Michael Bachmann a, Gabriele Schackert d, E. Peter Rieber a a

Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Fetscherstrasse 74, 01307 Dresden, Germany b Department of Medical Oncology, VU University Medical Center, HV1081 Amsterdam, The Netherlands c Eucodis GmbH, Brunner Str. 59, 1230 Vienna, Austria d Department of Neurological Surgery, Carl Gustav Carus University Hospital, Technical University of Dresden, Fetscherstrasse 74, 01307 Dresden, Germany Received 7 July 2006; received in revised form 14 September 2006; accepted 22 September 2006

Abstract Clinical studies have shown that nuclear expression of the inhibitor of apoptosis protein Survivin in tumor cells predicted a favorable prognosis whereas cytosolic-localized protein caused a decreased overall survival. Therefore Survivin’s subcellular localization may be important for its anti-apoptotic capacity. To address this question, we investigated localization and function of Survivin in normal human lung fibroblasts (NHLFs) and HeLa tumor cells. NHLFs of early passages expressed Survivin in the nucleus and were highly sensitive to C2 ceramide, which induces the mitochondrial apoptotic pathway. In contrast, NHLFs at higher passages relocated Survivin to the cytosol and became more resistant to C2 ceramide. Blocking nuclear export of Survivin by leptomycin B in HeLa cells increased susceptibility to C2 ceramide. In addition, transduction of HeLa cells with Survivin fused to a nuclear localization signal augmented basal expression levels of p53 and Bax and enhanced sensitivity for intrinsic apoptosis. Those findings suggest that a predominant nuclear localization of Survivin increases the sensitivity for pro-apoptotic stimuli, whereas nuclear export enables Survivin to fulfill its inhibitor of apoptosis function. A therapeutic intervention which holds Survivin in the nucleus of tumor cells might improve cancer therapy.  2006 Elsevier Ireland Ltd. All rights reserved. Keywords: IAP; Survivin; Bax; p53; Apoptosis; CRM1/exportin1; Leptomycin B

1. Introduction *

Corresponding author. Tel.: +49 351 7965751; fax: +49 351 7965750. E-mail address: [email protected] (A. Temme).

The induction of intrinsic apoptosis starts with a collapse of the mitochondrial membrane permeability leading to the efflux of cytochrome c into the

0304-3835/$ - see front matter  2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.09.020

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cytoplasm where it interacts with Apaf-1 and procaspase 9 to form an active ‘‘apoptosome’’ complex [1]. In this complex, procaspase 9 is cleaved into the active caspase 9 which starts a cascade of caspase activity and leads to cell death [1]. The cystein protease activity of the caspases can be hindered by a family of inhibitor of apoptosis (IAP) proteins, which contain from one to three conserved domains described as baculovirus inhibitor of apoptosis repeat (BIR) domain [2]. Survivin, a 16.5 kD protein, represents so far the smallest protein containing a BIR domain and therefore was included into the IAP family [3,4]. In accordance with its proposed function as an IAP, studies demonstrated that ectopic overexpression of Survivin protects cells against pro-apoptotic reagents [5,6]. This protective effect of overexpressed Survivin has been demonstrated to be due to its ability to bind caspase 9 [7]. A binding to other caspases is still disputed [8– 10]. Furthermore, an inhibition of Smac/DIABLO function [11], and a cell-protective impact on the caspase-independent apoptosis-inducing factor (AIF)-mediated cell death [12] have been described. Survivin was originally defined as a so called ‘‘universal tumor associated antigen’’ (TAA) because it was found to be highly expressed in a great variety of tumors [3,13]. However, beside its expression in neoplastic cells, Survivin can be detected in normal tissues and cells with high mitotic activity [14–19]. Further studies using HeLa cells revealed that the mRNA of Survivin is upregulated at the G2/M transition of the cell cycle [5], and that Survivin is a shortlived protein with a half-life time of approximately 30 min [20]. Inhibition of Survivin is associated with cell cycle defects [5] and disruption of its genetic locus causes an embryonic lethal phenotype in homozygous mice [21] which is in line with its just identified function in the spindle checkpoint [22,23]. Recently, further functions of Survivin in proliferation and cell senescence have been described. Strong expression of Survivin is able to augment the release of Cdk4 from its inhibitor p16ink4a, which facilitates the entry into S-phase of the cell cycle [24]. In addition, overexpressed Survivin was described to increase transcription of the gene for human telomere reverse transcriptase (hTERT) [25], postponing replicative senescence of cells. Most investigations revealed expression of Survivin protein in all stages of the cell cycle of tumor cells suggesting a deregulated expression pattern in these cells [26,27]. In retrospective studies, patients whose tumors overexpressed Survivin had a decreased

overall survival [28,29], a resistance to chemotherapy [30,31], and an increased rate of recurrence [32,33]. Furthermore, it has been shown that during interphase transformed cells transport Survivin by CRM1-/Exportin-1 from the nucleus into the cytosol [34]. This may confer resistance to chemotherapy or radiotherapy and could contribute to a more aggressive phenotype of tumors, whereas Survivin expression in the nucleus of tumor cells has been described to be a predictive of a favorable prognosis in gastric cancers, and lung cancer [35–37]. Since a cytosolic localization enables its IAP function we hypothesized that an enhanced nuclearexport of Survivin could be a mechanism of tumor cells to resist unfavorable conditions, i.e. hypoxia, radiation, or chemotherapy. Conversely, recent data suggest that nuclear Survivin influences the transcription of genes involved in development, senescence and apoptosis by an unknown mechanism leading to phosphorylation and activation of transcription factors like c-myc and specific protein 1 (SP1) [25,38]. Therefore, we hypothesized that a predominant nuclear localization could influence the susceptibility to apoptosis by increased transcription of pro-apoptotic genes. To test this hypothesis we investigated whether an enforced nuclear expression of Survivin in tumor cells increase the susceptibility to intrinsic apoptosis. 2. Materials and methods 2.1. Cell lines Normal human lung fibroblasts were obtained from Clonetics (BioWitthaker, Verviers, Belgium) and cultured according to the recommendation of the manufacturer, with the exception that the cells were grown in D10 medium (Dulbecco’s modified Eagle’s medium supplemented with fetal calf serum and 100 U/ml penicillin, 100 lg/ml streptomycin). These cells show after establishment of a monolayer contact inhibition of cell growth. Determination of DNA content was accomplished using propidium iodide staining of cells. Stained cells were analyzed using a Becton–Dickinson FACScan (Heidelberg, Germany) with at least 10,000 events/determination. The human breast cancer cell lines MCF7 (with wildtype p53 [39]; ATCC HTB22), SK-Br3 (with mutant p53 [39]; ATCC HTB30), the human pancreas carcinoma cell line Capan-1 (with mutant p53 [40]; ATCC HTB79), the hepatocellular carcinoma cell line HepG2 (with wild-type p53 [41]; ATCC HB8065), the colon adenocarcinoma cell line Colo205 (with mutant p53 [42]; ATCC CCL-222), HeLa cervix carcinoma cells, and the 293T human embryonic kidney cells were cultivated in D10-medium. U373

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(ATCC HTB-17) is a glioblastoma–astrocytoma derived cell line, H4 (ATCC HTB-148) is a glioma cell line, A172 (ATCC CRL1620), U343 [43], and U87-MG (ATCC HTB-14) are glioblastoma-derived cell lines. A172, U343 and U87-MG contain wild-type p53 [43,44], the U373 cell line expresses mutant p53 [45], and the near triploid H4 cell line contains two copies of p53 [46]. All cells were cultivated in Basal minimal Eagle’s medium(Invitrogen, Eggenstein, Germany) supplemented with 2 mM L-glutamine and 1% non-essential amino acids (Biochrom, Berlin, Germany). 2.2. Construction of retroviral vectors The vector pcz-CFG5.1-EGFP was made by introducing a BglII/HpaI-fragment containing the EGFP coding region of the vector pEGFP-1 (Clontech) into corresponding BamHI and HpaI restriction sites of pczCFG5.1-MCS [6]. The vector pcz-CFG5.1-EGFP-Survivin was generated by introducing the full coding region of Survivin [34] into the HindIII/BamHI-restriction sites of pcz-CFG5.1-EGFP. Survivin-NLS was made by PCR using wild-type Survivin [34] as template and the primers JAR6 (forward) and JAR44 (reverse). Primer sequences (5 0 –3 0 ): JAR6: TTG CCT CAA GCT TTT GCC ACC ATG GGT GCC CCG ACG TTG, JAR44: GAC TAA AGC GGC CGC GGA TCC TTA GAC TTT GCG CTT CTT CTT AGG CTT GTC GTC ATC GTC TTT GTA G. The PCR product was cloned as HindIII/BamHI fragment into pEYFP-C1 (BD Clontech, Heidelberg, Germany). The Survivin-NLS fragment was excised with HindIII/BamHI and ligated into the corresponding restriction sites of the vector pcz-CFG5.1-EGFP resulting in the vector pcz-CFG5.1-EGFP-Survivin-NLS. 2.3. Transduction of retroviral vectors into NHLF and HeLa cells Retroviral particles were generated as described previously [6]. Briefly, 293T cells were cotransfected with an expression construct for gag-pol (pHIT60), the MoMuLV-based retroviral vectors and the vesicular stomatitis virus G-protein (pMD.G2, kindly provided by D. Trono, University Geneva, Switzerland). Viral supernatants were harvested 48 and 72 h after transfection. 105 target cells were plated in 30 mm dishes a day before transduction and were transduced with retroviral supernatants at a multiplicity of infection (MOI) of 50. 2.4. Immunofluorescence and confocal laser scanning microscopy Cells were fixed in ice-cold paraformaldehyde for 20 min and incubated 30 min in phosphate buffered saline (PBS). They were permeabilized with 1% sodium citrate/ 0.1% Triton X-100 and washed three times with PBS.

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Then the cells were washed three times with PBS containing 0.1% bovine serum albumin. Cells were directly used for microscopy or were incubated for 1 h at room temperature with polyclonal anti-Survivin (R&D Systems, Wiesbaden, Germany, 1:120). In other experiments cells were incubated with TO-PRO-3 dye diluted 1:105 (Molecular Probes, Leiden, The Netherlands) or Hoechst 33342 (diluted 1:104) for detection of DNA. After repeated washing with PBS/0.1% BSA, the cells were incubated for 1 h with FITC-conjugated goat anti-rabbit-IgG (stock solution, dilution 1:80, as recommended by the supplier, Dianova, Hamburg, Germany). Finally, cells were washed three times in PBS/0.1% BSA and once in double distilled water, before being examined by confocal laser scanning microscopy (Leica NCS-NT, Germany) using the filters SP590, DD488/568, RSP580, BF530/30 for detection of FITC. TO-PRO3 nuclear staining was detected with the filters BP647, RSP660 and LP665. Image files were digitally processed for presentation using Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Tumor suppressor protein p53 was detected with a monoclonal anti-p53 antibody preparation (kindly provided by K.H. Scheidtmann, University of Bonn, Bonn, Germany, dilution 1:50). As secondary antibody we used rabbit anti-mouse coupled to Cy3 (1:50 stock solution; Dianova, Hamburg, Germany). Finally, cells were washed three times in PBS/0.1%BSA and once in double distilled water, before being examined by fluorescence microscopy (Olympus IX70, Hamburg, Germany). 2.5. Immunoblot analyses NHLF cells were cultured upon 30% or 100% confluency and nuclear and cytosolic proteins were prepared from 4 · 106 cells. Briefly, the cells were washed with PBS and trypsinized by using Trypsin–EDTA solution (Sigma, Taufkirchen, Germany). After washing with PBS cells were centrifuged at 400g, at 20 C for 10 min. The cell pellets were suspended in 250 ll hypotonic buffer A (10 mM KCl, 15 mM MgCl2, 10% (v/v) Hepes) (Biochrom, Berlin, Germany) containing 200 lg/ml PMSF, 5 lg/ml leupeptin and 2 lg/ml aprotinin and incubated for 20 min on ice. Then cells were transferred to a Dounce homogenisator and were disrupted. The cytosolic proteins were separated from nuclei and cell debris by centrifugation for 10 min at 8000g and 4 C. The supernatants containing cytsolic proteins were precipitated with 200 ll trichloroacetic acid and centrifuged for 10 min at 19,000g and 4 C. Resulting pellets of cytosolic proteins were suspended in 100 ll of 1· Laemmli buffer and 15 ll of the sample was immediately used for SDS–polyacrylamide gel electrophoresis (PAGE) or stored at 80 C. Pellets representing nuclear proteins were suspended in 100 ll of 1· Laemmli buffer and sonified in an ice cooled water bath before 7.5 ll were subjected to SDS–PAGE or stored at 80 C. In other experiments 4 · 106 HeLa cells

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expressing EGFP-Survivin, EGFP-Survivin-NLS and EGFP, respectively, were lysed with 1· RIPA buffer (50 mM Tris–HCl, pH 7,5, 150 mM NaCl, 1% Nonidet P40, 0,5% Natriumdesoxycholat, and 0,1% SDS) supplemented with 200 lg/ml PMSF, 5 lg/ml leupeptin and 2 lg/ml aprotinin or directly lysed in 2· Laemmli buffer. Equal amounts of protein probes were subjected to electrophoresis and blotted onto PVDF membranes (PALL, Dreieich, Germany). Survivin was detected by using monoclonal anti-Survivin 9B1D9 antibody [6], tubulin was detected by the use of monoclonal anti-tubulin (Sigma, clone DM 1A, dilution 1:500), PARP was detected using polyclonal goat anti PARP (R&D Systems, dilution 1:1000), and Bax was detected by using monoclonal antiBax (Chemicon International, Hampshire, United Kingdom, dilution 1:2000). The secondary rabbit anti-mouse antibody coupled to horseradish peroxidase (HRP) or rabbit anti-goat antibody coupled to HRP (both dilutions 1:2500, DAKO, Hamburg, Germany) were visualized with enhanced chemoluminescence (ECL) (Roche, Mannheim, Germany) as recommended by the supplier. 2.6. Leptomycin B-treatment and determination of C2 ceramide-induced apoptosis: AnnexinV-Alexa568 staining and analysis of cells undergoing internucleosomal DNA fragmentation To induce intrinsic apoptosis, 2 · 105 HeLa cells expressing EGFP, EGFP-Survivin, and EGFP-SurvivinNLS, respectively, were incubated for 4 h with 6 ng/ml leptomycin B prior incubation with new medium containing 50 lM C2 ceramide or were treated with C2 ceramide alone. The analysis of apoptosis was carried out by AnnexinVAlexa568 staining (Roche, Mannheim, Germany) and cytometer analysis as recommended by the supplier. In other experiments apoptosis was determined by FACS assisted analysis of the fraction of apoptotic cells undergoing internucleosomal DNA-fragmentation as described previously [47]. Briefly, the cells were washed in PBS, fixed in 70% (v/v) ethanol. After centrifugation of the cell at 600g at 20 C for 10 min the cell pellet was suspended in 0.5 ml DNA-extraction buffer (4 mM citric acid in 0.2 M Na2HPO4; pH 7.8). After 5 min incubation at RT the cells were spun down at 600g. The cells were then washed once with PBS, followed by incubation in PBS containing 40 lg/ ml propidium iodide (Sigma, Taufkirchen, Germany) and 200 lg/ml RNase A (Sigma, Dreieich, Germany) for 1 h at room temperature in the dark. Stained nuclei were then analyzed using a Becton–Dickinson FACScan (Heidelberg, Germany) with at least 10,000 events/determination. Statistical analysis was performed with Student’s T test.

analyzed by agarose gel electrophoresis. After DNA digestion (DNase I; Amersham Biosciences, Freiburg, Germany), we performed PCR analyses using intron-spanning primers for b-actin amplification (see Quantitative reverse transcription-PCR) to test for DNA contaminations in our RNA preparations. No amplification products were detected. cDNA synthesis was performed using 4 lg of total RNA and random hexamer primers in a standard 32-ll reaction according to the provider (Ready to Go You Prime First Strand Kit; Amersham Biosciences). 2.8. Quantitative light-cycler-PCR Cell specific mRNA expression was analyzed by a quantitative LC-based PCR assay. cDNAs form HeLa cells expressing EGFP, EGFP-Survivin and EGFP-Survivin fused to SV40 NLS, respectively, were analyzed by applying a real-time PCR protocol based on SYBR Green I detection (LC - FastStart DNA Master SYBR Green I; Roche Diagnostics, Mannheim, Germany) using the human Bax Real Time primer Set GHO0064 human p53 Real Time primer Set GHO0074 and human Bcl-2 PCR Primer Set GHO0014 (all Biosource International, Nivelles, Belgium) as recommended by the provider. For the quantification of Bad transcripts we used the primers Bad-for 5 0 GAGAG GCGGCGGCGGGAG-3 0 and Bad-realRev 5 0 -ATGATG GCTGCTGCTGGTTG-3 0 which allowed the amplification of an 189 bp fragment containing bad mRNA sequences from nucleotides 235–424. Forty cycles were performed for each probe. The SYBR Green I-based quantification of b-actin was performed using the primers actin-for 5 0 -G CCGTCTTCCCCTCCATCGTG-3 0 and actin-rev 5 0 -GG AGCCACACGCAGCTCATTGTAGA-3 0 . A calibration standard was established using serial dilutions (101–108 molecules) of a plasmid encoding human b-actin. The annealing temperature was 70 C. Forty cycles were performed. Serial dilutions of plasmids containing the bad, bcl-2, p53, or bax cDNA (the latter kindly provided by Dr. H. Abken, Laboratory for Tumor Genetics, University of Cologne) over eight log scales (101–108 molecules per capillary) were used as internal template standards (calculation via LC quantification software version 3.5; Roche). Each determination was carried out fourfold for each cDNA sample as independent PCR runs. The molecule ratios of bad, bax, bcl-2 and p53 to actin transcripts were calculated from the mean values. Statistical analyses were performed with Student’s T test. 3. Results 3.1. Normal human lung fibroblasts express Survivin in the interphase nucleus

2.7. RNA isolation and cDNA synthesis from cell lines Total RNA was extracted by standard procedures (Trizol LS Reagent; Invitrogen). The quality of the RNA was

Confocal laser scanning microscopy analyses of early passages of non-transformed human lung fibroblasts (NHLF) using a polyclonal serum against Survivin [6]

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revealed strong signals in nuclei of the cells during interphase and barely signals in the cytosol (Fig. 1A a, b). This contrasts the localization of Survivin in HeLa cervical car-

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cinoma cells, which express predominantly cytoplasmatic Survivin (Fig. 1A c, d). To confirm the nuclear expression of Survivin in NHLF we prepared cytosolic and nuclear extracts from arrested cells and proliferating cells, respectively. The arrested fibroblasts showed only a minor fraction of cells in S-phase and G2/M-phase (1%) and cells at 30–40% confluence showed a cell fraction of 30% in Sphase and G2/M-phase as revealed by propidium iodide staining and subsequent FACS analysis (data not shown). Western blot analyses revealed that non-proliferating cells did not express Survivin, whereas in proliferating cells a predominant signal for 16.5 kD Survivin was detected in the nuclear protein fraction (Fig. 1B). No Survivin protein was detected in the cytosolic protein preparations. Furthermore, we transduced an EGFP-tagged Survivin into NHLF cells and analyzed the subcellular localization of the transgenic protein by using fluorescence microscopy (Fig. 1C). This retroviral overexpressed EGFP-tagged Survivin was predominantly found in the nucleus of the transduced NHLF cells supporting the drown conclusion that endogenous Survivin is expressed in the interphase nucleus of NHLF. This again contrasts the subcellular localization of Survivin in HeLa cervical carcinoma cells, which lacked nucleus-localized Survivin during interphase. In addition, analyses of the subcellular localization of Survivin on a panel of human tumor cell lines, including the human breast cancer cell lines MCF7, SK-Br3, the human pancreas carcinoma cell line Capan-1, the hepatocellular carcinoma cell line HepG2, the colon adenocarcinoma cell line Colo205, and the glioblastoma cell lines A172, H4, U373, and U87-MG, all derived from solid tumors showed Survivin expression predominantly in the cytoplasm of interphase cells (Fig. 2). 3.2. Subcellular localization of Survivin during mitosis in early passages of normal human lung fibroblasts

Fig. 1. Expression of nuclear Survivin in NHLFs: (A, a) Confocal laser scan analyses of Survivin expression in NHLFs of passage number 9. Note the strong immunoreactivity in the nuclei of the cells. Arrow marks a stained midbody. (c) In HeLa tumor cells Survivin expression is predominantly detected in the cytosol. (b and d) DNA counterstaining was accomplished with TO-PRO-3. (B) Western blot analysis of nuclear (N) and cytosolic (C) protein preparations from growth arrested [NHLF(K)] and proliferating NHLFs [NHLF(P)]. Specific Survivin bands are only detected in the nuclear protein fraction of proliferating cells. As loading control for the nuclear protein fraction served PARP. Equal loading of the lanes is demonstrated by Coomassie blue staining of the blotted polyacrylamide gel. (C, a) Fluorescence microscopy of NHLF transduced with EGFPtagged Survivin. Note the predominantly nuclear localization of the EGFP-Survivin, which resembles the localization found for endogenous Survivin. (b) DNA counterstaining with Hoechst33342. Bars equal 20 lm.

As we found a different localization of Survivin in interphase of NHLF cells from early passages compared to interphase tumor cells it was of interest whether Survivin localizations during mitosis could also be different. Confocal laser scanning analyses of the subcellular localization of Survivin during the cell cycle in NHLF cells (Fig. 3A) and HeLa cells (Fig. 3B) were performed. In interphase NHLF cells Survivin was found in the nucleus of every cell, which concentrated to distinct spots of accumulated Survivin when the cell entered prophase (Fig. 3A a, a 0 (arrows)). At prometaphase/metaphase Survivin became associated with the centromere region of the chromosomes (Fig. 3A b, b 0 (arrowhead)). Survivin, which was located in the metaphase plate (Fig. 3A c, c 0 ), transmitted at anaphase/telophase to the cleavage furrow (Fig. 3A, d, d 0 , e, and e 0 (arrowhead)) and became concentrated in the midbody during cytokinesis (Fig. 3A f, f 0 (arrowhead)). These subcellular localizations of Survivin are similar to the situation described for HeLa tumor cells [6,21] (see

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Fig. 2. Expression of Survivin in tumor cells derived from solid cancers. Indirect immunofluorescence analyses of Survivin expression in (a) MCF7, (b) SK-Br3, (c) Capan-1, (d) HepG2, (e) Colo205, (f) U87-MG, (g) A172, (h) H4, and (i) U373. Bar equals 20 lm.

also Fig. 3B) with the exception that in late telophase of NHLF cells Survivin proteins moved into the developing nuclei of the sister cells. 3.3. Aging NHLFs relocate Survivin to the cytoplasm and show an increased resistance to C2 ceramide We observed an increase in the fraction of NHLFs bearing Survivin exclusively in the cytoplasm, when the cells were cultured after passage 12 suggesting that the aging of cells might influence the subcellular localization of Survivin. Cells of passage 9 had predominantly Survivin signals in the nucleus and some protein in the cytosol whereas cells of passage 31 relocalized Survivin completely to the cytosol (Fig. 4a–d). To test whether this different subcellular localizations affect the inhibitor of apoptosis function of Survivin we activated the intrinsic apoptotic pathway with C2 ceramide which is known to cause a dysregulated mitochondrial membrane potential leading to cytochrome c efflux and to activation of caspase 9 [48]. When we treated cells displaying nuclear localization of Survivin with C2 ceramide we found a pronounced (50%) apoptosis as determined by analysis of the fraction

of hypodiploid cells (Fig. 4e) whereas cells bearing cytosolic Survivin where less affected (7% hypodiploid cells) (Fig. 4e) resulting in a higher proportion of viable cells when compared to cells with nuclear localization of Survivin (Fig. 4f). 3.4. Leptomycin B-induced retention of Survivin in the cell nucleus sensitizes tumor cells for apoptosis Prompted by the observation that Survivin in all tested solid tumor cells was localized in the cytoplasm instead of the nucleus as observed in the normal human lung fibroblasts, we speculate that overexpression and the CRM1dependent nuclear export of Survivin could be a mechanism of malignant cells to counteract the intrinsic apoptotic pathway. We used leptomycin B to inhibit the CRM1mediated export of Survivin, which allowed an accumulation of Survivin into the nucleus of HeLa cells. We used HeLa cells transduced with a retroviral vector coding for EGFP-Survivin to express Survivin throughout the entire cell cycle. Furthermore, the use of this EGFPtagged protein allowed us to directly observe the accumulation of Survivin in the nucleus after addition of LMB.

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Fig. 3. Confocal laser scanning microscopy showing the dynamic distribution of Survivin during mitosis of NHLFs and HeLa cells: (A) Analysis of NHLF, (a) prophase, arrows marking Survivin accumulations on chromosomes. (b) Prometaphase and (c) metaphase kinetochores are loaded with Survivin (arrowheads). (d) At anaphase Survivin dissociates from the kinetochores. (e) Survivin is found at late anaphase/beginning telophase at the emerging cleavage furrow. (f) At the end of telophase Survivin is localized at the midbody (arrowhead) but also appears in the developing sister nuclei. (a 0 –f 0 ) Merge of FITC-signals with TO-PRO-3 DNA staining. (B) Analysis of HeLa cells. (a) Prophase, arrow marks Survivin accumulations on chromosome. (b) Metaphase (kinetochore marked with arrowhead). (c) At early anaphase Survivin has left the kinetochores and is found at the cleavage furrow (arrowhead) which is condensed during (d), early telophase and (e), late telophase. At the end of telophase Survivin is localized at the midbody (arrowhead) and does not accumulate in the sister nuclei. (f) HeLa cells in interphase. (a 0 –f 0 ) Merge of FITC-signals with TO-PRO-3 DNA staining. Bars equal 20 lm.

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Fig. 4. Cytoplasmic relocalization of Survivin and reduced sensitivity to intrinsic apoptosis in aging NHLFs. (a) Indirect immunofluorescence analysis of NHLFs of passage 9. (b) NHLFs of passage 31 relocated Survivin to the cytoplasm. Bar equals 40 lm. (e and f) NHLF cells expressing predominantly nuclear Survivin or expressing cytosolic Survivin were treated with C2 ceramide for 24 h. (e) Cell survival was investigated by analyzing the fraction of hypodiploid cells or by (f), trypan blue exclusion and counting of viable cells. At least 800 cells in three independent experiments were analyzed. Error bars represent SEM. *p < 0.01.

As a control we included HeLa cells transduced with EGFP. The blockade of CRM1 function led to a rapid diffusion of EGFP-Survivin into the nucleus. After 1 h EGFP-Survivin was detected in the nuclei of all cells. Apparently, the EGFP-Survivin proteins became more concentrated in the nucleus when compared to the cytosol (Fig. 5a) but still a prominent amount of transgenic EGFP-Survivin remained in the cytosol. To analyze the effects of nuclear relocalization of Survivin we treated HeLa cells expressing EGFP or expressing EGFP-Survivin with 6 ng/ml LMB for 4 h, washed the cells and added pro-apoptotic C2 ceramide. The short treatment with LMB alone already caused an increase in the amount of apoptotic cells. However, subsequent treatment with C2 ceramide further enhanced apoptosis (Fig. 5e and f). Here, we observed an approximately twofold increase in apoptosis in cells expressing EGFP and a

threefold rise in apoptosis in cells expressing EGFP-Survivin when compared to cells treated with C2 or LMB alone. 3.5. Forced expression of nuclear Survivin sensitizes HeLa tumor cells for C2 ceramide-mediated apoptosis The increased sensitivity for apoptosis in NHLF cells displaying nuclear-localized Survivin and in HeLa tumor cells treated with LMB suggested that a nuclear localization of Survivin may alter the resistance of HeLa cells to pro-apoptotic reagents affecting intrinsic apoptosis. We sought to verify the observed effects and wanted to exclude the cytotoxic influence of leptomycin B, which may non-specifically enhance the effects of C2 ceramide. To this end, we transduced HeLa cells with a vector coding for EGFP, a vector coding for EGFP-Survivin fused

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Fig. 5. Leptomycin B-induced nuclear retention of Survivin sensitizes tumor cells for apoptosis. (a–d) HeLa cells transduced with EGFP-Survivin were left untreated (a and c) or were treated with leptomycin B (LMB) (b and d). LMB led to a breakdown in CRM1-dependent nuclear export of proteins and allowed the diffusion of EGFP-Survivin into the nucleus. Bar equals 10 lm. (c and d) DNA staining was accomplished with Hoechst33342. (e) HeLa cells expressing EGFP or (f) EGFP-Survivin were left untreated or were treated for 4 h with LMB. Intrinsic apoptosis was induced by addition of C2 ceramide. Apoptosis was measured by FACS assisted analysis of the fraction of propidium iodide stained cells with hypodiploid DNA content. Error bars represent SEM. N = 8, *p < 0.01 when apoptotic mean value of cells doubled-treated with LMB and C2 ceramide were compared to mean values of all other groups.

to a nuclear localization signal of SV40 and a vector coding for EGFP-Survivin, respectively. All transductions with the different vectors were performed with 50 MOI.

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Subsequent analyses of the EGFP emission of the transduced cells revealed transduction efficacies about 95%, indicating comparable expression levels of the transgenic proteins (Fig. 6a). The mean fluorescence intensity of the EGFP control was approximately 2.5-fold higher than the MFI of the proteins fused to EGFP, which may be due to a diminished excitation or emission capacity of the EGFP-fusion proteins. The expression of EGFP-Survivin and the expression of EGFP-Survivin-NLS did not cause a significant increase in spontaneous apoptosis and did not result in a higher incidence of multinuclear cells, which sometimes is observed after gene transfer of wildtype Survivin or mutated Survivin [6,47]. We detected EGFP-Survivin-NLS fusion proteins at kinetochores but only faint EGFP signals at the cleavage furrow or in the midbody (data not shown). During interphase the EGFP-Survivin-NLS fusion protein specifically accumulates in the nuclei of the cells (Fig. 6b). However, we detected also a modest quantity of EGFP signals in the cytosol of the transduced cells, which might be due to the strong overexpression of EGFP-Survivin-NLS (Fig. 6b). Interestingly, indirect immunofluorescence analyses of endogenous and transgenic Survivin revealed that in EGFP-NLS-Survivin transduced cells a strong staining for Survivin also was present outside the nuclei, which indicates that endogenous Survivin is still located in the cytoplasm of these cells (Fig. 6b). EGFP-Survivin was found in the cytosol of transduced cells and also the indirect immunofluorescence analyses revealed a cytosolic localization of endogenous and transgenic Survivin (Fig. 6b). In order to investigate the resistance of cells for intrinsic apoptosis we treated the cells expressing EGFP, Survivin-EGFP and EGFP-Survivin-NLS, respectively, with C2 ceramide for 0, 4, 8 12, 24, 36, and 48 h. Analysis of the fraction of cells with fragmented DNA content showed between 12 and 48 h a significant increase in the fraction of apoptotic cells which expressed EGFPSurvivin-NLS when compared to cells expressing EGFPSurvivin (Fig. 6c). Cells expressing EGFP-NLS-Survivin were still more resistant to C2 ceramide induced apoptosis than HeLa cells stably transduced with only EGFP (Fig. 6c). Additional analyses of the fraction of AnnexinV-positive cells, an early marker of apoptosis confirmed that HeLa cells with expression of nuclear Survivin were more sensitive to C2 ceramide induced apoptosis than cells expressing EGFP-Survivin (Fig. 6d). 3.6. Nuclear Survivin increases p53 and Bax proteins through increased transcription The higher C2 ceramide-mediated apoptosis in NHLF cells displaying nuclear-localized Survivin and in HeLa tumor cells treated with LMB suggests that a nuclear localization of Survivin could modulate susceptibility to intrinsic apoptosis. We reasoned that nuclear-localized

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Fig. 6. Nuclear Survivin sensitizes HeLa tumor cells for apoptotic stimuli independent from leptomycin B treatment. (a) FACS analyses of HeLa cells transduced with EGFP-Survivin, EGFP-Survivin-NLS (NLS), or EGFP. White histograms represent mock-transduced cells; black histograms represent EGFP-Survivin-, EGFP-Survivin-NLS-, and EGFP-transduced cells. Note that due to strong EGFP-signals the FACS-setting for EGFP-transduced cells was modified. (b) Subcellular localizations of EGFP-Survivin-NLS and of EGFP-Survivin and indirect immunofluorescence analyses using a polyclonal anti-Survivin antibody (R&D Systems). DNA was stained with Hoechst33342. Bar equals 30 lm. (c) HeLa cells expressing EGFP (white circles) EGFP-Survivin (SE, white boxes) and EGFP-SurvivinNLS (NLS, black boxes) were treated with 50 lM C2 ceramide and the appearance of cells with hypodiploid DNA content was measured at the indicated time points. For better comparison the apoptotic index was calculated according to the formula. Apoptotic index, mean percentage of experimental apoptosis – mean percentage of spontaneous apoptosis. Spontaneous apoptosis was defined as apoptosis caused by transgenic protein expression. Mean of three independent experiments is shown. *p < 0.01 when cells expressing EGFPSurvivin-NLS were compared to cells transduced with EGFP and EGFP-Survivin, respectively. (d) HeLa cells expressing EGFP-Survivin (ES) and EGFP-Survivin-NLS (ES-NLS) were treated as above and apoptosis was determined by AnnexinV-Alexa568 staining of cells. One representative experiment out of three experiments is shown.

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Survivin could exert some effects on the expression of genes involved in apoptosis. We assayed the steady state mRNA expression of the p53 gene, the pro-apoptotic Bad and Bax genes, and the anti-apoptotic bcl-2 gene in HeLa cells by Real Time PCR analyses (Fig. 7a). Tran-

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script expression for the p53 gene was approximately 2.3-fold higher in HeLa cells expressing EGFP-SurvivinNLS when compared to cells expressing EGFP-Survivin and also higher than (1.6-fold) in cells expressing EGFP. Bad mRNA expression in cells with nuclear Survivin

Fig. 7. Nuclear Survivin increases p53 and Bax expression levels. (a) Quantitative PCR analyses of the expression levels of p53, Bad, Bax, and Bcl-2. Values were calculated as described in the material and method section. The mean of four independent light cycler PCR runs is shown. Error bars represent SEM. *p < 0.01 when p53 and BAD transcript levels of EGFP-Survivin-NLS expressing cells were compared to transcript levels of EGFP- and EGFP-Survivin-transduced cells. *p < 0.01 when Bax transcript levels of EGFP-Survivin-expressing cells were compared to transcript levels of EGFP- and EGFP-Survivin-NLS-transduced cells. (b) Western blot analyses of the expression of transgenic Survivin proteins and Bax in cell lysates from HeLa cells transduced with EGFP, EGFP-Survivin (SE), and EGFP-SurvivinNLS (NLS). Asterisks denote presumably degraded transgenic Survivin fusion proteins. Arrow marks endogenous Survivin bands at approximately 16.5 kD. Blots with the same amount of cell lysates were probed with an anti-Bax antibody. The blot subsequently was stripped and probed with an anti-tubulin antibody to confirm equal protein loading. (c) Densitrometric analysis of Bax expression. Signals were normalized to tubulin expression. Note the weak Bax expression in HeLa cells expressing EGFP-Survivin and the increased expression in cells transduced with EGFP-Survivin-NLS. Error bars represent SEM. *p < 0.01 when Bax protein levels of EGFP-SurvivinNLS-expressing cells were compared to controls.

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expression was on the average 1.5-fold higher than in cells transduced with EGFP and EGFP-Survivin, respectively. Interestingly, ectopic EGFP-Survivin expression was associated with a 4-fold decrease in the mRNA level of the Bax gene when compared to EGFP-Survivin-NLS expressing cells and also EGFP-expressing cells. On the other hand no significant differences in bcl-2 mRNA expression levels were detected in the differently transduced HeLa cells. Next, we investigated the protein expression levels of BAX. On the average we found a twofold higher steady state protein expression level in cells expressing EGFPSurvivin-NLS when compared to cells that were only transduced with EGFP (Fig. 7b). On the other hand, we monitored in cells expressing EGFP-Survivin-NLS an 8-fold increase in BAX protein levels when compared to cells expressing EGFP-Survivin (Fig. 7b and c). Since Bax transcription can be enhanced by the tumor suppressor p53 [49] we analyzed endogenous p53 protein levels by Western blot analyses and indirect immunofluorescence analyses. In line with the mRNA data the Western blot analyses showed an increased steady state level of p53 in HeLa cells transduced with EGFP-Survivin-NLS when compared to cells transduced with EGFP and EGFP-Survivin, respectively (Fig. 8A). We detected p53 signals predominantly in the cytosol of HeLa cells expressing EGFPSurvivin-NLS and barely signals in cells expressing EGFP-Survivin (Fig. 8B) indicating that elevated BAX expression levels in cells expressing EGFP-Survivin-NLS

were not directly linked to transcription-dependent functions of p53. However, in this experiment we could not completely rule out that residual nuclear p53 might exert transcriptional activity on target genes during induction of apoptosis by C2 ceramide. In order to elucidate whether C2 ceramide somehow activates the p53-dependent transcription of pro-apoptotic BAX in HeLa cells we investigated p53 protein expression levels at different time points after incubation with C2 ceramide. The analyses showed that directly upon C2 ceramide treatment p53 protein levels were significantly decreased (Fig. 9). Furthermore, no nuclear p53 protein was detected during the experiments (data not shown). Interestingly, BAX protein levels were gradually increased by a yet unknown mechanism during the time course of C2-treatment. However, this confirms the important role of BAX in C2-mediated apoptosis which already has been acknowledged in previous studies [50,51].

4. Discussion The intracellular localization of Survivin is thought to be crucial for its anti-apoptotic capacity. A cytosolic localization enables Survivin to interact with caspases or Smac/DIABLO and therefore can hinder intrinsic apoptosis [7,9,11]. A nuclear localization of Survivin has been described to be a predictive of a favourable prognosis in cancer [35–37].

Fig. 8. Increase in cytosolic p53 in HeLa cells transduced with NLS-Survivin. (A) Western blot analysis of p53 expression in HeLa cells transduced with EGFP, EGFP-Survivin and EGFP-Survivin-NLS, respectively. Note the increased expression of p53 in HeLa cells expressing EGFP-Survivin-NLS. A blot with the same amount of cell lysates was probed with an anti-tubulin antibody to confirm equal amounts of loaded proteins. (B, a–c) Analysis of HeLa cells expressing EGFP-Survivin-NLS and (d–e) HeLa cells expressing EGFPSurvivin. (a and d) EGFP signals of the transgenic fusion proteins. (b and e) Indirect immunfluorescence analysis of p53 expression shows cytosolic p53 in cells transduced with EGFP-Survivin-NLS but no signals in cells expressing EGFP-Survivin. (c and f) DNA staining was performed with Hoechst33342. Bar equals 80 lm.

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Fig. 9. Bax levels increase during C2 creamide-induced apoptosis whereas p53 expression levels are not induced. Western blot analyses of the expression levels of p53 and Bax after induction of C2-mediated apoptosis. Cells were treated as described in the material and methods section and total protein lysates were prepared at the indicated time points and subjected to SDS– PAGE. (a) Bax protein levels increase during C2-mediated apoptosis. (b) p53 steady state protein levels decreases after treatment with C2 ceramide. 293T cell lysate was included as positive control for p53 detection. Blot was overexposed in order to visualize the weaker p53 expression in HeLa cells. (c) The blot subsequently was stripped and probed with an anti-tubulin antibody to confirm equal protein loading.

However, also contradictory results on the subcellular localization of Survivin in non-small cell lung carcinoma tissue samples have been documented [52]. This recently published data may have been obtained by using different antibody preparations for immunochemistry. In particular, despite reactivity in Western blot, several commercially available antibodies to Survivin did not give the accepted pat-

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tern of centromere localization in human cells [53,54]. Here, we describe, that in contrast to various tumor cell lines derived from solid tumors, early passages of normal human lung fibroblasts showed a strong accumulation of Survivin in the nuclei of interphase cells. Despite evident Survivin expression the fibroblasts were tested to be highly sensitive to the pro-apoptotic reagent C2 ceramide, which induces the mitochondrial apoptotic pathway [48]. On the other hand, we observed that aging NHLFs exported Survivin into the cytosol which correlated with a stronger resistance to C2 ceramide-induced apoptosis. Driven by this observation we sought to increase the sensitivity to apoptosis of tumor cells by forcing Survivin into the nucleus through inhibition of CRM1/Exportin-1 by leptomycin B (LMB). For this experiment we stably transduced HeLa cells with EGFP and EGFP-Survivin, respectively. As shown in Fig. 5e and f the EGFP control cells were more susceptible to C2 ceramide treatment than cells with ectopic expression of EGFP-Survivin, which is in line with previous results showing that cytosolic overexpression of Survivin blocks apoptosis [5,6]. Next we used LMB to block nuclear export of Survivin. Since LMB can interfere with the E6mediated degradation of p53 by blocking the nuclear export of p53 [55] or other proteins involved in apoptosis [56] we treated the cells only for 4 h with LMB and washed the cells extensively before adding C2 ceramide. This short LMB-treatment also led to an increase in the amount of apoptotic cells which was in the same range when compared to the results of C2 ceramide-treatment (Fig. 4e and f). However, the successive incubation of cells expressing EGFP with C2 ceramide led to a twofold increase in apoptosis when compared to cells treated only with LMB- and C2 ceramide, respectively. Interestingly, a combined LMB and C2-treatment showed an approximately threefold increase in apoptosis in cells expressing EGFP-Survivin when compared to treatments with C2 ceramide or LMB, respectively, suggesting that a nuclear relocalization of Survivin preceding C2 ceramide treatment might further enhance the induction of the intrinsic apoptotic pathway (Fig. 4e). In order to exclude the observed cytotoxic influence of leptomycin B on the cells and to analyze the effects of nuclear Survivin in depth we transduced HeLa cells with a vector coding for EGFPSurvivin fused to a nuclear localization signal of

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SV40 and with EGFP-Survivin, respectively. Interestingly, Western blot analysis revealed approximately the same amount of transgenic protein expression but different degradation products. We assume that this observed result is caused by the additional NLS-sequence or is due to differences in cytoplasmic vs. nuclear proteasomal degradation of the fusion proteins. The transductions did not increase baseline apoptosis levels and did not result in a greater amount of cells bearing mitotic defects. However, when we induced intrinsic apoptosis in both cell populations a higher proportion of cells with ectopic expression of nuclear Survivin became apoptotic than cells with ectopic expression of cytosolic Survivin as determined by analyses of the fraction of cells undergoing internucleosomal DNA fragmentation and AnnexinV-staining (Fig. 5c and d). As an additional control we included HeLa cells transduced with EGFP. These cells were still more sensitive to C2 ceramide induced apoptosis when compared to cells expressing EGFP-Survivin-NLS as shown in FACS analysis of the content of cells with fragmented DNA. Due to problems with the compensation of bright EGFP emission we were not able to analyze the HeLa-EGFP cells in the AnnexinV-stainings. However, since overexpressed EGFP-Survivin-NLS, beside its pre-dominant nuclear localization, was also detected in the cytosol, we deduce that this amount of ectopic Survivin renders the cells less susceptible to C2 ceramide-induced apoptosis than the cells only transduced with EGFP. On the other hand, it seems logical that the suitable control for our experiments with the cells expressing Survivin fused to EGFP and the SV40 NLS are the cells expressing comparable amounts of EGFP-Survivin. Further experiments established that augmented basal transcription levels of p53 and pro-apoptotic Bax in the cells expressing nuclear Survivin obviously were responsible for this increased sensitivity to pro-apoptotic C2 ceramide. Subsequent Western blot analyses and indirect immunofluorescence investigations, respectively, confirmed that observed higher bax and p53 mRNA levels were appropriately translated into proteins. However, it is well known that HeLa cells, which are derived from a cervix carcinoma, bear HPV18 sequences in their genome. Typically, p53 levels of HeLa cells are repressed due to E6-mediated ubiquitination and nuclear export [57] which abolishes p53 transcriptional activity. In line with this, p53 was hardly detected in the cells expressing EGFP and

EGFP-Survivin, respectively. On the other hand, in cells transduced with EGFP-Survivin-NLS the increased p53 protein levels were detectable but obviously were translocated to the cytosol by the action of HPV E6 protein. That the C2-mediated induction of apoptosis in the HeLa cells depends on the transcription factor activity of p53 was rendered unlikely because the basal p53 protein levels decreased directly after C2 ceramide treatment. However, an increase of Bax was monitored which likely have been caused by conformational changes and oligomerization of Bax increasing its half-life time. Recently, it has been shown, that cytosolic p53 inhibits the anti-apoptotic capacity of BclXL and promotes cytochrome c release [58]. Another study has demonstrated that p53 can directly modulate Bax-oligomerization thereby priming cells for apoptosis by destabilizing the mitochondrial membranes [59]. In line with the latter finding, our data suggest a combined effect of cytosolic p53 and Bax, which could result in the increased sensitivity to intrinsic apoptosis in HeLa cells expressing nuclear Survivin. In addition, Survivin was described to be involved in histone modifications and in polymerase II-dependent transcription [60]. In fact, Survivin was described to augment transcription of the genes for Fas ligand [38] and for human telomere reverse transcriptase (hTERT) [25]. In these studies, Survivin led to phosphorylation and activation of transcription factors like the specific protein 1 (SP1) as well as the proto-oncogene c-Myc, the latter has been demonstrated to transactivate p53 transcription [61] and to cooperate with Bax to induce intrinsic apoptosis. Therefore, further studies are needed to investigate possible effects of Survivin on gene expression of proteins involved in cell cycle, senescence, and apoptosis. Furthermore, since most tumor cells have been described to express Survivin during the whole cell cycle and to export Survivin into the cytosol one might speculate that CRM1-dependent nuclear export of Survivin gives tumor cells a selection advantage. Therefore, therapeutic interventions which hold Survivin in the nucleus of tumor cells might improve cancer therapy.

Acknowledgements This work was supported by a grant from the Wilhelm Sander-Stiftung (Az. 2003.092.1) to A.T.

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