Human Molecular Genetics, 2002, Vol. 11, No. 1 69-76
© 2002 Oxford University Press
Transduction of wild-type merlin into human schwannoma cells decreases schwannoma cell growth and induces apoptosis
Molecular Neurobiology Laboratory, Department of Neurology and 1Department of Pediatric Hematology/Oncology, Heinrich-Heine University Medical Center, Düsseldorf, Germany and 2Zentrum für klinische Forschung, Department of Neurology, University of Ulm, Ulm, Germany
Received September 10, 2001; Revised and Accepted October 31, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in both alleles of the tumour suppressor gene coding for merlin/schwannomin, an ERM family protein, cause the hereditary disease neurofibromatosis type 2 (NF2). NF2 is characterized by the development of multiple nervous system tumours especially vestibular schwannomas. Efficient oncoretrovirus-mediated gene transfer of different merlin constructs was used to stably re-express wild-type merlin in primary cells derived from human schwannomas. Using two-parameter FACS analysis we show that expression of wild-type merlin in NF2 cells led to significant reduction of proliferation and G0/G1 arrest in transduced schwannoma cells. In addition, we show increased apoptosis of schwannoma cells transduced with wild-type merlin. Our findings in primary schwannoma cells from NF2 patients strongly support the hypothesis of merlin acting as a tumour suppressor and may help in understanding development of human schwannomas in NF2.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in both alleles of the gene coding for merlin/schwannomin, an ERM family protein of the protein 4.1 superfamily, cause the hereditary disease neurofibromatosis type 2 (NF2) (1,2). Clinically NF2 is characterized by the development of multiple nervous system tumours in particular vestibular schwannomas of the eighth cranial nerve. Up to 80% of NF2 patients also have spinal schwannomas and 50% have cutaneous schwannomas. NF2 patients also frequently suffer from meningiomas and to a lesser extent from gliomas and ependymomas (3). Additionally, all sporadic schwannomas (i.e. unrelated to NF2) as well as many sporadic meningiomas also carry mutations in the NF2 gene (3).
Development of tumours in NF2 patients follows the Knudson hypothesis on tumour suppressors (4), as NF2 patients carry a germline mutation in one merlin allele, a second hit in the other allele suffices to give rise to tumourigenesis. This concept is also supported by the fact that in sporadic schwannomas both hits arise spontaneously (1,2).
There is additional evidence from model systems that merlin/schwannomin acts as a tumour suppressor. Overexpression of merlin in oncogene-transformed cell lines reverts the oncogene-induced phenotype (5) and overexpression in NIH3T3 cells or RT4 and JS1 rat schwannoma cell lines reduces proliferation (6–8). In addition, suppression of merlin synthesis in tumour cell lines increases proliferation (9). Furthermore, NF2 knockout mice also develop multiple tumours (10,11).
This putative tumour suppressor function of merlin is remarkable as it is the only protein from the ERM family which has been described to act as a tumour suppressor. Merlin/schwannomin is localized in regions associated to cell motility and adhesion and has as binding partners mainly proteins associated to the plasma membrane or the cytoskeleton (12). Therefore, it is likely that merlin acts as a membrane–cytoskeletal linker similar to other members of the ERM family (13–16). Interestingly, Gutmann et al. (17) recently showed that DAL-1, a member of the protein 4.1 superfamily, also acts as a tumour suppressor in meningioma cell cultures.
Very few experimental data on patient-derived primary schwannoma cells exist due to the difficulty of growing human schwannoma cells in vitro. Primary human schwann cells cultured from NF2 schwannomas show partially increased proliferation, grow independently from growth factors and demonstrate morphological changes compared to normal human schwann cells (18,19). In a study focussing on gene transfer into human meningioma cells with retroviral, adenoviral and herpesviral vectors, Ikeda et al. (20) described reduced proliferation as a consequence of transient re-expression of wild-type merlin protein in these cells only with the latter vector system. They were not able to demonstrate differences in morphological appearance and apoptotic rate between merlin-transduced and -untransduced meningioma cells with any gene-transfer system.
Here we demonstrate for the first time that oncoretroviral vectors can be utilized efficiently to stably re-express wild-type merlin in primary human schwannoma cells derived from NF2 patients. In these experiments two merlin–EGFP fusion constructs and one merlin–IRES–EGFP construct were generated in order to localize merlin in transduced cells as well as to ascertain that merlin function was not altered by fusion to EGFP. The results show (i) efficient and stable re-expression of wild-type merlin in human schwannoma cells from three different NF2 patients, (ii) reduction of proliferation in these cell cultures with increased G0/G1 arrest and (iii) increased apoptosis of schwannoma cells transduced with wild-type merlin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transduction of human schwannoma cells with wild-type merlin
The retroviral constructs generated are shown in Figure 1. In order to introduce the merlin isoform I cDNA into NF2 schwannoma cells and easily identify transduced cells, a retroviral vector was constructed with the merlin and EGFP cDNAs being both expressed off the retroviral LTR as two independent proteins however linked via an encephalomyocarditis virus IRES site (LMIEG: LTR Merlin IRES EGFP; Fig. 1A). To visualize the intracellular localization of merlin, alternative constructs were generated in which the merlin cDNA was fused either at the N- or C-terminus to EGFP (LMEG: LTR Merlin EGFP, or LEGM: LTR EGFP Merlin; Fig. 1B and C). A vector only containing EGFP served as mock control in all experiments (LEG: LTR EGFP) and is described elsewhere (H.Hanenberg, S.Batish, K.Pollok, L.Vieten, C.Leurs, R. Cooper, K.Göttsche, L.Haneline, D.Clapp, S.Lobitz, D.Williams and A.Auerbach, submitted for publication).
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) are indicated. (A) LMIEG, (B) LMEG and (C) LEGM.These constructs were used to generate stable pg13 packaging cells (21), producing gibbon ape leukemia virus (GALV)-pseudotyped virus. Using these recombinant retroviruses at a multiplicity of infection (MOI) of
5, flow cytometry analysis of EGFP-positive cells revealed transduction efficiencies in primary schwannoma cells between 19 and 57% (Table 1). This high gene transfer efficiency was reached by successive infections (three times for 24 h each) in proliferation medium containing growth factors and by plating schwannoma cells at low density to allow maximal proliferation. Transduction with LMIEG measured as the percentage of EGFP positive cells by flow cytometry consistently achieved lower gene transfer efficiencies compared to those with the other vectors (Table 1). However, as all recombinant viruses were used at comparable MOIs and showed similar cell cycle effects on primary schwannoma cells (see below), this apparently lower gene transfer efficiency is most likely due to lower EGFP expression level caused by the IRES site (22).
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Microscopic analysis of the expression of the EGFP fusion proteins and a representative western blot analysis of cells from schwannoma 3 were used to demonstrate merlin expression in transduced schwannoma cells. On western blot, non-infected as well as mock-infected cells of schwannoma 3 showed no endogenous merlin expression (Fig. 2). This might be due to the fact that the germline mutation of schwannoma 3 (frameshift, exon 8) leads to an increased turnover of the mutated protein. Schwannomas infected with retrovirus containing the merlin–EGFP fusion constructs or the merlin–IRES–EGFP construct showed expression of merlin (
71 kDa) or merlin–EGFP fusion proteins (100 and 102 kDa, respectively) at the expected size (Fig. 2). The difference in size between the two merlin–EGFP fusion proteins visible in the western blot is caused by the cloning procedure. LMEG codes for a 13 amino acid longer protein than LEGM.
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By analysing EGFP fluorescence in LMEG/LEGM transduced schwannoma cells, we were able to demonstrate that merlin localization is accentuated at the plasma membrane but is also detectable in the cytoplasm (LMEG/LEGM; Fig. 3), whereas merlin-independent EGFP protein expression leads to a homogenous distribution of EGFP in the cell (LMIEG; Fig. 3). Merlin-directed localization of the fusion proteins is identical in NF2 schwannoma cells irrespectively whether merlin is fused N- or C-terminal to EGFP.
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Growth inhibition of human schwannoma cells due to wild-type merlin expression
To investigate whether the stable re-expression of wild-type merlin in NF2 schwannoma cells from three patients induces phenotypic changes we perform cell cycle analysis using flow cytometry. To this end, cells were transduced with the four recombinant retroviruses at early passages (passage 1 and 2) and then stained 5–7 days after the last infection with propidium iodide (PI) and FITC-labelled anti-BrdU antibody. A representative example for a two-parameter FACS analysis is shown in Figure 4 (R2, G0/G1 subpopulation; R4, G2/M subpopulation; R3, FITC-labelled S subpopulation).
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Re-introduction of wild-type merlin led to an increase of cells in G0/G1 phase and reduction of cells in the S phase of the cell cycle in primary cells from three NF2 patients (Fig. 5A). This effect was apparent when comparing infected cells with either non-infected or mock-infected cells. These changes in cell cycle distribution were found for all merlin constructs, regardless whether merlin was fused to EGFP (LMEG/LEGM) or expressed independently from EGFP (LMIEG) (Fig. 5). Thus, the results suggest that wild-type merlin inhibits schwannoma cell growth by the induction of G0/G1 arrest.
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Furthermore, this inhibitory effect of merlin on schwannoma cell growth was found in all tumours regardless of their genotype, as the mutation spectrum included nonsense, missense and frameshift mutations.
To investigate whether the re-expression of merlin is associated with morphological changes, transduced cells were analysed by phase contrast microscopy or by staining for actin filaments. The analysis revealed no differences between untransduced, mock-transduced or merlin re-expressing schwannoma cells (data not shown).
Because the ability of tumour cell populations to expand is not only determined by the rate of cell proliferation but also by the number of cells undergoing apoptosis, we finally examine apoptosis in schwannoma cells after reintroduction of wild-type merlin. In non-infected schwannoma cells apoptosis varies from 2.6 to 7.2% (Table 2). Cells infected with LMIEG (expressing merlin and EGFP independently) and also cells infected with LEGM (expressing the EGFP–merlin fusion protein), showed a doubling of the rate of apoptosis in all three infected schwannomas, whereas mock-infected cells (LEG) showed no alteration in the number of cells undergoing apoptosis (Table 2). Interestingly, infection with LMEG (expressing a merlin–EGFP fusion protein) did not show a clear tendency.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We were able to provide experimental data for merlin acting as a tumour suppressor in primary human schwannoma cells from NF2 patients. Using oncoretroviral vectors for stable expression, we show a clear reduction of proliferation in schwannoma cells re-expressing wild-type merlin compared to non-infected schwannoma cells. This was achieved by combining optimized transduction methods and cell culture conditions.
The cellular localization of the merlin–EGFP fusion proteins in transduced human schwannoma cells confirms with previous data on cellular localization of merlin in other cell types (13–15,23,24).
The suppression of cell growth through expression of merlin was detectable in three different schwannomas. As the germline mutations in these patients include missense, frameshift or nonsense mutations, the effects of merlin re-expression appear not to be restricted to certain patients or to a particular type of mutation. We found a clear reduction of proliferation in merlin re-expressing schwannoma cells compared to controls after transduction with all constructs regardless of whether merlin was fused N- or C-terminally to EGFP or expressed as a protein independently from EGFP. This indicates that the growth control function of merlin is not altered by fusion to EGFP. This is in agreement with data from Sherman et al. (7) who used a N-terminal flagged merlin in their growth suppression experiments on cell lines.
In our results, the percentage of schwannoma cells in the S phase was clearly decreased and that in G0/G1 phase increased upon re-expression of merlin, suggesting that the reduction of proliferation in transduced schwannoma cells is mainly caused by an increased arrest of merlin-transduced cells in G0/G1 phase. Our findings on merlin expression in human schwannoma cells are especially remarkable, as previous studies on merlin function were performed in cell lines and needed high cell densities to demonstrate any effect of merlin expression on proliferation (5,6,25,26). However, these authors used cell lines with a different genetic background. In addition, compatible to the results shown here for the effect of merlin expression in schwannoma cells, the analysis of non-corrected primary human schwannoma cells during logarithmic growth also demonstrated increased proliferation (18,19).
We did not find any change in the morphology of primary human schwannoma cells after re-introducing wild-type merlin (18,19,27,28). This could be due to the fact that human schwannoma cells in passage 1 and 2 appeared heterogeneously in tissue culture.
Herrlich et al. (25) recently showed that overexpression of merlin in primary rat schwann cells indeed increases apoptosis. Shaw et al. (29) only found apoptosis enhancement using the C-terminal but not the full-length form of merlin isoform 1 in NIH3T3 fibroblasts. We show for the first time that wild-type merlin isoform 1 expression is able to increase apoptosis in human NF2 schwannoma cells. This increased apoptosis was found in all our merlin transduced schwannoma cells.
Although all retroviral constructs achieved growth suppression and G0/G1 arrest, apoptosis was only enhanced when merlin was expressed independently from EGFP (LMIEG) or N-terminally fused to EGFP (LEGM), not when merlin was fused C-terminally to EGFP (LMEG). The growth suppression and pro-apoptotic effects we describe may thus involve different domains of merlin, the latter masked by fusing EGFP to the C-terminal end of merlin. It is interesting to note that the antibody C18 (Santa Cruz) directed to the C-terminus of merlin did not detect the merlin–EGFP fusion protein in western blot (data not shown).
The pro-apoptotic function of merlin is particularly interesting as it is known that many tumour cells develop a resistance to apoptosis-inducing signals and that other tumour suppressors such as the retinoblastoma protein and p53 play a major role in the control of cell cycle and apoptosis (30,31). Furthermore, there seems to be a link between apoptosis and cytoskeleton (32) as mutated ezrin, like merlin a member of the ERM family of proteins, is able to induce apoptosis in epithelial cells (33). In this context it is interesting to note that vestibular schwannomas arise at the border between areas in the eighth cranial nerve myelinated by central and peripheral glia. One could speculate that in this region, where two different cell types contact each other, precise apoptosis regulation is important and that decreased apoptosis due to merlin mutation leads to tumour formation with a predilection in this area.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Merlin vectors and retrovirus production
The cDNA for the isoform 1 of merlin (7) was removed from pBDNF2 (personal gift from Lan Kluwe) and cloned into the 5' and 3' MCS of MEG and in front of the IRES–EGFP cassette of MIEG 2 (Fig. 1). Both retroviral vectors are derivatives of MSCV 2.1 (mouse stem cell virus) (34) and are described elsewhere (S.Lobitz, L.Vieten and H.Hanenberg, unpublished data). In all constructs, merlin was expressed off the retroviral LTR. Cloning sites and PCR amplification products were sequence-verified using an ABI sequencer (Perkin Elmer).
Ecotropic envelope-pseudotyped retroviral vectors were made by transfecting ecotropic Phoenix packaging cells (http://www.stanford.edu/group/nolan/retroviral_systems/phx.html) with 20 µg of each plasmid and 60 µl of Fugene (Roche). Two days after transfection for virus containing medium was collected and filtered through 0.45 µm filters (Sartorius). To generate GALV-pseudotyped stable packaging cell lines, pg13 cells (21) were infected with ecotopic supernatants for 3 days in the presence of 5 µg of protaminsulfate (Sigma)/ml medium (35). Supernatants from pg13 packaging cell lines were harvested from confluent dishes every 24 h for 4 days (harvests 1–4), filtrated and stored at –80°C. The GALV-pseudotyped retroviral stocks were titred by infecting large T-immobilized human fibroblast cell line with limiting dilutions of each viral stock using three independent harvests and determining the EGFP expression for each dilution by flow cytometry on FACScan (Becton Dickinson).
Western blotting
To confirm the expression of merlin, subconfluent schwannoma cells (schwannoma 3) were collected and lysed by repeated freezing/thawing in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate). Protein concentration was estimated using a DC protein assay kit (Bio-RAD). Total proteins were separated by 5–10% SDS–PAGE and transferred to enhanced chemiluminescence nitrocellulose membrane (Amersham) by semidry blotting using a discontinous buffer system (36). Equivalency of protein loading was confirmed by Ponceau S red staining (Sigma). The NC membrane was blocked in 3% non-fat dry milk/1% BSA (Sigma) in PBS and probed with anti-merlin antibody (A18; Santa Cruz) directed to the N-terminus at a dilution of 1:500. Development was accomplished using goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Southern Biotechnology Associates) and ECL (Amersham).
Cell culture
NF2 schwannoma cells were isolated from surgically removed schwannomas of NF2 patients after informed consent as previously described by Rosebaum et al. (18). Schwannomas were preincubated for 1–7 days in DMEM (Gibco BRL) with 10% FCS (Roche), 2 µM forskolin (Sigma) and 500 U/ml penicillin/streptomycin (P/S; Gibco BRL) and then dissected into 1 mm long pieces in DMEM with 10% FCS containing 500 U/ml P/S, 160 U/ml collagenase type I (Sigma) and 1.25 U/ml dispase grade I (Roche). Tissue pieces were incubated in proteolytic enzymes for 24 h before they were completely dissociated by trituration with a narrowed Pasteur pipette. Cells were collected and resuspended in proliferation medium: DMEM/10% FCS, 500 U/ml P/S, 0.5 µM forskolin, 10 nM ß1-heregulin177–244 (Mark Sliwkowski, Genentech), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma), 2.5 µg/ml insulin (Sigma). The cell culture procedure described above resulted in 90–100% enriched cultures of human schwannoma cells (18,19) as confirmed by S100 immunostaining (data not shown).
Cells were seeded at a density of 10 000 cells/cm2 into six-well plates (Greiner) or eight-well LabTek slides (Nalgene Nunc International), precoated with 1mg/ml poly-L-lysine (Sigma) and 4 µg/ml natural mouse laminin (Gibco BRL). Proliferation medium was changed every 3–4 days and cells were passaged when confluent.
Gene transfer into schwannoma cells
Schwannoma cells were infected three times (MOI 5) for 24 h each at 50% confluency. During infection 0.5 µM forskolin, 10 nM ß1-heregulin177–244, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) and 2.5 µg/ml insulin was added to the viral supernatant. All infections were carried out in the presence of 5 µg/ml protaminsulfate (35). Titres were assessed after analysing the percentage of EGFP-positive cells on a FACS Calibur (Becton Dickinson) by limiting dilutions. In addition, EGFP fluorescence was visualized on a Nikon Eclipse TE 200 microscope using the Lucia 4.21 software (Nikon).
Cell cycle analysis
One to 2 days after plating, infected and non-infected schwannoma cells were incubated with 10 µM BrdU for 72 h. Cells were harvested by trypsinization, washed twice with 1% BSA/PBS and fixed in 70% ethanol at 4°C for at least 30 min. After fixation cells were pelleted, washed twice with 1% BSA/PBS and incubated for 15 min at 37°C in 100 µg/ml RNase A (Roth) to eliminate double-strand RNA. BrdU incorporated into the DNA was detected using FITC-conjugated anti-BrdU antibody (Pharmingen) according to the manufacturer’s instructions. For staining of the DNA content, cells were incubated with 5 µg/ml PI (Sigma) for 10 min in the dark. Cell cycle distribution was then calculated using two-parameter FACS analysis for DNA content and BrdU staining (Cell Quest software; Becton Dickinson) on a FACS Calibur. Infected cells were analysed in two independent experiments per merlin–EGFP construct.
Cell culture analysis
For actin staining, cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT), permeabilized with 0.2% Triton X-100 for 5 min at RT and incubated for 30 min at RT with 5 U/well (LabTek slides) Phalloidin stock solution (Molecular Probes) according to the manufacturer’s protocol. Nuclei were counterstained with DAPI (Roche).
Apoptotic cells were detected using the Tunel assay. Cells were plated on LabTek slides and analysed 7 (schwannoma 2) or 8 (schwannoma 1 and 3) days after infection. Briefly, cells were fixed for 1 h in 2% PFA (Sigma) and permeabilzed for 2 min with 1% Triton X-100/0.1% sodium citrate (Merck). The cells were incubated for 1 h with 25 U terminal deoxynucleotide transferase (Roche) and 0.3 nM rhodamine labelled dUTP (Roche). Cells were counterstained with DAPI. DNase I (Pharmacia)-treated cells served as positive controls. For each schwannoma and merlin–EGFP construct on average five microscopic fields with at least 500 cells were counted per experiment, respectively.
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ACKNOWLEDGEMENTS |
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We thank Drs L.Kluwe and V.F.Mautner for providing us with NF2 schwannomas. This work was supported by EC Grant no. Bio-CT92–2207 (C.O.H.); by the Forschungsverbund somatische Gentherapie des Bundesministeriums für Bildung und Forschung (beo 2103111661) and the Elterninitiative Kinderkrebsklinik e.V. (H.H.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 731 50033645; Fax: +49 731 50033609; Email: oliver.hanemann@medizin.uni-ulm.de
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