Human Molecular Genetics, 2000, Vol. 9, No. 20 3055-3064
© 2000 Oxford University Press
Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations
1Medical and Molecular Genetics Center-IRO, Hospital Duran i Reynals, Autovia de Castelldefels km 2.7 08907 L’Hospitalet de Llobregat, Barcelona, Spain, 2Department of Neuropediatrics, Heinrich-Heine-University, Children’s Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany and 3Department of Biochemistry, School of Health Sciences, International University of Catalonia, Sant Cugat del Vallès, Barcelona, Spain
Received 18 August 2000; Revised and Accepted 18 October 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Neurofibromas are one of the most characteristic features of neurofibromatosis type 1 (NF1), an inherited autosomal-dominant neurogenetic disorder affecting 1 in 3500 individuals worldwide. These benign tumors mainly consist of Schwann cells (SCs) and fibroblasts. Recent evidence demonstrates that somatic mutations at the NF1 gene are found in neurofibromas, but it has not been demonstrated whether SCs, fibroblasts and/or both cell types bear a somatic loss of NF1. We recently established a cell culture system that allows selective expansion of human SCs from neurofibromas. We cultured pure populations of SCs and fibroblasts derived from 10 neurofibromas with characterized NF1 mutations and found that SCs but not fibroblasts harbored a somatic mutation at the NF1 locus in all studied tumors. Furthermore, by culturing neurofibroma-derived SCs under different in vitro conditions we were able to obtain two genetically distinct SC subpopulations: NF1–/– and NF1+/–. These data strongly support the idea that NF1 mutations in SCs, but not in fibroblasts, correlate to neurofibroma formation and demonstrate that only a portion of SCs in neurofibromas have mutations in both NF1 alleles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Neurofibromas are benign peripheral nerve sheath tumors that are the most characteristic features of neurofibromatosis type 1 (NF1), an autosomal-dominant neurogenetic disorder affecting 1 in 3500 individuals worldwide (1,2). NF1 is caused by a germline mutation of the NF1 gene which contains 60 exons and spans
335 kb on 17q11.2 (3–6). This gene encodes a 2818 amino acid protein product termed neurofibromin, a GTPase activating protein for Ras. Neurofibromin negatively regulates Ras signaling in several cell types (7–14). Recently the Drosophila NF1 homolog product has also been implicated as a potential integrator of Ras and cyclic 3',5'-monophosphate (cAMP)-mediated signaling pathways (15,16). Mutations inactivating neurofibromin have been found in NF1-related and non-NF1-related tumors, supporting the hypothesis that NF1 gene functions as a tumor suppressor (17). There is accumulating evidence that the tumor suppressor hypothesis is also relevant to neurofibroma formation. Mutations in both NF1 alleles have been demonstrated for five neurofibromas (18–20). Several studies have found loss of heterozygosity (LOH) or point mutations in the NF1 gene in a considerable number of neurofibromas (19–24). In some tumors it has further been shown that LOH is located in the NF1 allele that does not segregate with the disease (22). Although these genetic studies indicate that the two-hit mechanism might be responsible for the development of neurofibromas they failed to define the cell type in which both NF1 alleles are mutant. Neurofibromas are made up of 60–80% Schwann cells (SCs) but also contain fibroblasts, fibroblast-derived perineurial cells, mast cells and axons (25,26). Despite this mixed cellular composition, it has been suggested that neurofibromas have a unicellular origin (27). Recently, cytogenetic abnormalities were identified in SC cultures derived from plexiform neurofibromas, but not from dermal neurofibromas (28). SCs from a single neurofibroma were shown to sustain the somatic NF1 loss (29) and in another report fibroblasts derived from several neurofibromas exhibited high NF1 mRNA expression, whereas this expression was absent in SCs derived from the same tumors (30). Although the idea that SCs are the primarily defective cell type in neurofibromas is supported by the high percentage of SCs in neurofibromas and by abnormal phenotypes of neurofibroma-derived and Nf1 knockout mice SCs (31,32), it has not been possible to clearly verify this hypothesis in a substantial number of cell strains from genetically defined tumors due to the absence of appropriate cell culture techniques to grow pure populations of human tumor-derived SCs.
Culture of neurofibroma-derived SCs has long been hampered by low proliferation, early senescence and fibroblast contamination. Based on the identification of ß-heregulins as potent mitogens of human SCs (33) and the development of improved culture conditions for these cells (34), we recently established a cell culture system that allows isolation and selective expansion of human SCs from neurofibromas (35). Here, we report our analysis of 10 neurofibromas in which the constitutional and somatic NF1 mutation were characterized and demonstrate that a subset of SCs but not fibroblasts harbor the somatic mutation at the NF1 locus. In addition we describe methods to separately generate NF1+/– SCs and NF1–/– SCs from neurofibromas.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Detection of germline and somatic NF1 mutations in the tumors studied
The aim of the present work was to identify and characterize the neurofibroma-derived cell type with mutations in both NF1 alleles. In order to achieve this purpose we studied 10 neurofibromas from six unrelated NF1 patients. Firstly, we identified the somatic NF1 mutations directly from the tumors either by microsatellite genotyping for LOH detection or by cDNA-single-strand conformation polymorphism (SSCP)–heteroduplex (HD) analysis for detecting point mutations (E. Serra et al., in preparation) (Table 1). Germline NF1 mutations were also identified as previously described (36) (Table 1). These somatic mutations would be used as markers for recognizing the NF1–/– neurofibroma-derived cells. In the majority of cases, we confirmed that the two NF1 mutations arose in the homologous NF1 chromosomes.
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The first evidence that two subpopulations of SCs, NF1+/– and NF1–/–, can be isolated from neurofibromas
First we cultured the two main cell types comprising neurofibromas, fibroblasts and SCs (26), from one of the tumors studied (CSG19N) in which both NF1 mutations had been characterized (Fig. 1a). Fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal calf serum (FCS) whereas SCs were grown following recently established conditions, here referred to as Schwann cell medium (SCM) (34,35) (see Materials and Methods). The percentage of SCs was determined by immunocytochemical staining using an S-100 antibody, a marker for SCs that does not label human fibroblasts. Once pure subpopulations were obtained, DNA was extracted from cells and detection of the somatic NF1 mutation was performed in order to confirm its presence or absence (Fig. 1b). The somatic mutation of CSG19N (2928del13) was almost undetectable in fibroblasts by passage two, indicating that this cell type was not harboring the ‘second hit’. On the contrary, it was present in a 98% pure SC culture by the third passage (Fig. 1b and c). Surprisingly, the amount of DNA harboring the somatic mutation in SCs at passage P3 appeared lower than in the original tumor and the aberrant somatic allele was absent in the same SC culture grown for a further three passages. These results suggested that, whereas SCs and not fibroblasts were the cells harboring the somatic NF1 mutation, cells with two mutant alleles were being lost with time in culture. We speculated that two genetically distinct subpopulations of SCs, NF1+/– and NF1–/–, were present in neurofibromas and that the in vitro conditions used gave NF1–/– SCs a growth disadvantage.
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Establishment of culture conditions for growing NF1–/– SCs
Based on these results we decided to assay a set of different culture conditions. For this purpose we chose tumor CCF1N derived from a patient with a large constitutional NF1 deletion. Therefore, only the expression of the allele non-associated with this deletion was detected, allowing us to quantify the proportion of NF1+/– and NF1–/– cells from this neurofibroma (Fig. 2a). Initially, cells were plated in SCM and after 24 h the medium was switched to serum-free (N2) medium for another 24 h. Then medium was changed to various experimental culture conditions for 3–4 days. The concentration of heregulin in the absence or presence of 0.5 µM forskolin was changed; the other SCM components remained unchanged. Heregulins are activators of erbB receptors and the MAP kinase pathway in SCs (32,37). Forskolin, an activator of adenylate cyclase (38), stimulates SC proliferation. Cells were maintained in specific media for a total of seven passages. At each passage some cells were used for RNA extraction, S-100 staining or further growth (Fig. 2).
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Addition or removal of forskolin led to remarkable differences in the growth of two SC subtypes (Fig. 2b, passage 5). When forskolin and heregulin were used in concentrations as in SCM (10 nM herß1 + forskolin), an enrichment of cells still bearing an active copy of the NF1 gene (NF1+/–) was observed in higher passages, as previously detected in tumor CSG19N (Fig. 1b). On the other hand, the removal of forskolin produced an enrichment of SCs with both mutated NF1 copies (NF1–/–) (Fig. 2b). Varying the concentration of heregulin did not alter this result.
Confirmation that SCs and not fibroblasts carry the somatic NF1 mutation and that two genetically distinct SC populations can be isolated from neurofibromas
Based on the ability to select NF1+/– and NF1–/– SCs, we selectively expanded both SC subtypes, as well as fibroblasts, from a further eight tumors from the six patients analyzed. Tumors were dissociated and cell suspensions were cultured under the following conditions. (i) In order to isolate NF1+/– SCs, part of the cell suspensions was plated in SCM and maintained under this condition through five passages. (ii) In order to obtain pure NF1–/– SCs, cell suspensions were plated in SCM, transferred to chemically defined serum-free N2 medium (39) after 24 h, and finally to SCM without forskolin. This procedure was repeated in subsequent passages (until passage 5) and enabled us to isolate SCs and obtain sufficient amounts of SCs for genetic test purposes. (iii) In order to obtain pure fibroblast cultures, cell suspensions were plated in DMEM and 10% FCS and grown for three passages (Fig. 3).
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G) in tumor CSG48N of patient CSG in samples corresponding to tumor and in NF1–/– SCs but not in NF1+/– SCs, fibroblasts and blood cells from this patient. (Bottom) SSCP analysis of exon 21 (E21) detecting the germline mutation of patient CSG. (d) (Top) Detection of the somatic mutation (Q756X) in tumor CSG29N of patient CSG by DNA SSCP of exon 14 (E14). Somatic mutation was only observed in samples from fresh tumor and NF1–/– SCs. (Bottom) SSCP analysis of exon 21 (E21) detecting the germline mutation of patient CSG.In all tumors SCs and not fibroblasts had mutations in both NF1 alleles (see Figs 1 and 3 for five of the studied tumors; data are not shown for the remaining tumors). Moreover, two NF1 genetically distinct SC populations were obtained from eight of 10 neurofibromas. In early cultures from individual tumors (e.g. from passage 2), the variable proportions of both SC types were inferred based on the abundance of the mutant allele, even though >97% of cells were S-100 positive (Fig. 4). These data indicate that the percentage of NF1+/– SCs was different in each tumor, although the precise proportion of both subtypes and their location in each tumor was not investigated. The results also showed that the procedures developed for the isolation of the different cell types are likely to be useful for most or all neurofibromas.
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Changes in the proliferative capacity and morphology of NF1–/– SCs under different forskolin conditions
In order to better understand why NF1–/– SCs do not proliferate in SCM, highly enriched NF1–/– SCs were submitted to previously defined culture conditions for growing (+/–) or (–/–) SCs. NF1–/– SCs were plated in SCM for 24 h, then the medium was switched to N2 which, after another 24 h, was replaced by either SCM or forskolin-free SCM. One set of these cultures was further processed for S-100 staining after 2–4 days in vitro (Fig. 5) and another set was used to determine the proliferation rate by immunocytochemical visualization of 5'-bromo-2'-deoxyuridine-5'-monophosphate (BrdU) incorporation (Table 2). Under SCM conditions NF1–/– SCs were characterized by a flat polygonal cell body—a morphology that became more pronounced with time—and proliferated at a relatively low rate, between 2.4 and 9.5%. In contrast, NF1–/– SCs showed an increased proliferation rate and exhibited the typical spindle-shaped bipolar morphology under forskolin-free conditions.
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Thus, it appeared that addition of forskolin to the culture medium led to a more differentiated cellular phenotype—correlated to a decreased proliferation rate—whereas the absence of forskolin resulted in a less differentiated phenotype with higher proliferative capacity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The work presented in this paper clearly demonstrates that SCs but not fibroblasts are the cells harboring a somatic mutation at the NF1 locus in benign neurofibromas, as suggested by data presented recently (29,30). Moreover, our results demonstrate for the first time the co-existence of two NF1 subtypes of SCs in these tumors: (+/–) and (–/–). Our findings were corroborated in experiments using cells from 10 different neurofibromas belonging to six unrelated NF1 patients. To achieve this demonstration, a complete study of the whole NF1 coding region had previously been performed in order to identify the somatic and germline mutation in each tumor. To date the complete characterization of both NF1 mutations has been reported in only five neurofibromas (18–20). The characterization of NF1 mutations in neurofibromas had been hampered by their mixed cell composition together with the large size of the gene and the absence of mutation hot spots. Several types of somatic mutation were identified in the 10 neurofibromas studied, including LOH, point mutations predicted to produce truncated proteins and mutations predicted to produce a protein slightly larger than the authentic neurofibromin due to the gain of several amino acids. In relation to the germline mutations detected in the patients studied, one patient had a large deletion that removes the whole NF1 gene, two had frameshift mutations and two had DNA mutations affecting the correct mRNA splicing. Three of these germline mutations will produce a truncated neurofibromin whereas one will produce a slightly shorter protein lacking 58 amino acids. In most of the neurofibromas excised from these patients we were able to isolate and selectively grow three different cell types: fibroblasts, NF1+/– SCs and NF1–/– SCs.
Fibroblasts were a possible candidate for the tumor-forming cell type in these tumors since Nf1–/– fibroblasts from E12.5 knockout mice had previously been shown to exhibit a greater in vitro proliferation capacity than normal or heterozygous fibroblasts (40,41). However, our results demonstrate that at least the vast majority of neurofibroma-derived fibroblasts do not bear a ‘second hit’ at the NF1 locus. Our data do not rule out the hypothesis that NF1+/– fibroblasts contributed to neurofibroma formation. Moreover, SCs were clearly identified as the cell type harboring the somatic NF1 mutation. This result is consistent with previous studies showing dysfunction in SCs with NF1 mutations (24,42,43) and Ras activation in neurofibroma extracts (44). Most of the neurofibromas examined consisted of a non-uniform SC population. NF1+/– and NF1–/– SCs could be isolated from these tumors. However, from two tumors (CSG51N and CSG42N) we were able to isolate only NF1–/– SCs and fibroblasts. The existence of two SC subtypes was recently suggested by analysis of Ras-GTP in single neurofibroma SCs; only a subpopulation of SCs at passage 1 showed elevated Ras-GTP (45). We were able to selectively grow and expand these SC subtypes by using different concentrations of forskolin. Therefore, we have been able to demonstrate for the first time that two genetically distinct SC subtypes are present in neurofibromas and that it is possible to isolate and selectively expand the putative tumor-forming NF1–/– SCs from neurofibromas. The relative proportion of NF1+/– and NF1–/– SCs in the original tumors analyzed is not known with certainty since only SCs from a second passage were screened for the presence of the mutation (Fig. 4). However, the impossibility of isolating NF1+/– SCs from two neurofibromas reflect that, at least in some tumors, the NF1–/– population may be the most abundant. Nevertheless, it has to be noted that the pool of neurofibromas presented in this work is probably slanted towards neurofibromas highly enriched with NF1–/– SCs, due to the fact that molecular analysis was performed previous to culturing.
It was surprising that the previously established SCM (35), containing heregulin and forskolin enabled selective expansion of normal or NF1+/– SCs but not NF1–/– SCs (Fig. 1). When we modified the concentration of forskolin we found that forskolin hampered the growth of NF1–/– SCs. cAMP has previously been shown to be an important second messenger involved in proliferation and differentiation of SCs (46,47). It appeared that the heregulin concentration was also interacting in this process. As shown in Figure 2b (P5), molecular analysis of SC cultures revealed that in the presence of forskolin high levels of heregulins increased the proportion of NF1+/– SCs earlier compared with low levels of heregulin. In cultures without forskolin and low levels of heregulins, NF1–/– SCs were purified in earlier passages despite their lower proliferation capacity (Fig. 2; data not shown). These data support the idea that forskolin-stimulated pathways and the heregulin-stimulated Raf–MEK–ERK pathway co-operate in the control of SC proliferation (32,37). We speculate that excessive signaling of one or the other pathway by external stimulation (culture conditions) and/or intrinsic activation (two NF1 mutations) results in altered proliferative capacity.
Interactions between neurofibromin and the cAMP–PKA pathway have previously been described in the Drosophila NF1 knockout. Small pupal size was rescued by expressing activated PKA and exposure to forskolin restored correct potassium currents driven by the neuropeptide PACAP38 in the neuromuscular junction (15,16). Whereas these results suggest that in Drosophila the cAMP–PKA pathway is positively regulated by neurofibromin, the different forskolin requirements of NF1+/– or NF1–/– SCs observed in our experiments point to a different possibility for human SCs. NF1–/– SCs were grown best when forskolin was removed from the culture medium 24 h after the cells had been plated. This indicates that SCs lacking neurofibromin function require some, but less forskolin-stimulated signaling than normal, or NF1+/– SCs. Table 2 and Figure 5 show a decrease in the proliferation rate and a change in morphology, respectively, of NF1–/– SCs when submitted to constant conditions containing this agent, which suggests that pathways stimulated by forskolin (e.g. the cAMP pathway) interfered with the growth capacity of neurofibromin-lacking SCs in the in vitro conditions used. Although cAMP signaling is necessary for SC proliferation (37), elevation of cAMP beyond a certain threshold has been shown to result in similar morphological changes and/or reduced proliferation in both rat and neurofibroma-derived SC (48–51). Moreover, it has been demonstrated that low concentrations of cAMP stimulate rat and mouse SC proliferation whereas high concentrations induce differentiation (47,49,50,52). In addition it has also been suggested that neurofibromin plays a role in SC differentiation (52,53). Although NF1+/– SCs can grow under SCM conditions, NF1–/– SCs reduce their proliferation capacity under the same conditions due to an excess of forskolin-stimulated signaling. We hypothesize that this is due to elevated cAMP levels in the NF1–/– cells.
Because Nf1–/– SCs derived from E12.5 knockout mice exhibit growth inhibition in vitro (42), it was postulated that further genetic events are required in this cell type for neurofibroma formation. However, the present study shows that growth of neurofibroma-derived NF1–/– SCs can be modulated depending on culture conditions. Thus, we present the possibility that these cells require only the lack of neurofibromin function in vivo in order to hyperproliferate and that precise culture conditions are required to reproduce this behavior in vitro.
By studying a set of neurofibromas bearing different germline and somatic NF1 mutations, we demonstrated that SCs but not fibroblasts are the NF1–/– cells in neurofibromas and that two different subpopulations of SCs are present in this type of tumor. The lower forskolin requirement for growing NF1–/– SCs suggests that neurofibromin is somehow related to the cAMP pathway in this cell type. The isolation and expansion of neurofibroma-derived SCs and their identification as the primarily defective cell type in this neoplasia will enable the development of new therapeutic approaches.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tumor acquisition and sample processing
Neurofibromas were kindly provided by NF1 patients after informed consent. Following removal of surrounding tissue, tumors were cut into smaller pieces. Part of each tumor was directly frozen for DNA and RNA isolation, the remaining tumor specimens were frozen in liquid nitrogen in complete medium with 10% DMSO solution and later used for cell culture.
DNA and RNA extraction and NF1 mutation detection
Total RNA was extracted from neurofibromas, neurofibroma-derived cell lines and peripheral blood lymphocytes using the Tripure isolation reagent (Boehringer Mannheim, Mannheim, Germany), according to the manufacturer’s instructions. Blood DNA was extracted following the standard ‘salting-out’ procedure; neurofibroma DNA was obtained as described elsewere (22). DNA from neurofibroma-derived cell lines was rapidly extracted by proteinase K digestion in PCR buffer, for 1 h at 55°C, followed by proteinase K inactivation. Two microliters of this product were directly used for PCR analysis.
Detection of large intragenic NF1 deletions was performed by genotyping of several microsatellite markers flanking and within the NF1 gene (22). Point mutations were detected as previously described by SSCP–HD analysis from RT–PCR products and confirmed at DNA level (36). Screening of the characterized mutations was performed by polyacrylamide gel electrophoresis (PAGE), using either DNA or RNA, followed by silver staining detection. Semi-quantitative PCR was performed after selecting the number of cycles in which the PCR product could be quantified but had not reached the stationary phase. To detect the somatic mutation in tumors CSG19N and CCF1N, 28 cycles were chosen. Specific primers for each mutation are available on request.
In order to confirm that both NF1 mutations in tumor CSG19N were in the two different homologous chromosomes, RNA from the tumor was retrotranscribed and a cDNA fragment ranging from exon 17 to 21 was subcloned in pGEM-T Easy (Promega, Madison, WI). A set of positive clones was automatically sequenced (ABI 377; Perkin Elmer, Foster City, CA) using specific forward and reverse oligonucleotides.
Isolation of SCs from neurofibromas
Frozen tumor pieces were thawed, mechanically dissociated and further digested in DMEM, 10% FCS (Gibco BRL, Paisley, UK), 500 U/ml penicillin/streptomycin (Sigma, St Louis, MO), 160 U/ml collagenase type 1 (Sigma) and 0.8 U/ml dispase grade 1 (Boehringer Mannheim). After an incubation period of 18–20 h at 37°C and 10% CO2 in this medium, tissue pieces were completely dissolved by trituration with a narrowed Pasteur pipette. The resulting cell suspensions were transferred to a 50 ml Falcon tube containing DMEM with 10% FCS, centrifuged at 3080 g for 10 min and resuspended in SCM composed of DMEM, 10% FCS, 500 U/ml penicillin/streptomycin, 0.5 mM 3-iso-butyl-L-methylxanthine (IBMX; Sigma), 10 nM ß1-heregulin177–244 (Mark Sliwkowski, Genentech, South San Francisco, CA), 0.5 µM forskolin and 2.5 µg/ml insulin (Sigma). SCM was freshly prepared for each feeding. Cells were seeded at a density of 25 000 cells/cm2 onto six-well plates (Falcon, Milville, NJ) or onto eight-well plastic labtek slides (Nunc, Rochester, NY); both coated with 1 mg/ml poly-L-lysine (Sigma) and 4 µg/ml natural mouse laminin (Gibco BRL). Cultures were incubated in a humidified atmosphere at 37°C and 10% CO2. SCM was changed twice a week and cells were passaged when cultures were confluent (usually after 5–7 days).
Selective expansion of NF1–/– SCs
Cell suspensions were plated in SCM as described above. After 24 h the medium was switched to serum-free N2 medium (39) for another 24 h period. Then N2-medium was replaced by a modified SCM which contains no forskolin. All other components remained unchanged. After 2–4 days cells were trypsinized, replated in SCM and again treated as described.
Immunocytochemical labeling of neurofibroma-derived SCs
The purity of neurofibroma-derived SC cultures was determined in each passage by immunostaining with an anti-cow S-100 antibody (Dako, Copenhagen, Denmark). Cells were fixed with 4% paraformaldehyde, 0.03% Triton X-100 for 10 min at room temperature. Subsequently, cells were rinsed three times in Tris–buffered saline (TBS), pre-incubated for 30 min with 10% normal goat serum (NGS; Sigma) in TBS, blocked with H2O2–TBS, rinsed again three times in TBS and then incubated with the anti-cow S-100 antibody, made in rabbit (1:5000 dilution) at 4°C overnight. Then the cells were rinsed three times in TBS and incubated with a biotinylated secondary anti-rabbit antibody (1:200; Vector, Peterborough, UK) for 1 h at room temperature. For detection of bound antibodies the Vectastain Elite ABC System (Vector) was used followed by DAB staining. Cell bodies were visualized using Meyer’s Hämalaun (Merck, Darmstadt, Germany). After a final wash, cultures were mounted using Aquatex (Merck). To determine the percentage of SCs, a total of 500 cells were counted in duplicate sets for each culture and the percentage of S-100-positive cells determined.
For immunofluorescent staining cells were fixed with 4% paraformaldehyde, rinsed and permeabilized with 10% NGS in phosphate-buffered saline (PBS) with 0.1% Triton X-100 for 30 min at room temperature, then incubated with a rabbit anti-cow S-100 antibody (1:1000; Dako) for 1 h at room temperature. This was followed by a 45 min incubation period with the fluorescent cy3rb (1:1500; Jackson ImmunoResearch, West Grove, PA) at room temperature.
Cell nuclei were visualized by a 1 min incubation with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI; Boehringer Mannheim). Finally, slides were rinsed three times in PBS and mounted with Citifluor (UKC Chemical Laboratory, Canterbury, UK). Immunostained cell cultures were analyzed on a Leitz Dialux 20 fluorescent microscope.
Proliferation of NF1–/– SCs under different culture conditions
To determine the percentage of proliferating SCs, incorporation of BrdU (Boehringer Mannheim) was visualized by immunocytochemistry. After NF1–/– SCs had been selectively expanded, they were split into two groups and seeded onto eight-well plastic labtek slides (Nunc) coated with 1 mg/ml poly-L-lysine (Sigma) and 4 µg/ml natural mouse laminin (Gibco BRL) at a density of 15 000 cells/well. SCs were allowed to settle down in SCM overnight, then the medium was replaced by chemically defined, serum-free N2-medium (39) to induce growth arrest. After another 24 h the N2-medium was replaced either by SCM with forskolin or by SCM lacking forskolin for a period of 5 days. Then BrdU was added to the medium and cells were fixed 18 h later as previously described (33,34). Immunocytochemical detection of BrdU incorporation was performed as described (54).
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ACKNOWLEDGEMENTS |
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The authors wish to thank the patients and clinicians that participated in this study. We are indebted to M.L. Arbonés, M. Carrió, R. de Cid, H. Kruyer, V. Nunes, M.A. Pujana, C. Rosenbaum, N. Ratner, A. Ruiz, N. Sala and L. Sumoy for critical reading and comments on the manuscript. Support of H.W. Müller, Molecular Neurobiology Laboratory and B. Royer-Pokora, Department of Human Genetics, University of Düsseldorf, is gratefully acknowledged. We, the Düsseldorf and the Barcelona group, would like to thank the NNFF Consortium for giving us the opportunity to meet each other at the International NNFF meeting held in Aspen in 1998 which finally led to a very pleasant and productive cooperation. This work was supported by grants of the Fondo de Investigaciones Sanitarias de la Seguridad Social (98-0992), the Fundació August Pi i Sunyer/Marató de TV3, the Institut Català de la Salut, the Ministerio de Educación y Ciencia (CICYT/SAF96-1787-E), the Generalitat de Catalunya (CIRIT/1997SGR-00085) and the Deutsche Krebshilfe (10-1622-Ro1). E.A. was a fellow of the Comissió Interdepartamental de Recerca i Innovació Tecnològica of the Generalitat de Catalunya. E.S. was a visiting researcher at the Department of Neuropediatrics, Heinrich-Heine-University, Düsseldorf by means of a fellowship from the Generalitat de Catalunya.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +34 93 260 7775; Fax: +34 93 260 7776; Email: clazaro@iro.es
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REFERENCES |
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