Positional and functional mapping of a neuroblastoma differentiation gene on chromosome 11
- Katleen De Preter†1Email author,
- Jo Vandesompele†1,
- Björn Menten1,
- Philippa Carr2,
- Heike Fiegler2,
- Anders Edsjö3,
- Nigel P Carter2,
- Nurten Yigit1,
- Wim Waelput4,
- Nadine Van Roy1,
- Scott Bader5,
- Sven Påhlman3 and
- Frank Speleman1
© De Preter et al; licensee BioMed Central Ltd. 2005
Received: 04 March 2005
Accepted: 06 July 2005
Published: 06 July 2005
Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11.
In a first step, we performed high-resolution arrayCGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene.
Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.
In addition to the well known group of high stage neuroblastomas with MYCN amplification and 1p-deletion, a second genetic subgroup of aggressive neuroblastomas has been delineated. This subgroup is characterised by the presence of 11q-deletions, often in association with 3p-deletions [1–5]. Both subgroups typically present with 17q-gain or a normal chromosome 17 copy number, which are the strongest independent genetic indicators of poor prognosis . Deletions of 11q mostly affect a large distal part of the long arm. Only a few small deletions have been identified which delineated a tentative SRO (shortest region of overlap) at 11q23 between markers D11S1340 and D11S1299, encompassing a region of approximately 3 Mb . More recently however, a neuroblastoma patient was reported with a constitutional 11q14.1-11q23.3 deletion that did not overlap with the proposed SRO . Consequently, the presumed localisation of the 11q neuroblastoma tumour suppressor gene (or genes) remains ill defined, thus hampering the selection of positional candidate genes. For the 11q23 region we proposed SDHD as a putative candidate neuroblastoma tumour suppressor, but only two bona fide mutations could be identified.
In addition to the observed losses of 11q in neuroblastoma, the existence of a tumour suppressor gene on 11q has also been supported by functional evidence obtained by microcell mediated chromosome 11 transfer (MMCT) experiments . Although these studies were initially aimed at investigating the role of chromosome 1p in tumour suppression, the control chromosome 11 transfer experiment unexpectedly produced clones with morphological features of differentiation. Introduction of chromosome 11 induced a more flattened and adherent morphology, with short neuritic processes, similar to the changes seen after a few days of growth in the presence of retinoic acid. As these microcell hybrids could be powerful models for the identification of candidate neuroblastoma suppressor or differentiation genes, we decided first to determine the genetic status of the chromosome 11 in the hybrid subclones prior to further experiments. To this purpose, the parental NGP cell line and the microcell hybrids after chromosome 11 transfer were analysed using high-resolution arrayCGH (microarray based comparative genomic hybridisation), FISH (fluorescence in situ hybridisation) and microsatellite heterozygosity mapping. Following the identification of a region on chromosome 11 with altered copy number, we measured the mRNA expression levels of genes in these regions in an attempt to find altered gene expression related to neurite outgrowth and differentiation.
Chromosome 11 status and morphology of the microcell hybrids (MCH) obtained after chromosome 11 transfer in parental NGP.1A.TR1 cells as determined by Bader and colleagues  and in this study
microcell hybrid subclone (NGP.1A.TR1 + chr 11)
chromosome 11 status (in addition to parental NGP.1A.TR1 11q-loss)
Bader et al. 
no additional changes
more flattened, adherent cells, some short neuritic processes
del(11)(q23.3) (MCT128.1, HBI 18P2)
more flattened, adherent cells, some short neuritic processes
non-adherent, spheroid cells, growing in cell clusters
Assessment of the organisation of the actin fibres using phalloidin staining confirmed the presence of neurites (and excluded stress fibres) in subclones MCH574c10 and MCH574c11.
ArrayCGH based chromosome 11 copy number assessment
Microsatellite heterozygosity mapping
To determine which of the chromosomes 11 exhibited loss of the 11pter->11p15.1, 11pter->11p13 and 11q25->11qter regions, microsatellite heterozygosity mapping in conjunction with FISH analysis of metaphase spreads was performed. Microsatellite markers D11S861 (on 11p15.2) and D11S1324 (on 11p14.1) were tested on NGP.1A.TR1, MCH574c3 and MCH574c10. These tests show that one of the two parental chromosomes 11 had lost the 11pter->11p15.1 region, while the 11pter->11p13 segment was lost in the transferred chromosome. FISH on metaphase spreads (clone RP11-545G16 on 11q25 in combination with RP11-206C1 on 11p15.1; clone RP11-709M17 on 11q25 in combination with clone RP11-4B7 on 11p15.2) demonstrated that the 11q25->11qter deletion occurred in the transferred chromosome 11, whereas the 11pter->11p15.1 deletion occurred in the normal parental chromosome 11 (and not in the parental der(11)t(2;11)) (Figure 2).
Breakpoint delineation of chromosome 11 deletions
mRNA expression profiling
In a search for candidate neuroblastoma genes located on chromosome arm 11q, we investigated microcell hybrids obtained by transfer of a normal chromosome 11 into NGP neuroblastoma cells with loss of 11q. Although initially designed as a control experiment, this transfer resulted in morphological changes in the obtained hybrids (without loss of tumorigenicity) and also yielded revertants after further culture . The induced differentiation that was observed in all but one microcell hybrid is consistent with the presence of a neuroblastoma differentiation gene on chromosome 11. We thus anticipated that these hybrids might be of interest for functional mapping of the regions on chromosome 11 critically involved in neuroblastoma pathogenesis. To investigate this, we performed arrayCGH copy number analysis of these microcell hybrids. This allowed us to assess the status of the introduced (and parental) chromosomes 11 and to validate these hybrids as model system for further functional assays. The obtained results were surprising and puzzling. One particular microcell hybrid that did not show the expected differentiation features upon chromosome 11 transfer was shown to carry an 11q25->11qter deletion in the transferred chromosome. In addition we found that all microcell hybrid subclones presented with an 11pter->11p15.1 deletion, and that the MCH574c3 hybrids presented with an additional 11pter->11p13 deletion.
As loss of distal 11q is a recurrent chromosomal aberration in MYCN single copy advanced stage neuroblastomas , we propose the 11q25->11qter chromosomal segment as the most likely candidate region for the presence of a differentiation gene. Despite efforts to define a shortest region of overlap (SRO) for 11q-loss in neuroblastoma by microsatellite heterozygosity mapping  and delineation of constitutional 11q-deletions [8, 16], a consensus region for loss of 11q in neuroblastoma has not been defined thus far. In the light of the uncertainty of the boundaries of the 11q SRO, the 11q25->11qter region must be considered as potentially harbouring a neuroblastoma suppressor or differentiation gene. This region is present in two copies in microcell hybrid subclones MCH574c4, c10, c11 and c13 with differentiated morphology, but only in one copy in the non-adherent, spheroid cells from NGP.1A.TR1 and MCH574c3. Six known genes, i.e. HNT, OPCML, JAM3, THY28, ACAD8 and B3GAT1 are located in this distal 11q segment, of which two genes are of particular interest. HNT (neurotrimin) is reported to promote neurite outgrowth and adhesion . B3GAT1 encodes for a protein that functions as the key enzyme in a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK1 (CD57) [18, 19], which is a carbohydrate expressed in developmentally immature neural crest cells . Interestingly, the expression of HNT is significantly increased in neuroblastoma cell lines that are induced to differentiate using RA (retinoic acid), RA plus BDNF (brain-derived neurotrophic factor), NGF (nerve growth factor) and TPA (12-O-tetradecanoyl-phorbol-13-acetate). However, HNT expression is not significantly different between the differentiated microcell hybrids and the parental cells. It is conceivable that the observed phenotypic changes are caused by small changes in expression that can not be reliably detected by Q-PCR. An alternative explanation is that the normal parental chromosome 11 harbours a mutated allele that is normally expressed at the mRNA level (Figure 5). Reintroduction of a wild type allele by chromosome transfer could repair the defect, leading to differentiation. This is in keeping with reversal to the non-adherent, spheroid morphology of the microcell hybrids that have lost the 11q25->11qter region of the transferred chromosome. Additional mutation, promoter hypermethylation and gene directed functional assays are needed to clarify which of the genes located within the deleted 11q25->11qter region are responsible for the differentiated phenotype.
While the 11q25 region is the best candidate region to harbour a differentiation gene, the observed deletions on the short arm of chromosome 11 may also account for the differentiated morphology. The observation of two independent deletion events along the distal part of chromosome arm 11p is suggestive for the involvement of this region. In particular, it is striking that all microcell hybrids in which chromosome 11 is transferred are characterised by the presence of an 11pter->11p15.1 deletion in the (prior to transfer) normal parental chromosome 11. This may either be the result of an early coincidental event during the transfer process, or indicative for a selection process against the presence of three copies of a growth suppressive gene in this region. The last hypothesis may be further supported by the presence of unbalanced 11p-deletions in 4% of neuroblastomas (14/394) [21, 22].
Apart from highlighting at candidate 11q regions involved in neuroblastoma pathogenesis, this study clearly shows that it is important to monitor the transfer of the desired chromosome, as well as the genetic background of the cell line before and after chromosome transfer experiments. Selective pressure processes may occur during or after transfer of a chromosome, e.g. by chromosomal loss in order to maintain the viability of the microcell hybrids. Hence, detailed information on the chromosome copy number status before and after transfer is required in order to correlate phenotypic changes with chromosomal alteration. ArrayCGH has been proven to be a valuable screening method for evaluation of the chromosome alterations and for delineation of possible deletion events, allowing fine-mapping of the candidate regions that harbour candidate suppressor genes.
Microsatellite marker heterozygosity analysis, FISH and (array)CGH based copy number in neuroblastoma tumour specimens and patients with constitutional deletions have thus far not identified a consensus SRO for 11q-deletion. Here, we present an alternative strategy to pinpoint chromosomal regions or genes that may be important in neuroblastoma pathogenesis. Chromosome 11 transfer, followed by phenotype scoring and high-resolution copy number analysis delineated putative regions on chromosome 11 involved in tumour differentiation. Further mutation and functional analyses are required to clarify the putative involvement of genes localised in these regions in neuroblastoma.
The parental cell line, NGP.1A.TR1, and the chromosome 11 microcell transfer derived subclones MCH574c3, c4, c10, c11 and c13 used in this study have been described previously .
Cell lines were cultured following standard procedures and were digitally photographed under an inverted (phase-contrast) microscope, pelleted, snap-frozen and stored at -80°C for further processing. DNA was isolated using the QIAamp DNA mini kit (Qiagen). RNA was isolated from the snap-frozen cell pellets using the RNeasy Mini kit (Qiagen) according to the manufacturer's guidelines, followed by RNase free DNase treatment on column (Qiagen).
Cell lines were fixed for 10 min in 4% paraformaldehyde/HEPES on ice. The excess of aldehydes is quenched for 5 min in 50 mM NH4Cl. After washing twice for 5 min in 1x PBS, extraction is performed for 5 min in acetone (20°C). The cells are washed again twice for 5 min in 1x PBS, followed by blocking in 0.2% Fish Skin Gelatine (FSG, Sigma)/PBS. During 60 min cells are incubated with Alexa 594-phalloidin (1 unit per section), dissolved in 0.2%FSG/PBS at 37°C. Cells are washed twice for 5 min in 1x PBS; nuclei are stained for 1 min with DAPI; sections are washed with 1x PBS and mounted in Vectashield.
ArrayCGH using 1 Mb BAC arrays was performed once for NGP.1A.TR1, MCH574c3 and MCH574c10 cells with normal female DNA as control. In addition, subclones MCH574c3 and MCH574c10 were hybridised to the same arrays with NGP.1A.TR1 DNA as control. Hybridisation of cell line and control DNA to the array was performed as described . Using our in-house developed analysis and visualisation software, arrayCGHbase, data were normalised to the median ratio, and replicate median ratio profiles visualised http://medgen.ugent.be/arrayCGHbase .
FISH and microsatellite marker analysis
BAC clones and microsatellite markers were selected based on their chromosomal position using the Ensembl genome browser http://www.ensembl.org, the UCSC human genome browser (July 2003 freeze, http://genome.ucsc.edu) or the Genome Database http://gdbwww.gdb.org. Labelling and FISH (fluorescence in situ hybridisation) was performed as described . Experimental conditions for the fluorescent based microsatellite screening can be obtained from the authors upon request.
Real-time quantitative RT-PCR based mRNA expression profiling
Primers were designed using Primer Express v2.0 (Applied Biosystems). Primer sequences are available in the public RTPrimerDB database http://medgen.UGent.be/rtprimerdb/: HNT (1078), OPCML (1079), JAM3 (1080), THY28 (1084), ACAD8 (1081), B3GAT1 (1082), HPRT1 (5), UBC (8) and GAPD (3) . Relative expression levels were determined using an optimized two-step SYBR Green I RT-PCR assay . PCR reagents were obtained from Eurogentec as SYBR Green I core reagents, prepared as 2x mastermixes, stored at -20°C and used according to the manufacturer's instructions. Reactions were run on an ABI5700 (Applied Biosystems). The comparative CT method was used for quantification. Gene expression levels were normalized using the geometric mean of the 3 most stable internal control genes in neuroblastoma (i.e. UBC, HPRT1 and GAPD) as reported previously .
- arrayCGH =:
microarray based comparative genomic hybridisation
- FISH =:
fluorescence in situ hybridisation
- MMCT =:
microcell mediated chromosome transfer
- SRO =:
shortest region of overlap
We greatly acknowledge Peter Degrave and Geert De Vos for the cell cultures, Anouck Waeytens and Isabel Rottiers for phalloidin staining, and Helén Nilsson for providing us with RNA from NTRK1-SH-SY5Y cells that were treated with differentiation inducers.
This text presents research results of the Belgian program of Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The scientific responsibility is assumed by the authors. KDP, JV and WW are supported by a post-doctoral grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT). NVR is a post-doctoral researcher with the FWO. This work was supported by FWO-grant G.0028.00, VEO-grant 011V1302, BOF-grant 011F1200 and 011B4300, and GOA-grant 12051203.
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