Human Molecular Genetics, 2000, Vol. 9, No. 5 757-763
© 2000 Oxford University Press
Retention of imprinting of the human apoptosis-related gene TSSC3 in human brain tumors
University Hospital Eppendorf, Department of Neurosurgery, Hamburg, Germany, 1Sequenom GmbH, Hamburg, Germany, 2University Children’s Hospital, Department of Haematology and Oncology, Cologne, Germany and 3German Cancer Research Center, Division of Cytogenetics, Heidelberg, Germany
Received 22 November 1999; Revised and Accepted 26 January 2000.
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ABSTRACT |
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Genomic imprinting is the result of a gamete-specific modification leading to parental origin-specific gene expression in somatic cells of the offspring. Several embryonal tumors show loss of imprinting of genes clustered in human chromosome 11p15.5, an important tumor suppressor gene region, harboring several normally imprinted genes. TSSC3, a gene homologous to mouse TDAG51, implicated in Fas-mediated apoptosis, is also located in this region between hNAP2 and p57KIP2. TSSC3 is the first apoptosis-related gene found to be imprinted in placenta, liver and fetal tissues where it is expressed from the maternal allele in normal human development. This study investigated the imprinting status of TSSC3 in human normal, adult brain and in human neuroblastomas, medulloblastomas and glioblastomas. A polymorphism in exon 1 at position 54 was used to analyze the allelic expression of the TSSC3 gene by a primer oligo base extension (PROBE) assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found that the TSSC3 gene is not imprinted in human normal, adult brain and blood. In contrast, strong allelic bias resembling imprinting could be detected in most examined tumor specimens. The results demonstrate for the first time that the tumors under investigation are associated with a retention of imprinting of a potential growth inhibitory gene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Epigenetic alterations to gene function are important in tumorigenesis (1). Of particular interest is genomic imprinting, a process where the two parental alleles of an autosomal gene are differentially expressed (2). A number of embryonal tumors show loss of imprinting (LOI) of genes located on human chromosome 11p15.5 (3,4). In many childhood tumors, such as nephroblastoma, rhabdomyosarcoma (5) and medullo- blastoma (6), but also in adult tumors such as gliomas, LOI of IGF2 and H19 has been observed (7,8). In contrast, normal monoallelic expression of both IGF2 and H19 has been demonstrated in neuroblastoma (9). Several novel imprinted genes (p57KIP2, KvQTL1, TSSC3) have recently been identified in chromosomal region 11p15.5. The LOI of several genes in this region occurs in Beckwith–Wiedemann syndrome (10) and a number of childhood tumors. Therefore, the importance of imprinting in oncogenesis is quite apparent.
TSSC3, a homologue of the mouse TDAG51 gene, is the first apoptosis-related gene found to be imprinted. Lee and Feinberg (11) found the maternal allele expressed in normal human development, except in heart and testis of one specimen. However, two postnatal kidney samples showed biallelic expression. In contrast, Qian et al. (12) found TSSC3 imprinted only in placenta and liver. The mouse homologue is essential for Fas (CD95, Apo-1) expression and susceptibility to apoptosis in a T lymphocyte cell line (13). The expression of CD95 and its ligand (CD95L) has been well investigated in glioblastomas where the expression increases from low grade to anaplastic astrocytoma and is highest in perinecrotic areas of glioblastoma (14,15). Although glioma cells coexpress CD95 and CD95L they do not undergo apoptosis. Fas is also expressed in primary neuroblastoma and medulloblastoma cell lines (16,17). The observations described above led us to examine whether the imprinting of TSSC3, a potential factor influencing the Fas-mediated apoptotic pathway, is changed in the different tumor specimens examined.
We analyzed the imprinting status of the TSSC3 gene using a primer oligo base extension (PROBE) assay combined with subsequent analysis of the products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Since its introduction (18), MALDI-TOF MS has emerged as a widely applicable method in molecular biology for fast and highly accurate DNA analysis (19,20) and its applicability in DNA diagnostics has been demonstrated by various groups (21–24).
Here this combination was applied the first time to the analysis of allele-specific expression patterns of a gene.
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RESULTS |
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Analysis of the imprinting status of the TSSC3 gene is based on a transcribed polymorphism at nucleotide 54 (Fig. 1) resulting in two differently sized extension products in the PROBE reaction (Table 1) referred to as C and T alleles, which then are detected by MALDI-TOF MS. Using this assay, we searched for tumor and cell line samples that are heterozygous for this polymorphism and thus are informative for imprinting analysis. All imprinting analysis was performed in duplicate to validate the resulting allele distributions.
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TSSC3 gene imprinting in human adult brain and blood
We analyzed three informative human normal, adult brain samples and five adult blood samples for imprinting of the TSSC3 gene. In all cases the TSSC3 gene was not imprinted and both alleles were expressed equally. A representative result of the imprinting analysis for human normal adult brain is shown in Figure 2. The even peak heights of the PROBE products specific for the T and C alleles (Fig. 2a), reflect their 1:1 allelic ratio at the genomic DNA level. All experiments were repeated twice to validate that the distribution of alleles is not affected by preferential amplification of one allele. Under this assumption any changes in the ratio of peak heights between C and T alleles analyzed at the cDNA level can be explained by changes in the expression profile. To evaluate the ability of our mass spectro- metric PROBE assay to detect biased allelic expression, C and T allele-specific PCR products were amplified from respective homozygous samples and mixed in different ratios. As can be deduced from Figure 3, the assay system enables the clear identification of the C allele even in a 1:30 mixture of C and T alleles. Accordingly, samples revealing an asymmetric peak intensity ratio of the alleles were classified as showing biased allelic expression and only those samples were classified as imprinted, where no mass signal representing either the C or T allele could be detected.
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Human neuroblastoma (primary tumors and cell lines)
Seven of twenty neuroblastomas analyzed were informative. Four (57%) of these showed reduced expression of the C allele, and in one of these cases (14%) the TSSC3 gene was completely imprinted. Of the 24 cell lines analyzed, 11 were informative. Four cell lines (36%) showed a reduced expression of the C allele and in one cell line the TSSC3 gene was totally imprinted. Typical assay results for neuroblastoma cell lines with a clearly reduced expression of the C allele are given together with the results of the respective genomic DNAs in Figure 4.
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Human medulloblastoma cell lines
All of the four cell lines analyzed were heterozygous for the C/T polymorphism. Imprinting of the TSSC3 gene was found in three (75%) of these cell lines. Only one cell line showed biallelic expression. A representative assay result for an imprinted medulloblastoma cell line is shown with the result from the respective genomic DNA in Figure 5a and b.
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Human glioblastoma/mixed glioma and glioma-derived cell lines
We examined 31 primary tumors and 26 cell lines for heterozygosity of the C/T polymorphism at nucleotide 54. Of 31 samples of glioblastomas/mixed gliomas 13 were informative. Eight cases (61%) showed biallelic expression of the TSSC3 gene, whereas five cases (39%) revealed clearly reduced expression of the C allele.
Of the 26 glioma cell lines analyzed, only four cases were heterozygous for the polymorphism. Three of them showed imprinting of the TSSC3 gene and one a reduced expression of the C allele. Thus, biased allelic expression was detected in 100% of the informative samples. A representative result for an imprinted glioblastoma cell line is given in Figure 5c and d.
Effect of DNA methylation-inhibiting agents
To determine whether imprinting or preferential expression of one allele of the TSSC3 gene depends on an unusual methylation pattern and can be reversed by altering DNA methylation, we treated the informative neuroblastomas, medulloblastomas and glioblastoma cell lines with 5-Aza-2'-deoxycytidine (5-azaCdR), a specific inhibitor of DNA methylation. The treatment with 5-azaCdR did not affect the status of imprinting in all cell lines tested.
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DISCUSSION |
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The TSSC3 gene maps to human chromosome 11p15.5. This region was not subject to genetic rearrangements in the tumor specimens analyzed, but it includes a domain with several genes subject to parental imprinting (11). The imprinting analysis of IGF2 and H19, two genes in this region, revealed a correlation between altered imprinting and tumor development (1). IGF2 is an autocrine growth factor, which is normally expressed from the paternal allele, whereas H19, an untranslated RNA expressed from the maternal allele, is thought to have a growth-inhibitory effect. IGF2 showed LOI in several analyzed glioblastomas, whereas H19 imprinting remained unchanged in these specimens (8). Thus, evidence for an association between LOI and development of human glioma was provided. Examination of the imprinting status of the IGF2 and H19 gene in a comparable series of other central nervous system tumors also showed the previously reported LOI of IGF2, which further indicates that this region is of specific interest in the relationship between imprinting status and tumorigenesis (S. Müller et al., unpublished data).
Within 11p15.5, TSSC3 is located between the known non-imprinted gene hNAP2 and the imprinted gene p57KIP2. Knowledge of the imprinting status of TSSC3 would be useful to define a boundary between imprinted and non-imprinted regions on 11p15.5. Even more interesting is the fact that TSSC3 is homologous to the mouse TDGA51, a gene implicated in Fas-mediated apoptosis. Analysis of the allelic expression pattern of this gene in fetal tissue revealed imprinting in almost all fetal tissues, except in heart and testis of one specimen. It was preferentially expressed from the maternal allele. Thus, TSSC3 was the first apoptosis-related gene found to be imprinted in normal development (11).
We analyzed imprinting of the TSSC3 gene in human normal, adult brain and blood, and found that TSSC3 expression is biallelic, and therefore the gene not imprinted in these tissues.
In contrast to this biallelic expression, the analyzed neuroblastoma, medulloblastoma and glioblastoma cell lines, as well as the primary tumors of glioblastomas and neuro- blastomas, showed either monoallelic expression of the TSSC3 gene or at least strong preferential expression from one allele in many cases (Table 2). Without exception, imprinting as well as allelic bias specifically diminished the C allele. Since the parental origin could not be determined as samples from the corresponding parents were not available, the significance of the restriction to the C allele remains to be explored.
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The percentage of tumors with imprinting or preferential expression from one allele of TSSC3 ranged from 38 to 100%. Thus, this report shows for the first time that a retention of imprinting of the apoptosis-related gene TSSC3 may be functionally significant in tumorigenesis and supports the notion that tumor development could be coupled to suppression of TSSC3 expression.
How can the retention of imprinting of the TSSC3 gene participate in tumor development?
The mouse homologue of TSSC3, TDAG51, is essential for Fas (CD95) receptor expression and therefore appears to be implicated in Fas-mediated apoptosis. It restored CD95 expression and susceptibility to apoptosis in a T lymphocyte cell line showing resistance to apoptosis as a result of a mutation in this gene (13). Furthermore, it was shown that CD95 and CD95L are co-expressed in gliomas, but the respective cells do not undergo suicide or fratricide through apoptosis (15). The expression of the CD95 ligand in tumor cells provides a way to escape the immune system by simply inducing apoptosis in T cells. That these tumor cells do not undergo suicide or fratricide at the same time can be explained if the expression of Fas is significantly reduced. The role of TSSC3 within this scheme could be seen as a factor influencing the expression of Fas. Retention of imprinting of TSSC3 in tumors, as revealed in this work, could then explain the silencing of the apoptotic pathway through a reduction of the Fas expression. Little is yet known about the protein encoded by the TSSC3 gene, but the homology to a mouse gene, which is essential for the expression of the Fas receptor, supports a possible role of TSSC3 in the regulation of CD95 expression.
In early reports about genomic imprinting a correlation between function of the gene within cell growth and development and imprinting was mentioned (11). According to this, LOI should be associated with genes having a potential growth-promoting effect, whereas loss of heterozygosity or imprinting should be of relevance for genes with a potential growth-inhibitory effect and for tumor suppressor genes (1). With the participation in Fas expression and thus in the apoptotic pathway, the TSSC3 gene has a potential growth-inhibitory effect. Retention of imprinting of TSSC3 in tumor development is therefore reasonable.
One of the mechanisms thought to control imprinting is methylation of cytosine within CpG dinucleotide islands. This could be essential for genomic imprinting as the methylation pattern probably marks the parental chromosomes differ- entially (25). Thus, altering the methylation patterns through inhibition of DNA methyltransferases, for example using 5-azaCdR, should have an effect on the allelic expression of imprinted genes. Indeed, the treatment of abnormally imprinted cells with 5-azaCdR showed that they are susceptible to epigenetic modification. Concerning the IGF2 gene, this treatment reversed the LOI of IGF2 to predominant expression of the paternal allele in the tumor cell lines analyzed. A complete imprinting of IGF2 in these cell lines was nevertheless not achieved by 5-azaCdR. The expression of H19 in the tumor cell lines analyzed switched as a consequence of the treatment from biallelic expression of H19 to mono- allelic (26). Hu et al. (27) showed that normal brain cells, after treatment with 5-azaCdR, switch from a normally monoallelic to a biallelic expression of IGF2. We analyzed several different cell lines for changes in the imprinting of the TSSC3 gene using treatment with 5-azaCdR, but in none of these cases did we observe any effect on the allelic expression of the TSSC3 gene. Thus, our results revealed that the imprinting status of the TSSC3 gene could not be influenced through the inhibition of DNA methylation with 5-azaCdR, and further investigations of the mechanisms will be necessary.
Of particular interest is the method used in this study. The combination of PROBE and MALDI-TOF MS allowed a clear determination of expression levels between maternal and paternal alleles up to a ratio of 1:30. The dynamic range of detectable expression ratios is extended significantly compared with conventional methods, which are sensitive only at ratios of 1:4 to 1:6. Thus, conventional methods could fail to detect subtle epigenetic modifications of imprinted genes involved in malignancies. The new MALDI-TOF MS-based method for the analysis of imprinting may possibly facilitate further studies on functional imprinting in growth regulation and elucidate the role of imprinted genes in oncogenesis. Nevertheless, further studies are necessary to clarify how such detected mRNA expression ratios translate to the protein level.
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MATERIALS AND METHODS |
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Cell cultures
Glioma cell lines (28) and medulloblastoma cell lines (obtained from T. Pietsch, University of Bonn, Germany) were maintained in Earle’s minimum essential medium, supplemented with 2 mM glutamine, 1 mM pyruvate, 2.5 mmol/ml amphotericin B (Gibco, Eggenstein, Germany), 40 mg/ml gentamycin (Merck, Darmstadt, Germany) and 10% fetal calf serum (FCS; Biochrom, Berlin, Germany). Neuroblastoma cell lines were maintained in RPMI 1640 medium (Gibco) supplemented with 10% FCS, 2.5 mmol/ml amphotericin B and 40 mg/ml gentamycin.
Tumor samples and RNA/DNA isolation
Neuroblastomas were collected at the Department of Hematology/Oncology at the Children’s University Hospital Cologne and glioblastomas at the Department of Neuro- surgery, University Hospital Eppendorf of Hamburg. All surgical specimens were frozen quickly in liquid nitrogen and kept at –80°C. Cells were grown to confluence in 100 mm dishes as described above.
Total RNA of cells and tumor samples was isolated by homogenization with Tri Star Reagent (acid-guanidinium method), followed by phase separation with chloroform and precipitation with isopropanol. The RNA was treated with RNase-free DNase (Boehringer Mannheim, Mannheim, Germany) for at least 3 h. Reverse transcription was carried out using the ‘Ready to go’ kit (Pharmacia, Uppsala, Sweden), random primed.
Genomic DNA of tissue and cells was isolated with the Qiamp Tissue kit (Qiagen, Düsseldorf, Germany) following the protocol provided by the manufacturer.
Polymerase chain reaction
The following primers flanking the polymorphism at nucleotide 54 were used: primer sense CCC GCG CTC GGC ACG ACA TGA AAT CC and antisense Biotin-GGG AAC AGG CTC AGG CGG TCG GAG GTG. The PCR reactions contained 100 ng of cDNA or genomic DNA, 0.2 U of AmpliTaq Gold Polymerase (Perkin Elmer, Weiterstadt, Germany), 0.2 mM Jump Start dNTPs, 1x PCR buffer, 20 pmol sense primer and 10 pmol antisense in 20 µl and were performed in a thermal cycler (Perkin Elmer) at temperatures as follows: 94°C for 16 min, 40 cycles of 94°C for 1 min, 64°C for 1 min and 72°C for 1 min, followed by 72°C for 10 min. One-tenth of the reaction was size separated by a 2% agarose gel electrophoresis to check the quality of the PCR products beforehand.
Template immobilization
To the unpurified PCR product, 0.200 mg of paramagnetic streptavidin beads (Dynal, Oslo, Norway) in 20 µl of 2x B/W buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 2 M NaCl) were added and incubated for 20 min at room temperature. The beads were then treated twice with 10 µl of 25% NH4OH for 2 min at room temperature, followed by three washes with 50 µl of 10 mM Tris–HCl pH 8.0. All steps were performed using the Dynal Magnetic Particle Collector.
PROBE reaction
Dideoxy termination reactions were carried out by adding 15 µl of reaction mix containing 1x reaction buffer (80 mM Tris–HCl pH 8.0, 2 mM MgCl2), 20 pmol of PROBE primer d(GAC GAG GTG CTA CGC GAG GGC), 50 µM of three dNTPs and one ddNTP, and 2.5 U of AmpliTaq FS. The reaction mixture was subjected to a non-cycling temperature profile comprising 80°C for 1 min, 55°C for 5 min and 72°C for 3 min. The reaction was allowed to cool to room temperature. PROBE products were conditioned by washing the beads twice with 50 µl of 10 mM Tris–HCl pH 8.0 and separated from the immobilized template by incubation of the beads with 5 µl of 25% NH4OH for 5 min at room temperature. The ammonium hydroxide supernatant was separated from the beads and transferred to a new tube.
MALDI-TOF MS measurement
A 600 nl aliqout of sample was pipetted onto the MALDI sample holder and mixed immediately with 850 nl of matrix [saturated solution (~0.5 M) of 3-hydroxypicolinic acid in 50% acetonitrile, 70 mM ammonium citrate]. This mixture was dried at ambient temperature and introduced into the mass spectrometer. All spectra were recorded in positive ion mode on a Bruker Reflex III DE mass spectrometer (Bruker, Bremen, Germany). Spectra were smoothed by a 15 point average (Savitsky–Golay method) and baseline correction was performed. Calibration was done externally using an oligonucleotide mixture.
Treatment with 5-azaCdR
Cells were cultered in 100 mm dishes as described above and were treated with 0.06–2 mM 5-azaCdR (Sigma, Deisenhofen, Germany). Samples were taken after 24, 72 and 120 h. Then the cells were washed in phosphate-buffered saline twice. Genomic DNA and RNA were isolated as described. The imprinting status of each sample was compared with the status in the untreated cell line.
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ACKNOWLEDGEMENTS |
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We thank Charles R. Cantor for valuable comments on the manuscript. This work was supported by the Werner-Otto-Stiftung Hamburg, Deutsche Forschungsgemeinschaft, Graduiertenkolleg Scha692/1-1 and the Kinderkrebshilfe Heidelberg. This work contains major parts of the doctoral thesis of S. Müller, submitted to the Fachbereich Medizin, University of Hamburg.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Universitätskrankenhaus Eppendorf, Neurochirugische Klinik, Martinistrasse 52, 20246 Hamburg, Germany. Tel: +49 40 42803 3751; Fax: +49 40 42803 4596
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