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Viewed and Published Immediately Upon Acceptance Cited in Pubmed and Archived on Pubmed Central Yours — You Keep the Copyright BMC Cancer BioMed Central Research article Open Access Loss of heterozygosity of TRIM3 in malignant gliomas Jean-Louis Boulay1, Urs Stiefel2, Elisabeth Taylor1, Béatrice Dolder1, Adrian Merlo*1 and Frank Hirth*2,3 Address: 1Department of Biomedicine, University Hospital, CH-4031 Basel, Switzerland, 2Institute of Zoology and Biocenter, University of Basel, CH-4056 Basel, Switzerland and 3MRC Centre for Neurodegeneration Research, King's College London, London, SE5 8AF, UK Email: Jean-Louis Boulay - [email protected]; Urs Stiefel - [email protected]; Elisabeth Taylor - [email protected]; Béatrice Dolder - [email protected]; Adrian Merlo* - [email protected]; Frank Hirth* - [email protected] * Corresponding authors Published: 27 February 2009 Received: 15 September 2008 Accepted: 27 February 2009 BMC Cancer 2009, 9:71 doi:10.1186/1471-2407-9-71 This article is available from: http://www.biomedcentral.com/1471-2407/9/71 © 2009 Boulay et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Malignant gliomas are frequent primary brain tumors associated with poor prognosis and very limited response to conventional chemo- and radio-therapies. Besides sharing common growth features with other types of solid tumors, gliomas are highly invasive into adjacent brain tissue, which renders them particularly aggressive and their surgical resection inefficient. Therefore, insights into glioma formation are of fundamental interest in order to provide novel molecular targets for diagnostic purposes and potential anti-cancer drugs. Human Tripartite motif protein 3 (TRIM3) encodes a structural homolog of Drosophila brain tumor (brat) implicated in progenitor cell proliferation control and cancer stem cell suppression. TRIM3 is located within the loss of allelic heterozygosity (LOH) hotspot of chromosome segment 11p15.5, indicating a potential role in tumor suppression. ... Methods: Here we analyze 70 primary human gliomas of all types and grades and report somatic deletion mapping as well as single nucleotide polymorphism analysis together with quantitative real- time PCR of chromosome segment 11p15.5. Results: Our analysis identifies LOH in 17 cases (24%) of primary human glioma which defines a common 130 kb-wide interval within the TRIM3 locus as a minimal area of loss. We further detect altered genomic dosage of TRIM3 in two glioma cases with LOH at 11p15.5, indicating homozygous deletions of TRIM3. Conclusion: Loss of heterozygosity of chromosome segment 11p15.5 in malignant gliomas suggests TRIM3 as a candidate brain tumor suppressor gene. Background ous infiltration of normal brain tissue. Especially glioblas- Malignant gliomas are brain tumors that represent devas- tomas appear notoriously resistant to therapy, which has tating and difficult-to-treat cancers with a mean patient been attributed to DNA-repair proficiency and deregu- survival of 10 months. Although surgical interventions lated molecular pathways [1,2]. More recently combined allow resection and improve local tumor control, the fur- chemoradiotherapy of concomitant and adjuvant temo- ther course of the disease remains dominated by re- zolomide and radiotherapy has been introduced which appearance of unscheduled cell proliferation and insidi- can lead to significant prolongation of survival, in partic- Page 1 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:71 http://www.biomedcentral.com/1471-2407/9/71 ular in patients with an epigenetically silenced DNA repair astrocytomas grades I to III, and 47 glioblastoma multi- gene [3]. However, the outcome remains poor. This ther- forme [see Additional file 1]. DNA was extracted from apeutic resistance has recently been attributed to tumor fresh frozen primary gliomas and peripheral blood mono- stem-like cells due to their unrestrained self-renewal nuclear cells (PBMCs) derived from the same patients, as capacity and the ability to maintain tumorigenic potential previously described [32,33]. Only material containing at the single cell level, thereby evading both resection and less than 30% residual amounts of non-neoplastic cells radiotherapy [4-7]. There is growing evidence that some was considered for further analysis. brain cancers arise either from normal stem cells or from progenitor cells in which self-renewal pathways have STS- and SNP-based LOH analysis become aberrantly activated [7-10]. Sequence tagged site (STS)-based LOH was performed essentially as described [32]. Briefly, DNA from 70 glioma Drosophila Brain tumor (brat) has been identified as a reg- specimens and PBMCs of the same patients was analysed ulator of progenitor cell proliferation control and cancer for loss of heterozygosity by amplification of microsatel- stem cell suppression [11-18]. Brat is expressed through- lite sequences [32]. Primers for these sequences were out Drosophila brain development and exerts an essential obtained from Microsynth (Balgach, Switzerland). Fluo- gate-keeper function in the binary switch between self- rescence based LOH mapping was employed with DNA renewal and differentiation of neural progenitor cells. from all gliomas. D11S4905 (located in TRIM3 intron 2) Neural progenitor cells mutated for Brat are unable to dif- and D11S1250 primers were FAM-labeled, D1S1318 and ferentiate but rather continue to proliferate, resulting in D11S1758 primers were HEX-labeled, and D11S1331 and brat mutant cells that display characteristic features of can- D11S1997 primers were TET-labeled. PCR product size cer-like stem cells. The resulting brain tumor tissue is char- fractionation and quantification were performed on ABI acterized by pleiomorphic cells, continued proliferation Prism 310 Genetic Analyzer (PE Applied Biosystems, Fos- and chromosome instability, as evidenced by a variety of ter City, CA, USA). The ratio of peak heights of both alleles karyotypic abnormalities [19]. was calculated for each tumor and PBMC DNA sample. For informative cases, allelic loss was scored if the ratio Homologues of brat have been recorded in various spe- between tumor and PBMC DNA was more than 1.5 (1/0) cies, with three human homologues, namely Tripartite or less than 0.66 (0/2). Single nucleotide polymorphism Motif Protein 2 (TRIM2), TRIM3, and TRIM32, located on (SNP)-based LOH was performed with the following chromosome 4q31.3, 11p15.5, and 9q33.1, respectively markers: rs11605881 (TRIM3 promoter), rs11607224 [20-23]. 11p15 represents the telomeric end of chromo- (exon1), rs1060067 (exon4) rs16913748 (exon6), some 11 which shows loss of allelic heterozygosity (LOH) rs11605141 (intron6), rs13343175 (intron7), rs3830325 in various types of tumors, indicating the presence of one (intron9), rs2306897 (exon10) and rs2723636 (exon13). or more tumor suppressor genes [24-27]. Homologues of SNPs were visualized on an ABI Prism 310 Genetic Ana- TRIM3 are primarily expressed in brain and may function lyzer (PE Applied Biosystems, Foster City, CA, USA). at the interface of proliferation and differentiation during the maturation of brain tissue [28-30]. Here we report Quantitative real-time PCR refined deletion mapping of chromosome 11p15.5 in Classification of the genetic status of TRIM3 was per- malignant gliomas. formed by quantitative real-time-PCR using the TaqMan ABI Prism® 7700 Sequence Detection System (PE Applied Methods Biosystems, Foster City, CA, USA) as previously described Biopsies and DNA extraction [34]. Gene-specific primers for the reference-control Tumor samples used in this study were obtained from 70 GAPDH, which is located on chromosome 12p13.3, as patients who underwent glioma resection at the Univer- well as primers for Intron1, Exon 3, Intron 6, Intron 11 sity Hospital of Basel between 1996 and 2005. The collec- and cDNA of TRIM3 were used as follows: tion of tumor samples has been approved by the Ethics Committee of Basel-Land and Basel-Stadt (EKBB). For GAPDH: Informed consent has been obtained together with the patient's permission to conduct open brain surgery, con- 5'-AATGGGACTGAGGCTCCAC (sense), senting to the use of biopsies for anonymous scientific research. This procedure follows the present recommen- 5'-TTATGGGAAAGCCAGTCCCC (antisense). dations of the Swiss Academy of Medical Sciences as pro- posed in 2008 and is in compliance with the Helsinki For TRIM3 Intron 1: Declaration. Tumors were classified according to the revised WHO classification of tumors of the nervous sys- 5'-CCCCAAGGGTGCGTTTGTATT (sense), tem [31], comprising 10 oligodendrogliomas grade II, 13 Page 2 of 9 (page number not for citation purposes) BMC Cancer 2009, 9:71 http://www.biomedcentral.com/1471-2407/9/71 5'-TGCTCTCACGGACATGGACA (antisense). cycles (Cts) obtained for the GADPH gene. In this approach the variable Ct is defined as the fractional cycle For TRIM3 Exon 3: number crossing a fixed threshold of fluorescence that has been generated by cleavage of the probe due to polymer- 5'-GCAGTTCCTGGTATGCAGCAT (sense), ase driven exonucleolytic activity. Differences in the Ct values between two genes were referred to the ΔCt value. 5'-TGCAGGCAAGGAAGAACCTT (antisense). After normalization of the tumor Ct values with the PBMC Ct values, the ΔCtvalues and the relative copy number of For TRIM3 Intron 6: Introns1, 6 and 11, as well
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