1: BMC Genomics. 2004 Feb 3;5(1):11. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons. De Preter K, Pattyn F, Berx G, Strumane K, Menten B, Van Roy F, De Paepe A, Speleman F, Vandesompele J. Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Gent, Belgium. katleen.depreter@ugent.be BACKGROUND: Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. RESULTS: As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. CONCLUSIONS: The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells. PMID: 15018647 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Cancer Lett. 2003 Feb 10;190(1):79-87. Characterization of the gene expression profile of neuroblastoma cell line IMR-5 using serial analysis of gene expression. Fischer M, Berthold F. Department of Pediatric Oncology, University Children's Hospital of Cologne, Joseph-Stelzmann-Strasse 9, D-50924 Cologne, Germany. matthias.fischer@medizin.uni-koeln.de The serial analysis of gene expression (SAGE) technique was used to generate a database of the most abundant transcripts of the MYCN-amplified neuroblastoma cell line IMR-5. A total of 8568 tags were sequenced and shown to represent 4034 unique tags, each of which corresponds to an individual transcript. Expression levels of genes are reflected by the frequency of occurrence of the respective tags. To validate fidelity of SAGE data, relative abundances of seven transcripts were evaluated by semiquantitative reverse transcriptase-polymerase chain reaction. Transcripts that were detected nine times or more (>0.1% of the total tag population) accounted for 36% of the total messenger RNA mass but only 3% of the total number of individual transcripts. A strong preponderance of genes involved in protein synthesis, in particular those encoding for ribosomal proteins, were observed among these high-abundance transcripts. Tags corresponding to the amplified gene DDX1 were conspicuously overrepresented in comparison to the other amplified genes MYCN, neuroblastoma amplified gene and MEIS1, which suggests an additional mechanism apart from genomic amplification contributing to the strong upregulation of this gene. This study provides a comprehensive gene expression profile of neuroblastoma cell line IMR-5 and may be used as a reference database for identification of candidate genes that are involved in etiology and pathogenesis of neuroblastoma. PMID: 12536080 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Eur J Cancer. 2000 Dec;36(18):2368-74. The homeobox gene MEIS1 is amplified in IMR-32 and highly expressed in other neuroblastoma cell lines. Jones TA, Flomen RH, Senger G, Nizetic D, Sheer D. Human Cytogenetics Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK. Neuroblastoma is a childhood tumour of the sympathetic nervous system that demonstrates striking clinical heterogeneity. In order to determine which genes are abnormally expressed in neuroblastoma, we screened regions of amplification from the short arm of chromosome 2 in the neuroblastoma cell line IMR-32 and found that the homeobox gene, myeloid ecotropic integration site 1 (MEIS1), is highly amplified. MEIS1 normally maps to chromosome band 2p14. High expression of MEIS1 without amplification was also found in other neuroblastoma cell lines, with and without MYCN amplification, and in medulloblastoma and crythroleukaemia cell lines. MEIS1 is highly expressed in cerebellum and ubiquitously expressed in normal immunohaematopoietic tissues and is thought to be important in cell proliferation and differentiation. While several lines of evidence point towards a role for homeobox genes in the development of other malignancies, this is the first report showing the amplification of a homeobox gene in neuroblastoma. Publication Types: Case Reports PMID: 11094311 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------