Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

Abstract

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.

Figure 1

Array CGH based haploid copy number of SSH clones mapping on chromosome 2: Base position of the SSH clones on chromosome 2 (with exception of fusion transcript clone g2h10) was determined according to the human genome browser at UCSC (April 2003 freeze [33]). Two clear amplification sites along the short arm emerge. Insert: detail of the array CGH (IMR-32 in red and control DNA in green), amplified clones are indicated.

Figure 2

Genomic position of known genes and 2p amplified SSH clones: These results were obtained by a human BLAT search (UCSC genome browser, April 2003 freeze [33]) (clones that were present on the microarray are marked in blue; RefSeq genes are marked in red and the initially misannotated gene NAG in grey). A: amplicon on chromosome band 2q13.3-14; B and C: amplicon on chromosome band 2p24.3 (acc. no. of SSH clone sequences between brackets).

Figure 3

FISH based visualisation of MYCN co-amplification with other genes on 2p in neuroblastoma cell line IMR-32: Amplification is present under the form of homogeneously staining regions. MYCN (in red) in combination with BAC clone RP11-85D18 (TEM8) (in green). Similar results (data not shown) were obtained with clone RP11-444B4 (MEIS1), clone RP11-314E10 (NSE1 and g10d12), clone RP11-422A6 (DDX1) and clone RP11-516B14 (NAG).

Figure 4

Relative expression levels obtained by real-time quantitative RT-PCR: Relative mRNA expression levels obtained by quantitative PCR in 30 neuroblastoma cell lines and 9 normal human tissue samples (samples with gene amplification are marked in red) (relative scale, rescaled to an average expression level of 1).