Profiling Paediatric Gliomas Figure 2. Genomic profiling of paediatric glioma cell lines. Copy number and loss of heterozygosity (LOH) profiles were generated by Affymetrix 500K SNP arrays. Log2 ratios are plotted (y axis) for each probeset according to chromosomal location (x axis). Loss (blue) and retention (yellow) of heterozygosity is depicted in the lower portion of the plots. Fluorescent in situ hybridisation validation of selected copy number changes is represented with clones for CDK4, RB1, CDKN2A/B, PDGFRA and PTEN (lCy5/SpectrumOrange) and appropriate centromeres (fluorescein/ SpectrumGreen). doi:10.1371/journal.pone.0005209.g002 Table 2. Methylation-specific multiplex ligation-dependent probe amplification of 39 genes in paediatric glioma cell lines. SF188 KNS42 UW479 Res259 Res186 Gene ASC APC ATM BRCA1 BRCA2 CASP8 CD44 CDH13 CDKN1B CDKN2A CDKN2B CHFR DAPK1 ESR1 FHIT GATA5 GSTP1 HIC1 IGSF4 MGMT MLH1 MLH3 MSH2 MSH3 MSH6 PAX5A PAX6 PMS2 PTEN RARB RB1 RASSF1A STK11 THBS1 TIMP3 TP53 TP73 VHL WT1 52% 2% 0% 1% 0% 1% 0% 2% 0% 2% 3% 0% 5% 25% 62% 10% 3% 2% 0% 11% 1% 0% 0% 0% 0% 43% 0% 0% 4% 5% 5% 95% 0% 5% 0% 1% 0% 0% 0% 66% 2% 2% 1% 0% 3% 2% 24% 0% 1% 4% 1% 0% 51% 0% 0% 5% 2% 0% 79% 1% 0% 0% 0% 0% 46% 0% 0% 1% 4% 0% 90% 2% 9% 16% 4% 0% 0% 88% 52% 100% 1% 2% 0% 71% 48% 94% 0% DEL DEL 81% 0% 31% 0% 14% 75% 7% 44% 26% 1% 0% 0% 3% 0% 99% 100% 3% 1% 56% 0% 100% 0% 9% 0% 3% 0% 0% 7% 81% 1% 1% 3% 0% 1% 1% 4% 0% DEL DEL 0% 0% 74% 56% 86% 0% 92% 0% 63% 1% 70% 0% 5% 7% 53% 0% 3% 2% 2% 0% 100% 0% 10% 1% 1% 0% 0% 69% 100% 2% 0% 3% 0% 3% 0% 0% 0% 3% 4% 0% 0% 55% 2% 94% 70% 0% 0% 57% 2% 0% 0% 7% 0% 100% 0% 0% DEL 0% 23% 100% 0% 0% 2% 22% 43% 0% 21% S6 activation in all lines, this was particularly pronounced in KNS42 and Res259, with very high levels of phospho-S6. Taken together, although variable, we observed constitutive activation of one or more of these signal transduction pathways in all the paediatric glioma lines. Expression signatures of functionally relevant genes distinguishes between high grade paediatric and adult glioma cell lines In order to investigate the differences between the paediatric- and adult-derived cell lines at the gene expression level, we carried out expression profiling using Affymetrix U133 Plus2.0 arrays. For the comparison, we excluded the lines Res259 and Res186, derived from low-grade tumours, in order to avoid the possible Values give percentage methylation at CpG islands in the genes promoter. DEL = gene deletion. n.d = not done. doi:10.1371/journal.pone.0005209.t002 Figure 3. Constitutive activation of key signalling pathways in glioma cell lines. Western blots analysis of c-Raf, phospho/total Erk1/ 2, phospho/total Akt, phospho/total GSK3b, phospho/total S6 and GAPDH as loading control in adult (LN229, A172, U118MG, U138MG, U87MG, SF268) and paediatric (SF188, KNS42, UW479, Res259, Res186) glioma cell lines. doi:10.1371/journal.pone.0005209.g003 PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5209