Profiling Paediatric Gliomas Figure 1. Immunophenotyping of paediatric glioma cell lines. All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, vimentin, synaptophysin, nestin and CD133. H&E – haematoxylin and eosin. All images original magnification 6400. doi:10.1371/journal.pone.0005209.g001 confirmed by Western blotting (data not shown). There was furthermore a focal deletion of NF1 at 17q11.2. KNS42 glioblastoma cells, by contrast, contained no genuine amplifica- tions, but instead its highly rearranged genome harboured low- level copy number gains at loci such as 3q26 (PIK3CA), and hemizygous deletions at known tumour suppressor loci such as 13q14 (RB1). FISH analysis confirmed that this was due to the loss of a single copy from an otherwise triploid genome. Homozygous deletion at the CDKN2A/B locus at 9p12 was observed in the anaplastic astrocytoma UW479 line, which additionally harboured numerous high level copy number changes including 3p11-p12, 6p21, 18p11 and 19q12. CDKN2A/B deletion was also observed in Res259 astrocytoma cells, which also contained the well-described 4q12 amplicon, resulting in high level gains of the oncogenes PDGFRA and KIT, and low-level gain of a region including KDR/ VEGFR2. This was found from FISH and M-FISH analysis to be due to an unbalanced translocation between chromosomes 4 and 19, resulting in a der(19)t(4;19). Finally, Res186, originally derived from a pilocytic astrocytoma, harboured the least complex genomic profile, although was found to have an intragenic homozygous deletion of PTEN at 10q23. Copy neutral and epigenetic events in paediatric high grade glioma cells In addition to the copy number changes, we also observed several copy neutral loss of heterozygosity (LOH) events (Figure 2). KNS42 cells exhibited focal copy neutral LOH at 19p13 (KLF2) and 21q21 (HEMK2), whilst larger regions were observed in UW479 cells at chromosome 2q11-qter and in Res186 at 7p11- pter and 17p13 (TP53). Although the significance of these events is not clear, it is expected that such loci may harbour genes in which the remaining allele may harbour a point mutation, as is the case for TP53. In order to explore epigenetic events in these lines we carried out methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) on 39 genes known to be hypermethy- lated at CpG islands in human cancer. Certain genes such as RASSF1A and ASC were found to be methylated in all cell lines tested (Table 2). There was specific promoter hypermethylation in FHIT in SF188 and Res259, HIC1 in Res259 and TP73 in Res186 cells. Interestingly, UW479 additionally harboured extensive promoter hypermethylation at a host of genes assayed including APC, CASP8, CD44, CDH13, CHFR, ESR1, GSTP1, IGSF4, MGMT, PAX5A, PAX6, and RARB. Of direct relevance to clinical resistance to alkylating agents were the observations of methyla- tion in the mismatch repair gene MLH3 in Res259 cells, which also harboured high levels of promoter hypermethylation of MGMT, along with KNS42 cells. Low to moderate MGMT methylation was also observed in UW479 and Res186 cells. Differential constitutive activation of PI3K, MAPK and downstream pathways Protein expression was measured by Western blot to examine constitutive pathway activation of several key signalling transduc- tion networks of importance in gliomagenesis (Figure 3). There was a modest activation of the MAPK pathway as determined by levels of phosphorylated Erk 1/2 in the paediatric high grade lines SF188 and UW479 as compared to KNS42 and the low grade lines (Res259, Res186). Although Akt pathway activation was seen in all paediatric cells, this was considerably lower than that observed in our panel of adult glioblastoma lines, except in the PTEN line Res186, which showed high levels of phospho-Akt. Phosphorylated GSK3b levels were low in UW479, Res259 and Res186, and almost entirely absent in the glioblastoma lines SF188 and KNS42. Although there was some degree of ribosomal protein PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5209