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Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

Bax DA, Little SE, Gaspar N, Perryman L, Marshall L, Viana-Pereira M, Jones TA, Williams RD, Grigoriadis A, Vassal G, Workman P, Sheer D, Reis RM, Pearson AD, Hargrave D, Jones C - PLoS ONE (2009)

Bottom Line: All lines proliferate as adherent monolayers and express glial markers.Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways.Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.

View Article: PubMed Central - PubMed

Affiliation: Paediatric Oncology, The Institute of Cancer Research, Sutton, United Kingdom.

ABSTRACT

Background: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines.

Principal findings: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.

Significance: These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

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Related in: MedlinePlus

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).
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pone-0005209-g002: 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).

Mentions: Affymetrix 500K SNP array analysis revealed all paediatric glioma cell lines to display complex genomes with numerous gross chromosomal copy number abnormalities and rearrangements. After exclusion of known copy number variants, this high-resolution copy number profiling additionally highlighted a number of focal copy number aberrations, which were confirmed by FISH on metaphase preparations (Figure 2). The glioblastoma cell line SF188 harboured high-level amplifications of several oncogenes known to be present in paediatric glioblastoma tumour samples. These included MYC at 8q24, CCND1 at 11q13, CDK4 at 12q14, all of which were due to extrachromosomal double minute formation, and overexpression of the corresponding proteins was 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 amplifications, 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.


Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

Bax DA, Little SE, Gaspar N, Perryman L, Marshall L, Viana-Pereira M, Jones TA, Williams RD, Grigoriadis A, Vassal G, Workman P, Sheer D, Reis RM, Pearson AD, Hargrave D, Jones C - PLoS ONE (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666263&req=5

pone-0005209-g002: 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).
Mentions: Affymetrix 500K SNP array analysis revealed all paediatric glioma cell lines to display complex genomes with numerous gross chromosomal copy number abnormalities and rearrangements. After exclusion of known copy number variants, this high-resolution copy number profiling additionally highlighted a number of focal copy number aberrations, which were confirmed by FISH on metaphase preparations (Figure 2). The glioblastoma cell line SF188 harboured high-level amplifications of several oncogenes known to be present in paediatric glioblastoma tumour samples. These included MYC at 8q24, CCND1 at 11q13, CDK4 at 12q14, all of which were due to extrachromosomal double minute formation, and overexpression of the corresponding proteins was 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 amplifications, 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.

Bottom Line: All lines proliferate as adherent monolayers and express glial markers.Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways.Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.

View Article: PubMed Central - PubMed

Affiliation: Paediatric Oncology, The Institute of Cancer Research, Sutton, United Kingdom.

ABSTRACT

Background: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines.

Principal findings: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.

Significance: These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

Show MeSH
Related in: MedlinePlus