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Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19.

ABSTRACT: Nearly 10% of human gliomas are oligodendrogliomas. Deletion of chromosome arm 19q, often in conjunction with deletion of 1p, has been observed in 65-80% of these tumors. This has suggested the presence of a tumor suppressor gene located on the 19q arm. Chromosome 19 deletion is also of interest due to the better prognosis of patients with deletion, including longer survival and better response to chemotherapy, compared with patients without deletion. Two glioma cell lines with deletion of 19q were used for chromosome 19 microcell-mediated transfer, to assess the effect of replacing the deleted segment. Complementation with chromosome 19 significantly reduced the growth rate of the hybrid cells compared with the parental cell lines. Affymetrix U133 Plus 2.0 Gene Chip analysis was performed to measure and compare the expression of the chromosome 19 genes in the chromosome 19 hybrid cell lines to the parental cell line. Probes were considered significantly different when a P value <0.01 was seen in all of the cell line comparisons. Of 345 probes within the commonly deleted 19q region, seven genes (APOE, RCN3, FLJ10781, SAE1, STRN4, CCDC8, and BCL2L12) were identified as potential candidate genes. RT-PCR analysis of primary tumor specimens showed that several genes had significant differences when stratified by tumor morphology or deletion status. This suggests that one or more of these candidates may play a role in glioma formation or progression.

SUBMITTER: Drucker KL

PROVIDER: S-EPMC3190979 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19.

Drucker Kristen L KL Kitange Gaspar J GJ Kollmeyer Thomas M TM Law Mark E ME Passe Sandra S Rynearson Amanda L AL Blair Hilary H Soderberg Cheryl L CL Morlan Bruce W BW Ballman Karla V KV Giannini Caterina C Jenkins Robert B RB

Genes, chromosomes & cancer 20091001 10

Nearly 10% of human gliomas are oligodendrogliomas. Deletion of chromosome arm 19q, often in conjunction with deletion of 1p, has been observed in 65-80% of these tumors. This has suggested the presence of a tumor suppressor gene located on the 19q arm. Chromosome 19 deletion is also of interest due to the better prognosis of patients with deletion, including longer survival and better response to chemotherapy, compared with patients without deletion. Two glioma cell lines with deletion of 19q w ...[more]

PMID: 19544381

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Project description:One sub-project within the global Chromosome 19 Consortium, part of the Chromosome-Centric Human Proteome Project, is to define chromosome 19 gene and protein expression in glioma-derived cancer stem cells (GSCs). Chromosome 19 is notoriously linked to glioma by 1p/19q co-deletions and clinical tests are established to detect that specific aberration. GSCs are tumor-initiating cells and are hypothesized to provide a repository of cells in tumors that can self-replicate and be refractory to radiation and chemotherapeutic agents developed for the treatment of tumors. In this pilot study, we performed RNA-Seq, label-free quantitative protein measurements in six GSC lines, and targeted transcriptomic analysis using a chromosome 19 specific microarray in an additional 6 GSC lines. Here, we present insights into differences in GSC gene and protein expression, including the identification of proteins listed as having no or low evidence at the protein level (such as small nuclear ribonucleoprotein G-like protein, RUXGL_HUMAN), as correlated to chromosome 19 and GSC subtype. Furthermore, the upregulation of proteins downstream of adenovirus-associated viral integration site 1 (AAVS1) in GSC11 in response to oncolytic adenovirus treatment was demonstrated. Taken together, our results may indicate new roles for chromosome 19, beyond the 1p/19q co-deletion, in the future of personalized medicine for glioma patients. Data analysis: MS files (.raw) were imported into Progenesis LC-MS (version 18.214.1528, Nonlinear Dynamics) for m/z and retention time alignment. The top 5 spectra for each feature were exported (charge deconvolution, top 1000 peaks) as a combined .mgf file for database searching in PEAKS (version 6, Bioinformatics Solutions Inc., Waterloo, ON) against the UniprotKB/Swissprot-Human database (July 2013 version, 20,264 proteins), appended with the cRAP contaminant database. PEAKS DB and Mascot (version 2.3.02, Matrix Science) searches were performed with a parent ion tolerance of 10 ppm, fragment ion tolerance of 0.025 Da, fixed carbamidomethyl cysteine, and variable modifications of oxidation (M), phosphorylation (STY), and deamidation (NQ). Trypsin was specified as the enzyme, allowing for 2 missed cleavages and a maximum of 3 PTMs per peptide. An additional search for unexpected modifications was performed with the entire Unimod database. Finally, homology searching was performed using the SPIDER algorithm to identify peptides resulting from nonspecific cleavages or amino acid substitutions. Mascot and PEAKS SPIDER searches were combined (inChorus), using a 1% false discovery rate cutoff for both search engines. The resulting peptide-spectrum matches (95% peptide probability) were imported into Progenesis LC-MS. Conflict resolution was performed manually to ensure that a single peptide sequence was assigned to each feature by removing lower scoring peptides. The resulting normalized peptide intensity data were exported, and the peptide list was filtered to remove non-unique peptides, methionine-containing peptides, and all modified peptides except cysteine carbamidomethylation. For quantification, the filtered list of peptide intensities was imported into DanteR (version 0.1.1), and intensities for peptides of the same sequence were combined to form a single entry. The resulting peptide intensities were log2 transformed and combined to protein abundances (RRollup) using the default settings, excluding one-hit wonders (50% minimum presence of at least one peptide, minimum dataset presence 3, p-value cutoff of 0.05 for Grubbs’ test, minimum of 5 peptides for Grubbs’ test). The resulting proteins were quantified by 1-way ANOVA relative to M37; p-value adjustment for multiple testing was performed according to Benjamini and Hochberg.

Project description:BackgroundAmbiguity in malignant transformation of glioma has made prognostic diagnosis very challenging. Tumor malignant transformation is closely correlated with specific alterations of the metabolic profile. Exploration of the underlying metabolic alterations in glioma cells of different malignant degree is therefore vital to develop metabolic biomarkers for prognosis monitoring.MethodsWe conducted (1)H nuclear magnetic resonance (NMR)-based metabolic analysis on cell lines (CHG5, SHG44, U87, U118, U251) developed from gliomas of different malignant grades (WHO II and WHO IV). Several methods were applied to analyze the (1)H-NMR spectral data of polar extracts of cell lines and to identify characteristic metabolites, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), fuzzy c-means clustering (FCM) analysis and orthogonal projection to latent structure with discriminant analysis (OPLS-DA). The expression analyses of glial fibrillary acidic protein (GFAP) and matrix metal proteinases (MMP-9) were used to assess malignant behaviors of cell lines. GeneGo pathway analysis was used to associate characteristic metabolites with malignant behavior protein markers GFAP and MMP-9.ResultsStable and distinct metabolic profiles of the five cell lines were obtained. The metabolic profiles of the low malignancy grade group (CHG5, SHG44) were clearly distinguished from those of the high malignancy grade group (U87, U118, U251). Seventeen characteristic metabolites were identified that could distinguish the metabolic profiles of the two groups, nine of which were mapped to processes related to GFAP and MMP-9. Furthermore, the results from both quantitative comparison and metabolic correlation analysis indicated that the significantly altered metabolites were primarily involved in perturbation of metabolic pathways of tricarboxylic acid (TCA) cycle anaplerotic flux, amino acid metabolism, anti-oxidant mechanism and choline metabolism, which could be correlated with the changes in the glioma cells' malignant behaviors.ConclusionsOur results reveal the metabolic heterogeneity of glioma cell lines with different degrees of malignancy. The obtained metabolic profiles and characteristic metabolites are closely associated with the malignant features of glioma cells, which may lay the basis for both determining the molecular mechanisms underlying glioma malignant transformation and exploiting non-invasive biomarkers for prognosis monitoring.

Dataset Information

Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19.

Publications

Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19.

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