Transcriptional profiling in an MPNST-derived cell line and normal human Schwann cells

Abstract

AbstractcDNA microarrays were utilized to identify abnormally expressed genes in a malignant peripheral nerve sheath tumor (MPNST)-derived cell line, T265, by comparing the mRNA abundance profiles with that of normal human Schwann cells (nhSCs). The findings characterize the molecular phenotype of this important cell-line model of MPNSTs, and elucidate the contribution of Schwann cells in MPNSTs. In total, 4608 cDNA sequences were screened and hybridizations replicated on custom cDNA microarrays. In order to verify the microarray data, a large selection of differentially expressed mRNA transcripts were subjected to semi-quantitative reverse transcription PCR (LightCycler). Western blotting was performed to investigate a selection of genes and signal transduction pathways, as a further validation of the microarray data. The data generated from multiple microarray screens, semi-quantitative RT-PCR and Western blotting are in broad agreement. This study represents a comprehensive gene-expression analysis of an MPNST-derived cell line and the first comprehensive global mRNA profile of nhSCs in culture. This study has identified ~900 genes that are expressed abnormally in the T265 cell line and detected many genes not previously reported to be expressed in nhSCs. The results provide crucial information on the T265 cells that is essential for investigation using this cell line in experimental studies in neurofibromatosis type I (NF1), and important information on normal human Schwann cells that is applicable to a wide range of studies on Schwann cells in cell culture.

High-Resolution DNA Copy Number Profiling of Malignant Peripheral Nerve Sheath Tumors Using Targeted Microarray-Based Comparative Genomic Hybridization

“…The array was constructed using a previously reported PCR-based approach for microarrays production (21,22). Briefly, genomic sequences from the 57 selected genes (Supplementary Table S2) were downloaded from Ensembl 8 (human genome build 35.1), and bioinformatic analysis was carried out using Sequence Allocator program. 9 The program combines and automates filtering out redundant stretches in the input genomic sequence and designing primers in the unique fragments.…”

A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation

“…Hallmarks of NF1 include a range of symptoms from caf e au lait spots of the skin to tumors of the peripheral nervous system. Schwann cells (SC) have been found to be the originating cell type in most of the benign tumors (Harrisingh and Lloyd, 2004;Zhu et al, 2002) and can also contribute to malignant peripheral nerve sheath tumors (Lee et al, 2004;Miller et al, 2006). Loss of heterozygosity (LOH) is frequently seen in NF1 tumors, particularly in the SC population (Zhu et al, 2002), although not all SCs in the tumors analyzed showed LOH (Rutkowski et al, 2000;Sawada et al, 1996;Serra et al, 2000).…”

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation

“…A few studies have performed cDNA microarray analysis to identify expression signatures of malignant peripheral nerve sheath tumor. 9,10,12,19 These studies have used frozen tissue or cell lines derived from tumors. They showed differences in the gene expression pattern between neurofibromas and unmatched malignant peripheral nerve sheath tumor samples from other patients.…”