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Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.

ABSTRACT: Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.

SUBMITTER: Koso H

PROVIDER: S-EPMC3497753 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.

Koso Hideto H Takeda Haruna H Yew Christopher Chin Kuan CC Ward Jerrold M JM Nariai Naoki N Ueno Kazuko K Nagasaki Masao M Watanabe Sumiko S Rust Alistair G AG Adams David J DJ Copeland Neal G NG Jenkins Nancy A NA

Proceedings of the National Academy of Sciences of the United States of America 20121008 44

Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in ...[more]

PMID: 23045694

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Project description:Pseudomonas nitroreducens TX1 is of special interest because of its ability to utilize 0.05% to 20% octylphenol polyethoxylates (OPEOn) as a sole source of carbon. In this study, a library containing 30,000 Tn5-insertion mutants of the wild-type strain TX1 was constructed and screened for OPEOn utilization, and 93 mutants were found to be unable to grow on OPEOn In total, 42 separate disrupted genes were identified, and the proteins encoded by the genes were then classified into various categories, namely, information storage and processing (14.3%), cellular processes and signaling (28.6%), metabolism (35.7%), and unknown proteins (21.4%). The individual deletion of genes encoding isocitrate lyase (aceA), malate synthase (aceB), and glycolate dehydrogenase (glcE) was carried out, and the requirement for aceA and aceB but not glcE confirmed the role of the glyoxylate cycle in OPEOn degradation. Furthermore, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase activity levels were 13.2- and 2.1-fold higher in TX1 cells grown on OPEOn than in TX1 cells grown on succinate, respectively. Growth of the various mutants on different carbon sources was tested, and based on these findings, a mechanism involving exoscission to liberate acetaldehyde from the end of the OPEOn chain during degradation is proposed for the breakdown of OPEOn IMPORTANCE: Octylphenol polyethoxylates belong to the alkylphenol polyethoxylate (APEOn) nonionic surfactant family. Evidence based on the analysis of intermediate metabolites suggested that the primary biodegradation of APEOn can be achieved by two possible pathways for the stepwise removal of the C2 ethoxylate units from the end of the chain. However, direct evidence for these hypotheses is still lacking. In this study, we described the use of transposon mutagenesis to identify genes critical to the catabolism of OPEOn by P. nitroreducens TX1. The exoscission of the ethoxylate chain leading to the liberation of acetaldehyde is proposed. Isocitrate lyase and malate synthase in glyoxylate cycle are required in the catabolism of ethoxylated surfactants. Our findings also provide many gene candidates that may help elucidate the mechanisms in stress responses to ethoxylated surfactants by bacteria.

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Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.

Publications

Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.

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