Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data from Control, AKT and RAS transduced cells

ABSTRACT: Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS, and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. More-over, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. We used microarrays to detail the global programme of gene expression after transduction of AKT and RAS IMR90 cells were transfected with Control, AKT and RAS retrovirus containing medium in 4 replicates. Fibroblasts were drug selected and kept in drug for duration of experiments.

ORGANISM(S): Homo sapiens

SUBMITTER: Tony McBryan

PROVIDER: E-GEOD-45276 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Activation of the PIK3CA/AKT pathway suppresses senescence induced by an activated RAS oncogene to promote tumorigenesis.

Kennedy Alyssa L AL Morton Jennifer P JP Manoharan Indrani I Nelson David M DM Jamieson Nigel B NB Pawlikowski Jeff S JS McBryan Tony T Doyle Brendan B McKay Colin C Oien Karin A KA Enders Greg H GH Zhang Rugang R Sansom Owen J OJ Adams Peter D PD

Molecular cell 20110401 1

Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS, and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. Moreover, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. In vivo, bypass of RAS-induced senesce ...[more]

PMID: 21474066

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Project description:Most adult patients have a D816V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus IT-PTD-mutants were introduced into rodent Ba/F3, EML, Rat2 and human TF1 cells to investigate their biological effect. ECD- and PTD-mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT pathways, whereas AKT pathway was only activated by ECD-mutants. Consistently, AKT inhibitor suppressed ECD-mutant-dependent proliferation, clonogenicity and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EMLPTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes. Each EML cell line (Del417-419insY mutant and D816V mutant) was cultured with or without 250 ng/mL SCF for 48h. Gene expression profile analysis was performed using whole-genome microarrays. RNA expression profiling of cell lines was done with Affymetrix M430 2.0 mouse oligonucleotide microarrays containing 45.101 probe sets, representing 21.408 transcripts and variants including 17.482 well-characterized mouse genes. Preparation of cRNA, hybridizations, washes, detection and quantification were done as recommended by the supplier. For each sample, synthesis of the first-strand cDNA was done from 3 μg total RNA by T7-oligo(dT) priming, followed by second-strand cDNA synthesis. After purification, in vitro transcription associated with amplification generated cRNA-containing biotinylated pseudouridine. Biotinylated cRNA was purified, quantified and chemically fragmented (95°C for 35 min), then hybridized to microarrays in 200 μL hybridization buffer at 45°C for 16 h. Automated washes and staining with streptavidin-phycoerythrin were done as recommended. Signal amplification was done by biotinylated antistreptavidin antibody with goat-IgG blocking antibody. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software.

Project description:The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. Using genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1, we find that repressed target genes are enriched at the nuclear periphery in myoblast cells. We further show that the interaction of Msx1 with the Polycomb repressive complex PRC2 is required for transcriptional repression and regulation of myoblast differentiation, and promotes increased tri-methylation of lysine 27 on histone H3 (H3K27me3) at Msx1 target genes. Furthermore, Msx1 genomic binding promotes the dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in developing embryos in vivo. Thus, our findings suggest a hitherto unappreciated spatial coordination of transcription factor binding, Polycomb recruitment, and subnuclear localization in regulation of developmentgene expression programs. In order to identify genes regulated by Msx1, we infected C2C12 myoblast cells with a retrovirus expressing a tamoxifen-regulated Msx1 protein, Msx1-ER (or with empty vector as a control), followed by induction with 0.2 nM of tamoxifen or vehicle (DMSO) for 6 hours. Regulated genes were identified as those that changed in expression upon tamoxifen induction of the Msx1-ER protein but did not change in the empty vector control. Experiments were performed in triplicate for each of the four experimental conditions (Msx1-ER + tamoxifen, Msx1-ER - tamoxifen, empty vector control + tamoxifen, empty vector control - tamoxifen), for a total of 12 independent array samples. This submission represents the transcriptome component of the study.

Dataset Information

Expression data from Control, AKT and RAS transduced cells

Publications

Activation of the PIK3CA/AKT pathway suppresses senescence induced by an activated RAS oncogene to promote tumorigenesis.

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