Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human T cell leukaemia cell line Jurkat that were retrovirally transduced with constitutievely active forms of Notch to identify novel transcriptional targets of Notch signalling

ABSTRACT: In this study we aimed to identify novel transcriptional targets of Notch signalling in the T cell leukaemia cell line, Jurkat. RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (Mock) or vectors containing constitutively active forms of Notch (N1deltaE or N3deltaE), and used for Affymetrix microarray analysis. As expected, several known transcriptional target of Notch, such as HES1 and HERP2, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1).

ORGANISM(S): Homo sapiens

SUBMITTER: Leo Zeef

PROVIDER: E-MEXP-1744 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of novel Notch target genes in T cell leukaemia.

Chadwick Nicholas N Zeef Leo L Portillo Virginia V Fennessy Carl C Warrander Fiona F Hoyle Sarah S Buckle Anne-Marie AM

Molecular cancer 20090609

<h4>Background</h4>Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat.<h4>Results</h4>RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-a ...[more]

PMID: 19508709

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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.

Dataset Information

Transcription profiling of human T cell leukaemia cell line Jurkat that were retrovirally transduced with constitutievely active forms of Notch to identify novel transcriptional targets of Notch signalling

Publications

Identification of novel Notch target genes in T cell leukaemia.

Similar Datasets

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