Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

0

MRNA Profiling of Polysome fractions from #483 T-ALL treated with 4EGI-1 or DMSO


ABSTRACT: High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). Here we investigated how 4EGI-1, a small inhibitor of cap-dependent translation, induces apoptosis in T-ALL clones displaying hyperactive PI3K-AKT-mTOR signaling. 4EGI-1 treatment reduced the ribosomal occupancy of transcripts that define the molecular difference between T-ALL blasts and normal thymocytes. These transcripts encoded proteins for the translational machinery, for mitochondrial metabolism as well as oncoproteins such as Cyclin D1, Bcl-2 and c-myc. Therefore, targeting translation initiation proves valuable in situations where mTOR is activated by multiple events. For each biological replicate 5 x 107 #483 T-ALL cells were treated with 100 µM 4EGI-1 or DMSO. After 2 h incubation at 37°C cells were washed in ice-cold PBS containing cycloheximide (0.1 mg/ml), pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM DTT, 5 mM MgCl2, 0.5 % nonidet-P40, 0.1 mg/ml cycloheximide, 10000 IU/ml dalteparin sodium) and incubated for 5 min on ice. All subsequent steps were carried out in the cold. After centrifugation for 10 min at 12000 x g, 0.4 ml supernatant was layered onto a 12 ml linear sucrose gradient (10 - 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 °C in an SW40Ti rotor (Beckman) at 35000 rpm without brake for 120 min. The gradients were harvested and absorbance profiles at 254 nm recorded (type 11 optical unit/UA-6 detector, ISCO). 1 ml fractions were collected and supplied with 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 °C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturer´s protocol. For microarray analysis RNA from polysome fractions were pooled, precipitated by adding LiCl to a final concentration of 2 M and resolubilized in 15 µl of RNase-free water. RNA concentrations were determined and the samples were stored at -80 °C.

ORGANISM(S): Mus musculus

SUBMITTER: Adrian Schwarzer 

PROVIDER: E-GEOD-41382 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

Similar Datasets

2015-02-01 | E-GEOD-56150 | biostudies-arrayexpress
2022-11-14 | PXD038080 |
2019-10-15 | GSE134102 | GEO
2006-03-23 | GSE4500 | GEO
2009-11-10 | GSE18834 | GEO
2023-10-27 | PXD044422 | Pride
2002-02-12 | GSE16 | GEO
2021-11-17 | GSE183083 | GEO
2019-07-02 | GSE133625 | GEO
2013-07-04 | GSE30457 | GEO