Project description:The purpose of this study was to compare changes in translation (using Gradient Encoding, described below) to changes in mRNA abundance. Lysates of wildtype v-Abl transformed pre-B cells harvested before and after 12 hours of treatment either with 2.5 uM imatinib, a v-Abl kinase inhibitor, or 10ng/mL (10.9 nM) rapamycin, an mTOR inhibitor, were fractionated by sedimentation through linear sucrose gradients. Gradient fractions were encoded such that the mRNA from successive fractions was labeled with increasing ratios of Cy5 to Cy3. mRNAs derived from fractions in the lighter portion of the gradient therefore have a lower Cy5 to Cy3 ratio, whereas those deeper in the gradient have a higher Cy5 to Cy3 ratio. The ratio of Cy5 to Cy3 for each mRNA therefore reflects its average position within the gradient. We thus encoded the sedimentation rate of each mRNA across the entire gradient. The resulting ratios were quantitatively measured for each mRNA species by hybridization to DNA microarrays, and related to the 260 nm absorbance peaks representing different numbers of ribosomes bound per mRNA Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE)
Project description:The purpose of this study was to compare changes in translation (using Gradient Encoding, described below) to changes in mRNA abundance. Lysates of wildtype v-Abl transformed pre-B cells harvested before and after 12 hours of treatment either with 2.5 uM imatinib, a v-Abl kinase inhibitor, or 10ng/mL (10.9 nM) rapamycin, an mTOR inhibitor, were fractionated by sedimentation through linear sucrose gradients. Gradient fractions were encoded such that the mRNA from successive fractions was labeled with increasing ratios of Cy5 to Cy3. mRNAs derived from fractions in the lighter portion of the gradient therefore have a lower Cy5 to Cy3 ratio, whereas those deeper in the gradient have a higher Cy5 to Cy3 ratio. The ratio of Cy5 to Cy3 for each mRNA therefore reflects its average position within the gradient. We thus encoded the sedimentation rate of each mRNA across the entire gradient. The resulting ratios were quantitatively measured for each mRNA species by hybridization to DNA microarrays, and related to the 260 nm absorbance peaks representing different numbers of ribosomes bound per mRNA Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE) compound_treatment_design
Project description:Measurement of mRNA abundance from the following cells lines (red) versus universal mouse reference RNA (green). Wildtype v-Abl transformed pre-B cells were treated for 12 hours with 2.5 uM imatinib mesylate, 10ng/mL rapamycin or nothing. Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE)
Project description:Measurement of mRNA abundance from the following cells lines (red) versus universal mouse reference RNA (green). Wildtype v-Abl transformed pre-B cells were treated for 12 hours with 2.5 uM imatinib mesylate, 10ng/mL rapamycin or nothing. Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE) compound_treatment_design
Project description:A single arm, open-label pilot study is designed to determine the safety, tolerability and effectiveness of personalized mRNA tumor vaccine encoding neoantigen in Patients with advanced esophageal squamous carcinoma, gastric adenocarcinoma, pancreatic adenocarcinoma and colorectal adenocarcinoma
Project description:Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Imatinib mesylate has emerged as the leading compound to treat chronic myeloid leukemia (CML), through its inhibition of Bcr- Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease and has necessitated the development of new BCR-ABL inhibitors. The synthesis of a new series of phenylaminopyrimidines, structurally related to imatinib showed large interest since the introduction of the nilotibin. To identify the cellular pathways affected by new synthesized compounds, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with imatinib and imatinib derivates. Further, the global proteome of the K562 cells treated with imatinib were quantitatively compared with the cells treated with the new compounds. This study enriched our knowledge about direct cellular targets of kinase selective drugs. Further the results offered important new knowledge for gaining insights into the structural effects of action of the new compounds. Samples were analyzed on a longer column (30cm) and a longer gradient (180min). Raw data files were processed with Mascot distiller 2.3. The mgf files were searched with Mascot daemon 2.3. The quantification was also done by Mascot Distiller. All data was stored in ms_lims. The manual validation of false peptide ratios was done with Rover (part of ms_lims). Fixed modifications: none. Variable modifications: acetylation of peptide N-terminus, pyroglutamate formation of N-terminal glutamine, methionine oxidation. Enzyme: trypsine with one missed cleavage allowed. Precursor mass tolerance: 10 ppm. Peptide fragment mass tolerance: 0.5 Da Quantitation method: SILAC arginine and lysine +6 Da. Overview of the 17 different analyses: B SK23 vs DMSO C Y22 vs DMSO D SK20 vs DMSO E Y18 vs DMSO I SK20 vs DMSO K Y18 vs DMSO O Y22 vs DMSO R Imatinib vs Water Z Imatinib vs Water J SK20 vs Imatinib M SK23 vs Imatinib N Y22 vs Imatinib P SK23 vs Imatinib Q Y18 vs Imatinib S Y22 vs Imatinib T SK20 vs Imatinib Y Y18 vs Imatinib
Project description:To investigate effects on mRNA expression in mouse lung tissue through treatment with Ly, Su, Imatinib, irradiation and combinations of them. mRNA expression was measured 24 days after irradiation. 2 biological replicates were analyzed for each treatment condition (control, Ly, Imatinib, Su, Ly+Imatinib, Ly+Su, Irradiation, Irradiation+Ly, Irradiation+Imatinib, Irradiation+Su, Irradiation+Ly+Imatinib, Irradiation+Ly+Su)
Project description:In Saccharomyces cerevisiae, deletion of genes encoding proteins of the large ribosomal subunit (RPLs) increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the mRNA and protein abundance, the ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and to increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations - including ribosomal protein (RP) gene deletions, calorie restriction, rapamycin treatment - and provide an explanation for its role in longevity and stress response. This SuperSeries is composed of the SubSeries listed below.