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Imatinib/Rapamycin Gradient Encoding

ABSTRACT: 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)

ORGANISM(S): Mus musculus

PROVIDER: GSE35039 | GEO | 2013/01/10

SECONDARY ACCESSION(S): PRJNA156187

REPOSITORIES: GEO

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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 identify Six1 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six1wt or VP16-Six1W171R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six1wt infected sample) or Cy5 (AxCAwt VP16-Six1W171R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. To identify Six4 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six4wt or VP16-Six4W263R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six4wt infected sample) or Cy5 (AxCAwt VP16-Six4W263R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. Keywords: other

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Imatinib/Rapamycin Gradient Encoding

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