RNA-seq of human EndoC-betaH1 cells treated with imatinib or dasatinib
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Homo sapiens
SUBMITTER: Nils Welsh
PROVIDER: E-MTAB-17592 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Gene expression profile variation between 4 BCR-Abl transduced cell lines: <br> 1-Mouse DA1-3b cell line as reference, <br> 2-DR1 imatinib resistant clone with E255K BCR-abl mutation<br> 3-DRBMSR 2 dasatinib resistant clone with E255K and T315I BCR-Abl mutation.<br> 4-DRBMSR 7 dasatinib resistant clone with E255K and T315I and V299L BCR-Abl mutation<br> <br> BCR-ABL is an oncogenic tyrosine kinase involved in the development of chronic myelogenous leukaemia (CML).
2007-08-01 | E-MEXP-772 | biostudies-arrayexpress
Project description:Characterization of structural genomic alteration in new-generated dasatinib and imatinib mouse BCR-ABL resistant cell line
2007-08-01 | E-MEXP-997 | biostudies-arrayexpress
Project description:Our objective is to decipher a miRNA expression signature associated with Chronic myeloid leukemia (CML), with or without Imatinib or Dasatinib and compared to normal blood leukocytes, and to determine potential target genes and signaling pathways affected by these signature miRNAs. Background/Aims: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. Results: Using miRNA microarrays we characterized the miRNAs expression profile of K562 cells in reference to healthy blood. We also looked into the expression profile of K562 cells treated with imatinib or dasatinib. We identified a miRNA signature that distinguishes K562 cells from healthy blood that included downregulation of miRNAs such as miR-31, miR-34a, miR-143, miR-145, miR-155, miR-196b and miR-564 and upregulation of miR-128. We also identified a miRNA signature that distinguishes untreated K562 cells from the treated ones that included downregulation of miRNAs such as miR-128 and upregulation of miR-564. Results: Using miRNA microarrays we characterized the miRNAs expression profile of K562 cells in reference to healthy blood. We also looked into the expression profile of K562 cells treated with imatinib or dasatinib. We identified a miRNA signature that distinguishes K562 cells from healthy blood that included downregulation of miRNAs such as miR-31, miR-34a, miR-143, miR-145, miR-155, miR-196b and miR-564 and upregulation of miR-128. We also identified a miRNA signature that distinguishes untreated K562 cells from the treated ones that included downregulation of miRNAs such as miR-128 and upregulation of miR-564. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, TGF-beta and Wnt were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the MAPK and TGF-beta signaling pathways and focal adhesion-related miRNAs. Conclusions: The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment. MiRNA profiling of CML: With the objective of deciphering a potential miRNA expression signature associated with CML, we analyzed the miRNAs expression profile of K562 in reference to a pool of 3 healthy blood samples using an miRNA-based microarray chip assay. In addition, to search for Bcr-Abl-dependent or Src-dependant miRNA expression patterns, K562 cells were treated or not with imatinib or with dasatinib to inhibit Bcr-Abl tyrosine kinase activity or Bcr-Abl and Src tyrosine kinase activity, respectively. In order to validate the microarray data and to ensure that the variability observed among samples was not technical, we carried out Taqman miRNA quantitative real-time PCR analysis (Applied Biosystems, USA) on miRNAs that we find to be most relevant, according to bioinformatics analysis (see below) and published data.
2011-10-20 | E-GEOD-28825 | biostudies-arrayexpress
Project description:RFX6 is a key transcription factor for the development of mouse pancreas, however the functional roles of RFX6 in human beta cells are poorly explored. Thus transcriptome analysis was perfomed to determine the functional targets of RFX6 in human beta cells using the recently developed human beta cell-line EndoC-M-NM-2H2. Transcriptome profile of human beta cell line (EndoC-M-NM-2H2 cells) following siRNA induced knockdown of RFX6 is compared with siControl (siNT) treated EndoC-M-NM-2H2 cells.
2014-12-22 | E-GEOD-59049 | biostudies-arrayexpress
Project description:Long-term treatment of Kasumi-1 cells at clinically attained doses of dasatinib led to decreased drug-sensitivity by means of IC50 values (relative to treatment-naive cells). Changes were paralled by profound alterations in c-KIT expression and cell signaling signatures. Upon brief discontinuation of dasatinib treatment, these alterations reversed and drug sensitivity was restored. We used gene expression profiling to examine reversal of dasatinib-resistance at the molecular/expression level. Kasumi-1 cells were either treated with dasatinib for 12 weeks (R48) or left untreated (K). Subsequently, dasatinib was withdrawn from R48 cells for 1 week (PR48). RNA was extracted from R48 and K cells and hybridized on Affymetrix microarrays after 12 weeks and PR48 after a total time span of 13 weeks.
2013-11-27 | E-GEOD-39073 | biostudies-arrayexpress
Project description:Single nucleotide replacement in the human TIA1 gene (E384K) in human EndoC-βH1 cells was achieved by PE according to a previous publication (Anzalone, Randolph, Davis et al., 2019). CRISPR PE guide RNA (pegRNA) and nicking sgRNA (ngRNA) sequences were designed using the web resources available at https://drugthatgene.pinellolab.partners.org/primevar. PE plasmid is pCMV-PE2-P2A-GFP (Addgene plasmid #132776) expressing Cas9 H840A with co-translational GFP expression. PegRNA plasmid was constructed by ligation of annealed oligonucleotides including sequences of spacer, scaffold, and 3' extension into BsaI-digested pU6-pegRNA-GG-acceptor (Addgene plasmid # 132777). Plasmids expressing ngRNAs were constructed by ligation of annealed oligonucleotides into U6-BsmBIcassette-Sp-sgRNA BPK1520 (Addgene plasmid # 65777) (Kleinstiver, Prew, Tsai et al., 2015). EndoC-βH1 cells were seeded in Geltrex Matrix coated T25 flasks. Cells were transfected at approximately 60% confluency with 5 uL lipofectamine 2000 (Thermo Fisher Scientific) together with 3.6 µg PE plasmid, 1.2 µg pegRNA plasmid, and 0.4 ug ngRNA plasmid according to the manufacturer’s instructions. Cells were cultured for three days followed by sorting of GFP-positive cells (approximately 10%) by flow cytometry. GFP-positive cells were seeded in Geltrex-coated 6-well plates at various cell densities, and cultured for another five weeks. At intermediate cell densities, individual colonies developed (five wildtype (WT 1-5 and three mutated (Mut 1-3) that were picked and re-seeded into a 96-well plate for seven days followed by reseeding into two wells of 96-well plates for each clone. One well of the cells from an individual clone was used for DNA isolation (Qiagen) and PCR amplification of the amplicon covering the TIA1 mutation site. The PCR product was then sequenced at Eurofins Genomics for detection and conformation of the homozygous point mutation (G>A on the sense strand, E384K).
2026-06-30 | E-MTAB-16316 | biostudies-arrayexpress
Project description:Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.
2014-05-12 | PXD000681 | Pride
Project description:Total RNA from 1e7 K562 cells either mock-treated with DMSO or treated with 100nM dasatinib in two biological repeats for 3 hours
2009-04-02 | E-MEXP-1561 | biostudies-arrayexpress
Project description:Access to an unlimited number of human pancreatic beta cells represents a major challenge in the field of diabetes to better dissect human beta cell functions and to make significant progress in drug discovery and cell replacement therapies. We previously reported the generation of the EndoC-bH1 human beta cell line that was generated by targeted oncogenesis in human fetal pancreases followed by in vivo cell differentiation in mice. Such cell line displayed many functional properties of adult beta cells. Here we devised a novel strategy to generate conditionally immortalized human beta cell lines based on CRE-mediated excision of immortalizing transgenes. The resulting EndoC-bH2 cell line can be massively amplified in vitro. Transgenes are next efficiently excised upon CRE expression leading to cell proliferation arrest and strong enhancement of beta cell specific features such as insulin expression, content and secretion. Excised EndoC-bH2 cells are close to authentic human beta cells and represent a unique tool to further study beta cell function and to understand why adult human beta cells are refractory to proliferation and how to achieve drug-dependent mobilization towards beta cell expansion. Expression profile of human beta cell lineEndoC-bH2 before and after excision of an immortalization cassette (SV40 LT and hTERT) is compared to human exocrine pancreas cell line SKPC and adult human islets from cadaveric donors. Three replicates were used for each sample group. The three adult human islets samples were taken from GEO series GSE40709 (GSM999550, GSM999551 and GSM999552) and normalized with H357 and SKPC cell line samples using RMA.
2014-03-21 | E-GEOD-48101 | biostudies-arrayexpress
Project description:Effect of Dasatinib on primary cancer-associated fibroblasts from lung cancer specimens.
2011-01-12 | E-MEXP-2546 | biostudies-arrayexpress