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Transcriptional profiling of Rapamycin treatment in yeast cells lacking the Ptc1 protein phosphatase.

ABSTRACT: We are interested in the study of the already reported sensitivity of yeast cells lacking the type 1 protein phosphatase Ptc1 to the drug rapamycin. In order to carry out our studies, we analyzed the changes in the transcription profile that a short-term incubation with rapamycin (200 ng/mL) has in both, wild type and ptc1 cells. It has been shown that inhibition of the TOR pathway by treatment with rapamycin has profound effects on the global transcriptional profile. We confirmed the previously described transcription changes induced by the drug in wild type cells. Under our working conditions rapamycin increased, in wild type cells, at least 2-fold the expression of 667 genes, whereas it decreased the mRNA level of 721 genes (13.8% and 14.9% of genes with measurable expression level, respectively). Gene ontology analysis shows that, as previously documented, many induced genes falls into the NCR, Msn2/Msn4 regulated stress response or Retrograde response categories, whereas genes encoding cytoplasmic, but not mitochondrial, ribosomal proteins and the so called RIBI regulon where largely repressed. Deletion of PTC1 decreases the number of genes induced or repressed at least 2-fold by rapamycin treatment. Remarkably, lack of Ptc1 seems to lead to a general attenuation of changes triggered by rapamycin.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE11530 | GEO | 2008/12/31

SECONDARY ACCESSION(S): PRJNA106249

REPOSITORIES: GEO

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Project description:Genomic rearrangements leading to intragenic gene fusion are mainly found in some types of haematopoietic malignancies and sarcomas. Recently they have been described also in carcinomas such as the papillary thyroid histotype (60%-70%) and the Hürthle thyroid tumours (58%). The presence of junction oncogene constitutes an area of exciting research for emerging therapy as targeting the RET-PTC1 fusion oncogene by using small interfering RNA (siRNA) strategies since it is present only in the tumour cells and not in the surrounding normal cells. Therefore, we developed a siRNA against RET-PTC1 junction and assess its efficiency on the human papillary thyroid carcinoma cell line TPC-1 which spontaneously harbours the RET-PTC1 oncogene. The targeted genes are assessed by microarray analysis by comparing the regulated genes by the siRNA_RET-PTC1 vs a siRNA_RET developed on the RET part of the mRNA minus the siRNA_control that contain four mutation within the RET-PTC1 sequence. To test the targeted genes in the TPC-1 cell line that spontaneously harbours RET-PTC1 junction of two siRNAs developed: Within the RET-PTC1 junction, and in the mRNA RET part. A non-specific siRNA harbouring 4 mutations within the RET-PTC1 sequence was used as negative control (siRNA_control). By real-time PCR (Q-RT-PCR) we demonstrated that both siRNAs (siRNA_RET-PTC1 and siRNA_RET) significantly reduce RET mRNA levels of about 85 % in TPC-1 cells. The negative control did not show an effect of RET mRNA levels. Three independent transfections were performed on TPC-1 cells using 5 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Cergy-Pontoise, France) and 50nM of i) siRNA_RET-PTC1 or ii) siRNA_RET or iii) siRNA_control that harbour 4 mutations within its sequence. Total RNAs of untreated cells and transfected cells were purified using the RNA cleanup and concentration kit (QIAGEN, Hilden, Germany) and gathered in 4 pools : 1) TPC-1 harbouring RET-PTC1 ; 2) TPC-1 silenced for RET-PTC1 with siRNA RET-PTC1 ; 3) TPC-1 silenced for siRNA RET ; 4) TPC-1 treated with the siRNA control.

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Transcriptional profiling of Rapamycin treatment in yeast cells lacking the Ptc1 protein phosphatase.

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