Human SKOV-3 cells, Input DNA vs ChIP DNA
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29649 | GEO | 2013/05/01
SECONDARY ACCESSION(S): PRJNA141235
REPOSITORIES: GEO
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Project description:Human paclitaxel-resistant (SKOV-3TR) ovarian cancer cell line and the parent SKOV-3 cell line were grown in RPMI medium containing 10% fetal calf serum. The experiment was done to compare the expression differences between the parent SKOV-3 cell line and the derived SKOV-3TR cell line. Cells were plated in T75 flasks. They were cultured until they were 80% confluent and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with the Tri-Reagent (SIgma) and purified using the Qiagen RNeasy Mini Kit reagents. The RNA (50 ug) was annealed with a random hexamer primer, and reverse-transcribed into cDNA with Powerscript (Clontech) reverse transcriptase for 2h at 42 degrees in the presence of amino-allyl dUTP. The cDNA from SKOV-3 RNA and SKOV-3TR RNA was covalently coupled separately with Cy3 and Cy5 monoreactive fluors in 50 mM sodium bicarbonate, pH 9.0. The Cy3 and Cy5 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and hybridized to the microarray using the Hybrite chamber (Vysis). Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray 4000 (Perkin Elmer) at a resolution of 10 um at maximum laser power and photo-multiplier tube voltage of 50 to 60%. Segmentation was performed using the histogram method of the QuantArray (Perkin Elmer) package. Data was transformed into log base 2 expression ratios and normalized using scaled loess normalization. Keywords: repeat sample
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Project description:Human paclitaxel-resistant (SKOV-3TR) ovarian cancer cell line and the parent SKOV-3 cell line were grown in RPMI medium containing 10% fetal calf serum. The experiment was done to compare the expression differences between the parent SKOV-3 cell line and the derived SKOV-3TR cell line. Cells were plated in T75 flasks. They were cultured until they were 80% confluent and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with the Tri-Reagent (SIgma) and purified using the Qiagen RNeasy Mini Kit reagents. The RNA (50 ug) was annealed with a random hexamer primer, and reverse-transcribed into cDNA with Powerscript (Clontech) reverse transcriptase for 2h at 42 degrees in the presence of amino-allyl dUTP. The cDNA from SKOV-3 RNA and SKOV-3TR RNA was covalently coupled separately with Cy3 and Cy5 monoreactive fluors in 50 mM sodium bicarbonate, pH 9.0. The Cy3 and Cy5 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and hybridized to the microarray using the Hybrite chamber (Vysis). Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray 4000 (Perkin Elmer) at a resolution of 10 um at maximum laser power and photo-multiplier tube voltage of 50 to 60%. Segmentation was performed using the histogram method of the QuantArray (Perkin Elmer) package. Data was transformed into log base 2 expression ratios and normalized using scaled loess normalization.
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Project description:With the onset of resistance, ovarian cancer cells display almost unpredictable adaptive potential. This may derive from genetic ancestry of the tumor cells and can be additionally tailored by post translational protein modifications (PTMs). The latter are profoundly shaped by the physical and chemical cues of the cancer microenvironment which, for ovarian cancer, is primarily the abdominal cavity. In this study, we took advantage of high-end proteome and phosphoproteome analyses combined with multiparametric morphometric profiling for the description of the proteome signatures of high-grade serous carcinomas (OVCAR-3) and non-serous carcinomas (SKOV-3) ovarian cancer cells. For the functional experiments, we applied two different protocols, representing typical stimuli of the peritoneal cavity and of the growing tumor mass: on the one side hypoxia (oxygen 1%) which develops within the tumor mass or is experienced during migration/extravasation in non-vascularized tissues. For comparison, fluid shear stress (2.8 dyn/cm2, orbital shaker method) which characterizes the tumor surface in peritoneal cavity or metastases spread in the bloodstream. After 3 hours incubation, treatment groups were clearly distinguishable by PCA analysis. Whereas basal proteome profile of SKOV-3 and OVCAR-3 cells appeared almost unchanged, phosphoproteome analysis revealed multiple regulations. These affected primarily cellular structure and proliferative potential and consolidated after 24h treatment. Albeit maintaining their individuality, hypoxia modified cell metabolism and morphology of both OVCAR-3 and SKOV-3: upon oxygen reduction cell size increased in concerted regulation of pathways related to Rho-GTPases and/or cytoskeletal elements (proteome and phosphoproteome). Also shear stress stimulation sharpened the response profile of SKOV-3 and OVCAR-3: this retraced their pathophysiological behavior and actively modified structural proteins as well as metabolism (e.g. delta(14)-sterol reductase, Kinesin-like proteins (KIF-22/20A) and Actin-related protein 2/3 complex). In conclusion, we characterized a biochemical and structural fingerprinting describing the adaptive potential of ovarian cancer cells to physical/chemical stressors typical for the abdominal cavity.
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