biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsDirk Kemmingtranscription profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-44354Initial screening for potential metastases suppressors down regulated by methylation was performed using breast cancer cell line models specific for site-specific metastasation. Gene expression profiling and qRT-PCR validations were conducted on tumor tissues from primary breast cancer (BC) and BCBM. CADM1 and RECK were further characterized for their methylation patterns and finally the protein expression of CADM1 was validated in a large number of BC and BCBM samples and correlated with clinico-pathologic parameters. A subclone of MDA-MB-231, which has a high metastatic potential for the brain (MDA-MB-231 BR), was compared to the parental MDA-MB-231 WT and to a bone-seeking subclone (MDA-MB-231 SA) in order to find genes, which might be specifically involved in brain metastasis formation. The cell lines were treated with 5-Aza-2'-deoxycytidine in order to find genes potentially down regulated by methylation. The non-tumorigenic epithelial cell line MCF 10A was used to control for stress response after the treatment with 5-Aza-2'-deoxycytidine.biostudies-arrayexpressScaning - Agilent Technologies Scanner G2505B; Agilent Scanner protocol GE1-v5_95_Feb07; Scan resolution 5 µm; XDR Hi 100% XDR Lo 10%Labeling - Cy3 and Cy5 labeled cRNA were prepared according to the standard Agilent protocol from 500ng total RNA.Nucleic Acid Extraction - Total RNA of the cells was isolated using RNAeasy kit (Qiagen, Hilden Germany)Hybridization - Following fragmentation, 825 ng of each cRNA were hybridized for 17 hr at 65C on Agilent 4x44k Whole Human Genome Array. Arrays were washed according to the manufacturer's protocol.Sample Treatment - Cells were seeded at 10% (MDA-MB-231 and subtypes) or 20% (MCF 10A) confluence. 5-Aza-2'-deoxycytidine was dissolved in sterile water. After 4 hours of plating the cells were cultured in absence or presence of 1 µM 5-Aza-CdR, and the medium was changed daily until the cells reached a confluence of 90-100%.Growth Protocol - The different MDA-MB-231 cell lines were grown in DMEM medium supplemented with 10% FBS, 2 mM Glutamine . MCF 10A cells were grown in DMEM/Ham's F-12 with 5% FBS, 10 μg/ml Insulin, 5 μg/ml Hydrocortisone, 20 ng/ml EGF, 100 ng/ml Cholera toxin and 2 mM L-Glutamine. All cells were maintained in a humidified incubator at 37°C and 10% CO2. All cells were tested free of mycoplasma.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsHarriet WikmanDirk KemmingData Transformation - Data extraction was done using Agilent Feature Extraction Software; raw Data have been median scaled; replicate spots have been averaged ID_REF = VALUE = normalized log10 ratio treatment/reference gProcessedSignal = rProcessedSignal = INV_VALUE = normalized log10 ratio (Cy5/Cy3)falseIdentification of novel metastases-suppressor genes involved in breast cancer brain metastases (BCBM) formationInitial screening for potential metastases suppressors down regulated by methylation was performed using breast cancer cell line models specific for site-specific metastasation. Gene expression profiling and qRT-PCR validations were conducted on tumor tissues from primary breast cancer (BC) and BCBM. CADM1 and RECK were further characterized for their methylation patterns and finally the protein expression of CADM1 was validated in a large number of BC and BCBM samples and correlated with clinico-pathologic parameters. A subclone of MDA-MB-231, which has a high metastatic potential for the brain (MDA-MB-231 BR), was compared to the parental MDA-MB-231 WT and to a bone-seeking subclone (MDA-MB-231 SA) in order to find genes, which might be specifically involved in brain metastasis formation. The cell lines were treated with 5-Aza-2'-deoxycytidine in order to find genes potentially down regulated by methylation. The non-tumorigenic epithelial cell line MCF 10A was used to control for stress response after the treatment with 5-Aza-2'-deoxycytidine.2014-03-04T00:00:00Z2023-08-26T19:16:15.79Z2021-09-28T10:53:28ZE-GEOD-44354GSE44354EFO_0002768