Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Antiangiogenic Antithrombin

ABSTRACT: Confluent passage-2 HUVECs in Falcon T12.5 flasks grown in the presence or absence of VEGF-165 (10ng/mL) (R&D systems) were treated with human native, cleaved or latent antithrombins (20 5g/mL) for 24 hours in 3 mL M-199 with 2% heat-inactivated FBS and antibiotics. Following this treatment, total RNA was extracted from the cells by the Trizol method (Invitrogen). The total RNA was subjected to one cycle of linear RNA amplification using the MessageAmp aRNA kit according to the manufacturers instructions (Ambion, Austin, TX). The quality of the antisense RNA was verified on a denaturing agarose gel. Only samples showing a distribution of sizes from 250-5500 nt with a peak centered at 1000-1500 nt were used to generate the test cDNA samples for the subsequent array experiments. DNA labeling and hybridizations were performed essentially as described (Pollack et al., 1999, Nat. Genet.), with slight modifications. Briefly, the test endothelial cell antisense RNA samples together with Universal Human Reference RNA (Stratagene, Cedar Creek, TX) were used to generate Cy3/Cy5 (Amersham Biosciences) labeled cDNA for array hybridization on the Stanford 43K human cDNA microarray (www.microarray.org/sfgf). The Stanford microarray used in this study consists of 18,416 named genes with UniGene symbol, 4,145 ESTs with known function, 19,365 ESTs with unknown function and ~1,000 repeated spots as internal controls. Hybridized arrays were scanned on a GenePix 4000B scanner (Axon Instruments), and fluorescence ratios (test/reference) calculated using the Stanford Microarray Database software (available at http://genomewww5. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment stimulus_or_stress_design

ORGANISM(S): Homo sapiens

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PROVIDER: E-GEOD-3956 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells.

Zhang Weiqing W Chuang Yung-Jen YJ Swanson Richard R Li Juan J Seo Kyunga K Leung Lawrence L Lau Lester F LF Olson Steven T ST

Blood 20031016 4

Antithrombin, a key serpin family regulator of blood coagulation proteases, is transformed into a potent antiangiogenic factor by limited proteolysis or mild heating. Here, we show by cDNA microarray, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting analyses that the expression of the proangiogenic heparan sulfate proteoglycan (HSPG), perlecan, but not other HSPGs, is dramatically down-regulated in human umbilical vein endothelial c ...[more]

PMID: 14563633

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Project description:Summary: Breast cancer cell lines have been used widely to investigate breast cancer pathobiology and new therapies. Breast cancer is a molecularly heterogeneous disease, and it is important to understand how well and which cell lines best model that diversity. In particular, microarray studies have identified molecular subtypes (luminal A, luminal B, ERBB2-associated, basal-like and normal-like) with characteristic gene-expression patterns and underlying DNA copy number alterations (CNAs). Here, we studied a collection of breast cancer cell lines to catalog molecular profiles and to assess their relation to breast cancer subtypes. Whole-genome DNA microarrays were used to profile gene expression and CNAs in a collection of 52 widely-used breast cancer cell lines, and comparisons were made to existing profiles of primary breast tumors. Hierarchical clustering was used to identify gene-expression subtypes, and Gene Set Enrichment Analysis (GSEA) to discover biological features of those subtypes. Genomic and transcriptional profiles were integrated to discover within high-amplitude CNAs candidate cancer genes with coordinately altered gene copy number and expression. Transcriptional profiling of breast cancer cell lines identified one luminal and two basal-like (A and B) subtypes. Luminal lines displayed an estrogen receptor (ER) signature and resembled luminal-A/B tumors, basal-A lines were associated with ETS-pathway and BRCA1 signatures and resembled basal-like tumors, and basal-B lines displayed mesenchymal and stem-cell characteristics. Compared to tumors, cell lines exhibited similar patterns of CNA, but an overall higher complexity of CNA (genetically simple luminal-A tumors were not represented), and only partial conservation of subtype-specific CNAs. We identified 80 high-level DNA amplifications and 13 presumptive homozygous deletions, and the resident genes with concomitantly altered gene-expression, highlighting known and novel candidate breast cancer genes. Overall, breast cancer cell lines were genetically more complex than tumors, but retained expression patterns with relevance to the luminal-basal subtype distinction. The compendium of molecular profiles defines cell lines suitable for investigations of subtype-specific pathobiology, biomarkers and therapies, and provides a resource for discovery of new breast cancer genes. HEEBO oligonucleotide microarrays from the Stanford Functional Genomics Facility were used to perform gene expression profiling of 50 human breast epithelial cell lines, in comparison to a universal RNA reference. Expression data were analyzed by hierarchical clustering to identify subgroups, and gene set enrichment analysis to identify subgroup-specific gene pathways.

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Dataset Information

Antiangiogenic Antithrombin

Publications

Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells.

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