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Title: Transcription profiling of human MCF10A cells subjected to ionizing radiation and treatment with transforming growth factor beta-1      
dateReleased:
11-08-2008
description:
Transforming growth factor beta-1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGFbeta (0.4 ng/ml), or double-treated. All double-treated (IR+TGFbeta) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, beta-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, even though IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression. Experiment Overall Design: Nonmalignant human mammary epithelial MCF10A cells (passages 106 and 108) were seeded at cloning density in 35mm dishes (10^5 cells/dish). Cell culture medium consisted of 3ml/dish of MGEM serum free medium (Cambrex Inc.), supplemented or not with 400pg/ml recombinant Transforming Growth Factor-beta. Cells were irradiated or not 5h post plating using 160 KV X-ray with a total dose of 2Gy. Sham, IR-treated, TGFbeta-treated and double-treated (IR+TGFbeta) MCF10A cells were harvested 8 days post-IR. Briefly, cells were washed with PBS, denatured in Trizol, scraped off the dish and subjected to chloroform extraction. After centrifugation, the upper phase was precipitated with an equal volume of isopropanol. RNA precipitates were resuspended in RNase free water and further purified on RNeasy columns (Qiagen, Germany). RNA quality was assessed on an Agilent Bio-Analyzer. The dataset analyzed by microarray included biological duplicates for each treatment in two independent experiments and three sham treated samples. Microarray data were generated at the Lawrence Berkeley National Laboratory Molecular Profiling Laboratory () using a high-throughput, automated GeneChip system (Affymetrix). Briefly, target preparation, HT_HG-U133A array plate hybridization setup, washing and staining were performed on an Affymetrix robotic system (GCAS) using version 2.1 protocols. Scanning (protocol version 2.2.09) was performed on a CCD-based high throughput scanner (Affymetrix). Samples were analyzed and clustered with the (UNO) One Color GenetrafficTM software version 3.2-12 (Iobion Informatics LLC, Stratagene, La Jolla, CA). Genes whose expression was specifically altered by treatment were defined as those in which dye ratio was more than 1.75-fold (|mean log2ratio|>0.8) from baseline in at least three out of the four treated samples compared to the three sham samples. Significance analysis tests (p<0.05) were performed using Excel between sham samples and either IR, TGFbeta or TGFbeta+IR samples.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-8240
refinement:
raw
alternateIdentifiers:
8240
dateSubmitted:
06-22-2007
keywords:
functional genomics
dateModified:
05-02-2014
creators:
Mary Helen Barcellos-Hoff
availability:
available
types:
gene expression
name:
Homo sapiens
name:
unknown experiment type
ID:
A-AFFY-113
name:
Affymetrix GeneChip HT Human Genome U133A HT_HG-U133A
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-8240/E-GEOD-8240.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-8240/E-GEOD-8240.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE8240
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress

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