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E-GEOD-8240 - Transcription profiling of human MCF10A cells subjected to ionizing radiation and treatment with transforming growth factor beta-1

Submitted on 22 June 2007, released on 8 November 2008, last updated on 2 May 2014
Homo sapiens
Samples (15)
Array (1)
Protocols (4)
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.
Experiment types
transcription profiling by array, unknown experiment type
Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition. Kumari L Andarawewa, Anna C Erickson, William S Chou, Sylvain V Costes, Philippe Gascard, Joni D Mott, Mina J Bissell, Mary Helen Barcellos-Hoff.
Investigation descriptionE-GEOD-8240.idf.txt
Sample and data relationshipE-GEOD-8240.sdrf.txt
Raw data (1)
Processed data (1)
Array designA-AFFY-113.adf.txt
R ExpressionSetE-GEOD-8240.eSet.r