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

Metadata Updated: August 1, 2018

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.

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Public: This dataset is intended for public access and use. License: U.S. Government Work

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Metadata Created Date August 1, 2018
Metadata Updated Date August 1, 2018
Data Update Frequency irregular

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Harvested from NASA Data.json

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Resource Type Dataset
Metadata Created Date August 1, 2018
Metadata Updated Date August 1, 2018
Publisher National Aeronautics and Space Administration
Unique Identifier nasa_genelab_GLDS-78
GeneLab Outreach
Maintainer Email
Public Access Level public
Data Update Frequency irregular
Bureau Code 026:00
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Schema Version
Catalog Describedby
Harvest Object Id 2ed2374f-ebfe-4254-ac5c-1043c39be667
Harvest Source Id 39e4ad2a-47ca-4507-8258-852babd0fd99
Harvest Source Title NASA Data.json
Language en-US
Data Last Modified 2018-04-05T00:00:00.000Z
Program Code 026:005
Source Datajson Identifier True
Source Hash e2de2aa63afc705d0f03e758f31c71f20abde0f2
Source Schema Version 1.1

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