Unknown,Transcriptomics,Genomics,Proteomics

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Involvement of JunD in the antiproliferative effect of 9-tetrahydrocannabinol on human breast cancer cells 1

ABSTRACT: THC induces an activation of JunD, by upregulation and translocation to the nuclear compartment, which is accompanied by a decrease in cell proliferation Keywords: THC, JUND Total RNA was isolated from cells treated with vehicle (DMSO), 3 or 5 ïM THC for 8 or 24h, and non-treated cells at the time of drug addition (t=0, reference) using RNeasy Mini Kit (Qiagen, Hilden, Germany). Double strand cDNA was amplify with Superscript Choice System and T7-(deoxythymidine)24 oligo-primers (Life Technologies, Inc., Gaithersburg, MD) and in vitro transcription was carried out with MegascriptTM T7 (Ambion, Austin, TX). For each condition, half of the replicates were labeled with Cy5-dUTP fluorochrome (Amersham, Uppsala, Sweden) and the reference with Cy3-dUTP fluorochrome (Amersham), and the other half were dye swapped. Hybridization was performed on a new version of the Spanish National Cancer Research Center (CNIO) Oncochip manufactured by the CNIO Genomic Unit (http://bioinfo.cnio.es/data/oncochip), which contains 9.726 clones corresponding to 6,386 different genes. Slides were scanned in a Scanarray 5000 XL scanner (GSI Lumonics Kanata, Ontario, Canada) and quantified using the GenePix Pro 6.0 program (Axon Instruments Inc., Union City, CA).

ORGANISM(S): Homo sapiens

SUBMITTER: Moreno-Bueno Gema

PROVIDER: E-GEOD-8502 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Loss of expression of the cell-cell adhesion molecule E-cadherin is an essential event for epithelial-mesenchymal transition (EMT), a process that allows cell migration in the developing embryo and during tumour invasion. Transcriptional repression has emerged as the main mechanism responsible for E-cadherin downregulation in most carcinomas. Recently, we have identified the class I HLH transcriptional regulator E2-2 (ITF2), in a yeast one hybrid screen designed to identify transcriptional repressors interacting with the E-pal element of the murine E-cadherin promoter. The E2-2 gene codifies two isoforms that differ in their N-terminal regions but their specific functions remain unknown. In the present work we show that both E2-2A and E2-2B induce a complete EMT, in MDCK cells, with loss of E-cadherin expression, gain of mesenchymal markers and acquisition of motile and invasive properties. Although both isoforms repress E-cadherin promoter in MDCK cells, only the E2-2B isoform does it in other epithelial cell lines. Notably, we found that E2-2B is upregulated at the mRNA level in MDCK cells after treatment with TGF-ï¢1, a key regulator of EMT. Upregulation of E2-2A/B factors was confirmed in MDCK cells overexpressing other EMT inducers, Snai1, Snai2 or E47. Interestingly, gene profiling studies indicate that bHLH E2-2 factors induce similar, yet distinct, genetic programs from those induced by bHLH E47 in MDCK cells. These results, together with the expression pattern observed in early mouse embryos, support a new role for E2-2A/B in E-cadherin regulation and EMT, and suggest an interesting interplay between bHLH factors and other E-cadherin repressors. Keywords: Genetic Modification (overexpression of E-cadherin repressors) The Oncochip microarray platform v2.0 contains 13,824 clones printed by duplicate corresponding to 9,300 different genes. Each of the MDCK transfectants (E22A, E22B) were labeled with dUTP-Cy5 and hybridized against the dUTP-Cy3-labeled MDCK-CMV controls. One additional hybridizations using reciprocal fluorochrome labeling were performed (dye-swap) in each clone. Thus, a total of two hybridizations were performed for each condition.

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Involvement of JunD in the antiproliferative effect of 9-tetrahydrocannabinol on human breast cancer cells 1

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