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

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 μM 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).