Overexpression of b-Arrestins in triple negative breast cancer cells.

Beta-Arrestins (βArrs) are intracellular signal regulating proteins. Their expression level varies in some cancers and they have significant impact on cancer cell function. In general, the significance of βArrs in cancer research comes from studies examining GPCR signalling. Given the diversity of different GPCR signals in cancer cell regulation, contradictory results are inevitable regarding the role of βArrs. Our approach examines the direct influence of βArrs on cellular function and gene expression profiles by changing their expression levels in breast cancer cells, MDA-MB-231. MDA-MB-231 human triple negative breast cancer cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and Eagle’s minimal essential medium (EMEM), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 5% CO2, 37˚C with 90-95 % humidity. The cells (2.5x105) were cultured in a 6 well plate and left for 24 h to reach 70-80 % confluency for cDNA transfections. DNA transfections were performed according to the manufacturer’s protocol using, for each well, 4 µl of TurboFectin Transfection 8.0 Reagent and 2µg of control nontarget DNA (Cloning vector PCMV6XL5), and Human βArr1cDNA or Human βArr2 cDNA. After transfection for 48 h at 37˚C, the medium was replaced, and the cells were cultured for an additional 12 h.