Impact of Targeting the Adenine- and Uracil-Rich Element of bcl-2 mRNA with Oligoribonucleotides on Apoptosis, Cell Cycle, and Neuronal Differentiation in SHSY-5Y Cells

We have identified previously a destabilizing adenine- and uracilrich element (ARE) in the 3-UTR of bcl-2 mRNA that interacted with ARE-binding proteins to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2-O-methyl oligoribonucleotides (ORNs) in both sense and antisense orientation with respect to the bcl-2 ARE that are able to regulate the bcl-2 mRNA half-life and Bcl-2 protein level in two different cell lines. Here we show that treatment of neuronal cell line (SHSY-5Y) with antisense ORNs targeting the bcl-2 ARE (bcl-2 ARE asORNs) prevents bcl-2 down-regulation in response to apoptotic stimuli with glucose/growth factor starvation (Locke medium) or oxygen deprivation and enhances the apoptotic threshold as evaluated by time-lapse videomicroscopy, fluorescence-activated cell sorting analysis, and caspase-3 activation. Additional effects of bcl-2 ARE asORNs included inhibition of cell cycle entry and a marked increase of cellular neurite number and length, a hallmark of neuronal differentiation resulting from bcl-2 up-regulation. The ability of bcl-2 ARE asORNs to enhance the apoptotic threshold and to induce neuronal differentiation implies their potential application as a novel informational tool to protect cells from ischemic damage and to prevent neuronal degeneration.