Received for publication May 22, 2007.
Revised November 6, 2007.
Accepted for publication November 6, 2007.

Impact of targeting the AU-rich element of bcl-2 mRNA with oligoribonucleotides on apoptosis, cell cycle and neuronal differentiation in SHSY-5Y cells

Abstract

We have previously identified a destabilizing AU-rich element (ARE) in the 3^'UTR of bcl-2 mRNA that interacted with ARE-binding proteins (AUBPs) to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2^'-O-methyl oligoribonucleotides both in sense and in 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, FACS 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.

Key words: Antisense, Mechanisms of cell killing/apoptosis