Abstract
We have identified previously a destabilizing adenine- and uracil-rich 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.
Footnotes
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This work was partially supported by Associazione Italiana per la Ricerca sul Cancro, Università di Firenze, Ente Cassa di Risparmio di Firenze, and the Visufarma srl and Ministero Italiano dell'Università e della Ricerca.
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ABBREVIATIONS: AU, adenine and uracil; ARE, adenine and uracil-rich element; AUBP, adenine and uracil-rich element binding proteins; ORN, 2′-O-methyl oligoribonucleotide; asORN, antisense 2′-O-methyl oligoribonucleotide; nt, nucleotide; degORN, degenerated 2′-O-methyl oligoribonucleotide; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; DRB, 5,5-dichloro-1-β-d-ribofuranosylbenzimidazole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; CFSE, carboxyfluorescein diacetate succinimidyl ester.
- Received May 21, 2007.
- Accepted November 6, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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