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G. Carella, A. Tempestini, L. Papucci, N. Schiavone, M. Donnini, A. Lapucci, E. Witort, R. Brancato, S. Capaccioli; Prevention of Retinal Cell Apoptosis by Antisense Modified Oligoribonucleotides That Upregulate Bcl-2 Expression by Targeting a Bcl-2 mRNA Destabilizing Element (ARE) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2343.
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Purpose: We previously identified in Bcl-2 mRNA 3'UTR a destabilizing A+U rich element (ARE) which downregulates Bcl-2 expression during apoptosis (Schiavone et al., The FASEB Journal 14:174-184, 2000) by interacting with specific ARE binding proteins (Lapucci et al., J. Biol. Chem. 277:16139-16146, 2002). The goal of this study was to ascertain whether lipotransfected modified antisense oligoribonucleotides (ORNs) targeting the Bcl-2 ARE were able to inactivate its destabilizing function and, thereby, to upregulate Bcl-2 expression and prevent apoptosis in cultured cells. Methods: A human retinal cell line (RPE), maintained in a standard culture medium supplemented with 10% FCS, was lipotransfected with three contiguous 26-mer 2'-O-methyl ORNs (5 µM each one) targeted to the core of Bcl-2 ARE (ARE ORNs), using DOTAP (15 µM) as transfection reagent (Fig 1). A fully degenerated 26-mer 2'-O-methyl ORN (Deg-ORN) was used as control. Three days following lipotransfection, RPE cells were either collected to quantify Bcl-2 mRNA and protein levels, or transferred to ARE-ORN-supplemented but serum-deprived Locke medium used as apoptotic stimulus. Apoptotic events were scored by time-lapse videomicroscopy or TUNEL. Results: Lipotransfected ARE-ORNs, but not the Deg-ORN, dramatically increased Bcl-2 mRNA and protein cellular levels and lowered the number of apoptotic events of RPE cells cultured in Locke medium. Conclusions: The ability of our lipotransfected ARE ORNs to upregulate Bcl-2 expression and to prevent apoptosis of RPE cells cultured in Locke medium, suggests their possible application as innovative antiapoptotic drug for treatment of all retinal diseases, included glaucoma, where excess of apoptosis is the main pathogenetic mechanism. Fig 1. The ORN-targeted Bcl-2 ARE core (bases 936-1021 GenBank M13994)
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