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 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.
Papucci
Laura
L
Department of Experimental Pathology and Oncology, University of Florence, Viale G.B. Morgagni 50, 50134 Florence, Italy.
Witort
Ewa
E
Bevilacqua
Anna Maria
AM
Donnini
Martino
M
Lulli
Matteo
M
Borchi
Elisabetta
E
Khabar
Khalid S A
KS
Tempestini
Alessio
A
Lapucci
Andrea
A
Schiavone
Nicola
N
Nicolin
Angelo
A
Capaccioli
Sergio
S
D000225:Adenine / Q000502:physiology*; D017209:Apoptosis / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D009843:Oligoribonucleotides / Q000235:genetics* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism*
D009843:Oligoribonucleotides; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014498:Uracil; D000225:Adenine