TY - JOUR
T1 - Complex host cell responses to antisense suppression of ACHE gene expression
AU - Galyam, N.
AU - Grisaru, D.
AU - Grifman, M.
AU - Melamed-Book, N.
AU - Eckstein, F.
AU - Seidman, S.
AU - Eldor, A.
AU - Soreq, H.
PY - 2001
Y1 - 2001
N2 - 3′-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2′-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.62-100 nM. PS-ODN was effective at 1-100 nM. Both AS-ODN displayed progressively decreased efficacy above 10 nM. In situ hybridization and confocal microscopy demonstrated dose-dependent decreases, then increases, in AChE mRNA. Moreover, labeling at nuclear foci suggested facilitated transcription or stabilization of AChE mRNA or both under AS-ODN. Intracellular concentrations of biotinylated oligonucleotide equaled those of target mRNA at extracellular concentrations of 0.02 nM yet increased only 6-fold at 1 μM ODN. Above 50 nM, sequence-independent swelling of cellular, but not nuclear, volume was observed. Our findings demonstrate suppressed AChE expression using extremely low concentrations of AS-ODN and attribute reduced efficacy at higher concentrations to complex host cell feedback responses.
AB - 3′-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2′-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.62-100 nM. PS-ODN was effective at 1-100 nM. Both AS-ODN displayed progressively decreased efficacy above 10 nM. In situ hybridization and confocal microscopy demonstrated dose-dependent decreases, then increases, in AChE mRNA. Moreover, labeling at nuclear foci suggested facilitated transcription or stabilization of AChE mRNA or both under AS-ODN. Intracellular concentrations of biotinylated oligonucleotide equaled those of target mRNA at extracellular concentrations of 0.02 nM yet increased only 6-fold at 1 μM ODN. Above 50 nM, sequence-independent swelling of cellular, but not nuclear, volume was observed. Our findings demonstrate suppressed AChE expression using extremely low concentrations of AS-ODN and attribute reduced efficacy at higher concentrations to complex host cell feedback responses.
UR - http://www.scopus.com/inward/record.url?scp=0035090623&partnerID=8YFLogxK
U2 - 10.1089/108729001750072128
DO - 10.1089/108729001750072128
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AN - SCOPUS:0035090623
SN - 1087-2906
VL - 11
SP - 51
EP - 57
JO - Antisense and Nucleic Acid Drug Development
JF - Antisense and Nucleic Acid Drug Development
IS - 1
ER -