Complex host cell responses to antisense suppression of ACHE gene expression.
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 0.02-100 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 microM 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.
Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel.
N
N
Galyam
D
D
Grisaru
M
M
Grifman
N
N
Melamed-Book
F
F
Eckstein
S
S
Seidman
A
A
Eldor
H
H
Soreq
D000110:Acetylcholinesterase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics* / Q000494:pharmacology*; D016716:PC12 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D000110:Acetylcholinesterase