Complex host cell responses to antisense suppression of ACHE gene expression

3'-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected wi th 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 decreas ed efficacy above 10 nM, In situ hybridization and confocal microscopy demo nstrated dose-dependent decreases, then increases, in AChE mRNA, Moreover, labeling at nuclear foci suggested facilitated transcription or stabilizati on of AChE mRNA or both under AS-ODN, Intracellular concentrations of bioti nylated oligonucleotide equaled those of target mRNA at extracellular conce ntrations of 0.02 nM yet increased only 6-fold at 1 muM ODN. Above 50 nM, s equence-independent swelling of cellular, but not nuclear, volume was obser ved. Our findings demonstrate suppressed AChE expression using extremely lo w concentrations of AS-ODN and attribute reduced efficacy at higher concent rations to complex host cell feedback responses.