DIFFERENTIATION INTENSIFIES THE SUSCEPTIBILITY OF PHEOCHROMOCYTOMA CELLS TO ANTISENSE OLIGODEOXYNUCLEOTIDE-DEPENDENT SUPPRESSION OF ACETYLCHOLINESTERASE ACTIVITY

To investigate the effect of neuronal differentiation on the capacity of antisense oligonucleotides (AS-ODNs) to suppress the production of acetylcholinesterase (AChE) in rat pheochromocytoma cells, we tested s even 3'-phosphorothioated AS-ODNs targeted to ACHEmRNA and two control ODNs. Three different administration protocols were used: oligonucleo tides were added at 1 mu M for 24 hours to nondifferentiated PC12 cell s, together with nerve growth factor (NGF) or 24 hours following NGF-i nduced cholinergic differentiation. The content of free thiol groups i n lysed cells was measured to evaluate cell number, therefore, surviva l, and the rate of acetylthiocholine hydrolysis was the measure of ACh E activity, Among nondifferentiated cells, over 95% survived treatment with 8 of 9 of the ODNs. Moreover, two AS-ODN suppressed AChE activit y in nondifferentiated PC12 cells by 16%-20% as compared with 10% supp ression by control ODNs (p less than or equal to 0.01), When added con currently with NGF, one other AS-ODN suppressed AChE activity signific antly better (28%) than the control ODNs (16%), Moreover, when added f ollowing NGF treatment, which induced a significant increase in AChE a ctivity, four different AS-ODNs but not the control ODNs suppressed 20 %-35% of the enhanced AChE activity (p less than or equal to 0.01), Re duced levels of AChE mRNA but no difference in actin mRNA levels were observed by following the kinetics of RT-PCR amplification in differen tiated PC12 cells treated with these four AS-ODNs, as compared with co ntrol cells, Our findings demonstrate a differentiation-related increa se in the susceptibility of PC12 cells to inhibition by specific AS-OD Ns, suggesting the use of this model system to select AS-ODNs for supp ression of AChE levels in the treatment of neurodegenerative diseases associated with cholinergic malfunction.