ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT 7:351-359 (1997) Mary Ann Liebert, Inc. Differentiation Intensifies the Susceptibility of Pheochromocytoma Cells to Antisense Oligodeoxynucleotide-Dependent Suppression of Acetylcholinesterase Activity MIRTA GRIFMAN and HERMONA SOREQ ABSTRACT 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 seven 3'- phosphorothioated AS-ODNs targeted to ACHEmRNA and two control ODNs. Three different administration protocols were used: oligonucleotides were added at 1 fiM for 24 hours to nondifferentiated PC12 cells, to- gether with nerve growth factor (NGF) or 24 hours following NGF-induced cholinergic differentiation. The content of free thiol groups in lysed cells was measured to evaluate cell number, therefore, survival, and the rate of acetylthiocholine hydrolysis was the measure of AChE activity. Among nondifferentiated cells, over 95% survived treatment with 8 of 9 of the ODNs. Moreover, two AS-ODN suppressed AChE activity in non- differentiated PC12 cells by 16%-20% as compared with 10% suppression by control ODNs (p < 0.01). When added concurrently with NGF, one other AS-ODN suppressed AChE activity significantly better (28%) than the control ODNs (16%). Moreover, when added following NGF treatment, which induced a significant in- crease in AChE activity, four different AS-ODNs but not the control ODNs suppressed 20%-35% of the en- hanced AChE activity (p<0.01). Reduced levels of AChE mRNA but no difference in actin mRNA levels were observed by following the kinetics of RT-PCR amplification in differentiated PC12 cells treated with these four AS-ODNs, as compared with control cells. Our findings demonstrate a differentiation-related increase in the susceptibility of PC 12 cells to inhibition by specific AS-ODNs, suggesting the use of this model system to select AS-ODNs for suppression of AChE levels in the treatment of neurodegenerative diseases associated with cholinergic malfunction. INTRODUCTION levels of the protein itself, it is important to develop anti- sense oligodeoxynucleotides (AS-ODN) targeted against Currently approved drugs for the treatment of ACHEmRNA (Grifman et al., 1997). By preventing the produc- Alzheimer's disease patients are designed to suppress the tion of AChE, anti-ACHE ODNs would exert two functions: catalytic activity of the acetylcholine hydrolyzing enzyme suppression of protein levels and reduction of enzyme activity, acetylcholinesterase (acetylcholine acetylhydrolase, EC To achieve this goal, the AS-ODNs in question should be non- 3.1.1.7, AChE) (Knapp et al., 1994). This is aimed at augment- toxic, highly selective for the ACHE gene, operate in a se- ing cholinergic neurotransmission, which is impaired in such quence-dependent manner, and exert their suppression activity patients because of a selective loss of cholinergic neurons. on fully differentiated neurons. However, such inhibitors do not reduce the amount of the The mammalian ACHE gene is transcribed and processed AChE protein, and there are recent reports of actions of AChE, into three alternatively spliced mRNAs (Li et al., 1991 ; Legay unrelated to its catalytic activity, in process extension (Small et al., 1993; Karpel et al., 1994). The mature transcript most et al., 1995; Layer and Wiellbold, 1995; Jones et al., 1995; abundant in brain is the one in which exon 4 is spliced to exon Darboux et al., 1996; Sternfeld et al., 1997) and amyloid 6 (E6), whereas the isoform in which exon 4 is spliced to exon fibril formation (Inestrosa et al., 1996). Therefore, to reduce 5 (E5) is found mainly in cells of hematopoietic lineages (Lev- Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel. 351