Due to
AF64A's structural similarity to
choline,
AF64A can selectively affect cholinergic neurons, which possess a high affinity
choline transport system for
acetylcholine synthesis. The mechanism by which
AF64A selectively produces its cytotoxic effect is unknown. However, based on previous studies that demonstrate that DNA lesions produced by
AF64A caused premature termination of N-myc transcription in vitro, it is possible that
AF64A may affect the transcription of genes necessary for developmental maintenance in
cholinergic cells. Using the LA-N-2 cells as a model to study the effects of
AF64A in a purely
cholinergic system, we investigated the effects of
AF64A on the expression of the N-myc gene and monitored cell growth.
AF64A produced a maximal decrease in N-myc
mRNA with a return to steady state levels at later time points. Moreover, a decrease in cell numbers in AF64A-treated cells was observed, and these cells did not double in number at their respective doubling time as compared to control. In other studies, a causal relationship between a reduction in N-myc and an inhibition of cell growth and replication has been reported. While these studies do not allow us to conclude that
AF64A is specific for N-myc, the data do, nevertheless, suggest that
AF64A affects cell growth and/or replication by down-regulating the expression of N-myc which is involved in differentiation and cell growth in
neuroblastomas. Presence of
choline or hemicholinium-3 prevented the AF64A-induced decrease of N-myc levels by competing with, or inhibiting the
choline transport mechanism by which
AF64A enters the cell, respectively.