Differentiation agents enhance cholinergic characteristics of LA-N-2 human neuroblastoma cells

LA-N-2, a cell line derived from a human peripheral neuroblastoma, has a partially cholinergic phenotype and is a potential in vitro model of cholinergic neurons. The object of this study was to enhance the cholinergic phenotype of these cells with differentiation agents to improve the cell line's usefulness as a convenient model of cholinergic function. I treated cells in the presence of serum with 10 microM 5-azacytidine, 2.5 microM bromodeoxyuridine, 2 nM ciliary neurotrophic factor, 1 mM dibutyryl cAMP, 0.25 nM leukemia inhibitory factor and/or 3.8 nM nerve growth factor, N-2 supplement (without serum), or 10 microM retinoic acid for 9-14 days. Treated cells were loaded with [3H]choline for 30 min at 37 degrees and washed. The amounts of cellular and released (5 min, room temperature), labeled and unlabeled acetylcholine and choline were determined by HPLC. None of the differentiation agents induced Ca(2+)-dependent release of [3H]acetylcholine, but 5-azacytidine, dibutyryl cAMP, N-2, and retinoic acid increased Ca(2+)-independent release that was specific for acetylcholine. In addition, 5-azacytidine, bromodeoxyuridine, leukemia inhibitory factor, and N-2 substantially increased [3H]acetylcholine levels, and these increases correlated highly with increases in total acetylcholine levels. Overall, LA-N-2 cells should prove to be a good model for studying cholinergic function.