Neuroblastoma clones were examined for choline acetyltransferase (EC 2.3.1.6), tyrosine hydroxylase (EC 1.14.3.a), acetylcholinesterase (EC 3 The neuroblastoma system established by Augusti-Tocco and Sato (1) provides an unusual opportunity to explore steps in neuron differentiation and function. The cells multiply rapidly in vitro, yet exhibit many properties characteristic of differentiated neurons (2-7). In this report, the properties of additional clones derived from the mouse neuroblastoma are described. Three cell types, cholinergic cells, adrenergic cells, and cells that do not synthesize acetylcholine or catecholamines, were detected.
METHODS AND MATERIALSCells. Mouse neuroblastoma C-1300 cells were grown as described (7). Some clones were obtained in two stages: first, a well-isolated colony of cells in agar was picked and then cloned by isolation of a single cell with a stainless-steel cylinder. In other cases, cells were added to petri dishes containing broken coverslips; each glass shard with a single cell was then transferred to a separate dish.Chromosomes were analyzed by incubation of cells in logarithmic growth for 6-12 hr with 15-300 1M colcemide (N-desacetyl-N-methyl-colchicine) obtained from Ciba;chromosomes were spread by the method of Merchant, Kahn, and Murphy (8).Choline Acetyltransferase (EC 2.3.1.6) Assay. Cell monolayers were washed 3 times with an isotonic salt solution; then cells and protein were harvested by scraping and washing with 10 mM potassium phosphate buffer (pH 6.8)-1 mM EDTA (potassium salt). The recovered suspension was sonicated for 5 min at 3°C, divided into small portions, and stored in a vapor-phase liquid-nitrogen freezer.Choline acetyltransferase activity was assayed by a method modified (manuscript in preparation) from that of Schrier and Shuster (9). Each reaction contained the following components in a final volume of 0.05 ml, except where noted: 50 mM potassium phosphate buffer (pH 6.8), 200 mM NaCl, 1 mM EDTA (potassium salt), 2.5 mM choline iodide, 0.5% Triton X-100 (Packard), 2.2 mM ['4C]acetyl CoA (10 Ci/mol), 0.1 mM neostigmine methylsulfate, and 0-0.5 mg of homogenate protein. Each reaction was incubated at 37°C for 10 min; then 0.5 ml of H20 at 3°C was added and the diluted reaction and 2 subsequent 1.0-ml washes were passed through a 0.5 X 5 cm column of Bio-Rad AG 1-X8 resin (C1-form, 100-200 mesh). Each eluate was collected in a scintillation vial; 10 ml of scintillation solution [1000 g Triton X-100-2 liters of toluene-165 ml Liquifluor (New England Nuclear Co.)] was added and radioactivity was determined. The counting efficiency for 14C was 80-90%. ,umol of unlabeled acetylcholine, and 0.2 ,umol of unlabeled acetylcarnitine were subjected to ascending paper chromatography for 16-24 hr with 1-propanol-0.1 N acetic acid 3:1. Chromatograms were dried and sprayed with the Dragendorf reagent (18) to visualize acetylcholine or acetylcarnitine. The chromatogram was cut into 1.0 X 0.5 cm segments, and the radioactivity of each was determined with a ...
