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Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F. & Rosen- baum, J. (1970) Proc.Nat. Acad. Sci. USA, 65, 129-136. 4. Anagnoste, B. F., Goldstein, M. & Broome, ...
Proc. Nat. Acad. Sci. USA Vol. 69, No. 1, pp. 258-263, January 1972

Neurotransmitter Synthesis by Neuroblastoma Clones (neuroblast differentiation/cell culture/choline acetyltransferase/acetylcholinesterase/ tyrosine hydroxylase/axons-dendrites)

TAKEHIKO AMANO, ELLIOTT RICHELSON, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014

Contributed by Marshall Nirenberg, November 15, 1971 Neuroblastoma clones were examined for ABSTRACT choline acetyltransferase (EC 2.3.1.6), tyrosine hydroxylase (EC 1.14.3.a), acetylcholinesterase (EC 3.1.1.7), and also for neurite formation. One clone does not form axons or dendrites. Three types of clones were found with respect to neurotransmitter synthesis: cholinergic, adrenergic, and clones that do not synthesize acetylcholine or catechols. All clones contain acetylcholinesterase. These results show that genes determining neurotransmitter species can be expressed in dividing cells, that the parental programs of gene expression are inherited, and that dividing cells can be programmed with respect to their ability to communicate with other cells.

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%. Duplicate or triplicate homogenates were prepared and each was assayed for choline acetyltransferase activity at 4 concentrations of protein. The rate of reaction was proportional to enzyme concentration within the range 5-350 pmol of [14C]acetylcholine formed per 10 min. Assay reproducibility with replicate homogenates was +15%. Each value reported is the average of values obtained with 2-3 homogenates. "4C-Labeled reaction products in column eluates were characterized by paper chromatography or electrophoresis. Reactions were modified so that the specific activity of the [I4C ]acetyl CoA was 40-50 Ci/mol, and choline chloride rather than choline iodide was used. Solutions containing 14C-labeled products (25-30 ul of a column eluate); 0.2 ,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 scintillation counter.

Elegant biological studies have yielded much information pertaining to the problem of how neural circuits form as the nervous system is assembled. However, virtually nothing is known about the molecular mechanisms for synapse formation. The problem ultimately must be defined in terms of the genetic program for generating different cell types and the steps that determine the specificity of neurons in forming functional synapses. 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 MATERIALS

Cells. 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)-

Acetylcholinesterase (EC 3.1.1.7) Assay. The enzyme was assayed as described by Blume et al. (7).

Tyrosine Hydroxylase (EC 1.14.3.a) Assay. Cell monolayers were washed, harvested, and sonicated as described

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Proc. Nat. Acad. Sci. USA 69

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except that cells and protein were harvested in 0.1 M potassium phosphate buffer, pH 6.2. Tyrosine hydroxylase activity was assayed by a modification of the methods described by Nagatsu, Levitt, and Udenfriend (10), and by Shiman, Akino, and Kaufman (11). Each reaction contained the following components in a final volume of 0.05 ml: 0.1 NI potassium phosphate buffer (pH 6.2), 0.5 mM L-[3,5-di 3H]tyrosine (12 Ci/mol, from Amersham-Searle), 0.3 mM 6,7-dimethyl-2-amino-4-hydroxy5,6,7,8-tetrahydropteridine (Calbiochem), 0.25 mM NADPH (sodium salt), about 27 jtg of sheep-liver dihydropteridine reductase protein [purified through the second ammonium sulfate-precipitation step of Kaufman (12)1, and 0-0.5 mg of homogenate protein. Reactions were incubated at 34WC for 10 min, and were stopped by the addition of 0.5 ml of 0.17 N acetic acid at 30C and assayed as described by Nagatsu etal. (10). 93% of the 3H+ released from i-[3,5-3H]tyrosine was recovered in the column eluate; appropriate corrections were applied to reported values. An internal standard of 3H OH then was added to each sample and radioactivity again was determined. The counting efficiency for 3H was about 30%. The rate of reaction was proportional to the concentration of homogenate protein for values reported. Duplicate homogenates were prepared and each was assayed for tyrosine hydroxylase at four protein concentrations; reproducibility was ± 25%. Average values are reported. Protein was assayed by a modification of the method of Lowry (13).

Characterization of the 3H-labeled product of the Tyrosine Hydroxylase Reaction. The tyrosine hydroxylase reaction contained 0.1 mM p-bromo-m-hydroxybenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, in addition to the components described above. 3H-Labeled products formed during incubation were adsorbed to alumina and separated from [3H]tyrosine as described by Nagatsu et al. (14), except that 3H-labeled products were eluted with 0.2 N HCl. Recovery of 3,4-dihydroxyphenylalanine was 78%. An appropriate correction was applied to values reported. The [3H]catecholamines then were characterized by paper and thin-layer chromatography with the following solvents: 1-butanol-glacial acetic acid-H20 12: 3: 5; methylethylketone-formic acid-H20 24:1:6; ethylacetate-glacial acetic acid-H20 15:15:10; and 1-butanol-1 N acetic acid-ethanol 35:10:10. Thin-layer chromatography was performed with Eastman Chromogram Sheet 6065 (20 X 20 cm). Spots were located after development by spraying with ethylenediamine ferricyanide solution (15) to locate catechols, or with ninhydrin to locate tyrosine. RESULTS Cell types

The specific activities of tyrosine hydroxylase, choline acetyltransferase, and acetylcholinesterase found with homogenates of the neuroblastoma tumor grown in vivo and different clonal cell lines derived from this tumor are shown in Table 1. Values obtained with mouse L-cells, a fibroblastic cell line, and mouse brain are also given for comparative purposes. The specific activity of choline acetyltransferase from tumor was about 2% that of mouse brain. Acetylcholine synthesis was detected with most cell extracts, including L-cells, and other established cell lines not shown here; however, the rate of acetylcholine synthesis was