Journal of Neuroscience Research 25:476-485 (1990) z Enzymatic Activities During Differentiation of the Human Neuroblastoma Cells, LA-N-1 and LA-N-2 I.N. Singh, C. Sorrentino, D.C. McCartney, zyxwv R. Massarelli, and J.N. Kanfer Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba. Winnipeg, Manitoba, Canada (I.N.S.. G.S.. D.G.M.. J.N.K.); CNRS Centre de Neurochimie, Strasbourg, Cedex. France (R.M.) The presence of M retinoic acid (RA) in the culture medium of LA-N-1, a catecholaminergic cell line, and LA-N-2, a cholinergic cell line, enhanced their morphological differentiation. Tyrosine hydrox- ylase (TH) activity of the LA-N-1 cells was increased in the RA-treated cells compared with control cul- tures at day zyxwvuts 4 and remained elevated. Choline acetyl- transferase (ChAT) activity in the LA-N-2 cells grad- ually increased until 8 days in vitro (DIV) both in the untreated control and the RA treated cultures. This activity in control and treated cells decreased gradu- ally to a constant level of activity, The ChAT activity at 8 DIV of RA-treated LA-N-2 cells was increased 2.1-fold (P<O.OOl) as compared to the control cul- tures. This increase in ChAT activity was accompa- nied by a 73% decrease of acetylcholinesterase (AChE) activity in LA-N-2 cells by 8 DIV. AChE ac- tivity of LA-N-1 cells was unchanged during the time course of the experiment. Phospholipase- A, (PL-A,) activity in RA-treated LA-N-2 cells was increased at day 4 as compared with the control cultures. There were no differences observed in phospholipase-D (PL- D), choline kinase and GPC-phosphodiesterases ac- tivities in RA-treated and -untreated LA-N-1 and LA- N-2 cells. Key words: differentiation, human neuroblastoma cells, LA-N-1, LA-N-2, enzymes, retinoic acid INTRODUCTION Neuroblastoma, presumed to be of neural crest or- igin, is a common solid tumor of children (Young and Miller, 1975); more than one half these occur within or near the adrenal gland (Beckwith and Perrin. 1963); Imashuku et al., 1976). More than a dozen of these tu- mors have been adapted to continuous cell culture and their morphological and biochemical properties deter- mined, including a partial characterization of the en- zymes involved in the synthesis of adrenergic and cho- linergic neurotransmitters (Imashuku et al., 1976; West et al., 1977; Biedler et al., 1978; Schlesinger. et al., 1976). These cells appear to have some biochemical characteristics similar to those of adrenergic and cholin- ergic neurons (Mains and Patterson, 1973; Bunge et al., 1978). Cultured human neuroblastoma cells can be in- duced to morphologically differentiate by retinoic acid (RA) (Sidell et al., 1983, 1986; Sidell and Horn, 1985; Robson and Sidell, 1985; Pahlman et al., 1984; Biedler et al., 1973; Bernal et al., 1983). Differentiation of neu- roblastoma cells is characterized by cessation of growth and the formation of extended neuritic processes (Prasad, 1975). It seemed useful to examine selected enzymatic ac- tivities of human neuroblastoma cultures as a function of time in culture both in the presence and absence of RA.' These studies were undertaken with both LA-N- I, a cat- echolaminergic, and LA-N-2, a cholinergic cell line, in an attempt to discriminate between enzymatic activity required for general cell metabolism from those that may contribute to the cholinergic characteristics. We mea- sured several of the enzymatic activities involved in the metabolism of acetylcholine (ACh) for cholinergic neu- rons, and tyrosine hydroxylasc for catecholaminergic neurons. Cholinergic neurons contain choline acetyl- transferase (ChAT) and acetylcholinesterase (AChE) (Biedler et al., 1978; Casper and Davies, 1988; Amano et a]., 1972; Ross et al., 198 zyxw 1 ; West et al., 1977; Ebel et al., 1974), while catecholaminergic cells have tyrosine hydroxylase (TH) (Amano et zyx id., 1972; Ross et al., Received June 9, 1989: revised November 10. 1Y8Y: accepted No- vember 13, 1989. Addresb reprint requests to Professor J. N. Kanfer. Department of Biochemistry and Molecular Biology. Faculty of Medicine. University of Manitoba, Winnipeg R3E OW3. Manitoba. zyx 0 1990 Wiley-Liss, Inc.