Glial cells release a factor into their culture medium that induces a high 4egree of morphological differentiation 'in neuroblastoma cells under normal growth conditions. This phenomenon is not correlated with a change; in intracellular adenosine 3': 5'-cyclic'monophosphate or in the rate of cell growth. Media from other cell lines tested induce less morphological differentiation or have no effect.Process formation by neuroblastoma cells in tissue culture has been termed morphological differentiation, and its importance as a model for neural differentiation has been pointed out by many authors (1-4). X-Irradiation (5), removal of serum (2), and addition of N6,02'-ibutyryl adenosine 3': 5'-cyclic monophosphate (But2cAMP) (3, 4) or 5-bromodeoxyuridine (BrdU) (6) can induce this process formation. All these treatments are unphysiological, even at low degree of morphological differentiation, and they all interfere with the growth rate of the cells. Considering the theoretical importance of cell to cell interactions in the central nervous system, we tried to influence process formation by using medium from glial cell cultures, We report that such a glial conditioned medium induces a high percentage of morphological differentiation under physiological conditions. MATERIALS AND METHODSNeuroblastoma' cells, clone NB-2a, derived from the C-1300 tumour cell line (7), and glial cells, clone C-6, derived from a N-nitrosomethylurea-induced rat glial tumour (8) To determine the percentage of cells undergoing morphological differentiation, 50,000 trypsinized neuroblastoma cells were plated per 60-mm dish. After 16 hr of incubation, the medium was replaced by the medium to be tested, and 48 hr later all cells with and without processes were counted in at least five randomly selected areas of each culture plate. Cells having processes longer than the diameter of the cell body were considered morphologically differentiated. A total of 300-400 cells were counted per dish. The values reported here represent the percentage of differentiated cells determined in three parallel cultures.For intracellular cAMP determination, the cells were quickly washed with Dulbecco's phosphate buffered saline (pH 7.2, 37°) then fixed with 1 ml of 0.05 N HCl. They were scraped with a rubber policeman and the suspension was heated for 10 min in boiling water. The samples were then neutralized with 0.1 N NaOH to pH 7.3 and treated with 50 Ml of 0.17 M BaSO4 and 50 Mu of 0.15 M Ba(OH)2. The solution was clarified with a glass-fiber filter and cAMP was determined in triplicate by the method of Gilman (14). For the early time points the cells of two or three plates were extracted in the same 1 ml of 0.05 N HCl. At each time point, three parallel cultures were washed, then solubilized in 0.5% sodium dodecyl sulfate for protein determinations (15). RESULTS AND DISCUSSIONThe induction of morphological differentiation of neuroblastoma cells by glial conditioned medium is illustrated by the micrographs in Fig. 1 (Table 1). Unlike those treatments, condi...

No abstract

Neuroblastoma cells have been found to extend neurites when gown in a medium su plemented with delipidated fetal calf serum. Fetal calf sera from different commercial sources give rise to marked differences in the degree of this spontaneous morphological differentiation. The phenomenon can be prevented by the addition of certain fatty acids; oleic acid is especially effective. The serum-free conditioned medium from glial cells can quantitatively antagonize the effect of oleic acid, suggesting that glial factor activity could be due to components [lipids and/or macromolecular factors(s)J that are able to modify the properties of the neuroblastoma cell membrane.The ability of cultured neuroblastoma cells to extend neurites under certain conditions has been adopted as a suitable model (for review see ref. 1) to study initial events in cellular neuronal differentiation. The study of such a model, which in its most fundamental form can be considered as one of morphological differentiation, should also give direction to an understanding of the biochemical mechanisms underlying neuronal plasticity. We have previously shown that glial cells in culture release a macromolecular factor that can induce morphological differentiation of neuroblastoma cells without affecting the growth rate of these cells (2). It has also been demonstrated (3) that this glial factor is distinct from the well-documented nerve growth factor. In this communication, we report that up to 60% of a neuroblastoma cell population is able to undergo spontaneous morphological differentiation when grown in a medium supplemented with delipidated fetal calf serum. Fatty acids, especially oleic acid, added to the delipidated serum can prevent this spontaneous morphological differentiation. Furthermore, the effect of fatty acids can be quantitatively overcome by the addition of serum-free glial-conditioned medium.MATERIALS AND METHODS Cells. Mouse neuroblastoma cells, clone NB2A, and rat glioma cells, clone C-6, were grown as described (2) with the exception that, if not specifically stated otherwise, 35-mm dishes from Falcon or Corning were inoculated with 20,000 EDTAdislodged neuroblastoma cells per dish containing 2 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Colorado). After 16 hr, the medium was replaced by 2 ml of the medium to be tested. At 24 and/or 48 hr after this change of medium, the cultures were fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS) containing (g/liter) NaCl, 8.0; KCI, 0.2; Na2HPO4-2H20, 1.44; KH2PO4, 0.2; CaCI2-2H20, 0.132; and MgCI2.6H20, 0.100. The extent of morphological differentiation, expressed as the per-The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (4), with 1-butanol/diisopropyl ether (40:60, vol/vol) as the organic phase. The precipitated material appearing at the interphase was d...

No abstract