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Neuroblasts-glia interaction
Experimental Cell Research 98 (1976) 191-203
NEUROBLASTS-GLIA The Effect of Co-cultivation of Neuroblastoma V. STEFANOVIC,’
INTERACTION upon Ecto-ATPase Activity and Glioma Cells
J. CIESIELSKI-TRESKA,
Centre de Neurochimie
A. EBEL and P. MANDEL
du CNRS, F-67085 Strasbourg
Cedex, France
SUMMARY The effect of cell contact and cell medium upon the ecto-enzymes, MgZ+- and Ca*+-dependent ATPase and 5’-nucleotidase were studied in nervous svstem cells in tissue culture. Conditions were worked out for co-culture and reseparation of glioblastoma and neuroblastoma cells so that the effects upon each of the co-cultured cell lines after interaction of these cells could be reliably determined. Co-cultivation of mouse neuroblastoma and glioma cell lines markedly enhanced Mg*+- and Ca*+-dependent ecto-ATPase activity. Evidence was obtained which indicates that increase in ecto-ATPase of co-cultured neuro- and glioblastoma cells occurs in both cell types. Ecto-ATPase was 500% of the orieinal level in clonal line NN astroblasts after co-culture with MI neuroblasts. This activity decreased over 50 transfers during the period of about a year. Increase in ecto-ATPase and morphological differentiation of Ml neuroblastoma cells after co-culture with NN astroblasts could also be brought about simply by treatment with the medium from NN cell cultures. Co-cultivation of neuroblastoma and glioma cells does not change significantly the specific activity of ecto-S’-nucleotidase.
The possibility of metabolic coupling between neurons and glia has been suggested in view of their close anatomical relationship, i.e. the ensheathing of some axons and synapses by glial cells, and the correlation of biochemical changes in neurons and glia. Several functions for glial cells have been considered, e.g. regulation of the ionic milieu of neurons [14], control of the level of neurotransmitters [5-71 and substrate available for protein synthesis [8, 91 in neurons and guidance of neuronal migration [lo]. Tissue culture methodology affords an opportunity to study molecular aspects of I Permanent address: Institut za Nefrologiju i Hemodijalizu, Faculty of Medicine, Nis, Yugoslavia. 13-761819
mutual interactions of dissociated cells of the nervous system and of clonal cell line of nervous origin. Okun [ 1l] has reported that chick embryo ganglion neurons survive in culture for several weeks in the apparent absence of glial cells. The work of several authors [12-161 has indicated that contact with glial cells enhances differentiation of dissociated neurons, and maintains it in long-term cultures. Flat embryonic brain cells in monolayer culture can be transformed into cells resembling mature astrocytes by a protein present in embryonic [ 171 or adult [ 181brain extract. Recently Monard et al. [19] reported on a factor which is released into the medium by glial cells and causes morphological differentiation of neuroblastoma cells upon addition to their Expr/ Cd Res 98 (1976)
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culture medium. Rat glioma cells contain a neuroblastoma and glial cells were seeded In Falcon containing IO ml DMEM and 10% foetal calf protein with properties similar to those of flasks serum. In order to separate neuroblastoma MI and astromouse salivary nerve growth [20]. In all of blast NN cells from mixed cultures, the cell monolayer these experiments only morphological char- was treated firstly with 0.02 % trypsin for 4 min. During this time most of the neuroblastoma cells were acteristics of cells have been studied. detached, whereas the astroblasts were dissociated by We have utilized neuroblastoma and glial incubation with a solution of 0.25 % trvusin. Harvested cell lines in culture as a model system to cells were diluted in complete medium and plated in low density, approx. IO@-300 cells/30 mm Falcon Petri study the surface membrane changes which dish. Ea& dish contained several broken coverslips may occur in cell-cell interaction. The ex- (approx. I mm 0). Those colonies with a small number of cells were removed and placed in microwells (vol ternal cell surface enzymes, ATPase (ATP I ml). For study of enzymatic activity, cells were placed in phosphohydrolase, EC 3.6.1.3) and 5’-nu60 mm Petri dishes containing 5 ml of growth medium. cleotidase (5’-ribonucleotide phospho- The medium was changed every other day. Tests for hydrolase, EC 3.1.3.5), were determined in infestation with mycoplasma were negative. a wide variety of mouse neuroblastoma and glial cell lines. In this paper, we report a Enzyme assays marked increase in cell surface ATPase ac- Enzymatic assays were performed on the day after the tivity in co-cultures of neuroblastoma and culture medium was changed. glial cells as well as in the different individual cells when separated. The effect of Assay of ATPase conditioned medium on neuroblastoma and Nutrient medium was carefully removed from Petri glial cell ecto-ATPase activity is also pre- dishes and discarded, and the cell monolaver was gently washed first with a medium containing 0.250 sented. mM EDTA, 0.125 mM EGTA, 30 mM Tris-HCI buffer I
METHODS Cell cultures Normal hamster astroblasts NN [21] and rat glioblastoma C6 cell lines r22 I were obtained from North American Biologicals, inc.-and American Type Culture Collection, respectively. Neuroblastoma clonal lines Nl8 and NlE-II5 were a gift from Dr M. Nirenberg (Laboratory of Biochemical Genetics, NIH, Bethesda, Md). Neuroblastoma clonal line Ml has been developed and characterized in our laboratory. Cells were grown in monolayer culture in Falcon tissue culture flasks with a growth area of 75 cm* in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with IO%foetal calfserum IGIBCo. Grand Island. NY) with 50 U of sodium penicillin G and 25 pg of streptomycin sulfate oer ml. The cultures were incubated at 37°C in a humidified atmosphere of 5 % CO, and 95 % air. The cells in stationary phase were subcultured after incubation with 0.25% trypsin (Difco l/250) for 2 min. Astroblasts from brain of newborn rats were cultured as previously described [23] and studied after 1720 days of cultivation. The growth medium was changed twice a week. Mixed cell cultures of neuroblastoma and glial lines were prepared as previously described [24]. Cultures of both cell lines were harvested by detaching cell monolayers with 0.25 % trypsin. Cell suspensions were diluted in complete medium, counted in a hemocytometer. Subsequently, equal parts of suspension of Exprl Cell Res 98 (1976)
.
(pH 7.8) and 250 mM sucrose, and afterwards 3 times with a total of about 10 ml of incubation solution without ATP. Incubation fluid contained 30 mM Tris-HCI buffer (pH 7.8), 5.5 mM glucose, 240 mM sucrose and 3 mM of CaCI, or MgCI,. Incubation was started by adding 3 mM Tris-ATP and conducted at 37°C for 2-15 min in a final volume of I.5 ml. The inorganic phosphate formed was determined calorimetrically according to the method of Gomori [25] modified by proportional reduction in reagent volumes. Appropriate controls without cells were also incubated to correct for small amounts of P, found in commercial oreparations of ATP or deriving from its spontaneous hidroIvsis. Another control with cells but without substrate was also included. These latter were consistently negative. In the kinetic studies, ATPase activity was determined in the presence of an ATP-regenerating system. For this assay, the incubation medium contained, in addition, I mM potassium salt of phosphoenolpyruvic acid and 6 U/ml of pyruvate kinase. Potassium was necessary for pyruvate kinase activation [26]. Since potassium al& activates MgZ+-dependeni ectoATPase [27], 0.1 mM ouabain was included in media used in this study. Although pyruvate kinase may be inhibited by Ca2+ [26], this ion had practically no effect on pyruvate kinase under our experimental conditions as the enzyme was present in many-fold excess. ATP breakdown products were studied by paper chromatography [28j, by enzymatic assays f&-A-TP 1291, ADP and AMP 1301, and by determining adenine nucleotides and IMP plus inosine by differential absorption spectra [3 I].
Ecto-ATPase
activity of neuroblastoma
and glioma cells
193
Assay of 5’-nucleotidase
Protein
Cells were washed as previously and incubated for 30 min at 37°C in a medium containing 30 mM Tris-HCI buffer (pH 7.4), 130 mM NaCl, 5.5 mM glucose, 1 mM 1 mM p-nitrophenylphosphate and 3 mM &Cl,, adenosine-5’-monophosphate (5’-AMP). Magnesium ensured cell attachment during the incubation period. The hydrolysis of 5’-AMP by non-specific phosphatases present on the surface of nervous system cells in culture was prevented by p-nitrophenylphosphate as described previously [32]. Pl liberated from 5’-AMP and p-nitrophenylphosphate and p-nitrophenol were determined on separated aliquots and the difference was taken as Pi enzymatically released from 5’-AMP. Choline acetyltransferase (ChAc, EC 2.3. I .6) was measured according to the microtechnique of McCaman & Hunt [3fl as modified by. Goldberg et al. r341 bv following the incornoration of rl-W-acetate from cl-“Cl-acetyl-CoA (NEN Chemicals; 59.2 mCi/ mmole) into acetylcholine. Acetylcholinesterase (AChE, EC 3.1.1.7) was determined by the method of McCaman et al. [35] which is based on the hydrolysis of [ I-IF]-acetylcholine (NEN Chemicals: 2.43 mCi/mmole). Non-specific cholinesterases were inhibited by the addition of iso-octamethyl-pyrophosphoramide ( 1OmB M) in the incubation medium. Histochemical demonstration of acetylcholinesterase was performed according to the method of Karnovsky & Roots [36].
Protein was determined by a modification of the method of Lowry et al. [40]. Specific activity of ATPase is expressed as pmoles Pi liberated/mg cell protein/h. In general, data are expressed as means +S.E.M.
Catecholamines Catecholamines were visualized according to Falk & Owman r371. Cell cultures in Falcon Petri dishes were used. After-removal of the culture medium, all preparations were frozen in dry ice and freeze-dried for 4 days at -35°C. They were then treated for 1 h with formaldehvde gas generated from uaraformaldehvde at 70°C [38]. The pamformaldehyde had been equilibrated with air at 70% relative humiditv f391. Cvtochemical controls were performed wiihout - freezedrying or exposure to formaldehyde vapour. A fluorescence microscope (Leitz Orthomat) with dark field condenser was used. The light source was an HBO-200 mercury lamp (Osram) with 5 mm B.A. 12 and 4 mm BG. 38 excitation filters. The fluorescence was observed through a secondary barrier filtre of 480 nm.
Chromosomal
analysis
Chromosome spreads were prepared from logarithmic phase cultures. After colchicine treatment (0.4 me/ml/ 3 h), osmotic shock was induced by dilution of themedium with distilled water (1 : 5/30 min/37”C). The cells were centrifuged, fixed in a freshly prepared mixture of acetic acid and methanol (1: 3) for 30 min, stained with Giemsa stain and photographed. A minimum of 8&90 metaphasal plates of each cell line was analysed to establish the modal chromosomal number and to identify the origin of the cells. 13*-761819
RESULTS Cultures of neuroblastoma
and glial cells
The morphological appearance of cultured neuroblastoma and glial cells has been previously described in detail [21-23, 41, 421. In the present study, we describe only the M 1 cell line. Neuroblastoma Ml clone was isolated in our laboratory and characterized by chromosomal and biochemical markers. Modal number of chromosomes of this cell line is 60. With regard to neurotransmitter synthesis neuroblastoma Ml clone was defined as adrenergic since it lacked ChAc activity and possessed catecholamines. AChE activity was rather low compared with other neuroblastoma clones but increased during the stationary phase of growth. Like other neuroblastoma cells, the Ml cell line proliferates rapidly in culture. During logarithmic phase of growth, the cells remain still small and without extensions. In the stationary phase, the cells attach firmly to the growth surface and the majority of cells extend neuron-like processes (fig. 1a). ATPase activio at external sutiace of neuroblastoma and glial cells
As it has been shown previously for NlE115 neuroblastoma cells [43], we found Mg2+- and Ca2+-dependent ATPase activity at the external surface of the N18 and Ml neuroblastoma clonal cell lines (table 1). The two indefinite glial cell lines, NN normal hamster astroblasts and C6 rat glioblastoma exhibited a very low Mg2+- as well as Ca2+-dependent ATPase activity. A difExptl Cell Res 98 (1976)
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kationary phase; (c) cokultu~e‘ok NN astroblasts Ml and NN cells. Phase contrast. x250.
ferent pattern of activation of neuro- Ecto-ATPase activity of the co-culture blastoma and glial cells ecto-ATPase by The results are summarized in table 2. After divalent cations is also evident. Mouse 15 days of co-culture of neuroblastoma Ml neuroblastoma clones had about two times cells and hamster astroblasts NN cells, an higher activity with Ca2+ than with Mg2+. increase of both Mg2+- and Ca2+-dependent Glial cell lines had higher Mg2+-dependent ecto-ATPase activities was observed. In ATPase activity. these co-cultures, Mg2+- and Ca2+-dependent ecto-ATPase activities were 2.24kO.03 Co-cultures of neuroblastoma and and 3.71 kO.08 pmoleslmg cell protein/h, glial cells respectively. When compared with the When mixed and placed into the culture Mg2+- and Ca2+-dependent ecto-ATPase acduring the first or on the second day neuro- tivity of the M 1 cell line, the increase is blastoma cells undergo a morphological about 4 times. Compared with the NN cell and biochemical differentiation [24]. An in- line, both ecto-ATPase activities increased crease in AChE activity of co-cultured Ml 8-10 times. In the co-cultures of hamster astroblasts cells was demonstrated by histochemical method (fig. 2). Catecholamine specific NN and neuroblastoma clonal lines N18 fluorescence was also increased (data are and NlE-115, a slight increase of Mg2+not presented). dependent ecto-ATPase activity appears, ExprlCellRes 98 (1976)
Ecto-ATPase
activity of neuroblastoma
2. Photomicrographs showing morphological aspect of neuroblastoma M 1 cells and Ml cells in coculture with NN cells, stained for acetylcholinesterase
Fig.
and glioma cells
195
activity. (a) Neuroblastoma MI cells; (6) co-culture of Ml and NN cells. (a) X200; (b) X250.
while the Ca2+-dependent ecto-ATPase ac- cell surface ATPase activity. Fig. 4 shows tivity is significantly higher. The values of the Mg*+- and Ca*+-dependent ATPase acMg*+-dependent ecto-ATPase activity in tivities of co-cultures of Ml and NN cells those co-cultures were similar to that of and their dependence on cell density. Both N18 and NIE-115 neuroblastoma clones. ATPase activities increased about twice Since in the co-cultures approx. 50% of the when the culture becomes confluent. At cell population were NN astroblasts with any period of the growth curve the ectoan originally low Mg*+-dependent ectoATPase activity, we may conclude that an increase of Mg*+-dependent activity of Table 1. Mg*+- and Ca*+dependent N18+NN and NlE-NllS+NN cell cul- ATPase activities at the external surface oj tures also occurred. neuroblastoma and glial cells in culture, exThe ecto-ATPase activity of the co-cul- pressed as voles Pi liberatedlmg cell protures of neuroblastoma Ml and rat glio- tein/h blastoma C6 cells were close to that of the Mg2+-dependent Ca*+-dependent original Ml clone and much higher than Cell line ATPase ATPase that of C6 clonal cell line. In the co-cultures 0.47+0.02 I. IO&O.03 of astroblasts from newborn rat brain and Ml 2.38kO.05 Nl8 I .20t0.04 M 1 neuroblastoma cells the Mg*+- and NIE-I I5 3.25f0.04 1.49+0.03 0.33+0.01 0.35 kO.02 Ca*+-dependent ecto-ATPase activities NN 0.23&0.01 0.27+0.02 C6 were close to that of the astroblasts in pri- Astroblasts in 15.86kO.65 primary culture 21.67+0.90 mary cell culture but manyfold higher than that of Ml cell line. NN and C6 glial cell lines and neuroblastoma clones were grown in Falcon flasks. For the study of enAmong several lines tested, neuro- zymatic activity the cells were subcultured in Petri blastoma Ml and astroblastoma NN cell dishes and enzymatic activity was determined after 4 Newborn rat astrocytes were grown as delines in co-culture were found to be the days. scribed under Methods. Each value is the mean of most potent in producing the increase in 54 experiments+S.E.M. Eiptl Cell Res 98 (1976)
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Fig. 3. Chromosomes of (a) mouse neuroblastoma cell line and (b) hamster astroblasts NN cell
Ml line.
ATPase activities exceeded by many times the respective activities of astroblast NN cells or neuroblastoma Ml cells.
Chromosome spreads Methods. x 1000.
prepared
as described
under
The heterospecific origin of the two cell lines make the chromosomal identification of neuroblasts and astroblasts easy. The modal chromosomal number of neuroblastoma Ml cells was 60 chromosomes with only 3 % of tetraploid cells in contrast to 40% of tetra- and octaploid astroblast NN cells. When mouse neuroblastoma cells have telocentric chromosomes with 2 or 3 submetacentric or mediocentric chromo-
Re-isolated NN cells and Ml cells One attempt was made to separate neuroblastoma and astroblast cells from the coculture. After 2 months in co-culture of neuroblastoma Ml and astroblasts NN cells, astroblasts NN cells were isolated. Neuroblastoma Ml cells were isolated after one Table 2. Mg2+- and Ca2+dependent ectoweek of co-culture with NN hamster astro- ATPase activities of neuroblastoma and glial cells in co-culture, expressed as blasts. The re-isolated neuroblastoma and glial voles Pi liberatedlmg cell protein/h cells were identified according to their MgY+-dependent Ca2+-dependent ATPase morphological, biochemical and chromo- Co-cultures ATPase somal properties. The morphological ap- MI+NN 2.24kO.03 3.71 LO.08 0.43~0.01 0.94?0.02 pearances of neuroblasts and astroblasts are MI+C6 MI +astroblasts shown in fig. 1 (a, b). Neuroblastoma cells in primary culture 25.82k2.80 15.47+0&l 1.30+_0.04 3.02kO.33 when compared with astroblasts arc small NII+NN NI IS+NN I .59+0.05 5.4810.23 and round. Some of the cells possess one or Neuroblastoma and glial cells were co-cultured in Falmore processes. Astroblasts cells after con flasks for IO days. For the study of enzymatic having formed a monolayer are greater in activity they were subcultured in Petri dishes and used after 4 days. Cultivation of newborn rat astrocytes is cell size, the nucleus is clearly visible with described under Methods. Each value is the mean of 5 one or more nucleoli. experiments+S.E.M. Expf/ Cell Res 98 (1976)
Ecto-ATPase activity of neuroblastoma and glioma cells
Figs 4, 5. Abscissa: time in subculture (days); ordinate: pmoles Pi liberated/mg prot/h. Fig. 4. Mgz+- and Ca*+-dependent ecto-ATPase activities of Ml and NN cells in co-culture. Equal parts of Ml neuroblastoma and NN astroblast cells were mixed and plated in Falcon flasks as described under Methods. After 15 days they were subcultured in Petri dishes and ATPase activity determined 2,4 and 6 days later. O-O, Mg2+-ATPase; O-O, Ca*+-ATPase. Fig. 5. Mg2+- and Caz+-dependent ecto-ATPase activities of NN astroblast cells and NN cells re-isolated after the co-cultivation with Ml neuroblastoma clone. Cells were cultured as described under Methods. NN
somes per mitosis, most of the hamster chromosomes are meta-, submeta- and mediocentric (fig. 3). The catecholamine-specific fluorescence was absent in re-isolated astroblast cells, while about 90% of Ml re-isolated cells contained catecholamines. Furthermore, ChAc and AChE activities of astroblasts before co-culture and separated thereafter were not changed. In addition, isoenzymatic patterns of MDH and LDH isoenzymes confirmed the identity of the original NN astroblasts and those re-isolated from co-cultures (Tholey et al., in preparation). Ecto-ATPase activity of re-isolated NN cells Re-isolated NN cells exhibited higher Mg2+- and Ca2+-dependent ecto-ATPase activities than the original NN cell line. ATPase activity at different periods of growth in original NN cells and re-isolated NN cells is presented in the fig. 5. The
197
cells: O-O, Mg2+-ATPase; O-O, Ca2+-ATPase. NN re-isolated cells: A-A, Mg2+-ATPase; A-A, CaZ+-ATPase. pH; ordinate: pmoles Pi liberated/mg prot/h. pH dependence of ATPase activity at the external surface of NN astroblast cells and NN cells re-isolated after the co-cultivation with Ml neuroblastoma clone. ATPase activities were determined on confluent cells. pH varied from 6.0-9.0. NN cells: O-0, Mg2+ATPase; B-m, Ca*+-ATPase. NN re-isolated cells; O-O, Mg*+-ATPase; O-O, Ca*+-ATPase.
Fig. 6. Abscissa:
specific ecto-ATPase activity increased about twice in both cell lines when the cultures become confluent. At each time, reisolated NN cells had about 5 times higher activities of both Mg2+- and Caz+-dependent ATPase than the original NN line. Some properties of ecto-ATPase activity of reisolated NN cells were studied, and compared with that of the original NN cell line. Substrate spec$city. It can be seen from table 3 that all of the nucleoside triphosphates tested are split by this ecto-enzyme at appreciable rates. In NN and re-isolated NN cells, purine nucleoside triphosphates are split at higher rates in the presence of Mg2+ than Ca2+. In re-isolated NN cells, hydrolysis of nucleoside diphosphates and monophosphates was much lower than hydrolysis of nucleoside triphosphates. The lowest hydrolysis, less than l/l5 of the ATPase activity, was found for other phosphate esters. Reaction products of ATP hydrolysis. As ATPases split only the terminal phosphate group from ATP, it was important to charExptl Cell Res 98 (1976)
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et al.
Table 3. Substrate specificity of ATPase activity at the external surface of NN and reisolated NN cells, expressed as pmoles Pi liberatedlmg cell protein/h Ca2+-dependent activity
Mg2+-dependent activity Substrate
NN
NN re-isolated
NN
NN re-isolated
ATP GTP CTP UTP ITP ADP GDP CDP UDP IDP AMP GMP Glucose-6phosphate Glycerophosphate p-Nitrophenylphosphate
0.34f0.01 0.29+0.01 0.31+0.01 0.26t0.01 0.29kO.00 0.09~0.00 0.1140.00 0.07+0.01 0.19~0.01 0.10+0.00 0.16+0.01 0.10~0.00
I .49+0.03 1.11~0.04 1.25*0.06 0.97&0.03 1.23kO.03 0.13f0.01 0.20f0.01 0. I I kO.01 0.25+0.01 0.13+0.01 0.12f0.00 0.08+0.00
0.28kO.01 0.26kO.01 0.32kO.02 0.28f0.01 0.25~0.01 0.17~0.01 0.16f0.00 0.17+0.01 0.26+0.01 0.17~0.01 0.17~0.01 0.15~0.01
l.llkO.06 0.86~0.05 1.3220.07 I .07+0.08 1.ot3+0.04 0.25+0.01 0.33+0.02 0.25+0.01 0.38kO.02 0.23+0.01 0.13~0.01 0.08f0.00
0.05+0.00 0.04~0.00
0.03 +o.OO 0.03 LO.01
0.09f0.00 0.1 I to.00
O.O8L-0.01 0.06f0.01
0.04~0.00
0.06+0.00
0.1 I +0.01
0.07~0.00
Nucleotides and phosphate esters were added to a final concentration of 3 mM. Details are given under Methods. Each value is the mean of 5 experiments?S.E.M.
acterize the reaction products of ATP breakdown. The enzymatic activity was usually determined by hydrolysing less than 10% of substrate, i.e. by estimating the initial reaction rates. A careful study using paper chromatography and enzymatic assays demonstrated that, under the conditions of our assays, the only products formed were ADP and Pi. Less than 0.8 % of AMP was found. NO PPil IMP or inosine could be detected. Cation effects. Mg2+ and Ca2+ ions have a strong activating effect and the enzyme is completely inactivated by ethylenediaminetetracetic acid (EDTA). The data presented in table 4 show that in the NN as well as in NN re-isolated cells divalent ions such as Mn2+, Co2+and Cd2+activate the enzyme to varying degrees. The relative activation was similar for both cell lines, in the order Mg2+ >Ca2+ >Mn2+ >Co2+ >Cd2f. Effect ofpH. The effect of pH on the enzymatic activity is presented in fig. 6. ReExptl Cell Res 98 (1976)
isolated NN cells have the pH optimum for Mgz+- and Ca2+-dependent ATPase activity in the alkaline range, at about 7.4-g. 1. NN original cell line exhibit similar pH optimum
Table 4. The effect of divalent cations on ecto-ATPase activity of NN and re-isolated NN cells, expressed as qnoles Pi liberated1 mg cell protein/h ATPase activity Cation
NN
NN re-isolated
Mg2+ Ca2+ MnZ+ Co2f Cd2+ Sr*+ cu*+ ZnZ+
0.47f0.02 0.35f0.02 0.26+0.01 0.16+0.01 0.12+0.02 0.09f0.00 0.12~0.01 0.08+0.01
1.64f0.04 1.41*0.03 1.02Yko.04 0.67kO.06 0.42kO.03 0.10+0.01 0.08+0.01 0.02+0.00
Divalent cations and ATP were present in the concentration of 3 mM each. ATPase activity determined as described under Methods. Each value is mean of 5 experimentsfS.E.M.
Ecto-ATPase
activity
on Table 5. The effect of subcultivation ecto-ATPase activity of re-isolated NN astroblast cells, expressed as prnoles Pi liberatedlmg cell protein/h Number of replications
Mg2+-dependent Ca2+-dependent ATPase ATPase
17 28 48
1.46+0&t 1.05+0.07 0.65f0.05
1.17~0.04 0.92+0&t 0.48jzO.05
ATPase activity determined on confluent cells as described under Methods. Each value is the mean of 6 experiments+S.E.M.
but the specific activity is 4-5 times lower than in NN cells re-isolated from the coculture. The effect of subcultivation on ATPase activity of re-isolated NN cells. The effect
of subcultivation of NN cells isolated from the co-culture of neuroblastoma clone Ml and hamster astroblasts NN on the ectoATPase activity is presented in the table 5. Separated from neuroblastoma cells, reisolated NN cells in monolayer culture, changed their surface ATPase activity. After 48 passages both Mg2+- and Ca2+dependent ATPase activities dropped but remained still higher than ATPase activity of the original NN clone. A kinetic study performed (fig. 7) showed that after 28 passages a decrease in V,,, took place without change in K,, followed by a decrease of both K, and V,,,. The observed phenomena can be a selective adaptive change in the nature of the enzyme or reflect a steric effect due to an unknown change in the surface geometry of the cell. One cannot distinguish between these two possibilities. However, it is clear from the V maxvalues that there is a decrease in the total enzyme activity upon successive replications of the cells.
of neuroblastoma
Ecto-ATPase Ml cells
and glioma cells
199
activity of re-isolated
The ecto-ATPase activity of Ml neuroblastoma cell line increased in parallel with cell density (fig. 8). Ecto-ATPase activity of Ml re-isolated cells showed a similar pattern, but both Mg2+- and Ca2+-dependent ATPase activities were, at each period of growth, 2-3 times higher than the respective activities of the original Ml cell line. Effect of conditioned medium on the ATPase activity of neuroblastoma and glial cells
The differentiation of neuroblastoma cells was induced by glia-conditioned medium [19]. Ecto-ATPase activity of neuroblastoma Ml clone cultured with normal as well as with glia-conditioned medium increased with the cell density (fig. 9). At any period of growth ATPase activity on the surface of neuroblastoma Ml cells cultivated in the glia-conditioned medium was significantly higher than ATPase activity of Ml cells cultivated in normal medium.
Fig. f. Abscissa: I/ATP (mM-r); ordinate: l/v. Lineweaver-Burk plots of ATPase activity at the external surface of NN ceils re-isolated after the cocultivation with Ml neuroblastoma clone. The effect of subcultivation: O-O, 18 replications; A-A, 28 replications; m-H, 48 replications. ATPase activity was determined in the presence of an ATP regenerating system. Mg2+-ATP varied from 0.1-3.0 mM (A) Mg*+-dependent ATPase; (i?) Ca*+-dependent ATPase. Exptl Cell Res 98 (1976)
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Figs S-10. Abscissa: time in subculture (days); ordinate: pmoles Pi liberated/mg prot/h. Fig. 8. Mg2+- and Ca*+-dependent ecto-ATPase activities of Ml cells re-isolated after the co-cultivation with astroblast NN cells. Cells were cultured as described under Methods. Ml cells: O-O, Mg2+ATPase; O-O, Ca*+-ATPase. Ml reisolated cells: A-A, Mg2+-ATPase; A-A, Ca2+-ATPase. Fig. 9. Ecto-ATPase activity of Ml neuroblastoma cells during the treatment by glia-conditioned medium. Dissociated neuroblastoma Ml cells were plated in Petri dishes as described under Methods. After I7 h the medium was replaced with alia-conditioned medium in the concentration of 505%. This medium was changed every other day. Glia-conditioned medium was prepared as described previously [l9]. Hamster astroblast NN were plated at the density required for reaching confluency in 72 h. After that time the me-
dium was collected and stored at -20°C. The thawed medium was centrifuged under sterile conditions for 2 h at 10000 rpm. M I cells grown in control medium: O-O, Mg*+-ATPase; O-O, Ca*+-ATPase. Ml cells treated by glia-conditioned medium: A-A, Mg*+ATPase; A-A, Ca2+-ATPase. Fig. 10. Ecto-ATPase activity of NN astroblast cells during the treatment by neuroblastoma-conditioned medium. NN cells were seeded in Petri dishes as described under Methods. After I7 h the medium was replaced with MI neuroblastoma-conditioned medium in the concentration of 20%. The medium was changed every other day. Neuroblastoma-conditioned medium was prepared by the procedure described in fig. 7. NN astroblasts grown in control medium: O-O, Mg2+ATPase; O-O, Ca*+-ATPase; NN astroblasts cultured with neuroblastoma-conditioned medium: A-A, Mg2+-ATPase; A-A. Ca2+-ATPase.
Neuroblastoma Ml cells were cultured in cultured with Ml-conditioned medium the the glia-conditioned medium for periods up increase was about 7-g times. The specific ecto-ATPase activity of NN to one month; Ca*+-dependent ectoATPase activity increased from 1.10+0.03 cells cultured with fresh medium and with to 2.25kO.20 and 5.62kO.20 pmoles Pi/mg Ml neuroblastoma-conditioned medium is presented in fig. 10. At any time during protein/h at 17 and 24 days, respectively. growth, Mg2+- and Ca*+-dependent ectoOn the contrary, neuroblastoma Ml-conditioned medium in a final concentration of ATPase activity of NN cells cultured with 20 % inhibits growth of NN cells. The aver- neuroblastoma-conditioned medium was age protein content of NN cells/Petri dish significantly lower than for NN cells grown was 0.59, 2.06 and 2.60 mg after 2, 4 and 6 in fresh medium. days in culture, respectively. NN cells cul5’-Nucleotidase of cultured neuroblastoma tured with M 1 neuroblastoma-conditioned medium, as described in the caption to fig. and glial cells 10, had a total protein content of 0.30, 0.41 Table 6 shows 5’-nucleotidase activity at and 0.35 mg, after 2,4 and 6 days in culture, the external surface of some neuroblastoma and glial cell lines in culture and in co-culrespectively. Ecto-ATPase activity of a whole culture tures of these cells. Relatively low 5’-nu(activity/dish) of control NN cells cultured cleotidase activity was found at the external for 6 days, increased about 15times as com- surface of all the neuroblastoma cell lines pared with activity at 2 days; for NN cells tested. NN astroblasts had 5’-nucleotidase Exptl Ceil Res 98 (1976)
Ecto-ATPase
activity of neuroblastoma
Table 6. 5’-Nucleotidase
at the external surface of neuroblastoma and glial cell lines in culture, expressed as voles Pi liberatedlmg cell proteinlh Cell line
5’-Nucleotidase activity
MI Nl8 NlE-115 NN Ml+NN N18+NN NlE-IIS+NN M 1 re-isolated NN re-isolated
0.065f0.003 0.016f0.003 0.027f0.002 0.146t0.005 0.110f0.006 0.068+0.002 0.109f0.006 0.074+0.005 0.118t0.004
The cells were cultured in Falcon flasks for 10 days. For the study of enzymatic activity they were subcultured in Petri dishes and used after 4 days. Details are given under Methods. Each value is the mean of 5 experiments+S.E.M.
activity which was twice as high as in the M 1 cell line. 5’-Nucleotidase activity of cocultures was below the activity of NN cells. Re-isolated Ml and NN cells had the same or lower .5’-nucleotidase activity than the original cell lines. DISCUSSION The importance of morphological and metabolic coupling between neurons and glia in the nervous system as expressed in differentiated function in the brain is well established. However, the elucidation of mechanisms involved in interaction of these cells is rather limited with regard to in vivo studies. The recently developed study [2 1, 22, 421 of nervous system clonal cell lines provides a tool for the investigation of effects produced in each cell type upon cellcell contact. By comparing physico-chemical and physiological properties before and after cell association many aspects of mutual cell interactions can be clarified. The data presented in this paper show that nervous system cells in co-culture un-
and glioma cells
201
dergo marked changes in their surface properties. Mg2+- and Ca2+-dependent ectoATPase activities of co-cultured neuroblastoma and glial cells increased manyfold over the level of the individual cell lines. When re-isolated from the co-culture both neuroblastoma and glioblastoma cells exhibited enhanced ATPase activity. After a certain number of generations, the ectoATPase activity of glial cells dropped but was still higher than in the original cell line. The effect of subcultivation on the reisolated Ml cells is under investigation. The enzymatic activity was monitored for more than 5 months. Thirty passages after separation from NN astroblasts, ectoATPase activity of M I re-isolated cells was still as high as immediately after isolation from co-culture. The data presented further support the view that in brain the untransformed counterparts of these nervous system cells exhibit a wide variety of functional interactions. In fact, communication from neurons to glia and also with the opposite polarity has been proposed, and mechanisms have been discussed in detail [l, 44, 451. The consequences of the interaction of nerve cells in tissue culture has begun to appear. Choline acetyltransferase activity was found more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord alone [46]. Choline acetyltransferase activity increased IOO-fold and acetylcholine synthesis lOO- to I OOO-foldin co-cultures of dissociated sympathetic neurons with non-neuronal cells from sympathetic ganglia [47]. A homogeneous population of neurons or glia is very attractive for biochemical study. However, separated each from the other, in monolayer culture, transformed neurons and glia may change from the respective corresponding cells of the nervous system. Exptl Cell Res 98 (1976)
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The data presented further support this view, e.g. astroblasts in primary culture had 60-80 times higher ecto-ATPase activity than the clonal cell line NN maintained for a long time in culture and probably more de-differentiated. When the NN cell line was co-cultured with Ml neuroblastoma cells and re-isolated thereafter, an increase up to 5-fold of both Mg2+- and Ca2+-ATPase activities was found. Several lines of evidence are presented linking external cell surface ATPase activity and differentiated function. Significant increase of ATPase activity in stationary phase of growth, when some percentage of cells differentiates, has been found. Furthermore, morphological differentiation of neuroblastoma cells by conditioned medium is accompanied by a striking increase of ATPase activity. The same increase in ATPase activity in neuroblastoma cells can be produced by the conditioned medium. Presumably glial cells in culture release a factor that produce increased Ca2+- and Mg2+-dependent ectoATPase activities in neuroblastoma cells. Treatment with glia-conditioned medium enlarges the surface of neuroblastoma cells. Morphological differentiation of neuroblastoma cells obtained by removal of serum and addition of either db-CAMP or BUdR, although it increases process formation and enlarges the cell surface, it does not increase specific activity of surface ATPase (unpublished data). Thus, it seems that increase of ecto-ATPase activity of neuroblastoma cells upon treatment with glia-conditioned medium cannot be attributed simply to the increased cell surface. Removal of serum and treatment by dbCAMP or BUdR are unphysiological and they all interfere with the growth rate of the cells [ 191. In the present study, no attempts were Exptl Cell Res 98 (1976)
made to explore in detail the properties of Mg2+- and Ca2+-dependent ATPase activities of Ml neuroblastoma cells and reisolated Ml cells. With this we will deal in a separate paper. The authors are grateful to Dr A. Rosenberg for valuable criticism during preparation of the manuscript. Miss A. Riehl and Miss M. Ostertag are thanked for their excellent technical assistance. A. E. is a Chargee de Recherche at CNRS.
REFERENCES 1. Kuffler, S W & Nicholls, J G, Ergebn physiol 57 (1966) I. 2. Trachtenberg, M C & Pollen, P A, Science 167 (1970) 1248. 3. Henn, F A, Haljamle, H & Hamberger, A, Brain res 43 (1972) 437. 4. Gill, T H, Young, 0 M & Tower, D B, J neurothem 23 (1974) loll. 5. Krjevic, K & Schwartz, S, Brain res 3 (1967)306. 6. Henn, F A & Hamberger, A, Proc natl acad sci US 68 (1971) 2686. 7. Schrier, B & Thompson, E J, J biol them 249 (1974) 1769. 8. Blomstrand, C & Hamberger, A, J neurochem 17 (1970) 1187. 9. Hamberger, A, Brain res 31 (1971) 169. IO. Rakic, P & Sidman, R L, Proc natl acad sci US 70 (1973) 240. 11. Okun. L M, J neurobiol3 (1972) I II. 12. Sensenbrenner, M, Lodin, Z, Treska, J, Jacob, M, Kaae, M P & Mandel, P, Z Zellforsch 98 (1969) 538: 13. Lodin, Z, Booher, J & Kasten, F H, Exp cell res 60 (1970) 27. 14. Varon. S & Rainbom. C. J neurocvtol I ( 1972)2 I I. 15. Luduena, M A, Dev bioi 33 (1973) 268. 16. Sensenbrenner. M & Mandel. P. Exp cell res 87 (1974) 159. 17. Sensenbrenner. M. Sorineer. N. Booher. J & Mandel, P, Neurobiology 2( 1972)49. 18. Lim, R & Mitsunobu, K, Science I85 (1974) 63. 19. Monard, D, Solomon, F. Rentsch, M & Gysin, R, Proc natl acad sci US 70 (1973) 1894. 20. Longo, A & Penhoet, E E, Proc natl acad sci US 71 (1974) 2347. 21. Shein, S H, Britva, A, Hess, H H & Selkoe, D J, Brain res I9 (1970) 497. 22. Benda, P, Lightbody, J, Sato, Cl, Levine, L & Sweet, W, Science I61 (1%8) 370. 23. Booher, J & Sensenbrenner, M, Neurobiology 2 (1972) 97. 24. Ciesielski-Treska. J. Hermetet. J C & Mandel, P. Compt rend sot biol 167(1973)’1971. 2s. Gomori. G. J lab clin med 27 (1942) 955. 26. Kachmar, J F & Boyer, P D, J‘biol them 200 (1953) 669. 27. Stefanovic, V, Ciesielski-Treska, J, Ebel, A & Mandel, P, Brain res 81 (1974) 427.
Ecto-ATPase activity of neuroblastoma and glioma cells 28. DunkJey, C R, Manery, J F & Dryden, E E, J cell physiol 68 (1%6) 241. 29. Adam, H, Methoden der enzymatischen Analyse (ed H U Bergmeyer) p. 539. Verlag Chemie, Weinheim (1965). 30. -Ibid, p.573. 31. Smillie, R M, Arch biochem biophys 67 (1957) 213. 32. DePierre, J W & Karnovsky, M L, Science 183 (1974) 1096. 33. McCaman, R E & Hunt, J M, J neurochem 12
39. Hamberger, B, Acta physiol Stand, suppl. 295
41. 42. 43. 44.
neurochem 16 (1969) 823. 35. McCaman, R W, Tomey, C R & McCaman, R E, Life sci 7 (1968) 233. 36. Kamovsky, M J, Roots, L, J histochem cytochem
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37. FaJck, B & Owman, C, Acta univ Lund 7 (1%5) 1.
(1967) 1. 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Amano, T, Richelson, E & Nirenberg, M, Proc natl acad sci US 69 (1972) 258. Augusti-Tocco, G & Sate, G, Proc natl acad sci US64(1969)311. Stefanovic, V, Ciesielski-Treska, J, Ebel, A & Mandel, P, FEBS lett 49 (1974) 43. Hydtn, H, The neuron. Elsevier, Amsterdam (1967). Gilman. A G & Nirenbere. M, Proc natl acad sci
40. Lowry,
(1965)253. 34. Goldberg, A M, Kaita, A A & McCaman, R E, J
12(1%4)219.
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& Nelson, P G, Science 182 (1973) 588. 47. Patterson, P H & Chun, L L Y, Proc natl acad sci us 71(1974)3607.
38. Caroddi, H & Jonsson, G, J histochem cytochem 15 (1967)65.
Received July 24, 1975
Exptl Cell Res 98 (1976)
NEUROBLASTS-GLIA The Effect of Co-cultivation of Neuroblastoma V. STEFANOVIC,’
INTERACTION upon Ecto-ATPase Activity and Glioma Cells
J. CIESIELSKI-TRESKA,
Centre de Neurochimie
A. EBEL and P. MANDEL
du CNRS, F-67085 Strasbourg
Cedex, France
SUMMARY The effect of cell contact and cell medium upon the ecto-enzymes, MgZ+- and Ca*+-dependent ATPase and 5’-nucleotidase were studied in nervous svstem cells in tissue culture. Conditions were worked out for co-culture and reseparation of glioblastoma and neuroblastoma cells so that the effects upon each of the co-cultured cell lines after interaction of these cells could be reliably determined. Co-cultivation of mouse neuroblastoma and glioma cell lines markedly enhanced Mg*+- and Ca*+-dependent ecto-ATPase activity. Evidence was obtained which indicates that increase in ecto-ATPase of co-cultured neuro- and glioblastoma cells occurs in both cell types. Ecto-ATPase was 500% of the orieinal level in clonal line NN astroblasts after co-culture with MI neuroblasts. This activity decreased over 50 transfers during the period of about a year. Increase in ecto-ATPase and morphological differentiation of Ml neuroblastoma cells after co-culture with NN astroblasts could also be brought about simply by treatment with the medium from NN cell cultures. Co-cultivation of neuroblastoma and glioma cells does not change significantly the specific activity of ecto-S’-nucleotidase.
The possibility of metabolic coupling between neurons and glia has been suggested in view of their close anatomical relationship, i.e. the ensheathing of some axons and synapses by glial cells, and the correlation of biochemical changes in neurons and glia. Several functions for glial cells have been considered, e.g. regulation of the ionic milieu of neurons [14], control of the level of neurotransmitters [5-71 and substrate available for protein synthesis [8, 91 in neurons and guidance of neuronal migration [lo]. Tissue culture methodology affords an opportunity to study molecular aspects of I Permanent address: Institut za Nefrologiju i Hemodijalizu, Faculty of Medicine, Nis, Yugoslavia. 13-761819
mutual interactions of dissociated cells of the nervous system and of clonal cell line of nervous origin. Okun [ 1l] has reported that chick embryo ganglion neurons survive in culture for several weeks in the apparent absence of glial cells. The work of several authors [12-161 has indicated that contact with glial cells enhances differentiation of dissociated neurons, and maintains it in long-term cultures. Flat embryonic brain cells in monolayer culture can be transformed into cells resembling mature astrocytes by a protein present in embryonic [ 171 or adult [ 181brain extract. Recently Monard et al. [19] reported on a factor which is released into the medium by glial cells and causes morphological differentiation of neuroblastoma cells upon addition to their Expr/ Cd Res 98 (1976)
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culture medium. Rat glioma cells contain a neuroblastoma and glial cells were seeded In Falcon containing IO ml DMEM and 10% foetal calf protein with properties similar to those of flasks serum. In order to separate neuroblastoma MI and astromouse salivary nerve growth [20]. In all of blast NN cells from mixed cultures, the cell monolayer these experiments only morphological char- was treated firstly with 0.02 % trypsin for 4 min. During this time most of the neuroblastoma cells were acteristics of cells have been studied. detached, whereas the astroblasts were dissociated by We have utilized neuroblastoma and glial incubation with a solution of 0.25 % trvusin. Harvested cell lines in culture as a model system to cells were diluted in complete medium and plated in low density, approx. IO@-300 cells/30 mm Falcon Petri study the surface membrane changes which dish. Ea& dish contained several broken coverslips may occur in cell-cell interaction. The ex- (approx. I mm 0). Those colonies with a small number of cells were removed and placed in microwells (vol ternal cell surface enzymes, ATPase (ATP I ml). For study of enzymatic activity, cells were placed in phosphohydrolase, EC 3.6.1.3) and 5’-nu60 mm Petri dishes containing 5 ml of growth medium. cleotidase (5’-ribonucleotide phospho- The medium was changed every other day. Tests for hydrolase, EC 3.1.3.5), were determined in infestation with mycoplasma were negative. a wide variety of mouse neuroblastoma and glial cell lines. In this paper, we report a Enzyme assays marked increase in cell surface ATPase ac- Enzymatic assays were performed on the day after the tivity in co-cultures of neuroblastoma and culture medium was changed. glial cells as well as in the different individual cells when separated. The effect of Assay of ATPase conditioned medium on neuroblastoma and Nutrient medium was carefully removed from Petri glial cell ecto-ATPase activity is also pre- dishes and discarded, and the cell monolaver was gently washed first with a medium containing 0.250 sented. mM EDTA, 0.125 mM EGTA, 30 mM Tris-HCI buffer I
METHODS Cell cultures Normal hamster astroblasts NN [21] and rat glioblastoma C6 cell lines r22 I were obtained from North American Biologicals, inc.-and American Type Culture Collection, respectively. Neuroblastoma clonal lines Nl8 and NlE-II5 were a gift from Dr M. Nirenberg (Laboratory of Biochemical Genetics, NIH, Bethesda, Md). Neuroblastoma clonal line Ml has been developed and characterized in our laboratory. Cells were grown in monolayer culture in Falcon tissue culture flasks with a growth area of 75 cm* in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with IO%foetal calfserum IGIBCo. Grand Island. NY) with 50 U of sodium penicillin G and 25 pg of streptomycin sulfate oer ml. The cultures were incubated at 37°C in a humidified atmosphere of 5 % CO, and 95 % air. The cells in stationary phase were subcultured after incubation with 0.25% trypsin (Difco l/250) for 2 min. Astroblasts from brain of newborn rats were cultured as previously described [23] and studied after 1720 days of cultivation. The growth medium was changed twice a week. Mixed cell cultures of neuroblastoma and glial lines were prepared as previously described [24]. Cultures of both cell lines were harvested by detaching cell monolayers with 0.25 % trypsin. Cell suspensions were diluted in complete medium, counted in a hemocytometer. Subsequently, equal parts of suspension of Exprl Cell Res 98 (1976)
.
(pH 7.8) and 250 mM sucrose, and afterwards 3 times with a total of about 10 ml of incubation solution without ATP. Incubation fluid contained 30 mM Tris-HCI buffer (pH 7.8), 5.5 mM glucose, 240 mM sucrose and 3 mM of CaCI, or MgCI,. Incubation was started by adding 3 mM Tris-ATP and conducted at 37°C for 2-15 min in a final volume of I.5 ml. The inorganic phosphate formed was determined calorimetrically according to the method of Gomori [25] modified by proportional reduction in reagent volumes. Appropriate controls without cells were also incubated to correct for small amounts of P, found in commercial oreparations of ATP or deriving from its spontaneous hidroIvsis. Another control with cells but without substrate was also included. These latter were consistently negative. In the kinetic studies, ATPase activity was determined in the presence of an ATP-regenerating system. For this assay, the incubation medium contained, in addition, I mM potassium salt of phosphoenolpyruvic acid and 6 U/ml of pyruvate kinase. Potassium was necessary for pyruvate kinase activation [26]. Since potassium al& activates MgZ+-dependeni ectoATPase [27], 0.1 mM ouabain was included in media used in this study. Although pyruvate kinase may be inhibited by Ca2+ [26], this ion had practically no effect on pyruvate kinase under our experimental conditions as the enzyme was present in many-fold excess. ATP breakdown products were studied by paper chromatography [28j, by enzymatic assays f&-A-TP 1291, ADP and AMP 1301, and by determining adenine nucleotides and IMP plus inosine by differential absorption spectra [3 I].
Ecto-ATPase
activity of neuroblastoma
and glioma cells
193
Assay of 5’-nucleotidase
Protein
Cells were washed as previously and incubated for 30 min at 37°C in a medium containing 30 mM Tris-HCI buffer (pH 7.4), 130 mM NaCl, 5.5 mM glucose, 1 mM 1 mM p-nitrophenylphosphate and 3 mM &Cl,, adenosine-5’-monophosphate (5’-AMP). Magnesium ensured cell attachment during the incubation period. The hydrolysis of 5’-AMP by non-specific phosphatases present on the surface of nervous system cells in culture was prevented by p-nitrophenylphosphate as described previously [32]. Pl liberated from 5’-AMP and p-nitrophenylphosphate and p-nitrophenol were determined on separated aliquots and the difference was taken as Pi enzymatically released from 5’-AMP. Choline acetyltransferase (ChAc, EC 2.3. I .6) was measured according to the microtechnique of McCaman & Hunt [3fl as modified by. Goldberg et al. r341 bv following the incornoration of rl-W-acetate from cl-“Cl-acetyl-CoA (NEN Chemicals; 59.2 mCi/ mmole) into acetylcholine. Acetylcholinesterase (AChE, EC 3.1.1.7) was determined by the method of McCaman et al. [35] which is based on the hydrolysis of [ I-IF]-acetylcholine (NEN Chemicals: 2.43 mCi/mmole). Non-specific cholinesterases were inhibited by the addition of iso-octamethyl-pyrophosphoramide ( 1OmB M) in the incubation medium. Histochemical demonstration of acetylcholinesterase was performed according to the method of Karnovsky & Roots [36].
Protein was determined by a modification of the method of Lowry et al. [40]. Specific activity of ATPase is expressed as pmoles Pi liberated/mg cell protein/h. In general, data are expressed as means +S.E.M.
Catecholamines Catecholamines were visualized according to Falk & Owman r371. Cell cultures in Falcon Petri dishes were used. After-removal of the culture medium, all preparations were frozen in dry ice and freeze-dried for 4 days at -35°C. They were then treated for 1 h with formaldehvde gas generated from uaraformaldehvde at 70°C [38]. The pamformaldehyde had been equilibrated with air at 70% relative humiditv f391. Cvtochemical controls were performed wiihout - freezedrying or exposure to formaldehyde vapour. A fluorescence microscope (Leitz Orthomat) with dark field condenser was used. The light source was an HBO-200 mercury lamp (Osram) with 5 mm B.A. 12 and 4 mm BG. 38 excitation filters. The fluorescence was observed through a secondary barrier filtre of 480 nm.
Chromosomal
analysis
Chromosome spreads were prepared from logarithmic phase cultures. After colchicine treatment (0.4 me/ml/ 3 h), osmotic shock was induced by dilution of themedium with distilled water (1 : 5/30 min/37”C). The cells were centrifuged, fixed in a freshly prepared mixture of acetic acid and methanol (1: 3) for 30 min, stained with Giemsa stain and photographed. A minimum of 8&90 metaphasal plates of each cell line was analysed to establish the modal chromosomal number and to identify the origin of the cells. 13*-761819
RESULTS Cultures of neuroblastoma
and glial cells
The morphological appearance of cultured neuroblastoma and glial cells has been previously described in detail [21-23, 41, 421. In the present study, we describe only the M 1 cell line. Neuroblastoma Ml clone was isolated in our laboratory and characterized by chromosomal and biochemical markers. Modal number of chromosomes of this cell line is 60. With regard to neurotransmitter synthesis neuroblastoma Ml clone was defined as adrenergic since it lacked ChAc activity and possessed catecholamines. AChE activity was rather low compared with other neuroblastoma clones but increased during the stationary phase of growth. Like other neuroblastoma cells, the Ml cell line proliferates rapidly in culture. During logarithmic phase of growth, the cells remain still small and without extensions. In the stationary phase, the cells attach firmly to the growth surface and the majority of cells extend neuron-like processes (fig. 1a). ATPase activio at external sutiace of neuroblastoma and glial cells
As it has been shown previously for NlE115 neuroblastoma cells [43], we found Mg2+- and Ca2+-dependent ATPase activity at the external surface of the N18 and Ml neuroblastoma clonal cell lines (table 1). The two indefinite glial cell lines, NN normal hamster astroblasts and C6 rat glioblastoma exhibited a very low Mg2+- as well as Ca2+-dependent ATPase activity. A difExptl Cell Res 98 (1976)
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kationary phase; (c) cokultu~e‘ok NN astroblasts Ml and NN cells. Phase contrast. x250.
ferent pattern of activation of neuro- Ecto-ATPase activity of the co-culture blastoma and glial cells ecto-ATPase by The results are summarized in table 2. After divalent cations is also evident. Mouse 15 days of co-culture of neuroblastoma Ml neuroblastoma clones had about two times cells and hamster astroblasts NN cells, an higher activity with Ca2+ than with Mg2+. increase of both Mg2+- and Ca2+-dependent Glial cell lines had higher Mg2+-dependent ecto-ATPase activities was observed. In ATPase activity. these co-cultures, Mg2+- and Ca2+-dependent ecto-ATPase activities were 2.24kO.03 Co-cultures of neuroblastoma and and 3.71 kO.08 pmoleslmg cell protein/h, glial cells respectively. When compared with the When mixed and placed into the culture Mg2+- and Ca2+-dependent ecto-ATPase acduring the first or on the second day neuro- tivity of the M 1 cell line, the increase is blastoma cells undergo a morphological about 4 times. Compared with the NN cell and biochemical differentiation [24]. An in- line, both ecto-ATPase activities increased crease in AChE activity of co-cultured Ml 8-10 times. In the co-cultures of hamster astroblasts cells was demonstrated by histochemical method (fig. 2). Catecholamine specific NN and neuroblastoma clonal lines N18 fluorescence was also increased (data are and NlE-115, a slight increase of Mg2+not presented). dependent ecto-ATPase activity appears, ExprlCellRes 98 (1976)
Ecto-ATPase
activity of neuroblastoma
2. Photomicrographs showing morphological aspect of neuroblastoma M 1 cells and Ml cells in coculture with NN cells, stained for acetylcholinesterase
Fig.
and glioma cells
195
activity. (a) Neuroblastoma MI cells; (6) co-culture of Ml and NN cells. (a) X200; (b) X250.
while the Ca2+-dependent ecto-ATPase ac- cell surface ATPase activity. Fig. 4 shows tivity is significantly higher. The values of the Mg*+- and Ca*+-dependent ATPase acMg*+-dependent ecto-ATPase activity in tivities of co-cultures of Ml and NN cells those co-cultures were similar to that of and their dependence on cell density. Both N18 and NIE-115 neuroblastoma clones. ATPase activities increased about twice Since in the co-cultures approx. 50% of the when the culture becomes confluent. At cell population were NN astroblasts with any period of the growth curve the ectoan originally low Mg*+-dependent ectoATPase activity, we may conclude that an increase of Mg*+-dependent activity of Table 1. Mg*+- and Ca*+dependent N18+NN and NlE-NllS+NN cell cul- ATPase activities at the external surface oj tures also occurred. neuroblastoma and glial cells in culture, exThe ecto-ATPase activity of the co-cul- pressed as voles Pi liberatedlmg cell protures of neuroblastoma Ml and rat glio- tein/h blastoma C6 cells were close to that of the Mg2+-dependent Ca*+-dependent original Ml clone and much higher than Cell line ATPase ATPase that of C6 clonal cell line. In the co-cultures 0.47+0.02 I. IO&O.03 of astroblasts from newborn rat brain and Ml 2.38kO.05 Nl8 I .20t0.04 M 1 neuroblastoma cells the Mg*+- and NIE-I I5 3.25f0.04 1.49+0.03 0.33+0.01 0.35 kO.02 Ca*+-dependent ecto-ATPase activities NN 0.23&0.01 0.27+0.02 C6 were close to that of the astroblasts in pri- Astroblasts in 15.86kO.65 primary culture 21.67+0.90 mary cell culture but manyfold higher than that of Ml cell line. NN and C6 glial cell lines and neuroblastoma clones were grown in Falcon flasks. For the study of enAmong several lines tested, neuro- zymatic activity the cells were subcultured in Petri blastoma Ml and astroblastoma NN cell dishes and enzymatic activity was determined after 4 Newborn rat astrocytes were grown as delines in co-culture were found to be the days. scribed under Methods. Each value is the mean of most potent in producing the increase in 54 experiments+S.E.M. Eiptl Cell Res 98 (1976)
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Fig. 3. Chromosomes of (a) mouse neuroblastoma cell line and (b) hamster astroblasts NN cell
Ml line.
ATPase activities exceeded by many times the respective activities of astroblast NN cells or neuroblastoma Ml cells.
Chromosome spreads Methods. x 1000.
prepared
as described
under
The heterospecific origin of the two cell lines make the chromosomal identification of neuroblasts and astroblasts easy. The modal chromosomal number of neuroblastoma Ml cells was 60 chromosomes with only 3 % of tetraploid cells in contrast to 40% of tetra- and octaploid astroblast NN cells. When mouse neuroblastoma cells have telocentric chromosomes with 2 or 3 submetacentric or mediocentric chromo-
Re-isolated NN cells and Ml cells One attempt was made to separate neuroblastoma and astroblast cells from the coculture. After 2 months in co-culture of neuroblastoma Ml and astroblasts NN cells, astroblasts NN cells were isolated. Neuroblastoma Ml cells were isolated after one Table 2. Mg2+- and Ca2+dependent ectoweek of co-culture with NN hamster astro- ATPase activities of neuroblastoma and glial cells in co-culture, expressed as blasts. The re-isolated neuroblastoma and glial voles Pi liberatedlmg cell protein/h cells were identified according to their MgY+-dependent Ca2+-dependent ATPase morphological, biochemical and chromo- Co-cultures ATPase somal properties. The morphological ap- MI+NN 2.24kO.03 3.71 LO.08 0.43~0.01 0.94?0.02 pearances of neuroblasts and astroblasts are MI+C6 MI +astroblasts shown in fig. 1 (a, b). Neuroblastoma cells in primary culture 25.82k2.80 15.47+0&l 1.30+_0.04 3.02kO.33 when compared with astroblasts arc small NII+NN NI IS+NN I .59+0.05 5.4810.23 and round. Some of the cells possess one or Neuroblastoma and glial cells were co-cultured in Falmore processes. Astroblasts cells after con flasks for IO days. For the study of enzymatic having formed a monolayer are greater in activity they were subcultured in Petri dishes and used after 4 days. Cultivation of newborn rat astrocytes is cell size, the nucleus is clearly visible with described under Methods. Each value is the mean of 5 one or more nucleoli. experiments+S.E.M. Expf/ Cell Res 98 (1976)
Ecto-ATPase activity of neuroblastoma and glioma cells
Figs 4, 5. Abscissa: time in subculture (days); ordinate: pmoles Pi liberated/mg prot/h. Fig. 4. Mgz+- and Ca*+-dependent ecto-ATPase activities of Ml and NN cells in co-culture. Equal parts of Ml neuroblastoma and NN astroblast cells were mixed and plated in Falcon flasks as described under Methods. After 15 days they were subcultured in Petri dishes and ATPase activity determined 2,4 and 6 days later. O-O, Mg2+-ATPase; O-O, Ca*+-ATPase. Fig. 5. Mg2+- and Caz+-dependent ecto-ATPase activities of NN astroblast cells and NN cells re-isolated after the co-cultivation with Ml neuroblastoma clone. Cells were cultured as described under Methods. NN
somes per mitosis, most of the hamster chromosomes are meta-, submeta- and mediocentric (fig. 3). The catecholamine-specific fluorescence was absent in re-isolated astroblast cells, while about 90% of Ml re-isolated cells contained catecholamines. Furthermore, ChAc and AChE activities of astroblasts before co-culture and separated thereafter were not changed. In addition, isoenzymatic patterns of MDH and LDH isoenzymes confirmed the identity of the original NN astroblasts and those re-isolated from co-cultures (Tholey et al., in preparation). Ecto-ATPase activity of re-isolated NN cells Re-isolated NN cells exhibited higher Mg2+- and Ca2+-dependent ecto-ATPase activities than the original NN cell line. ATPase activity at different periods of growth in original NN cells and re-isolated NN cells is presented in the fig. 5. The
197
cells: O-O, Mg2+-ATPase; O-O, Ca2+-ATPase. NN re-isolated cells: A-A, Mg2+-ATPase; A-A, CaZ+-ATPase. pH; ordinate: pmoles Pi liberated/mg prot/h. pH dependence of ATPase activity at the external surface of NN astroblast cells and NN cells re-isolated after the co-cultivation with Ml neuroblastoma clone. ATPase activities were determined on confluent cells. pH varied from 6.0-9.0. NN cells: O-0, Mg2+ATPase; B-m, Ca*+-ATPase. NN re-isolated cells; O-O, Mg*+-ATPase; O-O, Ca*+-ATPase.
Fig. 6. Abscissa:
specific ecto-ATPase activity increased about twice in both cell lines when the cultures become confluent. At each time, reisolated NN cells had about 5 times higher activities of both Mg2+- and Caz+-dependent ATPase than the original NN line. Some properties of ecto-ATPase activity of reisolated NN cells were studied, and compared with that of the original NN cell line. Substrate spec$city. It can be seen from table 3 that all of the nucleoside triphosphates tested are split by this ecto-enzyme at appreciable rates. In NN and re-isolated NN cells, purine nucleoside triphosphates are split at higher rates in the presence of Mg2+ than Ca2+. In re-isolated NN cells, hydrolysis of nucleoside diphosphates and monophosphates was much lower than hydrolysis of nucleoside triphosphates. The lowest hydrolysis, less than l/l5 of the ATPase activity, was found for other phosphate esters. Reaction products of ATP hydrolysis. As ATPases split only the terminal phosphate group from ATP, it was important to charExptl Cell Res 98 (1976)
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et al.
Table 3. Substrate specificity of ATPase activity at the external surface of NN and reisolated NN cells, expressed as pmoles Pi liberatedlmg cell protein/h Ca2+-dependent activity
Mg2+-dependent activity Substrate
NN
NN re-isolated
NN
NN re-isolated
ATP GTP CTP UTP ITP ADP GDP CDP UDP IDP AMP GMP Glucose-6phosphate Glycerophosphate p-Nitrophenylphosphate
0.34f0.01 0.29+0.01 0.31+0.01 0.26t0.01 0.29kO.00 0.09~0.00 0.1140.00 0.07+0.01 0.19~0.01 0.10+0.00 0.16+0.01 0.10~0.00
I .49+0.03 1.11~0.04 1.25*0.06 0.97&0.03 1.23kO.03 0.13f0.01 0.20f0.01 0. I I kO.01 0.25+0.01 0.13+0.01 0.12f0.00 0.08+0.00
0.28kO.01 0.26kO.01 0.32kO.02 0.28f0.01 0.25~0.01 0.17~0.01 0.16f0.00 0.17+0.01 0.26+0.01 0.17~0.01 0.17~0.01 0.15~0.01
l.llkO.06 0.86~0.05 1.3220.07 I .07+0.08 1.ot3+0.04 0.25+0.01 0.33+0.02 0.25+0.01 0.38kO.02 0.23+0.01 0.13~0.01 0.08f0.00
0.05+0.00 0.04~0.00
0.03 +o.OO 0.03 LO.01
0.09f0.00 0.1 I to.00
O.O8L-0.01 0.06f0.01
0.04~0.00
0.06+0.00
0.1 I +0.01
0.07~0.00
Nucleotides and phosphate esters were added to a final concentration of 3 mM. Details are given under Methods. Each value is the mean of 5 experiments?S.E.M.
acterize the reaction products of ATP breakdown. The enzymatic activity was usually determined by hydrolysing less than 10% of substrate, i.e. by estimating the initial reaction rates. A careful study using paper chromatography and enzymatic assays demonstrated that, under the conditions of our assays, the only products formed were ADP and Pi. Less than 0.8 % of AMP was found. NO PPil IMP or inosine could be detected. Cation effects. Mg2+ and Ca2+ ions have a strong activating effect and the enzyme is completely inactivated by ethylenediaminetetracetic acid (EDTA). The data presented in table 4 show that in the NN as well as in NN re-isolated cells divalent ions such as Mn2+, Co2+and Cd2+activate the enzyme to varying degrees. The relative activation was similar for both cell lines, in the order Mg2+ >Ca2+ >Mn2+ >Co2+ >Cd2f. Effect ofpH. The effect of pH on the enzymatic activity is presented in fig. 6. ReExptl Cell Res 98 (1976)
isolated NN cells have the pH optimum for Mgz+- and Ca2+-dependent ATPase activity in the alkaline range, at about 7.4-g. 1. NN original cell line exhibit similar pH optimum
Table 4. The effect of divalent cations on ecto-ATPase activity of NN and re-isolated NN cells, expressed as qnoles Pi liberated1 mg cell protein/h ATPase activity Cation
NN
NN re-isolated
Mg2+ Ca2+ MnZ+ Co2f Cd2+ Sr*+ cu*+ ZnZ+
0.47f0.02 0.35f0.02 0.26+0.01 0.16+0.01 0.12+0.02 0.09f0.00 0.12~0.01 0.08+0.01
1.64f0.04 1.41*0.03 1.02Yko.04 0.67kO.06 0.42kO.03 0.10+0.01 0.08+0.01 0.02+0.00
Divalent cations and ATP were present in the concentration of 3 mM each. ATPase activity determined as described under Methods. Each value is mean of 5 experimentsfS.E.M.
Ecto-ATPase
activity
on Table 5. The effect of subcultivation ecto-ATPase activity of re-isolated NN astroblast cells, expressed as prnoles Pi liberatedlmg cell protein/h Number of replications
Mg2+-dependent Ca2+-dependent ATPase ATPase
17 28 48
1.46+0&t 1.05+0.07 0.65f0.05
1.17~0.04 0.92+0&t 0.48jzO.05
ATPase activity determined on confluent cells as described under Methods. Each value is the mean of 6 experiments+S.E.M.
but the specific activity is 4-5 times lower than in NN cells re-isolated from the coculture. The effect of subcultivation on ATPase activity of re-isolated NN cells. The effect
of subcultivation of NN cells isolated from the co-culture of neuroblastoma clone Ml and hamster astroblasts NN on the ectoATPase activity is presented in the table 5. Separated from neuroblastoma cells, reisolated NN cells in monolayer culture, changed their surface ATPase activity. After 48 passages both Mg2+- and Ca2+dependent ATPase activities dropped but remained still higher than ATPase activity of the original NN clone. A kinetic study performed (fig. 7) showed that after 28 passages a decrease in V,,, took place without change in K,, followed by a decrease of both K, and V,,,. The observed phenomena can be a selective adaptive change in the nature of the enzyme or reflect a steric effect due to an unknown change in the surface geometry of the cell. One cannot distinguish between these two possibilities. However, it is clear from the V maxvalues that there is a decrease in the total enzyme activity upon successive replications of the cells.
of neuroblastoma
Ecto-ATPase Ml cells
and glioma cells
199
activity of re-isolated
The ecto-ATPase activity of Ml neuroblastoma cell line increased in parallel with cell density (fig. 8). Ecto-ATPase activity of Ml re-isolated cells showed a similar pattern, but both Mg2+- and Ca2+-dependent ATPase activities were, at each period of growth, 2-3 times higher than the respective activities of the original Ml cell line. Effect of conditioned medium on the ATPase activity of neuroblastoma and glial cells
The differentiation of neuroblastoma cells was induced by glia-conditioned medium [19]. Ecto-ATPase activity of neuroblastoma Ml clone cultured with normal as well as with glia-conditioned medium increased with the cell density (fig. 9). At any period of growth ATPase activity on the surface of neuroblastoma Ml cells cultivated in the glia-conditioned medium was significantly higher than ATPase activity of Ml cells cultivated in normal medium.
Fig. f. Abscissa: I/ATP (mM-r); ordinate: l/v. Lineweaver-Burk plots of ATPase activity at the external surface of NN ceils re-isolated after the cocultivation with Ml neuroblastoma clone. The effect of subcultivation: O-O, 18 replications; A-A, 28 replications; m-H, 48 replications. ATPase activity was determined in the presence of an ATP regenerating system. Mg2+-ATP varied from 0.1-3.0 mM (A) Mg*+-dependent ATPase; (i?) Ca*+-dependent ATPase. Exptl Cell Res 98 (1976)
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et al.
Figs S-10. Abscissa: time in subculture (days); ordinate: pmoles Pi liberated/mg prot/h. Fig. 8. Mg2+- and Ca*+-dependent ecto-ATPase activities of Ml cells re-isolated after the co-cultivation with astroblast NN cells. Cells were cultured as described under Methods. Ml cells: O-O, Mg2+ATPase; O-O, Ca*+-ATPase. Ml reisolated cells: A-A, Mg2+-ATPase; A-A, Ca2+-ATPase. Fig. 9. Ecto-ATPase activity of Ml neuroblastoma cells during the treatment by glia-conditioned medium. Dissociated neuroblastoma Ml cells were plated in Petri dishes as described under Methods. After I7 h the medium was replaced with alia-conditioned medium in the concentration of 505%. This medium was changed every other day. Glia-conditioned medium was prepared as described previously [l9]. Hamster astroblast NN were plated at the density required for reaching confluency in 72 h. After that time the me-
dium was collected and stored at -20°C. The thawed medium was centrifuged under sterile conditions for 2 h at 10000 rpm. M I cells grown in control medium: O-O, Mg*+-ATPase; O-O, Ca*+-ATPase. Ml cells treated by glia-conditioned medium: A-A, Mg*+ATPase; A-A, Ca2+-ATPase. Fig. 10. Ecto-ATPase activity of NN astroblast cells during the treatment by neuroblastoma-conditioned medium. NN cells were seeded in Petri dishes as described under Methods. After I7 h the medium was replaced with MI neuroblastoma-conditioned medium in the concentration of 20%. The medium was changed every other day. Neuroblastoma-conditioned medium was prepared by the procedure described in fig. 7. NN astroblasts grown in control medium: O-O, Mg2+ATPase; O-O, Ca*+-ATPase; NN astroblasts cultured with neuroblastoma-conditioned medium: A-A, Mg2+-ATPase; A-A. Ca2+-ATPase.
Neuroblastoma Ml cells were cultured in cultured with Ml-conditioned medium the the glia-conditioned medium for periods up increase was about 7-g times. The specific ecto-ATPase activity of NN to one month; Ca*+-dependent ectoATPase activity increased from 1.10+0.03 cells cultured with fresh medium and with to 2.25kO.20 and 5.62kO.20 pmoles Pi/mg Ml neuroblastoma-conditioned medium is presented in fig. 10. At any time during protein/h at 17 and 24 days, respectively. growth, Mg2+- and Ca*+-dependent ectoOn the contrary, neuroblastoma Ml-conditioned medium in a final concentration of ATPase activity of NN cells cultured with 20 % inhibits growth of NN cells. The aver- neuroblastoma-conditioned medium was age protein content of NN cells/Petri dish significantly lower than for NN cells grown was 0.59, 2.06 and 2.60 mg after 2, 4 and 6 in fresh medium. days in culture, respectively. NN cells cul5’-Nucleotidase of cultured neuroblastoma tured with M 1 neuroblastoma-conditioned medium, as described in the caption to fig. and glial cells 10, had a total protein content of 0.30, 0.41 Table 6 shows 5’-nucleotidase activity at and 0.35 mg, after 2,4 and 6 days in culture, the external surface of some neuroblastoma and glial cell lines in culture and in co-culrespectively. Ecto-ATPase activity of a whole culture tures of these cells. Relatively low 5’-nu(activity/dish) of control NN cells cultured cleotidase activity was found at the external for 6 days, increased about 15times as com- surface of all the neuroblastoma cell lines pared with activity at 2 days; for NN cells tested. NN astroblasts had 5’-nucleotidase Exptl Ceil Res 98 (1976)
Ecto-ATPase
activity of neuroblastoma
Table 6. 5’-Nucleotidase
at the external surface of neuroblastoma and glial cell lines in culture, expressed as voles Pi liberatedlmg cell proteinlh Cell line
5’-Nucleotidase activity
MI Nl8 NlE-115 NN Ml+NN N18+NN NlE-IIS+NN M 1 re-isolated NN re-isolated
0.065f0.003 0.016f0.003 0.027f0.002 0.146t0.005 0.110f0.006 0.068+0.002 0.109f0.006 0.074+0.005 0.118t0.004
The cells were cultured in Falcon flasks for 10 days. For the study of enzymatic activity they were subcultured in Petri dishes and used after 4 days. Details are given under Methods. Each value is the mean of 5 experiments+S.E.M.
activity which was twice as high as in the M 1 cell line. 5’-Nucleotidase activity of cocultures was below the activity of NN cells. Re-isolated Ml and NN cells had the same or lower .5’-nucleotidase activity than the original cell lines. DISCUSSION The importance of morphological and metabolic coupling between neurons and glia in the nervous system as expressed in differentiated function in the brain is well established. However, the elucidation of mechanisms involved in interaction of these cells is rather limited with regard to in vivo studies. The recently developed study [2 1, 22, 421 of nervous system clonal cell lines provides a tool for the investigation of effects produced in each cell type upon cellcell contact. By comparing physico-chemical and physiological properties before and after cell association many aspects of mutual cell interactions can be clarified. The data presented in this paper show that nervous system cells in co-culture un-
and glioma cells
201
dergo marked changes in their surface properties. Mg2+- and Ca2+-dependent ectoATPase activities of co-cultured neuroblastoma and glial cells increased manyfold over the level of the individual cell lines. When re-isolated from the co-culture both neuroblastoma and glioblastoma cells exhibited enhanced ATPase activity. After a certain number of generations, the ectoATPase activity of glial cells dropped but was still higher than in the original cell line. The effect of subcultivation on the reisolated Ml cells is under investigation. The enzymatic activity was monitored for more than 5 months. Thirty passages after separation from NN astroblasts, ectoATPase activity of M I re-isolated cells was still as high as immediately after isolation from co-culture. The data presented further support the view that in brain the untransformed counterparts of these nervous system cells exhibit a wide variety of functional interactions. In fact, communication from neurons to glia and also with the opposite polarity has been proposed, and mechanisms have been discussed in detail [l, 44, 451. The consequences of the interaction of nerve cells in tissue culture has begun to appear. Choline acetyltransferase activity was found more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord alone [46]. Choline acetyltransferase activity increased IOO-fold and acetylcholine synthesis lOO- to I OOO-foldin co-cultures of dissociated sympathetic neurons with non-neuronal cells from sympathetic ganglia [47]. A homogeneous population of neurons or glia is very attractive for biochemical study. However, separated each from the other, in monolayer culture, transformed neurons and glia may change from the respective corresponding cells of the nervous system. Exptl Cell Res 98 (1976)
202
Stefanovic et al.
The data presented further support this view, e.g. astroblasts in primary culture had 60-80 times higher ecto-ATPase activity than the clonal cell line NN maintained for a long time in culture and probably more de-differentiated. When the NN cell line was co-cultured with Ml neuroblastoma cells and re-isolated thereafter, an increase up to 5-fold of both Mg2+- and Ca2+-ATPase activities was found. Several lines of evidence are presented linking external cell surface ATPase activity and differentiated function. Significant increase of ATPase activity in stationary phase of growth, when some percentage of cells differentiates, has been found. Furthermore, morphological differentiation of neuroblastoma cells by conditioned medium is accompanied by a striking increase of ATPase activity. The same increase in ATPase activity in neuroblastoma cells can be produced by the conditioned medium. Presumably glial cells in culture release a factor that produce increased Ca2+- and Mg2+-dependent ectoATPase activities in neuroblastoma cells. Treatment with glia-conditioned medium enlarges the surface of neuroblastoma cells. Morphological differentiation of neuroblastoma cells obtained by removal of serum and addition of either db-CAMP or BUdR, although it increases process formation and enlarges the cell surface, it does not increase specific activity of surface ATPase (unpublished data). Thus, it seems that increase of ecto-ATPase activity of neuroblastoma cells upon treatment with glia-conditioned medium cannot be attributed simply to the increased cell surface. Removal of serum and treatment by dbCAMP or BUdR are unphysiological and they all interfere with the growth rate of the cells [ 191. In the present study, no attempts were Exptl Cell Res 98 (1976)
made to explore in detail the properties of Mg2+- and Ca2+-dependent ATPase activities of Ml neuroblastoma cells and reisolated Ml cells. With this we will deal in a separate paper. The authors are grateful to Dr A. Rosenberg for valuable criticism during preparation of the manuscript. Miss A. Riehl and Miss M. Ostertag are thanked for their excellent technical assistance. A. E. is a Chargee de Recherche at CNRS.
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Received July 24, 1975
Exptl Cell Res 98 (1976)