In vitro and in vivo growth characteristics of a new ascites-type neuroblastoma cell from mouse C1300 neuroblastoma

Neurochem. Int. Vol. 7, No. 1, pp. 29-36, 1985 Printed in Great Britain. All rights reserved

0197-0186/85 $3.00 + 0.00 Copyright © 1985 Pergamon Press Ltd

IN VITRO A N D IN VIVO GROWTH CHARACTERISTICS OF A NEW ASCITES-TYPE NEUROBLASTOMA CELL FROM MOUSE C1300 NEUROBLASTOMA TAIJI KATO, ITARU HORIUCHI, HmOSHI KATO, SHINGI SASAKI, HIDEHIKO TSUNOOKA, AKIRA MASAOKA, KUNIKO OKUMURA-NoJI, RYO TANAKA, HIROKO FUKAMI* a n d KYOKO KANO-TANAKA* Departments of Biochemistry and Surgery, Nagoya City University, Medical School, Mizuho-Ku, Nagoya 467 and *Aichi Cancer Center Inst., Nagoya 464, Japan (Received 28 February 1984; accepted 2 April 1984 Abstract--A clonal ascited type cell, NAs-1, was obtained in culture from a mouse neuroblastoma C 1300. The cells were adapted to anchorage-independently grow in the flask by the in vitro-in vivo alternate passage technique, and retained the ability of growing and producing ascites fluid when intraperitoneally injected into mice. Although the majority of growing cells in culture medium showed a small and round cell shape without any neuronal process, occasionally non-specific attachment onto the flask surface was observed, but devoid of the extrusion of processes. Karyotype analysis showed a homogeneous chromosome number, 40, with a marker chromosome [t(13 : 16)] and a minichromosome. Catecholamines, norepinephrine and dopamine, were found in the cell extracts and the contents of dopamine was particularly high as shown in another catecholaminergic neuroblastoma cell, N1E-115. Neuron specific enolase (?-subunit) was also detected. The treatment of the cells by dibutyryl cyclic AMP, prostaglandin E~, or BL191 (phosphodiesterase inhibitor) induced the biochemical differentiation in terms of catecholamine and cyclic AMP contents, but failed to promote typical morphological differentiations including the extension of process or the significant promotion of adherence onto the flask surface.

M a n y clonal cell lines isolated from m o u s e neur o b l a s t o m a C1300 have been described a n d widely used for m a n y neurological investigations ( A m a n o et aL, 1972; K n a p p a n d M a n d e l , 1974; P r a s a d et al., 1973). Using these cell lines such as N1E-115 (catecholaminergic clone), NS-20Y (cholinergic clone), N e u r o 2 a or N18 (inactive clones), o u r l a b o r a t o r y has exerted efforts in abolishing their tumorigenicity a n d p r o m o t i o n differentiations by cyclic A M P - r e l a t e d agents (dibutyryl c A M P , papaverine, p r o s t a g l a n d i n Et o r / a n d BL191) ( F u n a b i k i et al., 1979; K a t o H. et al., 1982a; K a t o H. et al., 1982b; K a t o T. et al., 1982; Sakazaki et al., 1983). Here we describe a new clonal ascites-type cell, NAs-1, which was characteristic in catecholaminergic functions, from m o u s e n e u r o b l a s t o m a C1300. The cells could be passaged in v i t r o - s y s t e m as well as in vivo-system. The in vitro b e h a v i o u r o f the cell is compared to t h a t o f a c o n v e n t i o n a l catecholaminergic cell, N1E-115.

culture consumables including Dulbecco's modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were obtained from Grand Island Biological Co. (Grand Island, New York). Methyl[3H]thymidine from New England Nuclear. Dibutyryl cyclic AMP from Boehringer Mannheim GmbH. Prostaglandin E I and BL191 were gifted by Ono Pharmaceutical Co. (Osaka, Japan) and Hoechst Japan Co. (Nagoya, Japan), respectively. Nerve growth factor (7s NGF) and fibronectin were purchased from Collaborative Res. (Waltham, MA.). Isolation and cultivation The tumor tissue of neuroblastoma was mechanically dissociated by a forceps in Ca 2÷- and Mg2+-free Tyrode solution and incubated for 30 min in the presence of 0.1% trypsin, All dissociated cells were grown in 25 cm 2 flasks and maintained in 5 ml of DMEM medium containing 20% FBS at 37°C in 10% CO 2 atmosphere. The medium was changed every 2-3 days. Cells were detached for subcultivation with 0.1% trypsin when the cell layer became confluent. The cells with 27 subcultivations in DMEM medium containing 10% FBS were transplanted in A/J mouse as parental cells. Inoculations into A / J mouse Each mouse for passages /n vivo received a single intraperitoneal injection of l x 107 viable cells prepared as for cell counting. Every 10 days after the inoculation grown cells in ascites were transplanted into another mouse. A part of the cells with the 6th passage /n vivo were distributed again into flasks, and then passaged every 2-3 days in F-10

EXPERIMENTAL PROCEDURES Materials The A/J mouse neuroblastoma, C1300, was gifted by Dr Imashuku, Kyoto Prefectural University of Medicine. Cell 29

30

TAIJI KATO et al.

medium containing 10~ FBS. Passage numbers of the cell in vit, o and in vitro are 31 and at this moment, respectively. Ka~votype analysis Conventional hypotonic methods were used for the karyotype analysis. The cells were arrested by 0.01-O.02#g/ml of colcemide solution for 3h at 37'C, swelled in 0.075 M KCI solution for 30 min at room temperature, fixed in methanol-acetic acid (3:1, v/v), and air-dried on slides. G-banding technique followed Deaven and Peterson (Deaven and Peterson, 1974). Plating eff4ciency Cell with 26 passages in vitro were distributed into the well (20 x 33 mm) of 8-well multiplate (Lux, No. 5218). Three days after the seeding, the number of floating cells in the medium through gentle pipetting by Pasteur pipette (5 times) to remove a weak and nonspecific adherence to plastic surface were counted by hemocytometer with trypan blue dye exclusion test. The remaining cells were finally detected by trypsinization for 15 min at 37C, and the number of which were also counted. Determination o f catecholamine contents The concentration of catecholamines, norepinephrine and dopamine, was determined by the use of a high-performance liquid chromatography with an electrochemical detector (Davis et al., 1981; Felice et al., 1978). The mechanically dislodged cells were exposed to cold water to give rise to the cell lysis. The lysate was deproteinated by the addition of 0.4 N perchloric acid and then centrifuged at 2,000 rpm for 5 min. The catecholamines in supernatant was adsorbed onto Amberlite CG-50 column and then eluted with 0.5 M HCI. 3,4-Dihydroxybenzylamine (DHBA) was used as the internal standard. One-hundred ,ul of the acid eluate was injected into a reversed-phase column (ODS-T Yanagimoto, Tokyo). The eluting buffer consisted of 0.1 M potassium phosphate buffer, pH 3.1 containing 50/~ M disodium ethylenediaminetetraacetic acid (EDTA), flowing at a rate of 0.8 ml/min under a pressure of 50 kg/cm 2. lntracellular cyclic nucleotides contents The contents of cyclic nucleotides, cAMP and cGMP, in cultured cells were measured by Steiner's radioimmunoassay method (Steiner et al., 1972). After removal of the medium the culture was washed twice with 2 ml of ice-cold 0.02 M Tris-HCl buffered saline, pH 7.4 and exposed to 50,~ trichloroacetic acid for 20 min on ice. The cells were scraped and suspended with a rubber policeman, and then pelleted by centrifugation. The pellet was used for protein determination by the method of Lowry et al. (Lowry et al., 1951). The supernatant was extracted 5 times with 3ml of H20-saturated ethyl ether. The water phase was collected in test tube and the remaining ether was evaporated by heating in a water bath at 60°C. Each cyclic nucleotide assay was conducted in triplicate. Neuron specific enolase Two forms of the enolase were determined with the sandwich-type enzyme immunoassay system described by Kato (Kato K. et al., 1981). The systems with the measurable sensitivity of 100 pg of both isozymes are composed of the antibody-bound solid-phase and the antibody Fab'-fl-D-galactosidase complex. The cells were sonically disrupted at 0°C and 15W of power for a total 2 min (using

15 s disruption periods followed by 15 s of cooling) by the use of the sonication cell disruptor (Heat systemUltrasonics, Inc.). The sonicate was diluted 30-fold with 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1 M NaC1, 1 mM MgCI 2, 0.1°.,, bovine serum albumin (fraction V from Armor), and 0.1°~, NaN 3. One-hundred #1 of each diluted sample were incubated in triplicate with the antibody-bound solid -phase at 4°C for 5 h with shaking. The solid-phase was washed twice with above buffer, and then incubated at 4'C overnight with the antibody Fab'-fl-o-galactosidase complex possessing 3 munits of fl-o-galactosidase. After washing out the unbound complex, the galactosidase activity associated with the solid-phase was fluorometrically assayed by measuring the produced 4-methylumbelliferon from 4-methylumbelliferyl-/~ -Bgalactoside as substrate. M o r p h o l o g y a n d in vitro g r m r t h characteristics

Figure 1 illustrated isolation procedures o f NAs-1 from mouse neuroblastoma C 1300. Although the parental cells with 27 subcultivations after primary culture of neuroblastoma C 1300 were able to attach and grow on the surface o f flasks, their characteristics were extremely transfigured to those of ascitic cells by only once intraperitoneal inoculation into A/J mouse. The ascitic cells could anchorage-independently grow in culture medium (F-10 medium containing 10'~i FBS). The p r e d o m i n a n t cell type was a small and round cell with a typical ascitic cell appearance on a smear (Fig. 2a). The cells, NAs-I, from 26 subcultivations o f ascites in ritro preserve a similar morphology to that o f the ascitic cells, but consisted o f a minor contaminating subclone, at a few percentage o f the total cell population, attaching firmly to the flask surface and extending short neuronal processses. A h o m o g e n e o u s population o f NAs-1 was easily separated from the minor subclone by exploiting these different adhesive properties. Separately NAs-I also could proliferate in vit,o with the same growth rate as parental cells by the inoculation i.p. into mice. During examining the best culture condition for the in vitro growth o f NAs-1 cells, it was revealed Neuroblastomo C1300 (Solid tumor in A/J mouse) Primary c u l t u r e

B Secondary culture

~ (27

Passages)

IntraPeritoneal injection Ascites-tYPe c e l l s

/

Passage ,:,~ ~£tw (26 passages)

-...,

Passage in ~i.o (30 passages)

Fig. 1. The procedures for establishment of NAs-I.

A new ascites-type neuroblastoma cell

Fig. 2. Morphology of original ascites type cells (a) and NAs-I with passages in vitro (b).

NC.I 7/I

C

31

32

TAIJ[ KATO et al.

KARYOTYPE

iI I| I( 1

2

ii I

tI

6

7

11

12

16

17

3

It

ti

4

5

ti

~t

ti

8

9

10

14

15

13

18

19

I

XY

O ml

M1 t(13:16)

Fig. 4. Karyotype of NAs-I from passage 26 of ascites type cells. The modal chromosome number was 40 and possessed a minichromosome (ml) and a marker chromosome (M l) derived from a translocation of No. 16 to 13 chromosome.

A new ascites-type neuroblastoma cell I0 z

NAs-I

33

could neither increase cell growth, nor promote morphological or biochemical differentiation observed in rat pheochromocytoma cell (PC-12) (Edgar and Thoenen, 1978; Green and Rein, 1977; Schubert and Whitlock, 1977).

,o'

Karyotype analysis z

lOS

100

1

50_~

o

l

~

~

DAYS AFTER SUBCULTURE

Fig. 3. Growth curve of NAs-I in F-10 medium containing 10~ FCS. 1 x 10Scells of NAs-1 (p26) were seeded in the well of 8-well multiplate. The number of ceils (--O--) and viability (--F-q--) were determined with a hemocytometer by trypan blue staining. Arrow indicates the time of replacement of the medium with F-10 medium containing 10~ FCS. Values are means of quadruplicate experiments with triplicate determinations for each experiment. The dispersion of the individual values is within 5~o of the mean. that Ham's F-10 medium was better than DMEM medium, and 5~o CO2 atmosphere was much more suitable than 10~o CO2. Since then F-10 medium containing 10~ FBS was employed as an ordinary growing medium for NAs-1 in vitro. As long as NAs-1 was growing under the exponential rate, the viability of cells was maintained over 95~. However, NAs-1 was noted to die easily in an ordinary culture well due to the shortage of nutrients as illustrated in Fig. 3, since the growing rate was so fast (doubling time: 21 h). The suspension such as spinner-culture is useful for obtaining a large quantity of cell volume. To determine the adhesiveness of NAs-1 onto the surface of plastic flask, multiplate, or petri dish, floating cells in the medium were collected together with weakly binding populations on the plate surface by gentle pipettings. Generally, 60-70~ of cells were counted as floating ceils with the anchorage-independent growth. Most of adhering cells (remaining 30-40~o of cells), however, preserve the original round cell-shape without any extrusion of neuronal process. It was most likely the adherence might be involved in GI phase through whole cell cycle of NAs-1, since the adherent cells also gradually detached from the surface and finally became to anchorage-independently growth in the medium. The contention was also supported by the result that fibronectin could give no effect on the adhesiveness of NAs-I (data not shown). The addition of nerve growth factor (0.25-2.5/~g/mi)

Chromosomes of NAs-1 were examined at the 20th subcultivation. The cells showed a homogeneous diploid chromosomes, and had a modal number of 40 with a minichromosome and a marker chromosome (M1) which might be transiocated from No. 16 to 13 chromosome (Fig. 4). The minor subclones of NAs-1, which had an adhesive property to the surface of flask, showed a wide distribution of chromosome numbers with polyploidy. In vivo growth characteristics NAs-1 failed to form tumors when implanted subcutaneously into mice, but tended to show infiltration into surrounding tissues. Intraperitoneal inoculation of NAs-1 induced detectable ascites 3-4 days, and the maximal amount (0.5-1.0 ml/animal) after 10 days. Curiously, thereafater, the ascites disappeared within 2-3 days, Most of NAs-1 invaded along peritoneum and also brought about the infiltrative metastasis in varied organs such as liver, kidney, lung and oesophagus. The host animal, however, survived for 3 months or longer, whereas occasionally accompanied by subcutaneous tumors. Figure 5 shows a typical infiltration of NAs-1 in the metastatic focus of liver. The infiltrating cells possessed a small and round shape with less cytoplasm, and corresponded to the morphology of the immature or embryonic type according to the classification of human neuroblastoma by Poore et al. (Poore et al., 1955).

Biochemical properties o f NAs-1 The biochemical characteristics of NAs-1 are outlined in Table 1 in comparison with those of another catecholaminergic cell from neuroblastoma C1300, N1E-I15. Comparably high contents of dopamine and norepinephrine were observed in NAs-1. The treatment of NAs-1 with 0.5mM dibutyryl cyclic AMP, which was high enough to bring out biochemical and morphological differentiations of conventional neuroblastoma cells (Neuro2a, NS-20Y, and N1E-I15), increased 1.4-fold dopamine level (1160 pg/107 cells), whereas norepinephrine level was not affected by the treatment. Neither marked adhesive property nor morphological differentiation of NAs-I was evoked by dibutyryl cyclic AMP treat-

34

TAIJI KATO et al.

Fig. 5. Focal metastasis in mouse liver. The metastatic legion comprises NAs-I cells with a small and round shape. Neurofibri[ and rosette formation, which are characteristic in differentiated neuroblastoma such as a sympathoblastoma are not detected. ment. Other differentiation-promoting drugs such as prostaglandin Et and BL191, which were an activator of adenylate cyclase and an inhibitor of phosphodiesterase, respectively, inhibited DNA syntheses of NAs-1, but cell growth rate was lowered by only prostaglandin E~ (Table 2). Intracellular cyclic AMP contents in the treated NAs-1 by such drugs elevated up to about 2-fold higher level. These drugs gave an adhesive property to NAs-I to some extent (20~,0 increase in the maximum), but failed to promote the morphological differentiation. The contents of isozyme of enolase, ~- and ?,-subunits, were determined by a sandwich-type enzyme immunoassay system. ?-Subunit, neuronspecific enolase which corresponds to 14-3-2 protein (Bock and Dissing, 1975; Marangos et al., 1976), were detected in both NAs-1 and N IE-115 cells. ~-Subunit widely distributing in nonneuronal cells

was observed in both two cell lines in fairly large quantities. The existence of ~-subunit in these cells was consistent with the results on other mouse neuroblastoma cell lines reported by Kato et al. (Kato K. et al., 1981). The significance of ~ -subunit enolase in neuroblastoma cell has not yet been elucidated. The availability of this ascites-type neuroblastoma cell offered the opportunity to apply the simple and stable culture-passage techniques in vitro as well as in vivo system, which were extremely convenient to analyse cytochemical alteration of the cells exposed to antitumor drugs. Moreover, the cells will be also useful for defining the dynamic process of differentiation of neuroblastoma cells in coculture system with nonneuronal cells such as glial cells in regard to the direct mutual interactions through cell-surface glycoproteins or trophic factor(s) (Kato

Table 1. Neurochemical properties of NAs-I in comparison with those of NIE-115

Cl0n e NAs-I NIE-115

Catecholamine contents (pg/107cells) Norepinephrine Dopamine 420 + 135 893 ± 225

837 ± 230 1287 ± 364

Enolase isozyme (ng,mg prot.) :~-subunit -;-subunit 46.7 + 5. I 39.3 ± 4.6

1.3 ± 0.5 45.0 ± 8.4

Cyclic nucleotides (pmol/mg prot.) cAMP cGMP 0.68 ± 0.08 9.40 _+0.60

N.D.* 19.4 +_ 5.20

*N.D.: not detectable. Both lines of neuroblastoma cells, 106 cells of which were seeded in Petri dishes (9.0 cm), were cultured for 4 days with a medium replacement on 3rd day in subculture, and harvested for each assay according to the method described in the text. All data indicate the mean and standard deviation of quadruplicate experiments.

A new ascites-type neuroblastoma cell

35

Table 2. Influence of cAMP related drugs on the cell biological expressions of NAs-1 Dibutyryl cAMP Prostaglandin E I BLI91 Control (0.5 raM) (0.5/~g/ml) (1 mM) Cell number 152.4 __.12.4×k04 40.8 + 5.0 101.2 __.12.3 141.4 + 25.4 (~o Inhibition) (0) (73.2) (33.6) (7.2) DNA synthesis 1410 _+235corn 1065 5:138 1047 _+57 1156 -t- 304 (~ Inhibition) (0) (24.5) (25.8) (18.1) Cell viability 96.5~o 88.2 95.1 93.6 Adherence 68.7~o 87.7 90.4 78.3 pmol/mg prot. cAMP contents 0.68-0.08 -1.15 + 0.26 1.59 _+0.40 NAs-1 cells (1 × 105cells) were stimulated on the 2nd and 3rd days in subculture by drugs concurrently with the replacement by F-10 medium. The cells for the determination of DNA synthesis were simultaneously labeled for 24h with 0.1/iCi[3H]thymidine per well at the second stimulation. The cell number and viability were determined with a hemocytometer by trypan blue staining. The percentage of adherent cell to plastic surface and the contents of cyclic AMP were determined by the methods described in the text.

T . et al., 1982; S a k a z a k i et al., 1983). In this r e s p e c t f u r t h e r w o r k is in progress.

Acknowledgements--We are indebted to Dr K. Kato, Institute for Developmental Research, Aichi Prefectural Colony, for generously supplying enolase enzymeimmunoassay system. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Japan.

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T. (1982) Experimental study on maturation therapy of neuroblastoma. Pediat. Oncology 16, 45-46. Kato H., Kato T., Sakazaki Y., Yamakawa Y., Naganawa N., Funabiki J., Kato T., Masaoka A., Tanaka R. and Tsunooka H. (1982) Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin. Neurochem. Int. 4, 419-426. Kato K., Suzuki F. and Umeda Y. (1981) Highly sensitive immunoassays for three forms of rat brain enolase. 3.. Neurochem. 36, 793-797. Kato T., Sakazaki Y., Yamakawa Y., Kato H., Naganawa N., Kato T., Tsunooka H., Masaoka A. and Tanaka R. (1982) Inhibition of growth of mouse neuroblastoma cells by protein factor derived from rat glioblasts. Devl Brain Res. 3, 645-651. Knapp S. and Mandel A. J. (1974) Serotonin biosynthetic capacity of mouse c-1300 neuroblastoma ceils in culture. Brain Res. 66, 547-551. Lowry O. H., Rosenbrough N. H., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Marangos P. J., Zomzely-Neurath C. and York C. (1976) Determination and characterization of neuron specific protein (NSP) associated enolase activity. Biochem. biophys. Res. Commun. 68 (1976) 1309-1316. Poore T. N., Dockerty M. B., Kennedy R. L. J. and Waiters W. (1951) Abdominal neuroblastoma. Surg. Clin. North. Am. 31, 1121-1141. Prasad K. N., Mandel B., Waymire J. C., Lees G. J., Varnadkis A. and Weiner N. (1973) Basal levels of neurotransmitter synthesizing enzymes and effect of cAMP agents on morphological differentiation of isolated neuroblastoma clones. Nature New Biol. 241, 117-119. Sakazaki Y., Kato T., Kato H., Ito J., Tanaka R., Naganawa N., Kato T., Masaoka A. and Tsunooka H. (1983) Characterization and partial purification of N G I F from culture medium of glioblasts. Brain Res. 262, 125-135. Shubert D. and Whitlock C. (1977) Alteration of cellular adhesion by nerve growth factor. Proc. natn. Acad. Sci., U.S.A. 74, 4055~,058. Steiner A. L., Parker C. W. and Kipnis D. M. (1972) Radioimmunoassay for cyclic nucleotides. 3. biol. Chem. 247, 1106-1113.