Human neuroblastoma growth inhibitory factor (h-NGIF), derived from human astrocytoma conditioned meduim, has neurotrophic properties

BRAIN RESEARCH ELSEVIER

Brain Research 644 (1994) 282-290

Research Report

Human neuroblastoma growth inhibitory factor (h-NGIF), derived from human astrocytoma conditioned medium, has neurotrophic properties Y a m a n Z. Eksioglu a, Junko Iida b Kiyofumi Asai a Takatoshi Ueki a Keiko Nakanishi a Ichiro Isobe at Kazuo Yamagata b, Taiji Kato a,, " Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-Ku, Nago~'a 467, Japan h Sumitomo Metal Industries Biomedical Dit,ision, 5 Hikaridai .~'-Chome, Seika-Cho Souraku-Gun, Kvoto 619-02, Japan (Accepted 18 January 1994)

Abstract

Investigations on the general characteristics of human astrocytoma cell line NAC-1 revealed neuroblastoma growth inhibitory activity in conditioned medium. Neuroblastoma growth inhibitory factor (NGIF) was partially purified by Econo Q, Econo CM, and Superose 12 column chromatography. The protein is weakly basic with an estimated M r of 120,000, possibly having an M r 60,000 dimeric structure. NGIF inhibits the growth of human neuroblastoma cell lines but has no effect on morphology nor does it produce any change in the growth of human glioblastoma cell lines. Interestingly, NGIF appears to promote survival and neurite outgrowth of embryonal rat cortical neurons. These neurotrophic properties suggest a role for NGIF in the development of the nervous system. Key words: Human NGIF; Astrocytoma; Neuroblastoma growth inhibitor; Neurotrophic factor; Neuron-gila interaction

1. Introduction

In the developing nervous system, a great number of neurons die while others develop normally. This phenomenon of natural neuronal death is the result of competition for limited amounts of target-derived neurotrophic factors, nerve growth factor ( N G F ) [8,12,23, 33,50,51] being an extensively studied example. N G F was first proposed as an essential factor for development and maintenance of peripheral sensory and sympathetic ganglia. Subsequently, it has been shown to possess neurotrophic action, in the CNS, on cholinergic neurons of the basal forebrain [57]. NGF, however, cannot explain the survival and differentiation of all neuronal populations. This has encouraged research leading to the identification of other neurotrophic factors such as brain-derived neurotrophic factor (BDNF) [7,25], ciliary neurotrophic factor (CNTF) [1,56], fibroblast growth factors (FGF) [19,20, 41,52,54], as well as novel neurotrophins (NT-3, N T - 4 / NT-5) [9,22,26,37].

* Corresponding author. Fax: (81) (52) 842-3316. 0006-8993/94/$07.00 g> 1994 Elsevier Science B.V. All rights reserved SSDl 0 0 ( } 6 - 8 9 9 3 ( 9 3 ) 0 0 1 1 7 - U

Many investigators have pointed to glial cells as mediators of neurotrophic factors [6,39], drawing attention to trophic interactions between astrocytes and neurons. Recent studies provide evidence for the astrocyte production of N G F [13], C N T F [34,44], gila maturation factor (GMF) [35], acidic and basic F G F [2,24], and glia-derived neurotrophic factor ( G D N F ) [36]. Additionally, it has been demonstrated that the neurotrophic effects of acidic and basic F G F are mediated through an unknown factor produced by growing astrocytes [15]. These and many yet unidentified factors may play a role in nervous system development and regeneration. Hence, neuron-glia neurotrophic interactions have become a primary focus to better understand neuronal survival and axonal guidance. Considering glia as key elements in regulatory pathways, we have studied protein factors produced in these cells and have demonstrated several gliotropic and neurotrophic factors such as G M F [30,31] and neurotrophic gliostatin [3,4]. Investigating GMF-like activity in glioma cells in the early 1980s we discovered a factor in rat astrocyoma cells and conditioned medium of glioblasts which inhibited mouse neuroblastoma cell growth. We character-

Y.Z. Eksioglu et al. ,' Brain Research 044 (1994) 282-290

ized and described this factor as mouse neuroblastoma growth inhibitory factor (m-NGIF) [32,45]. Our group was then investigating neuroblastoma therapy using agents promoting tumor cell differentiation [29]. Phosphodiesterase inhibitors (papaverine, BL199) and adenylate cyclase activators (prostaglandin E~, cAMP) were determined clinically unsuitable and m-NGIF's highly specific neuroblastoma growth inhibition was intriguing. To investigate NGIF's potential to arrest or prevent malignant growth of neuroblastomas, we have partially characterized and here report initial purification of human-NGIF (h-NGIF) produced by human NAC-1 astrocytoma cells. We further demonstrate the neurotrophic action of h-NGIF on cultured embryonal rat cortical neurons.

2. Materials and methods 2.1. Culture of glial and neuronal cells Human neuroblastoma cell lines GOTO [46], NAGAI [46], TGW [46], SKN-DZ [49], YT-nu [28], human osteosarcoma cell line OST [58], and human gastric cancer cell line MKN-45 [27] (obtained from Japanese Cancer Research Resources Bank (JCRB)) were maintained in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Microbiological Associates). Human neuroblastoma cell line IMR-32 [53] from the American Type Culture Collection (ATCC) and ascites type mouse neuroblastoma cell line NAs-1 (our laboratory), and human glioblastoma cell lines A-172 [18] (from JCRB), T98G [48] (from JCRB), GB-1, and NAC-1 human astrocytoma cell lines (both established in our laboratory) were maintained in Ham's F-10 medium (F-10, Gibco) containing 10% FBS. Cortical neuron cultures were prepared from rat embryonal cortex (El6) as described by Banker and Cowan [5]. Cerebral cortices were dissected and digested with papain (2 mg /ml) in Ca 2÷ and Mg e+ free Tyrode's solution (CMF-Tyrode) containing bovine serum albumin (2 mg/ml) and cysteine (2 mg/ml) for 30 min. Dissociated cells were collected by centrifugation and re-suspended in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% heat-inactivated horse serum (Microbiological Associates) and 1 mM sodium pyruvate. After gentle trituration for a homogeneous suspension, cells were filtered through sterile lens paper. Filtered cells were collected by centrifugation and re-suspended in serum free growth medium (N2) [10] of Ham's F-12 medium (F-12, Gibco)/DulbeccoVogt modification of Eagle's medium (DMEM, Gibco) (1/1; v/v) supplemented with 5 p,g/ml insulin, 100 /zg/ml transferrin, 20 nM progesterone, 100 p,M putrescine and 10 nM selenium (as Na2SeO3). Cells were cultured in poly-L-lysine coated 48-well dishes (1.0 cmZ/well, Costar 3548) at a density of 5×104 cells/well. Media were routinely supplemented with penicillin (100 units/ml) and streptomycin (100 #g/ml). Cultures were maintained at 37°C, 5% CO 2 and saturated humidity, except primary neuronal cultures for which CO2 concentration was 7%.

2.2. Collection of serum-free conditioned medium Serum-free conditioned medium (CM) was prepared from NAC-I cells. Cells were cultivated in 10% FBS supplemented F-10 medium. Upon reaching a 50% confluency, serum-containing medium was removed, and cells were washed twice in a 1:l mixture of F12 and DMEM, followed by the addition of N2 medium. A medium change

with N2 was carried out after 48 tl. After the .~cc~md 48-h cuharc period, CM designated as Fraction I (FIt was collected. Fractkm I1 (FI1) was collected 48 h subsequently. Protease inhibitors, leupeptinc (10 #M), pepstatin (10 #M), and phenylmethylenesuffonyl fluoride (PMSF, I mM) were added to collected CM fractions before storage at - 80°C. In all experiments presented in this study, CM haction 11 was used, since it elicited the highest growth inhibition of human neuroblastoma cells. Conditioned media from other human glial cell lines (T98G, A-172, and GB-1) were collected in the same way, Also. fraction II of each conditioned medium was used in related experiments.

2.3. Preparation of crude h-NGIF from NAC-1 conditioned medium CM fractions 1 and 2 from NAC-I human astrocytoma cells were centrifuged at 4,000 x g to remove insoluble material. Amicon Diaflo membranes with molecular weight cut-offs of 5 kDa (YM 5) or 10 kDa (PM 10) were employed to concentrate the samples before a subsequent wash with three-fold &02 M Tris-HCl buffer, pH 7:4. Crude NGIF was further purified by column chromatography. Conditioned media from T98G, A-172, and GB-1 were concentrated, employing the same procedures that have been used for NAC-1 CM, before using them in experiments to evaluate the production of human neuroblastoma growth inhibitory activity.

2.4. Column chromatography for partial purification of h-NGIF An Econo Pac Q Cartridge (Econo Q, Bio Rad; 5 ml bed volume), equilibrated with 0.02 M Tris-HCl buffer, pH 7.4, was used for the initial purification step. Adsorbed proteins were eluted by a

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1000

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Volume

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Fig. l. Dose-dependent growth inhibition of neuroblastoma cell lines by crude NGIF ( > 10 kDa). Human neuroblastoma TGW cells (5 x 104 cells/well, o ) and mouse ascites type neuroblastoma NAs-1 cells (5 x 104cells/well, e) were plated in 96-well microtiter tissue culture plates and cultured for 4 h in RPMI supplemented with 10% FBS and 0.02 M HEPES buffer. Following this, 0-20 /zl crude NGIF/well (4.25 mg protein/ml) was applied just prior to addition of 2.96 kBq/well [3H]dThd. After an incubation of 16 h, cells were harvested and cell-associated 3H radioactivity was determined by liquid scintillation counting. Values represent DPM means + S.E.M. of triplicate experiments.

284

Y.Z. Eksioglu et al. / Brain Research 644 (1994) 282-290

Table 1 Effects of conditioned media and extracts of various cells on T G W Cell line NAC-1 T98G A-172 GB-I

a b a b a b a b

Control

5/xl

10 ,al

15 #1

2269.0_+86.2 1329.4 + 37.0 2269.0 + 86.2 1329.4 _+ 37.0 2269.0 + 86.2 1329.4 _+ 37.0 2269.0 + 86.2 1329.4 +_ 37.0

2238.7+ 62.2 1667.3 + 68.3 1760.6 +_ 69.5 1938.0 + 76.9 3031.7 ± 150.8 1479.2 4-_ 46.4 2645.9 + 61.5 2461.1 ± 136.4

1735.0_+110.8 2178.0 + 55.2 1679.5 -+ 2.9 2526.0 -+ 120.7 2723.5 + 129.6 1793.7 4__ 75.1 2697.7 -+ 84.1 2/)91.6 4__ 145.7

1382.0+ 66.9 1772.4 -+_ 31.7 1696.4 + 47.3 4413.1 + 165.0 2996.2 + 86.6 1987.4 ± 99.1 2732.6 -+ 252.(l 2649.6 -+ 116.0

Dose-dependent response of T G W h u m a n neuroblastoma cells to conditioned media and cell extracts of various cell lines. T G W cells (5 × 104 cells/well) were plated in 96-well plates in 100 Ixl RPMI supplemented with 10% FBS and 0.02 M HEPES buffer. This was followed by treatment with NAC-1, T98G, A-172, GB-I (a) conditioned media, (b) cell extracts, and buffer control (Tris-HCl 0.02 M, pH 7.4 was used as control, since CM, concentrated by ultrafiltration, and cell extracts were w a s h e d / d e s a l t e d using this buffer) in accordance with the bioassay procedure described in Section 2. All values indicate the mean -+ S.E.M. of triplicate experiments.

linear gradient of 0 to 1 M NaCl in 0.02 M Tris-HC1, pH 7.4. Active fractions were acidified to pH 6.0 using a 1 M HCI stock solution and loaded onto an Econo Pac CM Cartridge (5 ml bed volume, Econo CM, Bio Rad), equilibrated with 0.02 M MES buffer, pH 6.0. Proteins were similarly eluted with a linear NaC1 gradient of 0 to 1 M. Prior to bioassay, fractions were desalted using Bio (}el P-2 minicolumns (1 ml bed volume, Bio Rad) prewashed with 0.02 M Tris-HCI, pH 7.4. To determine the molecular size of h-NGIF, bioactive fractions from Econo CM were applied to a Superose 12 gel filtration column ( 1 × 2 2 cm) with 0.2 M Tris-HCl, pH 7.4 containing isotonic NaCI buffer. Molecular weight standards for column calibration were: alcohol dehydrogenase (M~ 150,000), bovine serum albumin (M~ 67,000), hen egg white ovalbumin (M~ 45,000), and carbonic anhydrase (M~ 29,000). Chromatography was carried out at 24°C on a Bio-Rad Econo System.

method of McLeester and Hall [38] was developed. T G W cells, at a density of 5 × 104 cells/well in 100 /xl of RPMI medium containing 111% FBS, were plated in microtiter wells. After incubation for 4 h, 20-/xl samples purified by Econo Q or Econo CM chromatography were added to the wells in triplicate. An equivalent volume of 0.02 M Tris-HCl buffer, pH 7.4 was supplied for controls. 2.96 kBeq/well [3H]dThd ( A m e r s h a m ) i n CMF-Tyrode solution was added to monitor growth inhibition. Following incubation of 16 h, cells were harvested on glass fiber filters using a multiple cell harvester (Labo mash). Filters were air-dried and placed in vials containing 5 ml of AL-I scintillation fluid (Dojindo Laboratories). Cell-associated 3H was detected on a model 5801 liquid scintillation counter (Beckman). [3H]dThd incorporation of experimental and control samples were used for relative D N A synthesis estimates. Each experiment was performed in triplicate, and results were evaluated after calculation of mean and standard error values.

2.5. NGIF bioassay 2.6. Eraluation of neurotrophic action 96-well microtiter tissue culture plates (0.33 cme/well, Nunc 1-67008) were used for neuroblastoma growth inhibitory assays. A multiwell system giving comparable results to the conventional

1800

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Neurotrophic action was evaluated by morphological and quantitative analysis of embryonal rat cortical neurons in culture. Surviving

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Fraclion Number Fig. 2. Elution profile of h-NGIF Econo Q cartridge chromatography. Crude NGIF (85 mg protein) was applied to an Econo Q cartridge (5 ml), developed with 0.02 M Tris-HCl buffer, pH 7.4. Bound proteins were eluted with a linear gradient of NaCI from 0 to 1 M at 1 m l / m i n . 2-ml fractions were collected and subjected to bioassay after desalting on Bio Gel P2 mini columns ((o) [3H]dThd incorporation of T G W cells, ( o ) UV absorbance).

}'Z. l:'k~'iogh¢ ctal. Brain Research 044 (1994) 282-290

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Fraction Number Fig. 3. Elution profile of h-NGIF Econo CM cartridge chromatography. Pooled active fractions from Econo Q separation (2.72 mg protein) were acidified with 1 M HC1 to pH 6.0 and applied on an Econo CM cartridge (5 ml) equilibrated with 0.02 M MES buffer, pH 6.0. A linear NacI gradient from 0 to 0.7 M in 30 rain followed by a linear slope increase to 1.0 M in 10 rain was utilized for elution. 2 ml fractions were collected at 1 ml/min and bioassayed after desalting on Bio Gel P2 columns. [3H]dThd incorporation of TGW cells is denoted by (e), and (o) indicates UV absorbance.

and neurite-bearing neurons were examined and quantified by counting 4 visual fields/well (under 200-fold magnification), in 48-well plates (1.0 cm2/well), 10 days after cell plating.

1°61

2. 7, General

Protein content of samples was determined with the BCA protein assay kit (Pierce) using bovine serum albumin as standard.

Table 2 Protease susceptibility of human NG1F Treatment

,5,

% Reduction in DNA synthesis Econo Q

10 5

10 4 2.2

2.4

2.16

•

218

I

3.0

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312

•

314

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3.6

Elution Volume (ml/3) Fig. 4. Molecular size determination of h-NGIF. Human NGIF purified on Econo Q and Econo CM columns was applied to a 1 × 22 cm Superose 12 column. Factor elution corresponded to two peaks at 60 and 120 kDa, as depicted on the molecular weight standard curve. Molecular weight markers used for the standard curve were: (1) alcoholic dehydrogenase (M r 150,000), (2) bovine serum albumin (M r 66,000), (3) hen egg white ovalbumin (M r 45,000), (4) carbonic anhydrase (M, 29,000).

Trypsin Control NGIF fraction Trypsin digest in PMSF S1 NGIF in PMSF Trypsin in PMSF PMSF Crude NGIF tteat (90°C) t0 min 20 rain 30 min

0.0±4.0 36.3±3.9 3.6±8.5 10.0±7.0 4.6±6.5 -9.6f0.9 43.3±4.4

Econo CM 0.0± 2.3 32.3± 5.7 1.7± 6.8 11.0± 4.2 3.8±10.0 - O . l ± 6.0

- 8 . 8 ± 6.6 - 7 . 1 ± 7.8 - 1 2 . 0 ± 2.5

Partially purified h-NGIF from Econo Q and Econo CM columns (140 ~g protein/ml) was incubated with trypsin at an enzyme/substrate ratio of 1:50 (w/w) for 3 h at 37°C. The enzymatic reaction was stopped with PMSF (1 mM final concentration). Trypsin digests and controls were subjected to a neuroblastoma growth inhibitory bioassay. Heat stability was determined by incubating samples at 90°C for 10, 20 and 30 min respectively, followed by bioassay (SI NGIF: spontaneously inactivated h-NGIF fraction in PMSF in the absence of trypsin). All values indicate the mean_+ S.E.M. of triplicate experiments.

Y.Z. Eksioglu et al. /Brain Research 644 (1994) 282-290

286

3. Results

10 6 •

3.1. Neuroblastoma growth inhibitors in the conditioned medium of human glioma Serum-free conditioned medium (fraction II) from NAC-I cells displayed a dose-dependent inhibition of

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Fig. 6. I n h i b i t i o n o f neuroblastoma cell g r o w t h by p a r t i a l l y p u r i f i e d

40

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i

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Volume

(#1)

h-NGIF (CM fractions). T G W cells (5 x 104 ) were cultured in 24-well plates (Costar, 1.8 cm2/well) in 500/xl of RPM1 supplemented with 10% FBS and 10% ttEPES buffer. Arrowheads indicate daily h-NGIF application. Culture medium was changed at 48-h intervals. (Tell density and viability were determined by hemocytometer on the first, second, fourth, sixth, eighth and tenth day after subculture. All values indicate means +_S.E.M. of quadruplicate experiments.

B 140

0-----

GB-1

T G W human neuroblastoma cell proliferation, as measured by [3H]dThd incorporation, whereas inhibitory activity on NAs-1 ascites type mouse neuroblastoma cells was not significant (Fig. 1). Before further investigation we surveyed conditioned media and cell extracts from various human glial cell lines (T98G, A-172, GB-1) for a better source of neuroblastoma growth inhibition. However, except for a slightly weaker growth inhibitory activity in T98G CM, none of the CM from latter cells showed any inhibitory effect on T G W cells. Moreover, cell extracts from all glioma cell lines mentioned, including that of NAC-1, augmented proliferation of T G W cells, in contrast with the unique inhibitory action of NAC-I and T98G conditioned media (see Table 1).

Tg8G A172

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80

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Volume

(~l)

Fig. 5. H u m a n - N G I F dose d e p e n d e n t responses of various h u m a n cell lines. Cells plated in 96-well microtiter plates (5 x 104cells/well) were treated with 0 to 20 p.l/well of partially purified h-NGIF (pooled CM chromatography fractions) and subjected to bioassay. [3H]dThd incorporation of (a) h u m a n neuroblastoma cell lines T G W (o), NAGA1 (e), YT-nu (D), and S K N - D Z (A); (b) h u m a n glioblastoma cell lines GB-1 (©), T 98 G (e), A 172 (D), OST h u m a n osteosarcoma cells ( • ), and MKN-45 h u m a n gastric carcinoma cells (zx).

3.2. Partial purification and characterization of h-NGIF Crude fraction II concentrated to 85 mg protein was applied to an Econo Q cartridge. N G I F activity passed through the column, indicating its basic property in a neutral pH environment (Fig. 2). At this step, we investigated protease susceptibility and observed inhibition loss at a trypsin/substrate ratio of 1:50 (Table 2). Fractions (Nos. 3-1{)) were pooled and applied to an Econo CM column (Fig. 3). Active fractions eluted at approximately 0.35 M NaCI.

Ez. Ekstoglu et u/. /Brain Research 044 (1994) 282-290

Econo CM active fractions were pooled and used for further characterization. To identify activator or stabilizer substances of h - N G I F activity we tried metal ions (Ca 2+, Mg 2+ or Mn2+), their chelators ( E D T A or EGTA), and thiol reducing agents. None were found to be effective. H - N G I F activity was lost after trypsin digestion and heat treatment, reconfirming its proteinaceous nature (Table 2). To estimate the molecular size of h-NGIF, we applied the h - N G I F fraction from Econo CM separation to a Superose-12 column equilibrated with 0.02 M Tris-saline buffer, p H 7.4. H - N G I F eluted in two peaks with molecular weights of 60,000 + 5,000 and 120,000 + 5,000, possibly indicating a dimeric structure (Fig. 4). 3.3. Biological effects o f partially purified h - N G I F on l~arious cell lines

Proliferation of human neuroblastoma cell lines TGW, N A G A I , YT-nu and S K N - D Z were inhibited in a dose-dependent manner by partially purified h - N G I F (Fig. 5a). Neither human glioblastoma cells GB-1,

(a)

~-"

T98G, or A-172 nor human ostesarcoma cell line OST responded to h-NGIF, however (Fig. 5b). Curiously, h - N G I F was found to stimulate the proliferation of MKN-45 human gastric carcinoma ceils (Fig. 5b). To confirm and further evaluate our method of [~H]dThd incorporation as an indicator of cell proliferation [55], the effects of h - N G I F on T G W cell number were determined. Culture populations were sampled using a hemacytometer to determine cell number and D P M correlation (Fig. 6). A slight decrease in early exponential cell growth rate, denoted by elongation of doubling time from 24 to 36 h, was noted in h - N G I F treated cells. In addition, a two-fold decrease in saturation cell density, after the ninth day in subculture, favored strong inhibition. Although h - N G I F administration was discontinued beyond day 7, no recoveryfrom inhibition was observed through day 10. 3.4. Neurotrophic action o f h - N G I F on cortical neurons

Since m - N G I F was initially detected in the conditioned medium of normal rat astrocytes and was specu-

(b)

Fig. 7. Neurotrophic effect of h-NG1F on rat cortical neurons. Embryonal day 16-rat cortical neurons were dissociated by papain and seeded on poly-L-lysine coated 48-well plates in 250/zl N2 medium (5 x 10 4 cells/well). After an incubation of 24 h cells were treated daily by either (a) Tris-HCI 0.02 M, pH 7.4 (25/zl; control) or (b) h-NGIF (3.5/xg protein/25/zl; experimental). Photos were taken 10 days following cell plating, under 200-fold magnification.

EZ. Eksiogh~ et al. / Brain Research 644 (1994) 282-290

288 500

400

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300

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200

100

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Fig. 8. Quantitative analysis of neuronal survival in response to h-NGIF, Embryonal rat cortical neurons were cultured and treated without (control) or with h-NGIF (NGIF) as in Fig. 7. Surviving neurons bearing neurites were counted in four visual fields/well (under 200-fold magnification) on day 10 following cell plating. Values indicate means + S.E.M. of quadruplicate cell counts.

lated to be involved in the developmental processes of the central nervous system, we evaluated possible neurotrophic action of h - N G I F on neurons. Cortical neurons from E16-rat embryos were stimulated daily with 25 /xl (3.5 Ixg protein) of factor. As seen in Fig. 7, h - N G I F promoted survival as well as neurite extension of cortical neurons after 10 days in culture, a time when control cultures were senescing. Neuritc-bearing neurons were counted 10 days after plating cells in order to evaluate morphological observations quantitatively. Control cultures had 218_+ 4 cells in one microscopic area, while 423 +_ 3 cells were counted in wells treated with h-NGIF, thus confirming its supportive effect on neuronal survival (Fig. 8).

4. Discussion

Our present study demonstrates the presence of h - N G I F in the conditioned medium of human glial cell lines. Characterization of h - N G I F has elucidated structural and biophysical features of a macromolecular protein. H - N G I F is a basic, protease-sensitive, and thermolabile protein of 120 kDa, showing a possible dimeric structure of two 60 kDa subunits. In addition to a slight suppression in early exponential cell growth, the inhibitory action of h - N G I F was found to decrease saturation density of T G W cells to 50% of controls. Interestingly, no recovery from growth inhibition

through day 10 was observed after removal of h - N G I F beyond day 7 (Fig. 7). The identification of a human growth inhibitory factor acting specifically on neuroblastoma cells suggests its possible use as a therapeutic agent in preventing or arresting neuroblastoma malignancies. The target specificity of h - N G I F as a neuroblastoma growth inhibitor is promising for clinical applications. This contrasts with agents promoting differentiation of tumor cells (papaverine, BL191, prostaglandin E 1, dibutyryl cyclic adenosine 3' : 5'-monophosphate) which have broad-based and severe side effects [16,29,32]. Apart from therapeutic interests, h - N G I F is shown to be a novel astrocytoma-derived neurotrophic factor promoting survival and neurite outgrowth of cortical neurons. It has been well documented that N G F [40,42], basic F G F [42], and gila-derived nexin [21,39,43] induce the growth a n d / o r differentiation of human ncuroblastoma cells. In this regard, the inhibitory activity of h - N G I F on various human neuroblastoma cell lines, without any associated morphological changes, is a distinct biological property. Though biochemical similarities between h - N G I F and other known neurotrophic factors like NGF, basic FGF, BDNF, and NT-3 [47], do not seem to be in favor of such a distinction, careful evaluation of h - N G I F ' s biochemical properties supports our view. The growth factor content of the original crude source and the stability of the factor under harsh conditions are two fundamental elements underlying success in protein purification [11]. Unfortunately, hN G I F is present only in trace amounts in NAC-1 conditioned medium and is very unstable. Although wc screened the conditioned media and cell extracts of various human glial cell lines for a better source of N G I F , none showed higher production than that of NAC-1 cells. In addition, metal ions, their chelators, or thiol reducing agents were not efficient in activating or stabilizing the factor. Interestingly, the cell extracts of human glial cell lines, including NAC-1, had no inhibitory activity on human neuroblastoma cells. On the contrary, they induced neuroblastoma cell proliferation (see Table 1). The fact that h - N G I F has virtually no effect on glial cell lines, with respect to cell growth and D N A synthesis, indicates a strict target specificity. Taken together with its neurotrophic action on normal cortical neurons, h - N G I F may play an essential role in neural differentiation following the termination of proliferation during the development of the brain. Neuron-gila interactions have been demonstrated to play important roles in neuronal proliferation putting gila forward as a key modulator. Some recent publications describe gila-derived suppression of neuroblast proliferation and draw attention to the regulatory role of gila in the terminal differentiation of CNS progenitor cells [14,17].

Y..Z. Eksioglu et al. /Brain Research 644 (1994) 282 290

However, studies are necessary to further evaluate h - N G I F ' s role in neuron-gila interactions and developmental events.

Acknowledgements We would like to thank Drs. Ryo Tanaka and mkihiko Moriyama for constructive discussions in the course of this study and Mark Hodgson for critical reading of the manuscript. This work was supported by the following grants: the Grant-in-Aid for Scientific Research on Priority Areas; for Cancer Research from the Ministry of Education, Science and Culture, Japan; Grant (1A-2) for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan; the Japan Health Sciences Foundation; and Japan Research Foundation for Clinical Pharmacology.

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