Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside

Experimental

Cell Research 181 (1989) 226-237

Morphologic and Phenotypic Changes of Human Neuroblastoma Cells in Culture Induced by Cytosine Arabinoside M. PONZONI, Pediatric

Oncology

M. LANCIOTTI, A. MELODIA, and P. CORNAGLIA-FERRARIS’ Research

Laboratory,

G. Gaslini

Children’s

A. CASALARO, Hospital,

16148 Genoa,

Italy

The effects of cytosine-arabinoside (ABA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. LOW doses of ABA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 h after a dose of ABA-C as low as 0.1 l&g/ml (about lOOO-fold lower than the conventional clinic dose). The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in [‘Hlthymidine incorporation was also observed within 24 h and the cell growth was completely inhibited on the sixth day. Moreover, ABA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ABA-C. At the same time ABA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ABA-C do promote the differentiation of G&ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

0 1989 Academic

Press, Inc.

The dismal prognosis for the majority of children with advanced-stage neuroblastomas (NB) highlights the need for more effective therapies. Research on NB cell differentiation has been stimulated by two clinical peculiarities: NB has the highest overall rate of spontaneous regression of any malignant neoplasia (approximately 7 % of all cases) [ 1,2] and is capable of spontaneously differentiating or maturing to a benign ganglioneuroma [3]. Even though such differentiation is rare, the possibility that one therapeutic strategy for this tumor might involve induction of differentiation, thereby interfering with tumor growth, should be carefully tested. Continuously growing human and murine NB cell lines have become extremely important in establishing in vitro experimental models of this tumor [4-71. The mouse Cl300 neuroblastoma cell line [8] and its subclones have been widely used for such studies [9]. A variety of substances and culture conditions including dibutyryl-CAMP, nerve growth factor, dimethyl sulfoxide, prostaglandin El, sodium butyrate, and serum-free medium have been reported to induce morphological and biochemical differentiation in the Cl300 cells [l&15]. Moreover, several cytotoxic agents, including adriamycin, cytosine arabinoside, sulfur mustards, cisplatin, daunomycin, and aracytin [16-201 have ’ To whom reprint requests should be addressed. Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved 0014~4827189 $03.00

226

ARA-C induces diflerentiation

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proved effective inducers of morphological and biochemical differentiation in both murine and human models; however, more experiments should be done in order to confirm this for the various human NB cell lines available. In the present report the histological generality of these phenomena were tested by determining the effects of ARA-C on a new human NB cell line, GI-ME-N, established and cytogenetically and molecularly characterized in our laboratory [21, 221 from a bone marrow sample of a 3-year-old girl with stage IV NB, according to Evan’s staging system [23]. MATERIALS

AND

METHODS

Chemicals. Tris base, Bis-HCl, EDTA, Nonidet-P40 (NP-40), Tween-20 (TW-20), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Calbiochem (La Jolla, CA). Acrylamide, bisacrylamide, and sodium dodecyl sulfate (SDS) were obtained from Bio-Rad (Richmond, CA). Glytine, 2-mercaptoethanol, methanol, 4-Cl-1-naphthol, ARA-C were purchased from Sigma Chemical So. (St Louis, MO). Cell cultures. Cells were maintained in the logarithmic phase of growth in 75-cm2 plastic tissue culture flasks (Falcon Plastic, Oxnard, CA) in RPM1 1640 medium (Flow Laboratories, Milan, Italy) supplemented with 15% heat-inactivated fetal calf serum (FCS) (Flow), sodium penicillin G (50 HI/ml), and streptomycin sulfate (50 &ml) (complete medium) at 37°C in humidified 5% CO,95% air. GI-ME-N cells were passaged following treatment with 0.05 % trypsin and 1 m&f EDTA in Hanks’ salt solution (Flow) washed, counted, and replated in fresh medium. Assay for inhibition of cefl growth. Cells (10’) were seeded into T-25 flasks (Falcon) with 5 ml of culture medium. Two days after plating, the culture medium was replaced with medium containing ARA-C or with solvent control. The cultures were refed every 4 days with only solvent-containing fresh medium until the day of counting, and at that time the cells were detached with the trypsin solutions, counted with a hemacytometer, and microscopically using a Turk solution. Cell viability was determined by the trypan blue exclusion test. Assay for inhibition of [3H]thymidine incorporation. Cells, 103/well, were plated in quadruplicate wells of flat-bottom microtest plates (Costar, Cambridge, MA). At various intervals after adding ARA-C, the plates were pulsed with 0.5 uCi [‘H]thymidine/well (Amersham International, Buckinghamshire, UK). After an additional 18 h incubation at 37°C the cells were trypsinized and harvested on strips of fiberglass filter paper with the use of a multiple automated sample harvester (Flow) and the radioactivity associated with individual samples was measured in a liquid scintillation counter (IX-Carb 4530, Packard Instrument Company, Downers Grove, IL). Colonyformation in soft ngar. The ability of the cells to form colonies in soft agar in the presence of 0.1 &ml ARA-C was determined as described elsewere for retinoic acid [24]. Immunojlourescence analysis. Surface antigens were detected with indirect membrane immunofluorescence using the following monoclonal antibodies: Ab 390, Ab 459, Ab 126.4, generously provided by B. Seeger [25-271. Adherent cells were scraped off the flasks with a disposable plastic cell-scraper (Costar); 15 pl of the cell suspension containing 1 x106 cells was incubated with 15 pl of the appropriately diluted monoclonal antibody for 30 min at 4°C. After cells were washed twice the reaction was developed by a second incubation with GAM-FITC (Coulter Electronics Ltd., Luton, UK) for 30 min at 4°C. All reagents contained sodium azide (0.1% by volume) to prevent antigenic modulation. The cells were washed twice and observed under a microscope (Leitz Orthoplan, Leitz GmbH, Wetzlar, Germany) equipped with a uv 100-W mercury bulb. At least 200 cells per sample were counted and the percentage of stained cells showing either partial of complete ring fluorescence was determined (means f SD). Neurofilaments were detected using MoAb antineurofdament 68, 160, and 200 kDa (BoehringerMannheim GmbH, Mannheim, West Germany). The cells were seeded at 25x103/ml into multiwell slides and incubated under conditions identical to those of the assay for growth inhibition. At the defined times the slides were fixed in acetone for 10 min at -20°C and incubated for 1 h at 37°C in a humid chamber with 10 ul of the appropriately diluted monoclonal antibodies. After being washed three times with PBS for 3 min at room temperature, a second incubation with GAM-FITC was performed. The samples were counted as for surface antigens analysis. Analysis of cytoskeleton: (A) Protein extraction. Five petri dishes each containing 5X 10’ cells in complete medium were prepared and kept in a water jacketed incubator. When the cells were

228 Ponzoni et al. cot&tent, the medium was removed and 3 ml of PBS was added just before scraping. After centrifugation at 8OOg for 5 min, the pellet was resuspended in 5 ml of the extraction buffer (50 mM Tris-HCl, pH 6.8, 2 mit4 EDTA, pH 8, 0.5 % NP-40, and 1 mM PMSF). The nuclei were then eliminated by centrifugation at 5OOg for 5 min. The supematant containing cytoskeletal proteins was recovered and centrifuged at 30,OOOgfor 30 min. The pellet was resuspended in 50-100 pl of buffer (50 nut4 Ttis-HCl, pH 6.8, 1% SDS, 2 % 2-mercaptoethanol) transferred into Eppendorf tubes, and boiled for 5 min. (B) Western blot. Electrophoresis of 50 ug of protein, obtained as described above, was carried out onto 7.5 or 10% acrylamide slab gels [28] using a Mini-Protean Dual Slab Cell (Bio-Rad) at 120 V for 1 h. Electrotransblotting on a nitrocellulose filter (Millipore Co. Bedford, MA) was performed in transfer buffer (25 r&f ‘Iris, pH 8.3, 192 m&4 glycine, 20% methanol) overnight, applying a current of 200 mA. The filter was preincubated with PBS containing 2 % BSA for 1 h at 37°C and then incubated for 1 h at 37°C with specific diluted human monoclonal antibodies (Boehringer). After being washed three times for 10 min in PBS TW-20, biotinylated antibody (Amersham) diluted 1: 100 was applied for 1 h. The filter, after being washed in PBS, was incubated for 30 min with horseradish peroxidase-streptavidin conjugate (Amersham) diluted 1: 100, washed with PBS, and finally incubated with a solution containing 4-Cl-1-naphthol in order to evidenziate the immune complex. Ceil morphology. GI-ME-N cells were plated and treated with ARA-C as in the cell growth inhibition assay. Starting with Day 1 of ARA-C administration, 200 cells/culture from at least three diierent random regions were examined daily with a phase-contrast microscope (Olympus IMT-2). Alternativity, GI-ME-N cells (103) were plated directly on 8-well multitest slides (Flow) and treated with ARA-C as in the cell growth inhibition assay. Cultures were scored for extent of morphologic differentiation after May-Grumwald-Giemsa staining. Catechokzmines detection. Sample of 3 x 10’ cells were harvested from the petri dishes by brief treatment with EDTA and washed in PBS. The cells were sonicated three times with an amplitude of 9 urn using an MSE sonicator. Aliquots were withdrawn for protein measurements [29]. The catecholamine assay was as described by Endert [30].

RESULTS Dose-response relationship of ARA-C-induced growth inhibition. The dosedependent effects of ARA-C on the proliferation of G&ME-N cells were detected by cell counts after 8 days and by incorporation of [3H]thymidine after 6 days. Figures 1 and 2 show that cellular proliferation as assessed by both criteria was inhibited in a concentration-dependent manner over a range of 0.005 to 0.1 &ml ARA-C. In these systems, about 50% inhibition of cell growth and [3H]thymidine incorporation was achieved with ARA-C at a concentration of 0.05 ug/ml. No decrease in the percentage of viable cells was detected in the treated cultures as compared with the control cultures. The effects of ARA-C on the ability of the cells to form colonies in soft agar were also evaluated. After 10 days of treatment, 0.1 ug/ml ARA-C induced inhibition of colony formation by at least 7040%. In fact, the clonogenicity in semisolid medium was 826 colonies/well (+19 SD) for medium-treated cells and 198 colonies/well (+9 SD) for RA-treated cells (P
ARA-C induces diflerentiation

ARA-C

of NB cells

229

( rig/ml )

Fig. 1. Dose-response curve of ARA-C-induced inhibition of GI-ME-N cell proliferation as assessed by viable cell counts after 8 days of treatment. Values are the means + SD of four different experiments each done in duplicate.

ARA-C-induced modulation of cytoskeletat proteins. The cytoskeletal proteins expressed by ARA-C-treated and untreated G&ME-N cells are summarized in Table 2. Western blot analysis confirmed that the neurofilament pattern did not change after ARA-C treatment, although the 200-kDa neurofdaments were quan-

ARA-C

( ngiml )

Fig. 2. Dose-response curve of ARA-C-induced inhibition of GI-ME-N cell proliferation as assessed by incorporation of [3H]thymidine after 6 days of treatment. Values are the means + SD of four different experiments each done in quadruplicate.

230 Ponzoni et al.

TABLE

1

EfSects of ARA-C on membrane and intracytoplasmic markers of G&ME-N evaluated by immunojluorescence assay after 6 days of treatment

cells

Neurofilaments Sample Control m-c, ARA-C, Al&C, ARA-C,

0.005 @nl 0.01 @ml 0.05 &ml 0.1 &ml

Ab 390

Ab 126.4

Ab 459

68 kDa

160 kDa

200 kDa

64+4 60f4 37?6 19+4 5+3

42f7 38+3 13rt2 a+2 5+1

65+5 60+4 45+5 10+4 6+5

Neg. Neg. Neg. Neg. Neg.

Pos. Pos. Pos. Pos. Pos.

Pos. Pos. Pos. Pos. Pos.

Note. The data are the means *SD of five different experiments.

titative up-modulated after 6 days of treatment of GI-ME-N cells with 0.1 ug/ml ARA-C. By laser scanning of the slides from the nitrocellulose filters, there was a 170% (163-178 % in two different experiments) increase in the band corresponding to 200~kDa neurofdaments in ARA-C-treated ceils compared to control cells. In contrast, in the same experiments vimentin was down-modulated: the 57kDa band was reduced to 41% (37-45 % in two separate experiments) of control level by 6 days of treatment with ARA-C. Time-course of ARA-C-induced growth inhibition. The growth curves of GIME-N cells in the absence and in the presence of 0.1 Ctg/ml ARA-C are shown in Fig. 3. The number of viable cells in both the ARA-C-treated and control cultures were similar for 24-48 h. However, from 48 to 72 h cellular growth in the presence of ARA-C was completely inhibited, with few changes in cell count even after an additional 4 days of incubation. This inhibition persisted for at least 8 days after replacement with fresh medium, being independent of the addition of extra drug to the culture (data not shown). Figure 4 shows the daily [3H]thymi-

TABLE

2

EfSects of 0.1 pglml ARA-C on cytoskeletal proteins of GI-ME-N cells assessed by Western blot analysis after 6 days of treatment Cytoskeletal proteins Neurofilaments Neurofilaments Neurofilaments Vimentin Chromogranin GFAP Synaptophysin Note. (-) negative; (+/-)

Control 68 kDa 160 kDa 200 kDa

+ + + -

AU-C,

0.1 &ml + ++ +I-

weakly positive; (+) positive; (++) strongly positive.

ARA-C induces differentiation

104’ 0

1

2

3 Days

4

after ARA-C

5

6

7

of NB cells

231

8

treatment

Fig. 3. Time course of GI-ME-N cell growth in the absence (0) or presence (@ of 0.1 &ml ARAC. Values are the means + SD of three different experiments.

dine incorporation of the ARA-C-treated and control cultures. Results were analogous to those obtained by direct cell counts except that inhibition of the incorporated isotope could be detected earlier (within 24 h), and was more evident after the addition of 0.1 p@rnl ARA-C. ARA-C-induced morphologic difirentiation. Treatment of GI-ME-N cells with

103

0

1

2 Days

3 after ARA-C

4

5

6

treatment

Fig. 4. Time course of [3H]thymidine incorporation by GI-ME-N cells in the absence (A) or presence (A) of 0.1 pg/ml ARA-C. Values are the means f SD of three different experiments, each done in quadruplicate.

232 Ponzoni et al.

Fig. 5. ARA-C-induced morphologic differentiation of GI-ME-N cells. Phase-contrast micrographs. Bar, 10 pm. (A) Medium-treated controls; (B) cells cultured in the presence of 0.1 drnl ARA-C for 48 h; (C) cells after 4 days of ARA-C-treatment; (0) cells at the 10th day after the removal of the drug. (E) Nontreated and (F) ARA-C treated cells after 4 days of culture at a lower cell density and at a lower magnitude (bar, 30 v).

ARA-C induces differentiation

Fig.

5-Continued.

of NB cells

233

234 Ponzoni et al.

Fig.

5-Continued.

ARA-C caused a dramatic morphologic alteration as evidenced by formation of large flattened epithelial-like cells with elongated neuritic processes (Fig. 5). The ARA-C concentration causing maximum increase in the percentage of differentiated cells ranged from 0.05 to 0.1 &ml. The time course of morphologic differentiation in the absence and presence of 0.1 ug/ml ARA-C is shown in Fig. 5. A significant increase in the number of differentiated cells could be detected

ARA-C

induces differentiation

of NB cells

235

within 48 h (Fig. 5 B); the maximum extent of differentiation occurred after approximately 4 days of treatment (Figs. 5 C and F). The morphologic differentiation induced by ARA-C was retained by the GI-ME-N cells in the absence of the drug. When ARA-C was removed after 4 days of treatment, most cells retained their morphologic alterations for at least 10 days (Fig. 5D). Catecholamines in morphologically differentiated cells. The effects of ARA-C on the ability of the cells to express catecholamines were evaluated after 5 days of treatment. The addition of 0.1 ug/ml ARA-C to the cells slighly affected the noradrenaline levels [0.038 pmol/mg protein (f0.009 SD) for untreated cells and 0.067 pmol/mg protein (+O.Ol 1 SD) for ARA-C-treated cells] whereas it seemed to increase the adrenaline concentrations [O.OOS pmoYmg protein (&2 SD) for control cells and 0.015 pmol/mg protein (+3 SD) for ARA-C treated cells]. DISCUSSION In contrast to the advances achieved in the treatment of other tumors, the situation for NB patients has not significantly improved in the last 10 years. In most cases, NB behaves as a highly malignant tumor, resistant and/or relapsing after irradiation and/or chemotherapy [l]. On the other hand, neuroblastoma is known, in same cases, to undergo spontaneous regression in vivo [l, 21. One mechanism for the regression has been suggested to be maturation of the tumour cells into terminally differentiated, non-proliferative, ganglion-like cells [3 11. Even though such differentiation is rare, it has raised the possibility that one therapeutic strategy for this tumor might involve induction of differentiation, thereby interfering with tumor growth. So one attractive approach to future treatment of patients with NB would be to identify drugs which induce terminal differentiation in vivo thus arresting the tumor cells in the nonproliferative phase of the cell cycle. The successful establishment of both mouse and human NB cell lines in vitro in recent years has made it possible to initiate studies on induced differentiation in a well-defined and well-controlled environment. This report provides evidence for the ability of ARA-C to regulate the phenotypic and cytoskeleton expression and the morphologic differentiation of a new human NB cell line, GI-ME-N, recently established and characterized in our laboratory [21, 221. The results of this study demonstrate that ARA-C induces concentration-dependent growth inhibition, morphologic alterations, and membrane antigen and cytoskeletal protein modulation of the GI-ME-N cells. The maximum effect was obtained using a dose of ARA-C as low as 0.1 ug/ml, about lOOO-fold lower than clinically used conventional doses [32], and a 3-fold lower “low-dose” cytosine arabinoside [33]. The fact that ARA-C treatment of GI-MEN cells started one day after plating with no subsequent decrease in cell viability in the cultures suggests that its effects were not the result of selection of more differentiated cells by differences in plating efficiency or ARA-C cytotoxicity. This conclusion was supported by time-lapse photography, which clearly showed individual ARA-C-treated GI-ME-N cells undergoing morphologic differentiation

236 Ponzoni et al. (unpublished data). ARA-C-induced inhibition of cell growth of GI-ME-N cells could be detected after about 24 h when assayed by incorporation of [3H]thymidine. The elapsed time before reduction of [3H]thymidine incorporation was observed corresponds to the time of ARA-C treatment before an increase in morphologic differentiation of GI-ME-N cells was observed. This is consistent with the reduced rate of DNA synthesis generally associated with a differentiation process [16] and supports the contention that true differentiation is occurring. Moreover, the inhibition of cell growth by treatment with ARA-C was also evident by testing colony formation in soft agar. The ARA-C sensitivity of GIME-N cells in this system, in conjunction with their sensitivity to ARA-C in monolayer, seems to indicate a very high chemosensitivity of both monolayer cultures and those subpopulations of cells able to form colonies. The dose-dependent down-modulation of membrane markers specific for NB cells, detected after 5 days of ARA-C treatment of GI-ME-N cells, further on suggests the hypothesis that a true differentiation occurred. The latter hypothesis was ultimately confirmed by down-modulating vimentin, a cytoskeletal protein specific for undifferentiated neuroblast and up-modulating the 200-kDa neurofilament, typically expressed by more differentiated NB cells. In contrast, the presence of the two subclasses of neurofilaments (160-200 kDa) on untreated GIME-N cells leaves open the possibility that G&ME-N cells are at least partially mature cells. Moreover, the proliferation of the GI-ME-N cell population was not completely inhibited after ARA-C treatment, indicating that only some of the cells had traversed the differentiation pathway to the terminally differentiated, nonproliferative state. This agrees with the findings that the catecholamine levels in the ARA-C-treated cells were not dramatically increased and were lower than expected for mature, normal neurons [34, 351. Very little is known about control of nerve cell differentiation in humans. In neuroblastoma tissue cultures, ARA-C appears to be a potent compound for promoting differentiation, inhibiting cell growth, and perhaps reducing tumorigenicity. Further studies of the sequence of cellular events associated with ARA-Cinduced differentiation of NB should help to clarify the potential of various chemotherapeutic drugs for reverting the malignant phenotype. In conclusion this report presents a new NB cell model appearing to be suitable to study the differentiating activity of antitumor agents. This system becomes especially appealing when one considers the morphologic, biochemical, and electrophysiologic modes of differentiation that might be evaluated in NB cells [16]. These findings should encourage further studies of the possible role of antitumor drugs in differentiating human NB cells. We are grateful to Mrs. Laura Malacrida supported by A.I.R.C. (Italian Association

for her excellent secretarial for Cancer Research).

assistance.

This

work

was

Press,

New

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