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In vitro differentiation of human neuroblastoma cells induced by sodium phenylacetate
Cancer Letters, 70 (1993) 15-24 Elsevier Scientific Publishers Ireland
15 Ltd.
In vitro differentiation sodium phenylacetate
of human neuroblastoma
cells induced by
Jindrich Cinatl”, Jaroslav CinatlaVb, Marion Mainke b, Albrecht Weiflflog b Holger Rabenaua, Bernhard Kornhuberb and Hans-Wilhelm Doerra “Centre of Hygiene, Department of Medical Virology. bCentre of Pediatrics, Department of Haematology and Oncology, J. W. GoetheUniversity, Frankfurt a. M. (Germany) (Received (Accepted
11 January 3 February
1993) 1993)
Summary
Introduction
Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 6 mM stimulated morphological differentiation of two human neuroblastoma cell lines IMR-32 and UKF-NB-3. These concentrations inhibited growth and DNA synthesis of the cells in a dose dependent manner without significant effect on cell viability. The differentiated cells showed pseudoganglia formation and extension of cellular processes. The morphological differentiation in both cell lines was accompanied by decreased expression of N-myc oncoprotein. These results suggest that NaPA at concentrations, which have been achieved in humans with no significant adverse effects, promotes differentiation of cultured human neuroblastoma cells in association with the reduced expression of the malignant phenotype.
Neuroblastoma (NB) is a neural crest-derived childhood tumour that has the highest overall rate of spontaneous regression of any malignant neoplasia and is capable of spontaneously differentiating or maturing to a benign ganglioneuroma [ 1,2,7]. The biological mechanisms underlying these changes are unknown. This has, however, stressed the importance of the finding that some NB cell lines will differentiate in vitro under the influence of certain substances. The most commonly used agents have been retinoic acid [22], forscolin [lo], phorbol esters [14], dibutyryl CAMP [25], nerve growth factor [15] and vasoactive intestinal peptide [16]. The availability of such substances encouraged testing to show whether one therapeutic strategy for this tumour might involve induction of differentiation, thereby interfering with tumour growth. Treatment with retinoic acid was shown to be beneficial to some patients with NB u71. Phenylacetate is a naturally occurring plasma component capable of conjugating glutamine to yield phenylacetylglutamine, which is subsequently excreted in the urine [9]. This was the basis for using sodium phenylacetate (NaPA) in the treatment of hyperammonemia [4,11,23]. Moreover, phenylacetate was proposed as a potential antitumour agent [12], considering the unique dependence of tumour cells on circulating glutamine
Keywords: neuroblastoma; differentiation; phenylacetate; retinoic acid; N-myc
Correspondence to: Jindrich Cinatl, Zentrum der Hygiene, Abt. fiir Med. Virologie, Universitltskhnikum, J. W. GoetheUniversitat, Paul-Ehrlich-Str. 40. D-6000 Frankfurt a. M. 70. Germany.
0304-3835/93/%06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
Ltd.
16
[27]. In fact, NaPA inhibited cell proliferation in cell lines established from some solid tumours and affected growth and differentiation of leukemic and immortalized mesenchymal cells in vitro at concentrations that have been achieved in humans with no significant adverse effects [ 191.In the present study, we examined effects of NaPA on growth and differentiation of two human neuroblastoma cell lines. Materials and Methods Cell culture
NB cell line IMR-32 [26] was obtained from American Type Culture Collection (Rockville, MD). NB cell line designated UKF-NB-3 was established from bone marrow metastasis harvested in relapse in one of our patients with Evans stage 4 NB as described previously in preliminary form [6]. All culture media and media supplements were purchased from Seromed (Berlin, Germany). Both NB cell lines were maintained in IMDM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 &ml streptomycin at 37°C in a humidified 5% CO* incubator. The cells were routinely tested for mycoplasma by the HOECHST 33238 staining method [3] and found to be free of contamination.
ment viable cells were counted using a hemocytometer. The viability of the cells was determined by the dye exclusion method after staining with 0.5% trypan blue solution. DNA synthesis was measured by the incorporation of BrdU (5-brom-2’-deoxyuridine) into cellular DNA on day 8 of treatment with NaPA or RA. Incorporated BrdU was detected using the immunoenzymatical alkaline phosphatase-antialkaline phosphatase method. Anti-BrdU monoclonal antibody and other chemicals were obtained as assay kits from Boehringer (Mannheim, Germany). The procedure was performed according to the manufacturer’s instruction. Labelling indices were determined by counting 1000 cells. Assessment of morphological differentiation
NB cells were seeded in 60-mm tissue culture dishes (Nunc) at a density of 3 x lo4 cells in 5 ml culture medium. NaPA or RA were added at different concentrations on the second day after cell seeding. The cells were examined daily with a phase-contrast microscope for assessment of morphological changes. The quantification of differentiated cells was performed on day 8 by scoring 400 random non-clumped cells. The cells were regarded as differentiated if they possessed one or more processes at least twice as long as the soma diameter [20].
Chemical agents
Immunocytochemical investigations
All-trans retinoic acid (RA), phenylacetate, L-(aS,SS)-a-amino-3-chloro-4,5-dihydro-5isoxazolacetic acid (acivicin) and 6-diazo-5-oxo-Lnorleucine acid (DON) were purchased from Sigma (Deisenhofen, Germany). To prepare NaPA solution, phenyl acetate was dissolved in distilled water and brought to pH 7.0 by the addition of NaOH. RA was dissolved in dimethylsulfoxide. Stock solutions were stored at -20°C.
NB cells grown 5 days on glass slides in medium without or with differentiation agents were used for immunoperoxidase staining. Monoclonal antibody directed against N-myc were purchased from Dianova (Hamburg, Germany). Immunocytochemical analysis was performed as described by Hsu et al. [8]. Cell fixation and incubation with primary and secondary antibody was performed on ice.
Assessment of cell proliferation and DNA synthesis
Results
NB cells were seeded at a density of 1.5 x lo4 per 1 ml of culture medium in 35-mm culture dishes (Nunc, Wiesbaden, Germany). NaPA or RA were added at different concentrations on the second day after cell seeding. On day 8 of treat-
Proliferation and DNA synthesis of human NB cell lines UKF-NB-3 and IMR-32 was inhibited by NaPA at concentrations ranging from 2 to 10 mM in a dose dependent manner (Fig. 1). These effects were similar in both NB cell lines. The data
17
UKF-NB-3
NaPA (mM)
00; I
0
2
&PA
(md)
8
UKF-NB-3
T
10
Fig. 1. Effect of NaPA on UKF-NB-3 and IMR-32 cells as measured by cell count (m---m and BrdU incorporation (+-+) after 8 days of treatment. Values represent the mean f S.E.M. of triplicate culture from two independent experiments.
presented in Fig. 1 also suggest that cell division was more sensitive to inhibition than DNA synthesis. The cells of both NB cell lines treated with concentrations ranging from 2 to 6 mM showed cell viability comparable to that of untreated control cultures (over 95%). In contrast, NaPA at a concentration of 8 mM decreased cell viability by about 15% and 12% in UKF-NB-3 and IMR-32 cell lines, respectively. NaPA at a concentration of 10 mM decreased cell viability by 23% and 16% in UKF-NB-3 and IMR-32 cell lines, respectively. Morphological differentiation was observed in both NB cell lines after treatment with non-toxic concentrations of NaPA ranging from 2-6 mM (Fig. 2). UKF-NB-3 cells showed pseudoganglia formation and neurite extension (Fig. 3). The
Effect of NaPA or RA on morphological differentiation of UKF-NB-3 and IMR-32 cells as measured by neurite extension after 8 days of treatment. Values represent the mean f S.E.M. of triplicate culture from two independent experiments. Fig. 2.
number of differentiated cells stimulated in UKFNB-3 cultures by NaPA was similar to that stimulated by RA (Fig. 2). A maximum of 70% of UKFNB-3 cells showed morphological differentiation in cultures treated both with NaPA and RA. Morphological changes stimulated by NaPA in IMR32 cultures were similar to those in UKF-NB-3 cells, i.e. pseudoganglia formation and neurite extension (Fig. 4). The extent of differentiation was lower in IMR-32 than in UKF-NB-3 cells (Fig. 2). In IMR-32 cultures, RA was a more potent differentiation agent than NaPA; a maximum of 35% cells were differentiated with NaPA while 47% cells were differentiated with RA (Fig. 2). When NaPA or RA was removed from the medium (after an 8-day treatment), cultures of either cell line
Fig. 3. Morphology of UKF-NB-3 neuroblastoma cells in the absence and presence of RA or NaPA. Cells H,ere treated for 8 days with solvent control (A), I pM RA (B) or 6 mM NaPA (C). Both NaPA and RA stimulated morphological differentiation crf the cells. Note pseudoganglia formation and extension of cellular processes. Native preparation. phase contrast, magnification 3 00x.
19
resumed normal exponential growth, and the established morphological differentiation disappeared (data not shown). To demonstrate whether morphological differentiation of NB cells may be due to the ability of NaPA to deplete glutamine from culture medium, the cells were grown in glutamine-free medium supplemented with dialyzed FBS or in medium supplemented with glutamine antagonists including acivicin (l-4 pg/ml) or DON (5-25 &ml). The results showed that these culture conditions did not stimulate morphological transformation in NB cells. Immunocytochemical investigations revealed that treatment with NaPA significantly decreased expression of N-myc oncoprotein both in IMR-32 and UKF-NB-3 cells (Fig. 5). In IMR-32 and UKF-NB-3 cell cultures treated with 6 mM NaPA, 25% and 21% cells stained with monoclonal antibody directed against N-myc, respectively. IMR32 and UKF-NB-3 untreated cultures showed 92% and 95% stained cells, respectively. Moreover, cells in treated cultures stained less intensively than those of untreated controls.
Discussion
In the present study we demonstrated that NaPA at non-toxic concentrations promoted morphological differentiation in the human NB cell lines UKF-NB-3 and IMR-32. The differentiated cells of both cell lines showed formation of pseudoganglia and extension of cellular processes. The differentiation of either cell line was reversible after a short course (8 days) of treatment with NaPA or RA. Previous studies dealing with the reversibility of in vitro differentiation of NB cells treated with different agents suggested that the changes are reversible after a short period of treatment [3,16,21]. In contrast, Robson and Side11 showed that in long-term (up to 3 weeks) cultures of LAN-5 in the presence of RA, the differentiation persists and there is no evidence of overgrowth by a population of resistant cells [ 181. It remains to be established whether NaPA may promote irreversible changes in long-term cultures of NB cells. The morphological differentiation of human NB cells in culture has been shown to correlate with
Fig. 4. Morphology of IMR-32 neuroblastoma cells in the absence and presence of RA or NaPA. Cells were treated for 8 days with solvent control (A). I PM RA (B) or 6 mM NaPA (C). Both NaPA and RA stimulated morphological diffc zrentiation of the cells. Note pseudoganglia formation and extension of cellular processes. Native preparation. phase contrast. mag :nification 300 x
21
other indicators of differentiation such as neurotransmitter synthesis, increased activity of acetylcholinesterase and decreased expression of N-myc [ 17,24,25]. We demonstrated decreased expression of N-myc oncoprotein in IMR-32 and UKF-NB-3 cells treated with NaPA. In addition, acetylcholinesterase activity was increased in the treated cells (data not shown). These findings suggest that NaPA does promote the differentiation of NB cells in association with reduced expression of the malignant phenotype. Only recently, NaPA was shown to stimulate differentiation in human leukemic HL-60 cells and in mouse immortalized embryonic mesenchymal C3H 10T l/2 cells [19]. Differentiation-of these cell lines was time dependent and efficient at a concentration of 5- 10 mM. No cytotoxicity was observed in doses twice as high. We observed that NaPA decreased viability at 10 mM concentration by 26% and 15% in UKF-NB-3 and IMR-32 cells, respectively. NaPA at I5 mM concentration decreased cell viability by more than 80% in both NB cell lines (data not shown). Pharmacokinetic studies in children with urea cycle disorders show-
ed that phenylacetate plasma levels of 3-6 mM can be achieved with no significant adverse effects [4,23]. Therefore, it should be tested whether some NaPA concentrations may kill NB cells in animal models without toxic side effects. The mechanism by which NaPA promoted differentiation of UKF-NB-3 and IMR-32 cells is not clear. Phenylacetate was first proposed by Neish as a potential antitumour agent due to its ability to deplete glutamine [ 121. However, glutamine depletion does not seem to account for NaPA activity, because glutamine starvation alone was not sufficient to promote differentiation of NB cells. Similar results were obtained with the glutamine antagonists DON and acivicin. In contrast, glutamine starvation or glutamine antagonists promoted differentiation of human leukemic HL-60 cells [ 13,191. A mere depletion of extracellular glutamine or inhibition of glutamine-utilizing enzymes by DON and acivicin caused a transient maturation of HL-60 cells into monocytes, while the cells treated with NaPA converted primarily into granulocytes. The resuits dealing with downmodulation of N-myc oncoprotein in NB cells
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treated with NaPA suggest that NaPA may influence gene expression. Previously down-regulation of myc gene expression was found in HL-60 cells treated with NaPA [19]. It is of interest, to compare mechanisms by which NaPA influence gene expression in different malignant cells. NaPA has already been established as safe and effective in the treatment of hyperammonemia [4,11,23], and has been proposed as a possible agent for cancer prevention and therapy [ 12,191. A therapeutic strategy for NB involving the induction of differentiation was followed using RA [ 171. However, beneficial effects resulting from RA treatment remained limited to some patients, while no response was achieved in patients with gross measurable disease. The antitumour activities, lack of toxicity, and easy administration (oral) strongly encourage further testing to show whether NaPA may be used as a chemotherapeutic agent in patients with NB.
neuroblastoma cell culture. Suppl.. 116. 9.
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Acknowledgement 14
This research was supported in part by the organization ‘Verein fiir krebskranke Kinder, Frankfurt/M. e. V.‘. We are grateful to Gabriele Steigmann for excellent technical assistance and Alena Cinatlava for the microphotographs.
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References Adam, A. and Hochholzer. L. (1981) Ganglioneuroblastoma of the posterior mediastinum: a clinicopathologic review of 80 cases. Cancer, 47. 373-381. Bolande, R.P. (1985) Spontaneous regression and cytodifferentiation of cancer in early life: the oncogenic grace period. Surv. Synth. Pathol. Res., 4, 296-31 I. Bottenstein, J.E. (1981) Differentiated properties of neuronal cell lines. In: Functionally Differentiated Cell Lines, pp. 155-184. Editor: G.H. Sato. Alan R. Liss, New York. Brusilow, S.W., Danney, M., Waber, L.J., Batshaw, M., Burton, B., Levitski, L., Roth, K.. McKeethren, C. and Ward, J. (1984) Treatment of episodic hyperammonemia in children with inborn errors of urea synthesis. N. Engl. J. Med., 310, 1630-1634. Chen, T.R. (1977) In situ detection of mycoplasma contamination of cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res.. 104. 255-262. Cinatl, J.. Gussetis, E.S.. Cinatl. J. Jr., Ebener, U., Mainke, M., Schwabe, D.. Doerr, H.W., Kornhuber, B. V. (1990) Differentiation arrest in and Gerein,
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Everson, T.C. (1964) Spontaneous regression of cancer. Ann. N.Y. Acad. Sci., 114, 721-735. Hsu, S.M., Raine, L. and Fanger, H. (1981) Use of avidinbiotin-peroxidase complex in immunoperoxidase techniques. J. Histochem. Cytochem.. 29. 577-580. James, M.O., Smith, R.L., Williams, F.R.S. and Reidenberg. M. (1972) The conjugation of phenylacetic acid in man. sub-human primates and some non-primate species. Proc. R. Sot. Lond. B. 182, 23-35. Lando, M., Abemayor, E. and Sidell, N. (1989) In vitro effects of cyclic-AMP-elevating agents and retinoic acid on the growth, differentiation and cyclic APM content of human neuroblastoma cells. Proc. Am. Assoc. Cancer Res.. 30. 42. Mendenhall, C.L., Rouster, S.. Marshall. L. and Weesner, R.A. (1986) A new therapy for portal systemic encephalopathy. Am. J. Gastroenterol., 81. 540-543. Neish. W.J.P. (1971) Phenylacetic acid as a potential therapeutic agent for the treatment of human cancer. Experientia (Basel), 27, 860-661. Nichols, K.E. and Weinberg, J.B. (1989) Essential amino acid deprivation induces monocytic differentiation of the human HL-60 myeloid leukemia cell line. Blood, 73. 1298- 1306. Pahlman. S., Ruusala, A.1.. Abrahamsson. L., Mattsson, M.E.K. and Esscherr, T. (1984) Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbol ester-induced differentiation. Cell Differ., 14. 135-144. Pavelic, K. and Spaventi, S. (1987) Nerve growth factor induced differentiation of human neuroblastoma cells. Int. J. Biochem.. 19. 1237-1240. Pence, J.C. and Shorter, N.A. (1990) In vitro differentiation of human neuroblastoma cells caused by vasoactive intestinal peptide. Cancer Res., 30, 5177-5183. Reynolds, C., Kane, D. and Einhorn. P. (1991) Response of neuroblastoma to retinoic acid in vitro and in vivo. In: Advances in Neuroblastoma Research, pp. 203-211. Editors: A.E. Evans, G.J. D’Augro and R.C. Seeger. Alan R. Liss, New York. Robson, J.A. and Sidell. N. (1985) Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid. Neuroscience, 14. 1 149- 1162. Samid. D.. Shack, S. and Sherman. L.T. (1992) Phenylacetate: a novel nontoxic inducer of tumor cell differentiation Cancer Res., 52. 1988-1992. Sandquist, D., Williams. T.H., Sahu, S.K. and Kataoka S. (1978) Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone. Exp. Cell Res.. 1113. 375-381. Sidell. N. (1982) Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. J. Natl. Cancer Inst.. 68. 589-593. Sidell. N.. Altman. A.. Haussler. M. and Seeger. R.C. (1983) Effects of retinoic acid on the growth and
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phenotypic expression of several human neuroblastoma lines. Exp. Cell Res., 148, 21-30. Simell, O., Sipila, I., Rajantie, J.. Valle, D.L. and Brusilow. S.W. (1986) Waste nitrogen excretion via amino acid acylation: benzoate and phenylacetate in lysinuric protein intolerance. Pediatr. Res., 20, I 117-l 121. Thiele, C.J., Reynolds. C.P. and Israel, M.A. (1985) Decreased expression of N-myc precedes retinoic acidinduced morphological differentiation of human neuroblastoma. Nature, 313, 404-406.
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Tsokos, M., Scarpa, S., Ross, R.A. and Triche, T. (1987) Differentiation of human neuroblastoma recapitulates neural crest development. Am. J. Pathol.. 128, 484-496. Tumilowicz, J.J., Nichols, W.W., Cholon, J.J. and Green A.E. (1970) Definition of a continuous human cell line derived from neuroblastoma. Cancer Res., 30. 21 IO-21 18. Weber. G. (1983) Biochemical strategy of cancer cells and the design of chemotherapy: G.H.A. Clowes Memorial Lecture. Cancer Res.. 43, 3466-3499.
15 Ltd.
In vitro differentiation sodium phenylacetate
of human neuroblastoma
cells induced by
Jindrich Cinatl”, Jaroslav CinatlaVb, Marion Mainke b, Albrecht Weiflflog b Holger Rabenaua, Bernhard Kornhuberb and Hans-Wilhelm Doerra “Centre of Hygiene, Department of Medical Virology. bCentre of Pediatrics, Department of Haematology and Oncology, J. W. GoetheUniversity, Frankfurt a. M. (Germany) (Received (Accepted
11 January 3 February
1993) 1993)
Summary
Introduction
Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 6 mM stimulated morphological differentiation of two human neuroblastoma cell lines IMR-32 and UKF-NB-3. These concentrations inhibited growth and DNA synthesis of the cells in a dose dependent manner without significant effect on cell viability. The differentiated cells showed pseudoganglia formation and extension of cellular processes. The morphological differentiation in both cell lines was accompanied by decreased expression of N-myc oncoprotein. These results suggest that NaPA at concentrations, which have been achieved in humans with no significant adverse effects, promotes differentiation of cultured human neuroblastoma cells in association with the reduced expression of the malignant phenotype.
Neuroblastoma (NB) is a neural crest-derived childhood tumour that has the highest overall rate of spontaneous regression of any malignant neoplasia and is capable of spontaneously differentiating or maturing to a benign ganglioneuroma [ 1,2,7]. The biological mechanisms underlying these changes are unknown. This has, however, stressed the importance of the finding that some NB cell lines will differentiate in vitro under the influence of certain substances. The most commonly used agents have been retinoic acid [22], forscolin [lo], phorbol esters [14], dibutyryl CAMP [25], nerve growth factor [15] and vasoactive intestinal peptide [16]. The availability of such substances encouraged testing to show whether one therapeutic strategy for this tumour might involve induction of differentiation, thereby interfering with tumour growth. Treatment with retinoic acid was shown to be beneficial to some patients with NB u71. Phenylacetate is a naturally occurring plasma component capable of conjugating glutamine to yield phenylacetylglutamine, which is subsequently excreted in the urine [9]. This was the basis for using sodium phenylacetate (NaPA) in the treatment of hyperammonemia [4,11,23]. Moreover, phenylacetate was proposed as a potential antitumour agent [12], considering the unique dependence of tumour cells on circulating glutamine
Keywords: neuroblastoma; differentiation; phenylacetate; retinoic acid; N-myc
Correspondence to: Jindrich Cinatl, Zentrum der Hygiene, Abt. fiir Med. Virologie, Universitltskhnikum, J. W. GoetheUniversitat, Paul-Ehrlich-Str. 40. D-6000 Frankfurt a. M. 70. Germany.
0304-3835/93/%06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
Ltd.
16
[27]. In fact, NaPA inhibited cell proliferation in cell lines established from some solid tumours and affected growth and differentiation of leukemic and immortalized mesenchymal cells in vitro at concentrations that have been achieved in humans with no significant adverse effects [ 191.In the present study, we examined effects of NaPA on growth and differentiation of two human neuroblastoma cell lines. Materials and Methods Cell culture
NB cell line IMR-32 [26] was obtained from American Type Culture Collection (Rockville, MD). NB cell line designated UKF-NB-3 was established from bone marrow metastasis harvested in relapse in one of our patients with Evans stage 4 NB as described previously in preliminary form [6]. All culture media and media supplements were purchased from Seromed (Berlin, Germany). Both NB cell lines were maintained in IMDM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 &ml streptomycin at 37°C in a humidified 5% CO* incubator. The cells were routinely tested for mycoplasma by the HOECHST 33238 staining method [3] and found to be free of contamination.
ment viable cells were counted using a hemocytometer. The viability of the cells was determined by the dye exclusion method after staining with 0.5% trypan blue solution. DNA synthesis was measured by the incorporation of BrdU (5-brom-2’-deoxyuridine) into cellular DNA on day 8 of treatment with NaPA or RA. Incorporated BrdU was detected using the immunoenzymatical alkaline phosphatase-antialkaline phosphatase method. Anti-BrdU monoclonal antibody and other chemicals were obtained as assay kits from Boehringer (Mannheim, Germany). The procedure was performed according to the manufacturer’s instruction. Labelling indices were determined by counting 1000 cells. Assessment of morphological differentiation
NB cells were seeded in 60-mm tissue culture dishes (Nunc) at a density of 3 x lo4 cells in 5 ml culture medium. NaPA or RA were added at different concentrations on the second day after cell seeding. The cells were examined daily with a phase-contrast microscope for assessment of morphological changes. The quantification of differentiated cells was performed on day 8 by scoring 400 random non-clumped cells. The cells were regarded as differentiated if they possessed one or more processes at least twice as long as the soma diameter [20].
Chemical agents
Immunocytochemical investigations
All-trans retinoic acid (RA), phenylacetate, L-(aS,SS)-a-amino-3-chloro-4,5-dihydro-5isoxazolacetic acid (acivicin) and 6-diazo-5-oxo-Lnorleucine acid (DON) were purchased from Sigma (Deisenhofen, Germany). To prepare NaPA solution, phenyl acetate was dissolved in distilled water and brought to pH 7.0 by the addition of NaOH. RA was dissolved in dimethylsulfoxide. Stock solutions were stored at -20°C.
NB cells grown 5 days on glass slides in medium without or with differentiation agents were used for immunoperoxidase staining. Monoclonal antibody directed against N-myc were purchased from Dianova (Hamburg, Germany). Immunocytochemical analysis was performed as described by Hsu et al. [8]. Cell fixation and incubation with primary and secondary antibody was performed on ice.
Assessment of cell proliferation and DNA synthesis
Results
NB cells were seeded at a density of 1.5 x lo4 per 1 ml of culture medium in 35-mm culture dishes (Nunc, Wiesbaden, Germany). NaPA or RA were added at different concentrations on the second day after cell seeding. On day 8 of treat-
Proliferation and DNA synthesis of human NB cell lines UKF-NB-3 and IMR-32 was inhibited by NaPA at concentrations ranging from 2 to 10 mM in a dose dependent manner (Fig. 1). These effects were similar in both NB cell lines. The data
17
UKF-NB-3
NaPA (mM)
00; I
0
2
&PA
(md)
8
UKF-NB-3
T
10
Fig. 1. Effect of NaPA on UKF-NB-3 and IMR-32 cells as measured by cell count (m---m and BrdU incorporation (+-+) after 8 days of treatment. Values represent the mean f S.E.M. of triplicate culture from two independent experiments.
presented in Fig. 1 also suggest that cell division was more sensitive to inhibition than DNA synthesis. The cells of both NB cell lines treated with concentrations ranging from 2 to 6 mM showed cell viability comparable to that of untreated control cultures (over 95%). In contrast, NaPA at a concentration of 8 mM decreased cell viability by about 15% and 12% in UKF-NB-3 and IMR-32 cell lines, respectively. NaPA at a concentration of 10 mM decreased cell viability by 23% and 16% in UKF-NB-3 and IMR-32 cell lines, respectively. Morphological differentiation was observed in both NB cell lines after treatment with non-toxic concentrations of NaPA ranging from 2-6 mM (Fig. 2). UKF-NB-3 cells showed pseudoganglia formation and neurite extension (Fig. 3). The
Effect of NaPA or RA on morphological differentiation of UKF-NB-3 and IMR-32 cells as measured by neurite extension after 8 days of treatment. Values represent the mean f S.E.M. of triplicate culture from two independent experiments. Fig. 2.
number of differentiated cells stimulated in UKFNB-3 cultures by NaPA was similar to that stimulated by RA (Fig. 2). A maximum of 70% of UKFNB-3 cells showed morphological differentiation in cultures treated both with NaPA and RA. Morphological changes stimulated by NaPA in IMR32 cultures were similar to those in UKF-NB-3 cells, i.e. pseudoganglia formation and neurite extension (Fig. 4). The extent of differentiation was lower in IMR-32 than in UKF-NB-3 cells (Fig. 2). In IMR-32 cultures, RA was a more potent differentiation agent than NaPA; a maximum of 35% cells were differentiated with NaPA while 47% cells were differentiated with RA (Fig. 2). When NaPA or RA was removed from the medium (after an 8-day treatment), cultures of either cell line
Fig. 3. Morphology of UKF-NB-3 neuroblastoma cells in the absence and presence of RA or NaPA. Cells H,ere treated for 8 days with solvent control (A), I pM RA (B) or 6 mM NaPA (C). Both NaPA and RA stimulated morphological differentiation crf the cells. Note pseudoganglia formation and extension of cellular processes. Native preparation. phase contrast, magnification 3 00x.
19
resumed normal exponential growth, and the established morphological differentiation disappeared (data not shown). To demonstrate whether morphological differentiation of NB cells may be due to the ability of NaPA to deplete glutamine from culture medium, the cells were grown in glutamine-free medium supplemented with dialyzed FBS or in medium supplemented with glutamine antagonists including acivicin (l-4 pg/ml) or DON (5-25 &ml). The results showed that these culture conditions did not stimulate morphological transformation in NB cells. Immunocytochemical investigations revealed that treatment with NaPA significantly decreased expression of N-myc oncoprotein both in IMR-32 and UKF-NB-3 cells (Fig. 5). In IMR-32 and UKF-NB-3 cell cultures treated with 6 mM NaPA, 25% and 21% cells stained with monoclonal antibody directed against N-myc, respectively. IMR32 and UKF-NB-3 untreated cultures showed 92% and 95% stained cells, respectively. Moreover, cells in treated cultures stained less intensively than those of untreated controls.
Discussion
In the present study we demonstrated that NaPA at non-toxic concentrations promoted morphological differentiation in the human NB cell lines UKF-NB-3 and IMR-32. The differentiated cells of both cell lines showed formation of pseudoganglia and extension of cellular processes. The differentiation of either cell line was reversible after a short course (8 days) of treatment with NaPA or RA. Previous studies dealing with the reversibility of in vitro differentiation of NB cells treated with different agents suggested that the changes are reversible after a short period of treatment [3,16,21]. In contrast, Robson and Side11 showed that in long-term (up to 3 weeks) cultures of LAN-5 in the presence of RA, the differentiation persists and there is no evidence of overgrowth by a population of resistant cells [ 181. It remains to be established whether NaPA may promote irreversible changes in long-term cultures of NB cells. The morphological differentiation of human NB cells in culture has been shown to correlate with
Fig. 4. Morphology of IMR-32 neuroblastoma cells in the absence and presence of RA or NaPA. Cells were treated for 8 days with solvent control (A). I PM RA (B) or 6 mM NaPA (C). Both NaPA and RA stimulated morphological diffc zrentiation of the cells. Note pseudoganglia formation and extension of cellular processes. Native preparation. phase contrast. mag :nification 300 x
21
other indicators of differentiation such as neurotransmitter synthesis, increased activity of acetylcholinesterase and decreased expression of N-myc [ 17,24,25]. We demonstrated decreased expression of N-myc oncoprotein in IMR-32 and UKF-NB-3 cells treated with NaPA. In addition, acetylcholinesterase activity was increased in the treated cells (data not shown). These findings suggest that NaPA does promote the differentiation of NB cells in association with reduced expression of the malignant phenotype. Only recently, NaPA was shown to stimulate differentiation in human leukemic HL-60 cells and in mouse immortalized embryonic mesenchymal C3H 10T l/2 cells [19]. Differentiation-of these cell lines was time dependent and efficient at a concentration of 5- 10 mM. No cytotoxicity was observed in doses twice as high. We observed that NaPA decreased viability at 10 mM concentration by 26% and 15% in UKF-NB-3 and IMR-32 cells, respectively. NaPA at I5 mM concentration decreased cell viability by more than 80% in both NB cell lines (data not shown). Pharmacokinetic studies in children with urea cycle disorders show-
ed that phenylacetate plasma levels of 3-6 mM can be achieved with no significant adverse effects [4,23]. Therefore, it should be tested whether some NaPA concentrations may kill NB cells in animal models without toxic side effects. The mechanism by which NaPA promoted differentiation of UKF-NB-3 and IMR-32 cells is not clear. Phenylacetate was first proposed by Neish as a potential antitumour agent due to its ability to deplete glutamine [ 121. However, glutamine depletion does not seem to account for NaPA activity, because glutamine starvation alone was not sufficient to promote differentiation of NB cells. Similar results were obtained with the glutamine antagonists DON and acivicin. In contrast, glutamine starvation or glutamine antagonists promoted differentiation of human leukemic HL-60 cells [ 13,191. A mere depletion of extracellular glutamine or inhibition of glutamine-utilizing enzymes by DON and acivicin caused a transient maturation of HL-60 cells into monocytes, while the cells treated with NaPA converted primarily into granulocytes. The resuits dealing with downmodulation of N-myc oncoprotein in NB cells
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treated with NaPA suggest that NaPA may influence gene expression. Previously down-regulation of myc gene expression was found in HL-60 cells treated with NaPA [19]. It is of interest, to compare mechanisms by which NaPA influence gene expression in different malignant cells. NaPA has already been established as safe and effective in the treatment of hyperammonemia [4,11,23], and has been proposed as a possible agent for cancer prevention and therapy [ 12,191. A therapeutic strategy for NB involving the induction of differentiation was followed using RA [ 171. However, beneficial effects resulting from RA treatment remained limited to some patients, while no response was achieved in patients with gross measurable disease. The antitumour activities, lack of toxicity, and easy administration (oral) strongly encourage further testing to show whether NaPA may be used as a chemotherapeutic agent in patients with NB.
neuroblastoma cell culture. Suppl.. 116. 9.
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Acknowledgement 14
This research was supported in part by the organization ‘Verein fiir krebskranke Kinder, Frankfurt/M. e. V.‘. We are grateful to Gabriele Steigmann for excellent technical assistance and Alena Cinatlava for the microphotographs.
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