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Establishment and characterization of a human neuroectodermal cell line (TB) from a cerebrospinal fluid specimen
Brain Research 827 Ž1999. 205–209
Short communication
Establishment and characterization of a human neuroectodermal cell line ŽTB . from a cerebrospinal fluid specimen Giuseppe Sorrentino a, ) , Maria R. Monsurro` a , Guido Pettinato b , Roberta Vanni c , Alessandro Zuddas d , Umberto Di Porzio e , Vincenzo Bonavita a a
Institute of Neurological Sciences, Faculty of Medicine, 2nd UniÕersity of Naples, Via S. Pansini 5, 80131 Naples, Italy b Institute of Pathology, UniÕersity ‘Federico II’, Naples, Italy c Institute of Biology, UniÕersity of Cagliari, Cagliari, Italy d Department of Neuroscience, UniÕersity of Cagliari, Cagliari, Italy e International Institute of Genetics and Biophysics, CNR, Naples, Italy Accepted 26 January 1999
Abstract We have established a cell line ŽTB. from a cerebrospinal fluid ŽCSF. specimen of a patient with a primary leptomeningeal melanomatosis. TB cell line was immunoreactive with the antibodies for low molecular weight neurofilament protein, vimentin, neuron-specific enolase, chromogranin, synaptophysin and HMB-45 Žan antibody sensitive and specific for melanoma.. When TB cells were transplanted into nude mice, the same immunohistochemical pattern present in cultured cells was found but surprisingly, a positive staining for desmin was observed. Significant amounts of serotonin and its metabolite were detectable. Retinoic acid but not nerve growth factor was able to induce differentiation towards a neuronal phenotype. In summary, TB cells represent primitive neuroectodermal cells having the potential for neuronal, myoblastic and possibly melanoblastic differentiation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Leptomeningeal melanoma; Neural crest; Neuroendocrine differentiation; Neurofilament; Retinoic acid
The neural crest appearing transiently during vertebrate embryogenesis is a mass of the primordium that gives rise to a variety of cell types such as neurocytic elements, supportive tissues, neuroendocrine cells and melanoblasts w8x. Faulty development of these cell types may result in formation of neural crest tumors w20x. The leptomeninges, together with cranial vasculature, dermal connective tissue, and uveal tract are all derivatives of the neural crest and can contain melanocytes w16x. Because of their common embryological origin, these tissues are frequently the sites of primary melanomas after the malignant transformation of melanocytes. We report the establishment of a novel human cell line, designated as TB cell line, derived from a cerebrospinal fluid ŽCSF. specimen of a patient with clinical diagnosis of primary leptomeningeal melanomatosis. TB cell line expresses features clearly indicating its neuroectodermal origin, notably from the neural crest.
)
Corresponding [email protected]
author.
Fax:
q 39-081-5666787;
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A 5-ml CSF specimen from a patient with primary leptomeningeal melanomatosis was cultured according to Sorrentino et al. w22x. After 4 weeks, a highly uniform population of growing cells was observed Ždoubling time s 18 " 2 h.. The analyses that are presented were carried out on cells ranging from passages 8 to 15 when not otherwise specified. Cell differentiation was obtained by using 10 mM all trans-retinoic acid ŽRA; Sigma. or 100 ngrml nerve growth factor ŽNGF; a gift from Dr. Mercanti. that was added to the culture medium 24 h after plating Ž7.5 = 10 4 cellsrcm2 .. The immunological and ultrastructural characterization was carried out on both TB cell line grown for 4 days in vitro Ždiv. and xenograft tissue obtained from mice injected with the established cells Ž10 6 . as described by Fiszman et al. w4x and Pietsch et al. w18x, respectively. The following monoclonal antibodies were used: glial fibrillary acid protein ŽGFAP; diluted 1:300., neuron specific enolase ŽNSE; 1:400., synaptophysin Ž1:50., chromogranin Ž1:300., S-100 protein Ž1:500., HMB-45 Ž1:100., cytocheratin Ž1:200., carcino-embryonic antigen ŽCEA; 1:500., ep-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 7 - X
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G. Sorrentino et al.r Brain Research 827 (1999) 205–209
ithelial membrane antigen ŽEMA; 1:100., leukocyte common antigen ŽLCA; 1:200., vimentin Ž1:50., desmin Ž1:200., factor VIIIrvon Willenbrand-related antigen ŽF VIIIrvW-RA; 1:100., all obtained from Dakopatts, Denmark. The antibody to the low molecular weight neurofilament protein ŽNF-L. was a purified mouse antibody Ždiluted 1:400. and the antibodies to the middle and high molecular weight neurofilaments ŽNF-M, NF-H. were polyclonal rabbit antisera Ždiluted 1:50 and 1:250, respectively.; NF-L and NF-H were obtained from Sigma, NF-M from Affiniti ŽNottingham, UK.. The antibody binding sites were detected and visualized according to Hsu et al. w7x. Secondary antibodies alone were used as control. Western blot analysis on 4 and 7 div control and RA or NGF differentiated cells was performed according to Pernas Alonso et al. w17x. A mix of monoclonal antibody anti NF-L ŽSigma. Ždiluted 1:50. and polyclonal antisera anti NF-M and NF-H ŽAffiniti. Ždiluted 1:2000. was used. The same filters were further incubated with antiserum anti NSE diluted 1:2000 obtained from Genosys ŽCambridge, UK. and visualized as above. Choline acetyltranferase ŽChAT. and tyrosinase activities were measured according to the procedures of Fonnum w5x and Pomerantz w19x, respectively. Monoamine determination was performed as previously described w26x. Cytogenetic analysis was performed on the established cell line at passages 15, 18 and 67 as described w25x. Chromosome analysis followed the ISCN recommendation w9x. Statistical comparisons were made applying analysis of variance ŽANOVA. followed by Scheffe’s F-test. The results of the immunocytochemical analysis are summarized in Table 1. As shown on the CSF specimen, the cell line expressed vimentin ŽFig. 1A., but not cytocheratin and GFAP. NF-L was the only neurofilament to be present ŽFig. 1B.. Neuroendocrine cell markers like NSE, chromogranin and synaptophysin were detected ŽFig. 1C,D,E.. Furthermore, TB cell line showed immunoreactivity for HMB-45, a monoclonal antibody which was reported to be highly sensitive and specific for melanoma w6,24x ŽFig. 1F.. S-100 protein, which was detected on CSF cells, was not present in cultured TB cell. The lack of CEA, EMA, LCA, F-VIII staining ruled out the epithelial, haematopoietic and endothelial derivation. To confirm the presence of NF-L in the TB cell line, a Western blot analysis on cell extracts was performed. In order to establish if the presence of NGF or RA was able to induce differentiation towards a neuronal phenotype, the presence of NFs in TB cells differentiated for 4 and 7 days was examined. The presence of NF-L, -M and -H in the different growth conditions is shown in Fig. 2. The NF-L was present in each condition. The NF-M was clearly evident only in the RA treated cells but not in control and NGF treated cells. The NF-H was only present in the cells grown for 7 days in the presence of RA, whereas the control and 7 div NGF treated cells did not contain NF-H.
Table 1 Comparison of immunohistochemical results on original cells from CSF, TB cell line and transplant CSF
TB cells
Xenograft
Neuronal and glial markers Neurofilament L n.d. Neurofilament M n.d. Neurofilament H n.d. GFAP y
q y y y
q y y y
Neuroendocrine cell markers NSE n.d. Chromogranin n.d. Synaptophysin n.d.
q q q
q q q
Melanotic cell markers S-100 protein HMB-45
q n.d.
y q
y y
Epithelial cell markers Cytocheratin CEA EMA
y n.d. n.d.
y y y
y y y
Haematopoietic cell marker LCA Žcd-45. y
y
y
Endothelial cell markers F VIIIrvW-RA n.d.
y
y
Muscle cell markers Desmin
y
q
q
q
n.d.
Mesenchimal cell markers Vimentin q
y: Negative staining; q: positive staining; n.d.: not determined. GFAP: glial fibrillary acidic protein; NSE: neuron specific enolase; CEA: carcino-embryonic antigen; EMA: epithelial membrane antigen; LCA: leukocyte common antigen; F VIIIrvW-RA: factor VIIIrvon Willenbrand-related antigen.
Furthermore, the treatment with RA for 7 days increased the presence of NF-L. NSE did not change in any condition. When examined by transmission electron microscopy, TB cells showed few features of differentiated cells. Furthermore, typical dense-core neurosecretory granules were found Ždata not shown.. The results of the immunohistochemical ŽTable 1. and ultrastructural study on the xenograft transplant were highly consistent with those obtained with the cell line. Surprisingly, the xenograft sections showed a scattered staining for desmin Ždata not shown.. Tyrosinase activity was barely detectable, whereas no ChAT activity was found. After 1 div, significant amounts of serotonin Ž5HT. and its metabolite, 5-hydroxy indoleacetic acid Ž5HIAA., were detected in the cell homogenate Ž10.8 " 1.3 and 12.0 " 0.6 ngrmg protein, respectively.. Indolamine content decreased with time. At 4 div, it was 3.8 " 0.7 and 5.1 " 0.3 ngrmg of protein for serotonin and 5HIAA, respectively. No indolamines were
G. Sorrentino et al.r Brain Research 827 (1999) 205–209
207
Fig. 1. Indirect immunostaining of TB cells. TB cells, grown on glass coverslips, were fixed and subjected to immunostaining by using antibodies specific for vimentin ŽA., low molecular weight neurofilament ŽB., neuron specific enolase ŽC., chromogranin ŽD., synaptophysin ŽE., HMB-45 ŽF.. Color X development was with 3, 3 -diaminobenzidine Ž=400..
detectable after 10 div. Dopamine, norepinephrine or their metabolites were not found. At 1 div, pargyline increased serotonin content to 14.8 " 0.7 ngrmg protein Ž37.0% of control. and decreased 5HIAA to 6.9 " 0.3 Ž57.5% of control., indicating that the 5HT peak is indeed serotonin. Retinoic acid did not change the serotonin and 5HIAA content. The chromosome analysis showed very complex karyotypes with modal chromosome number basically in the hypotetraploid range Ž74–78 in passage 15 and 18, 78–83 in passage 67. and with a number of structurally rearranged chromosomes, some of which were clonally present. The origin of few clonal chromosome markers was established: addŽ1.Žp32.; tŽ2;5.Žp23;q11.2.; iŽ5.Žp10.;
addŽ9.Žp24.; derŽ14.tŽ14;15.Žq32;p11. in passage 15, addŽ1.Žp32.; tŽ14;22.Žq10q10. in passage 18, addŽ1.Žp32.; delŽ3.Žq21.; addŽ6.Žp25. in passage 67. Of the three developmentally regulated neurofilaments, TB cells normally expressed only NF-L, suggesting that they represent an early stage of neural differentiation. Furthermore, TB cell line expressed vimentin, the first intermediate filament ŽIF. to appear in embryological development, regardless of cell type, that is later replaced by the IF protein specific for each particular cell type. In some cases, vimentin coexists with NF proteins, namely in developing neuronal cells as well as in some neoplastic cells w1x. Several markers indicative of neuroendocrine differentiation such as NSE, chromogranin and synapto-
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G. Sorrentino et al.r Brain Research 827 (1999) 205–209
Fig. 2. Western blot of TB cell line for the three neurofilaments. Lane 2 through 4 are extracts from 4 div cells; lane 5 through 7 are extracts from 7 div cells. Lane 1 molecular weight markers in kilodaltons; lane 2 and 5 control; lane 3 and 6 NGF treated cells; lane 4 and 7 RA treated cells; lane 8 rat brain extract.
physin were clearly present in TB cells supporting a neuroendocrine origin of this cell line w13x. Further evidence of the neuroendocrine nature of the cell line was provided by the presence of many dense-core granules both in vitro and in xenografts. Serotonin and its metabolite 5HIAA accumulated in TB cell line. Serotonin is expressed by normal, dysplastic and neoplastic neuroendocrine cell population w3x. Furthermore, an in vitro study using quail neural crest cells showed that these cells can be serotonin-immunoreactive w21x. A quite unexpected finding was the presence of staining for desmin in the xenograft transplant. Desmin, the muscle-cell type of intermediate filament protein, has a restricted distribution in striated and most smooth muscle cells. It has been suggested that neoplastic primitive neuroectodermal cells have intrinsic rhabdomyoblastic potential, corresponding to the normal mesectodermal differentiation of neural crest tissues into striated muscle in head and neck region w2x. It is possible that a set of human neural crest cells migrates to soft tissue and differentiates into other mesenchymal cells. In this respect, various heterotopic grafting experiments suggest that neural crest cells differentiate along one or another phenotypic pathway in response to local environmental stimuli w15x. Recent studies have shown that RA plays a major role during neuronal differentiation. A potential source of RA and the mechanisms whereby cells can respond to RA are both present during CNS development w11,12,23x. In TB cell line, the NF-L, -M and -H are upregulated by RA treatment in a temporal pattern resembling that in the developing brain. Among the identified structurally rearranged chromosomes, the derŽ1. was present in all the examined pas-
sages. Rearrangement of the 1p32 band is commonly found in a number of tumors, however, it is specifically associated to neuroblastoma and is very frequently observed in melanoma w14x. Although no conclusion can be drawn, it should be noted that these results support the hypothesis of a tumor of neural crest origin. A series of studies have shown that the neural crest is a heterogeneous population of pluripotent cells, partially restricted cells, and cells committed to a particular cell lineage w10x. The availability of a cell line that is committed to differentiate in response to specific stimuli would enable detailed studies of the molecular basis of the complex process, whereby cells acquire the mature phenotype. TB cell line can thus provide a useful tool to analyze differentiation and dedifferentiation of neural crest derivatives as well as their tumorigenicity.
Acknowledgements We would like to thank Dr. D. Mercanti, Institute of Neurobiology, CNR, Rome, for his generous gift of NGF. This work was supported by grants from Consiglio Nazionale delle Ricerche ŽGS., North Atlantic Treaty Organization ŽGS. and Associazione Italiana per la Ricerca contro il Cancro ŽUDP..
References w1x A. Bignami, T. Raju, D. Dahl, Localization of vimentin, the nonspecific intermediate filament protein, in embryonal glia and in early differentiating neurons, Dev. Biol. 91 Ž1982. 286–295. w2x D.W. Dickson, M.N. Hart, A. Menezes, P.A. Cancilla, Medulloblas-
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w16x R. O’Rahilly, F. Muller, The meninges in human development, J. ¨ Neuropathol. Exp. Neurol. 45 Ž1986. 588–608. w17x R. Pernas Alonso, A.E. Schaffner, C. Perrone Capano, A. Orlando, F. Morelli, C.T. Hansen, J.L. Barker, B. Esposito, F. Cacucci, U. Di Porzio, Early upregulation of medium neurofilament gene expression in developing spinal cord of the wobbler mouse mutant, Brain Res. Mol. Brain Res. 38 Ž1996. 267–275. w18x T. Pietsch, T. Scharmann, C. Fonatsch, D. Schmidt, R. Ockler, D. Freihoff, S. Albrecht, O.W. Wiestler, P. Zeltzer, H. Riehm, Characterization of five new cell lines derived from human primitive neuroectodermal tumors of the central nervous system, Cancer Res. 54 Ž1994. 3278–3287. w19x S.H. Pomerantz, Tyrosine hydroxylation catalyzed by mammalian tyrosinase: an improved method of assay, Biochem. Biophy. Res. Commun. 16 Ž1964. 188–194. w20x R.N. Schimke, Tumors of the neural crest system, in: J.J. Mulvihill, et al. ŽEds.., Genetics of Human Cancer, Raven Press, New York, 1977, pp. 179–198. w21x M. Sieber-Blum, W. Reed, H.G. Lidov, Serotoninergic differentiation of quail neural crest cells in vitro, Dev. Biol. 99 Ž1983. 352–359. w22x G. Sorrentino, I.N. Singh, R. Massarelli, J.N. Kanfer, Stimulation of phospholipase C activity by norepinephrine, t-ACPD and bombesin in LA-N-2 cells, Eur. J. Pharmacol. 308 Ž1996. 81–86. w23x M. Wagner, C. Thaller, T. Jessell, G. Eichele, Polarizing activity and retinoid synthesis in the floor plate of the neural tube, Nature 345 Ž1990. 819–822. w24x M.R. Wick, P.E. Swanson, A. Rocomora, Recognition of malignant melanoma by monoclonal antibody HMB-45. An immunohistochemical study of 200 paraffin-embedded cutaneous tumors, J. Cutan. Pathol. 15 Ž1988. 201–207. w25x J.J. Yunis, New chromosome techniques in the study of human neoplasia, Hum. Pathol. 5 Ž1981. 540–550. w26x A. Zuddas, G.U. Corsini, S. Schinelli, J.N. Johannessen, U. Di Porzio, J. Kopin, MPTP treatment combined with ethanol or acetaldehyde selectively destroyed dopaminergic neuron in mouse substantia nigra, Brain Res. 501 Ž1989. 1–10.
Short communication
Establishment and characterization of a human neuroectodermal cell line ŽTB . from a cerebrospinal fluid specimen Giuseppe Sorrentino a, ) , Maria R. Monsurro` a , Guido Pettinato b , Roberta Vanni c , Alessandro Zuddas d , Umberto Di Porzio e , Vincenzo Bonavita a a
Institute of Neurological Sciences, Faculty of Medicine, 2nd UniÕersity of Naples, Via S. Pansini 5, 80131 Naples, Italy b Institute of Pathology, UniÕersity ‘Federico II’, Naples, Italy c Institute of Biology, UniÕersity of Cagliari, Cagliari, Italy d Department of Neuroscience, UniÕersity of Cagliari, Cagliari, Italy e International Institute of Genetics and Biophysics, CNR, Naples, Italy Accepted 26 January 1999
Abstract We have established a cell line ŽTB. from a cerebrospinal fluid ŽCSF. specimen of a patient with a primary leptomeningeal melanomatosis. TB cell line was immunoreactive with the antibodies for low molecular weight neurofilament protein, vimentin, neuron-specific enolase, chromogranin, synaptophysin and HMB-45 Žan antibody sensitive and specific for melanoma.. When TB cells were transplanted into nude mice, the same immunohistochemical pattern present in cultured cells was found but surprisingly, a positive staining for desmin was observed. Significant amounts of serotonin and its metabolite were detectable. Retinoic acid but not nerve growth factor was able to induce differentiation towards a neuronal phenotype. In summary, TB cells represent primitive neuroectodermal cells having the potential for neuronal, myoblastic and possibly melanoblastic differentiation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Leptomeningeal melanoma; Neural crest; Neuroendocrine differentiation; Neurofilament; Retinoic acid
The neural crest appearing transiently during vertebrate embryogenesis is a mass of the primordium that gives rise to a variety of cell types such as neurocytic elements, supportive tissues, neuroendocrine cells and melanoblasts w8x. Faulty development of these cell types may result in formation of neural crest tumors w20x. The leptomeninges, together with cranial vasculature, dermal connective tissue, and uveal tract are all derivatives of the neural crest and can contain melanocytes w16x. Because of their common embryological origin, these tissues are frequently the sites of primary melanomas after the malignant transformation of melanocytes. We report the establishment of a novel human cell line, designated as TB cell line, derived from a cerebrospinal fluid ŽCSF. specimen of a patient with clinical diagnosis of primary leptomeningeal melanomatosis. TB cell line expresses features clearly indicating its neuroectodermal origin, notably from the neural crest.
)
Corresponding [email protected]
author.
Fax:
q 39-081-5666787;
E-mail:
A 5-ml CSF specimen from a patient with primary leptomeningeal melanomatosis was cultured according to Sorrentino et al. w22x. After 4 weeks, a highly uniform population of growing cells was observed Ždoubling time s 18 " 2 h.. The analyses that are presented were carried out on cells ranging from passages 8 to 15 when not otherwise specified. Cell differentiation was obtained by using 10 mM all trans-retinoic acid ŽRA; Sigma. or 100 ngrml nerve growth factor ŽNGF; a gift from Dr. Mercanti. that was added to the culture medium 24 h after plating Ž7.5 = 10 4 cellsrcm2 .. The immunological and ultrastructural characterization was carried out on both TB cell line grown for 4 days in vitro Ždiv. and xenograft tissue obtained from mice injected with the established cells Ž10 6 . as described by Fiszman et al. w4x and Pietsch et al. w18x, respectively. The following monoclonal antibodies were used: glial fibrillary acid protein ŽGFAP; diluted 1:300., neuron specific enolase ŽNSE; 1:400., synaptophysin Ž1:50., chromogranin Ž1:300., S-100 protein Ž1:500., HMB-45 Ž1:100., cytocheratin Ž1:200., carcino-embryonic antigen ŽCEA; 1:500., ep-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 7 - X
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G. Sorrentino et al.r Brain Research 827 (1999) 205–209
ithelial membrane antigen ŽEMA; 1:100., leukocyte common antigen ŽLCA; 1:200., vimentin Ž1:50., desmin Ž1:200., factor VIIIrvon Willenbrand-related antigen ŽF VIIIrvW-RA; 1:100., all obtained from Dakopatts, Denmark. The antibody to the low molecular weight neurofilament protein ŽNF-L. was a purified mouse antibody Ždiluted 1:400. and the antibodies to the middle and high molecular weight neurofilaments ŽNF-M, NF-H. were polyclonal rabbit antisera Ždiluted 1:50 and 1:250, respectively.; NF-L and NF-H were obtained from Sigma, NF-M from Affiniti ŽNottingham, UK.. The antibody binding sites were detected and visualized according to Hsu et al. w7x. Secondary antibodies alone were used as control. Western blot analysis on 4 and 7 div control and RA or NGF differentiated cells was performed according to Pernas Alonso et al. w17x. A mix of monoclonal antibody anti NF-L ŽSigma. Ždiluted 1:50. and polyclonal antisera anti NF-M and NF-H ŽAffiniti. Ždiluted 1:2000. was used. The same filters were further incubated with antiserum anti NSE diluted 1:2000 obtained from Genosys ŽCambridge, UK. and visualized as above. Choline acetyltranferase ŽChAT. and tyrosinase activities were measured according to the procedures of Fonnum w5x and Pomerantz w19x, respectively. Monoamine determination was performed as previously described w26x. Cytogenetic analysis was performed on the established cell line at passages 15, 18 and 67 as described w25x. Chromosome analysis followed the ISCN recommendation w9x. Statistical comparisons were made applying analysis of variance ŽANOVA. followed by Scheffe’s F-test. The results of the immunocytochemical analysis are summarized in Table 1. As shown on the CSF specimen, the cell line expressed vimentin ŽFig. 1A., but not cytocheratin and GFAP. NF-L was the only neurofilament to be present ŽFig. 1B.. Neuroendocrine cell markers like NSE, chromogranin and synaptophysin were detected ŽFig. 1C,D,E.. Furthermore, TB cell line showed immunoreactivity for HMB-45, a monoclonal antibody which was reported to be highly sensitive and specific for melanoma w6,24x ŽFig. 1F.. S-100 protein, which was detected on CSF cells, was not present in cultured TB cell. The lack of CEA, EMA, LCA, F-VIII staining ruled out the epithelial, haematopoietic and endothelial derivation. To confirm the presence of NF-L in the TB cell line, a Western blot analysis on cell extracts was performed. In order to establish if the presence of NGF or RA was able to induce differentiation towards a neuronal phenotype, the presence of NFs in TB cells differentiated for 4 and 7 days was examined. The presence of NF-L, -M and -H in the different growth conditions is shown in Fig. 2. The NF-L was present in each condition. The NF-M was clearly evident only in the RA treated cells but not in control and NGF treated cells. The NF-H was only present in the cells grown for 7 days in the presence of RA, whereas the control and 7 div NGF treated cells did not contain NF-H.
Table 1 Comparison of immunohistochemical results on original cells from CSF, TB cell line and transplant CSF
TB cells
Xenograft
Neuronal and glial markers Neurofilament L n.d. Neurofilament M n.d. Neurofilament H n.d. GFAP y
q y y y
q y y y
Neuroendocrine cell markers NSE n.d. Chromogranin n.d. Synaptophysin n.d.
q q q
q q q
Melanotic cell markers S-100 protein HMB-45
q n.d.
y q
y y
Epithelial cell markers Cytocheratin CEA EMA
y n.d. n.d.
y y y
y y y
Haematopoietic cell marker LCA Žcd-45. y
y
y
Endothelial cell markers F VIIIrvW-RA n.d.
y
y
Muscle cell markers Desmin
y
q
q
q
n.d.
Mesenchimal cell markers Vimentin q
y: Negative staining; q: positive staining; n.d.: not determined. GFAP: glial fibrillary acidic protein; NSE: neuron specific enolase; CEA: carcino-embryonic antigen; EMA: epithelial membrane antigen; LCA: leukocyte common antigen; F VIIIrvW-RA: factor VIIIrvon Willenbrand-related antigen.
Furthermore, the treatment with RA for 7 days increased the presence of NF-L. NSE did not change in any condition. When examined by transmission electron microscopy, TB cells showed few features of differentiated cells. Furthermore, typical dense-core neurosecretory granules were found Ždata not shown.. The results of the immunohistochemical ŽTable 1. and ultrastructural study on the xenograft transplant were highly consistent with those obtained with the cell line. Surprisingly, the xenograft sections showed a scattered staining for desmin Ždata not shown.. Tyrosinase activity was barely detectable, whereas no ChAT activity was found. After 1 div, significant amounts of serotonin Ž5HT. and its metabolite, 5-hydroxy indoleacetic acid Ž5HIAA., were detected in the cell homogenate Ž10.8 " 1.3 and 12.0 " 0.6 ngrmg protein, respectively.. Indolamine content decreased with time. At 4 div, it was 3.8 " 0.7 and 5.1 " 0.3 ngrmg of protein for serotonin and 5HIAA, respectively. No indolamines were
G. Sorrentino et al.r Brain Research 827 (1999) 205–209
207
Fig. 1. Indirect immunostaining of TB cells. TB cells, grown on glass coverslips, were fixed and subjected to immunostaining by using antibodies specific for vimentin ŽA., low molecular weight neurofilament ŽB., neuron specific enolase ŽC., chromogranin ŽD., synaptophysin ŽE., HMB-45 ŽF.. Color X development was with 3, 3 -diaminobenzidine Ž=400..
detectable after 10 div. Dopamine, norepinephrine or their metabolites were not found. At 1 div, pargyline increased serotonin content to 14.8 " 0.7 ngrmg protein Ž37.0% of control. and decreased 5HIAA to 6.9 " 0.3 Ž57.5% of control., indicating that the 5HT peak is indeed serotonin. Retinoic acid did not change the serotonin and 5HIAA content. The chromosome analysis showed very complex karyotypes with modal chromosome number basically in the hypotetraploid range Ž74–78 in passage 15 and 18, 78–83 in passage 67. and with a number of structurally rearranged chromosomes, some of which were clonally present. The origin of few clonal chromosome markers was established: addŽ1.Žp32.; tŽ2;5.Žp23;q11.2.; iŽ5.Žp10.;
addŽ9.Žp24.; derŽ14.tŽ14;15.Žq32;p11. in passage 15, addŽ1.Žp32.; tŽ14;22.Žq10q10. in passage 18, addŽ1.Žp32.; delŽ3.Žq21.; addŽ6.Žp25. in passage 67. Of the three developmentally regulated neurofilaments, TB cells normally expressed only NF-L, suggesting that they represent an early stage of neural differentiation. Furthermore, TB cell line expressed vimentin, the first intermediate filament ŽIF. to appear in embryological development, regardless of cell type, that is later replaced by the IF protein specific for each particular cell type. In some cases, vimentin coexists with NF proteins, namely in developing neuronal cells as well as in some neoplastic cells w1x. Several markers indicative of neuroendocrine differentiation such as NSE, chromogranin and synapto-
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Fig. 2. Western blot of TB cell line for the three neurofilaments. Lane 2 through 4 are extracts from 4 div cells; lane 5 through 7 are extracts from 7 div cells. Lane 1 molecular weight markers in kilodaltons; lane 2 and 5 control; lane 3 and 6 NGF treated cells; lane 4 and 7 RA treated cells; lane 8 rat brain extract.
physin were clearly present in TB cells supporting a neuroendocrine origin of this cell line w13x. Further evidence of the neuroendocrine nature of the cell line was provided by the presence of many dense-core granules both in vitro and in xenografts. Serotonin and its metabolite 5HIAA accumulated in TB cell line. Serotonin is expressed by normal, dysplastic and neoplastic neuroendocrine cell population w3x. Furthermore, an in vitro study using quail neural crest cells showed that these cells can be serotonin-immunoreactive w21x. A quite unexpected finding was the presence of staining for desmin in the xenograft transplant. Desmin, the muscle-cell type of intermediate filament protein, has a restricted distribution in striated and most smooth muscle cells. It has been suggested that neoplastic primitive neuroectodermal cells have intrinsic rhabdomyoblastic potential, corresponding to the normal mesectodermal differentiation of neural crest tissues into striated muscle in head and neck region w2x. It is possible that a set of human neural crest cells migrates to soft tissue and differentiates into other mesenchymal cells. In this respect, various heterotopic grafting experiments suggest that neural crest cells differentiate along one or another phenotypic pathway in response to local environmental stimuli w15x. Recent studies have shown that RA plays a major role during neuronal differentiation. A potential source of RA and the mechanisms whereby cells can respond to RA are both present during CNS development w11,12,23x. In TB cell line, the NF-L, -M and -H are upregulated by RA treatment in a temporal pattern resembling that in the developing brain. Among the identified structurally rearranged chromosomes, the derŽ1. was present in all the examined pas-
sages. Rearrangement of the 1p32 band is commonly found in a number of tumors, however, it is specifically associated to neuroblastoma and is very frequently observed in melanoma w14x. Although no conclusion can be drawn, it should be noted that these results support the hypothesis of a tumor of neural crest origin. A series of studies have shown that the neural crest is a heterogeneous population of pluripotent cells, partially restricted cells, and cells committed to a particular cell lineage w10x. The availability of a cell line that is committed to differentiate in response to specific stimuli would enable detailed studies of the molecular basis of the complex process, whereby cells acquire the mature phenotype. TB cell line can thus provide a useful tool to analyze differentiation and dedifferentiation of neural crest derivatives as well as their tumorigenicity.
Acknowledgements We would like to thank Dr. D. Mercanti, Institute of Neurobiology, CNR, Rome, for his generous gift of NGF. This work was supported by grants from Consiglio Nazionale delle Ricerche ŽGS., North Atlantic Treaty Organization ŽGS. and Associazione Italiana per la Ricerca contro il Cancro ŽUDP..
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