Aeta Neuropathol (1992) 84:52 - 58
kta NeumpatlmltQ ~) Springer-Verlag1992
A continuous cell line (KK-2) from a supratentorial primitive neuroectodermal tumor* H. Ito 1,2, T. Kameya 1, T. Suwa 2, C. Wada 3, and N. Kawano 2 Departments of 1Pathology, 2Neurosurgery, and 3Clinical Pathology Kitasato University School of Medicine, Sagamihara, Kanagawa 228, Japan Received August 5, 1991/Revised, accepted February 6, 1992
Summary. Tumor tissue located in the occipital lobe with hemorrhage was obtained from a 19-year-old patient. Histological examination indicated it to consist of undifferentiated small, round cells without neuronal or glial differentiation, and possibly to be a type of primitive neuror tumor. The tumor cells were cultured for 3 years and a continuous cell line (KK-2) was established. KK-2 was transplantable to nude mice. With immunocytochemistry, neuron-specific enolase, protein gene product 9.5, vimentin, TUJ1 (a monoclonal antibody specific for neuron-associated class III ~-tubulin isotype) and 6H7 (a monoclonal antibody to N C A M produced by us) were detected. None of the following could be found: glial fibrillary acidic protein, S-100 protein, neurofilament and synaptophysin, calcitonin gene-related peptide, gastrin releasing peptide corticotropin-releasing factor, substance R somatostatin, chromogranin, aromatic L-amino acid decarboxylase and tyrosine hydroxylase, The original tumor and KK-2 cells obtained after 3 years of culture and transplants in nude mice displayed essentially the same ultrastructural and immunohistochemical characteristics. KK-2 cells showed no differentiation to mature neuronal, glial or ependymal cells. This cell line may possibly serve as a useful model for studying cellular differentiation of human neuroectodermal tumors and normal neuronal development. Key words: Primitive neuroectodermal t u m o r - Cell line - Characterization - Establishment
The category of primitive neuroectodermal tumors (PNET) was established by Hart and Earle in 1973 [13], who defined P N E T as a group of undifferentiated small, round cell tumors of the central nervous system, not * Supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare (1-5), Japan Correspondence to: H. Ito (address see 2 above)
diagnosable specifically as medulloepithelioma, central neuroblastoma, polar spongioblastoma, ependymoblastoma, pineal parenchymal tumor, cerebellar medulloblastoma, or retinoblastoma. These tumors were thought to derive from fetal neuroectodermal precursor cells. Rorke [23, 24] proposed terminology for five P N E T subtypes of undifferentiated neuroepithelial round cell tumors and included all undifferentiated small, round cell tumors mentioned above. However, Rubinstein [25, 26] was critical of these P N E T subtypes, since they fail to take into consideration histological patterns previously described. To avoid confusion, the authors feel that " P N E T " should be used only for tumors presenting no histological differentiation based on neuronal and/or glial lines and for the category originally established by Hart and Earle [13]. We investigated a t u m o r which could be diagnosed as P N E T by the absence of morphological neuronal and glial differentiation. It was considered to be a new continuous supratentorial P N E T cell line, which we designated as KK-2. Its features were examined and its potential for differentiation in P N E T based on the above criteria [13] was assessed.
Materials and methods Clinical s u m m a r y
The patient was a 19-year-old female who was referred to our clinic in June, 1984. CT showed an intracranial tumor situated in the occipital lobe with hemorrhage. At the time of operation, the tumor was relatively well circumscribed, and attached to the dura mater. Following the complete removal of the tumor, radiation therapy was performed. There were subsequently two episodes of recurrence and on each occasion, tumor was removed surgically. Finally multiple tumors developed in the brain and the patient died in May, 1988. Cell line establishment
For the primary culture, tumor tissue was obtained from material from a second operation in February, 1988.Tissue fragments were
53
Table 1. Antibodies used in the study Antibody NSE PGP9.5 GFAP NFoL NF-H TUJ1 S-100 Vim CG CGRP SS SP Syn GRP CRF AADC TH 6H7
anti-rabbit anti-rabbit anti-mouse anti-mouse anti-mouse anti-mouse anti-rabbit anti-mouse anti-mouse anti -rabbit anti-rabbit anti-rabbit anti-mouse anti-rabbit anti-rabbit anti-rabbit anti-rabbit anti-mouse
Dilution
Source
1 : 400 1:400 1: 200 1: 400 1 : 400 1:100 1: 500 1 : 800 1: 500 1:1000 1: 300 1:1 1:2 1: 800 1: 200 1:1000 1 : 400 1 : 75
DAKO, Copenhagen, Denmark, code A589 UltraClone, England Labsystems, Helsinki, Finland, code 6400 100 Amersham, Little Chalsont, England, code RPNll03 Labsystems, code FE500 Donated by Dr A. Frankfurter, Dept Biol, Univ Virginia School of Medicine DAKO, code Z311 Amersham Lipshaw, Detroit, USA, code C34200 Amersham, code RPN1842 DAKO, code A566 Nichirei, Japan, code 292505 Boehringer Mannheim Biochemica, FRG, code 106445 Donated by Dr K. Yamaguchi, Natl. Cancer Center Research Institute, Toyko, Japan Peptide Institute Inc., Osaka, Japan, code 862-360218 Eugene Tech. International, Court Alendale, N J, USA Eugene Tech. International Produced by us [17]
NSE, neuron-specific enolase; PGP 9.5, protein gene product 9.5; GFAR glial fibrillary acidic protein; NF-H, 200 kDa neurofilament; NF-L, 68 kDa neurofilament; TUJ1, monoclonal antibody for neuron-specific [3-tubulin; S-100, S 100 protein; Vim, vimentin; CG, chromogranin; CGRP, calcitonin gene-related peptide; SS, somatostatin; SE substance P; Syn, synaptophysin; GRP, gastrin releasing peptide; CRF. corticotropin-releasing factor; AADC, aromatic L-amino acid decarboxylase; TH, tyrosine hydroxylase; 6H7, monoclonal antibody for a neural cell adhesion molecule produced by us
put in a 60-ram plastic dish (Falcon Primaria, Becton Dickinson, Calif.) containing RPMI 1640 (Nissui Pharmaceutical Co., Toyko, Japan) with 10 % fetal calf serum (Flow Lab., Lot No. 29101954, North Ryde, Australia). A cell line consisting of one cell type was obtained within 15 months and designated as the KK-2 cell line. Cells (1 • 108) were inoculated subcutaneously into the backs of 5-8 week-old BALB/c female nude mice (nu/nu genotype; Clea Japan Inc., Japan). The karyotype of the KK-2 cells was determined by using the Giemsa-trypsin banding method [29]. Specimens of the original tumor and xenografts fixed in 10 % formalin were stained with hematoxylin and eosin (H&E) and immunostained by the avidin-biotin-complex (ABC) method [I5]. For indirect immunofluorescence, the cells were cytcentrifuged at 100 rpm for 10 min and then fixed in cold acteone at - 2 0 ~ for 20 rain for subsequent use.Western blotting was conducted on the cell line [31]. Cultured cells were lysed in Laemmli's sample buffer by brief sonication and diluted to a concentration of 1 mg protein/ml in the same buffer. The cell lysate was then heated in boiling water for 3 min. SDS-PAGE was carried out on a 4 O~/o-12 O concave gradient polyacrylamide gel. The proteins were then transferred to polyvinylidene difluoride membranes (Immobilon PVDF Transfer Membrane; Millipore, Bedford, Mass.) and immunostained by the ABC method. The antibodies and antisera shown in Table 1 were used for phenotypic characterization of the cell line. For transmission electron microscopy (TEM), the specimens were fixed in 2.5 % glutaraldehyde, and then 1% osmium tetroxide, dehydrated in a graded series of alcohol and embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate, and examined with a TEM (Hitachi H-600). Human y-enolase was assayed in a sandwich enzyme immunoassay system by Dr. K, Kato, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi [16].
Results H i s t o l o g i c a l e x a m i n a t i o n (Fig. 1) s h o w e d t h a t t h e original t u m o r c o n s i s t e d o f s m a l l , r o u n d n u c l e i s u r r o u n d e d
Fig, 1, H&E-stained section of the original tumor. The tumor consists of undifferentiated small, round cells with chromatin-rich nuclei. The cells in the small area form Homer Wright rosettes (arrows). x 400
54
by a minimal rim of cytoplasm. The nuclei were rich in chromatin and contained small single or double nucleoli. The cells formed Homer Wright rosettes in small areas. Otherwise, there were neither fibrillary areas nor ganglionic cells. Mitotic figures were conspicuous and numerous. Immunohistochemistry of the paraffin sections indicated neuron-specific enolase (NSE) to be weakly and diffusely positive, whereas glial fibrillary acidic protein (GFAP), S-100 protein (S-100), and neurofilament proteins (NF) were negative in tumor cells. Electron microscopy showed the original tumor to be comprised of undifferentiated cells with large nuclei, free ribosomes to be abundant, and elements of rough endoplasmic reticulum and Golgi complex to be scant. Cilia and basal bodies were occasionally present, and parallel microtubules and synapsis elements were absent. No definite morphological differentiation to mature neuronal or glial cells could be detected in the tumor cells, and a final diagnosis of PNET was thus made. Our KK-2 cell line has been growing well as suspension cell clusters or spheroids for the past 3 years by repeated subculturing. The cell clusters measured 50 to 1800 ~tm in diameter. The population doubling time of the KK-2 cells is 96 h. After being transplanted into nude mice, they elicited tumor growth of about
1 0 m m • 1 5 m m • 10mm in size within 3months. Chromosomes of the KK-2 cells were examined I year after the primary culture. The modal number of chromosomes was 46XX; 6q-, 10q- and 15q- were observed in all cells, and 11q-, 12p-, 22q-, 6p+ in some cells. Light microscopy showed that the KK-2 cells possess scanty cytoplasm, round and oval nuclei and that mitosis rarely occurs. The center of a large cell cluster was necrotic. In TEM, the cultured cells had little cytoplasm and chromatin-rich nuclei (Fig. 2a). Cytoplasmic processes were seen to connect to each other by junctional apparatus (Fig. 2b). Very rarely, these processes contained microtubules and a few dense-cored granules (Fig. 2c). In other respects, the cultured cells and those of the transplants were morphologically quite similar to those of the original tumor. By immunohistochemistry, only NSE and protein gene product (PGP) 9.5 were detected in paraffin sections of original tumor and xenografts fixed with formalin. However, in cultured cells, PGP 9.5 (27 kDa), NSE (48kDa), a monoclonal antibody specific for neuron-associated class III ~-tubulin isotype (TUJ1; 54 kDa), and vimentin (Vim; 57 kDa) were detected by Western blotting (Fig. 3), and NSE, PGP 9.5,Vim and a monoclonal antibody to a neural cell adhesion molecule (NCAM) (6H7) were positive in nearly all cells as
Fig. 2a-c. Transmission electron microscopy of the cultured cells. a The cultured cells possess scanty cytoplasm and chromatin-rich nuclei, b High magnification of the cultured cells. Cytoplasmic processes are connected to each other by a cell junction (arrow)and
there are some dense-cored granules (arrowheads) in cytoplasmic processes, e Cytoplasmic processes contain parallel microtubules (arrowheads) interspersed with a few cored granules (arrow). a • 5000; b,e x 30000
55 indicated by immunofluorescence (Fig. 4). GFAP, S100, NF, synaptophysin (Syn), and neuropeptides such as calcitonin gene-related peptide (CGRP), gastrin releasing peptide (GRP), corticotropin-releasing factor (CRF), substance P (SP) and somatostatin (SS), and neuroendocrine markers such as chromogranin (CG), aromatic L-amino acid decarboxylase ( A A D C ) and tyrosine hydrolase (TH) were all negative (Table 2). NSE content detected by immunoassay of KK-2 cells 20 months following the establishment of the line was 820 ng/mg protein.
Discussion
Fig. 3. Western blots of the cultured cells. Protein gene product (PGP 9.5; 27 kDa; lane A), neuron-specific enolase (NSE; 48 kDa; lane B), Vimentin (Vim; 57 kDa; lane C), and TUJ1 (54 kDa; lane D) are detected
Fig. 4a-d. Indirect immunofluorescence on cytocentrifuged preparations. KK-2 cells are immunostained with PGP 9.5 (a), NSE (b), Vim (c) and 6H7 (d) antibodies. PGP 9.5, NSE and Vim are
A continuous cell line of supratentorial P N E T without definite glial, ependymal or neuronal differentiation, as far as could be determined by light and electron microscopy, was established. Some cell lines from medulloblastoma [6, 7, 18] and neuroblastoma [1, 10, 27, 30, 32] have been established. In medulloblastoma cell lines, TE-671 [18] and D 283 Med [6] show indication of
positive in scanty cytoplasm, but 6H7 stains the cell surface only. • 400
56 Table 2. Comparison of immunohistochemical results on original tumor, transplant, and KK-2 cell linea
Original tumor fixed by formalinb Immunofluorescence of KK-2 fixed by a c e t o n e T r a n s p l a n t o f KK-2 f i x e d by f o r m a l i n b W e s t e r n b l o t t i n g o f KK-2
NSE PGP9.5
Vim
6H7
NF-L NF-H
+
+
-
ND
.
+
++
+
+
.
+
+
-
ND
.
+
++
+
ND
-
.
TUJ1 .
. .
. .
GFAP
.
+
. .
. .
.
TH
.
.
.
AADC
.
.
. -
S-100
.
.
. .
Syn
CGRP
GRP
CRF
ND
-
-
-
-
-
-
ND
ND
ND
. ND
.
.
SP
SS
CG
.
ND ND ND
a+, positive; + + , strongly positive; - , negative; ND, not done b I m m u n o s t a i n e d by A B C m e t h o d
neuronal differentiation. However, to our knowledge, the present cell line is the first continuous cell line of supratentorial PNET without definite glial, ependymal, or neuronal differentiation. The population doubling time of KK-2 was 96 h; this is somewhat longer than that of other cell lines (44.5 to 57.6 h [6, 27,321. Immunohistochemically, NSE was present in KK-2 cells. In an immunochemical assay, the NSE content of KK-2 cells was shown to be 820 ng/mg protein, much less than in a series of neuroblastoma and ganglioneuroblastoma (2060 + 890 ng/mg protein, n = 7) [16]. In the neoplastic state, the specificity of NSE as indication of neuronal origin or differentiation cannot be assumed in all cases [3] since immunopositivity for NSE has been demonstrated in glioblastoma multiforme [33] and numerous non-neural cells [28]. However, the antibody against NSE has proved useful as a marker for developing embryonal neuronal cells and for the recognition of peripheral neuroblastomas, melanomas, amine precursor uptake and decarboxylation (APUD) cells, neuroendocrine cells and their tumors [3]. PGP 9.5 was the most strongly positive in all cells in the present tumor. PGP 9.5 is a 27 kDa soluble protein that has been isolated from the human brain and is found in neurons, nerve axons and all neuroendocrine cells in normal adult human tissues and immature neural tissues at early embryonic stages [5]. The distribution of immunoreactive PGP 9.5 in tissues is virutally the same as that of NSE [34]. PGP 9.5 has been found to serve reliably as a marker in PNET [22]. Positive staining for PGP 9.5 was evident in 16/21 tumors, and the results of staining with antibody to PGP 9.5 were clear-cut and without background staining [12]. No NF proteins could be detected using two monoclonal antibodies (anti-68 and 200 kDa NF) by immunoblotting or immunofiuorescence. In their study of a less differentiated human medulloblastoma cell line (D341 Med), Friedman et al. [7] detected NF proteins of high and middle molecular weight by both immunoblotting and immunohistochemistry. Differences in sensitivity of detection cannot be compared for the two studies, since the antibodies used were different. However, most tumors of PNET and continuous cell lines of medulloblastoma express NF proteins [6, 7, 11], our cell line may possibly belong to the least differentiated neuroectodermal tumor. Gould et al. [11] detected NF in surgical specimens from 16 out of 22 PNET tumors. Their
diagnosis of tumors was made based on the 1985 W H O classification [24], and their PNET included medulloblastoma and neuroblastoma. The epitope recognized by TUJ1, specific for neuronassociated class III ~-tubulin isotype, was detected in KK-2 by Western blotting analysis but not immunostaining. Class III ~-tubulin is expressed by neurons and not by glia. TUJ1 stains neuronal cell bodies, dendrites, axons and axonal terminations [9, 14]. Primitive neuroepithelial structures resembling neural tubes (medulloepithelial rosettes) contain single or small groups of cells which react with TUJ1. Immature neuroblasts and maturing polar neurons also show immunoreactivity with TUJ1, whereas reactivity for microtubule-associated protein 2, tau, the 200 kDa isoform of NF, NSE and Syn is restricted for the most part ot maturing neurons [4]. Our cell line contained vimentin, the first intermediate filament (IF) protein to appear in embryologic development, regardless of cell type. IF protein is later replaced by several IF proteins specific for each particular cell type [3]. The presence of vimentin has been confirmed in early differentiating neurons in one report [2], which indicated that vimentin-positive structures first appear in the embryo on day 11, but that the neural tube is still be negative at this stage. On day 12, vimentin-positive fibers appear throughout the central nervous system. Vimentin and NF proteins continue to be present together in neurons during a limited period of embryonal development. On the other hand, GFAP and NF, IF proteins specific for glial and neuron, respectively, were not detected in KK-2 cells. The cells may, thus, be analogous to undifferentiated cells at the primitive stage of embryonic neuronal development. A monoclonal antibody, 6H7, was produced by the immunization of small cell carcinoma of the lung and characterized at our laboratory [20]. The cell surface antigen recognized by this antibody, belonging to the cluster-1 monoclonal series, was found to correspond to NCAM [8, 21]. NCAM are expressed in various tissues at an early stage of embryogenesis, but its expression becomes restricted to tissues of neuroectodermal origin as development progresses. NCAM on cell membranes are believed involved in cell-cell interactions and may possibly be essential to neural embryogenesis and development. This pan-neuroectodermal antigen is expressed in most neuroectodermal small, round cell
57
tumors including neuroblastoma, retinoblastoma, ependymoblastoma and small cell carcinoma of the lung [211. The expression of neuropeptides, such as SS and SP has been reported in central PNET [11]. SS, SP, CGRP, G R P and C R F were negative in KK-2 cells by i m m u n o histochemistry. KK-2 was also e x a m i n e d for the expression of CG, A A D C and T H , all d e t e c t e d f r e q u e n t l y or occasionally in A P U D cells and t u m o r s including small cell lung c a n c e r [17, 19]. T h e s e m a r k e r s were all negative. B a s e d o n the findings o f positivity for N C A M antigen, v i m e n t i n , N S E , T U J 1 and P G P 9.5, and negative results for N F p r o t e i n s and GFAP, t h e K K - 2 m a y be c o n s i d e r e d a v e r y primitive n e u r o e c t o d e r m a l t u m o r cell line. I n c o n s i d e r a t i o n o f the original m e a n i n g o f P N E T , the diagnosis of " P N E T " s h o u l d be m a d e only for t u m o r s p r e s e n t i n g no indication of m o r p h o l o g i c a l l y n e u r o n a l or glial differentiation, as o b s e r v e d for the tumor. To o u r k n o w l e d g e , t h e r e is no o t h e r c o n t i n u o u s s u p r a t e n t o r i a l P N E T cell line which fulfills these criteria. U s e o f this cell line m a y facilitate clarification of the origin o f P N E T as well as the p r o p e r t i e s o f h u m a n n e u r o e c t o d e r m a l t u m o r s and n o r m a l n e u r o n a l developments.
Acknowledgements. The authors thank Dr. A. Frankfurter for generously providing the TUJ1 used in this study.We are grateful to Mrs Kuniko Kadoya of the Morphology Division at our institute for her technical assistance. The members of the Electron Microscope Laboratory Center of the Kitasato University School of Medicine are thanked for preparing electron micrographs.
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8. Fukayama M, Funata N, Hayashi Y, Maeda Y, Koike M, Watanabe J, Okabe T (1990) Brain-associated small-cell lung cancer antigen (BASCA) is expressed in developing lung: an immunohistochemicat and immunoelcctron microscopic study. J Histochem Cytochem 38:51-57 9. Geisert EE, Frankfurter A (1989) The neuronal response to injury as visualized by immunostaining of class III [3-tubulin in the rat. Neurosci Lett 102:137-141 10. Gerson J, Schlesinger HR, Sereni E Moorhead PS, Hummeler K (1977) Isolation and characterization of a neuroblastoma cell line from peripheral blood in a patient with disseminated disease. Cancer 39:2508-2512 11. Gould VE, Jansson DS, Molenaar WM, Rorke LB,Trojanowski JQ, Lee VMY, Packer RJ, Franke WW (1990) Primitive neuroectodermal tumors of the central nervous system. Lab Invest 62:498-509 12. Harris MD, Moore IE, Steart PV, Weller RO (1990) Protein gene product (PGP) 9.5 as a reliable marker in primitive neuroectodermal tumors: an immunohistochemical study of 21 childhood cases. Histopathology 16:271-277 13. Hart MN, Earle KM (1973) Primitive neuroectodermal tumors of the brain in children. Cancer 32:890-897 14. Herman MM, Perentes E, Katsetos CD, Darcel F, Frankfurter A, Collins VR Donoso LA, Eng LF, Marangos PJ, Wiechmann AF, May EE, Thomas CB, Rubinstein LJ (1989) Neuroblastic differentiation potential of the human retinoblastoma cell lines Y-79 and WERI-Rbl maintained in an organ culture system. Am J Pathol 134:115-132 15. Hsu SM, Raine L, Fanger H (1981) A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying peptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75:734-738 16. Ishiguro Y, Kato K, Ito T, Horisawa M, Nagaya M (1984) Enolase isozymes as markers for differential diagnosis of neuroblastoma, rhabdomyosarcoma, and Wilms' tumor. Gann 75: 53 -60 17. Jensen SM, Gazdar AF, Cuttitta F, Russell EK, Linnoila I (1990) A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines. Cancer Res 50:6068-6074 18. McAllister R, Isaages H, Rongey R, Peer M, Au W, Sooukkup SW, Gardner MB (1977) Establishment of a human medulloblastoma cell line. Int J Cancer 20:206-212 19. Nagatsu ~1~ Ichinose H, Kojima K, Kameya T, Shimase J, Kodama T, Simosato Y (1985) Aromatic L-amino acid decarboxylase activities in human lung tissues: comparison between normal lung and lung carcinomas. Biochem Med 34:52-59 20. Okuda T, Kasai K, Kameya % Saito S, Takase N (1988) Monoclonal antibody directed against neuroendocrine properties of both normal and malignant cells. Hybridoma 7:569-581 21. Patel K, Moore SE, Dickson G, Rossell RJ, Beverley PC, Kemshead JT, Walsh FS (1989) Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma. Int J Cancer 44:573-578 22. Rode J, Dhillon AR Doran JF, Jackson P,Thompson RJ (1985) PGP 9.5, a new marker for human neuroendocrine tumors. Histopathology 9:147-158 23. Rorke LB (1983) The cerebellar medulloblastoma and its relationship to primitive neuroectodermal tumors. J Neuropathol Exp Neurol 42:1-15 24. Rorke LB, Gelles FH, Davis RL, Becket LE (1985) Revision of the World Health Organization classification of brain tumors for childhood brain tumors. Cancer 56 [Suppl] : 1869-1886 25. Rubinstein LJ (1985) A commentary on the proposed revision of the World Health Organization classification of brain tumors for childhood brain tumors. Cancer 56 [SupplJ : 1887-1888
58 26. Rubinstein LJ (1985) Embryonal central neuroepithelial tumors and their differentiating potential. J Neurosurg 62: 795 -805 27. Schlesinger HR, Gerson JM, Moorehead PS, Maguire H, Hummeler K (1976) Establishment and characterization of human neuroblastoma cell lines. Cancer Res 36:3094-3100 28. Schmechel DE (1985) Gamma-subunit of the glycolytic enzyme enolase: non specific or neuron specific. Lab Invest 52: 239-242 29. Seabright M (1971) A rapid banding technique for human chromosomes. Lancet I:971-972 30. Seeget RC, Rayner SA, Banergee A, Chung H, Laug WE, Neustein HB, Benedict WF (1977) Morphology, growth, chromosomal pattern and fibrinolytic activity of two new human neuroblastoma cell lines. Cancer Res 37:1364-1371
31. Tobin H, StaehelinT, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354 32. Tumilowicz JJ, Nichols WW, Cholon JJ, Greene AE (1970) Definition of a continuous human cell line derived from neuroblastoma. Cancer Res 30:2110-2118 33. Vinores SA, Rubinstein LJ (1985) Simultaneous expression of glial fibrillary acidic (GFA) protein and neuron-specific enolase (NSE) by the same reactive or neoplastic astrocytes. Neuropathol Appt Neurobiol 11:349-359 34. Wilson POG, Barber PC, Harold QA, Power BF, Dhillon AR Rode J, Day INM, Thompson RJ, Polak JM (1988) The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol 69:91-104
kta NeumpatlmltQ ~) Springer-Verlag1992
A continuous cell line (KK-2) from a supratentorial primitive neuroectodermal tumor* H. Ito 1,2, T. Kameya 1, T. Suwa 2, C. Wada 3, and N. Kawano 2 Departments of 1Pathology, 2Neurosurgery, and 3Clinical Pathology Kitasato University School of Medicine, Sagamihara, Kanagawa 228, Japan Received August 5, 1991/Revised, accepted February 6, 1992
Summary. Tumor tissue located in the occipital lobe with hemorrhage was obtained from a 19-year-old patient. Histological examination indicated it to consist of undifferentiated small, round cells without neuronal or glial differentiation, and possibly to be a type of primitive neuror tumor. The tumor cells were cultured for 3 years and a continuous cell line (KK-2) was established. KK-2 was transplantable to nude mice. With immunocytochemistry, neuron-specific enolase, protein gene product 9.5, vimentin, TUJ1 (a monoclonal antibody specific for neuron-associated class III ~-tubulin isotype) and 6H7 (a monoclonal antibody to N C A M produced by us) were detected. None of the following could be found: glial fibrillary acidic protein, S-100 protein, neurofilament and synaptophysin, calcitonin gene-related peptide, gastrin releasing peptide corticotropin-releasing factor, substance R somatostatin, chromogranin, aromatic L-amino acid decarboxylase and tyrosine hydroxylase, The original tumor and KK-2 cells obtained after 3 years of culture and transplants in nude mice displayed essentially the same ultrastructural and immunohistochemical characteristics. KK-2 cells showed no differentiation to mature neuronal, glial or ependymal cells. This cell line may possibly serve as a useful model for studying cellular differentiation of human neuroectodermal tumors and normal neuronal development. Key words: Primitive neuroectodermal t u m o r - Cell line - Characterization - Establishment
The category of primitive neuroectodermal tumors (PNET) was established by Hart and Earle in 1973 [13], who defined P N E T as a group of undifferentiated small, round cell tumors of the central nervous system, not * Supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare (1-5), Japan Correspondence to: H. Ito (address see 2 above)
diagnosable specifically as medulloepithelioma, central neuroblastoma, polar spongioblastoma, ependymoblastoma, pineal parenchymal tumor, cerebellar medulloblastoma, or retinoblastoma. These tumors were thought to derive from fetal neuroectodermal precursor cells. Rorke [23, 24] proposed terminology for five P N E T subtypes of undifferentiated neuroepithelial round cell tumors and included all undifferentiated small, round cell tumors mentioned above. However, Rubinstein [25, 26] was critical of these P N E T subtypes, since they fail to take into consideration histological patterns previously described. To avoid confusion, the authors feel that " P N E T " should be used only for tumors presenting no histological differentiation based on neuronal and/or glial lines and for the category originally established by Hart and Earle [13]. We investigated a t u m o r which could be diagnosed as P N E T by the absence of morphological neuronal and glial differentiation. It was considered to be a new continuous supratentorial P N E T cell line, which we designated as KK-2. Its features were examined and its potential for differentiation in P N E T based on the above criteria [13] was assessed.
Materials and methods Clinical s u m m a r y
The patient was a 19-year-old female who was referred to our clinic in June, 1984. CT showed an intracranial tumor situated in the occipital lobe with hemorrhage. At the time of operation, the tumor was relatively well circumscribed, and attached to the dura mater. Following the complete removal of the tumor, radiation therapy was performed. There were subsequently two episodes of recurrence and on each occasion, tumor was removed surgically. Finally multiple tumors developed in the brain and the patient died in May, 1988. Cell line establishment
For the primary culture, tumor tissue was obtained from material from a second operation in February, 1988.Tissue fragments were
53
Table 1. Antibodies used in the study Antibody NSE PGP9.5 GFAP NFoL NF-H TUJ1 S-100 Vim CG CGRP SS SP Syn GRP CRF AADC TH 6H7
anti-rabbit anti-rabbit anti-mouse anti-mouse anti-mouse anti-mouse anti-rabbit anti-mouse anti-mouse anti -rabbit anti-rabbit anti-rabbit anti-mouse anti-rabbit anti-rabbit anti-rabbit anti-rabbit anti-mouse
Dilution
Source
1 : 400 1:400 1: 200 1: 400 1 : 400 1:100 1: 500 1 : 800 1: 500 1:1000 1: 300 1:1 1:2 1: 800 1: 200 1:1000 1 : 400 1 : 75
DAKO, Copenhagen, Denmark, code A589 UltraClone, England Labsystems, Helsinki, Finland, code 6400 100 Amersham, Little Chalsont, England, code RPNll03 Labsystems, code FE500 Donated by Dr A. Frankfurter, Dept Biol, Univ Virginia School of Medicine DAKO, code Z311 Amersham Lipshaw, Detroit, USA, code C34200 Amersham, code RPN1842 DAKO, code A566 Nichirei, Japan, code 292505 Boehringer Mannheim Biochemica, FRG, code 106445 Donated by Dr K. Yamaguchi, Natl. Cancer Center Research Institute, Toyko, Japan Peptide Institute Inc., Osaka, Japan, code 862-360218 Eugene Tech. International, Court Alendale, N J, USA Eugene Tech. International Produced by us [17]
NSE, neuron-specific enolase; PGP 9.5, protein gene product 9.5; GFAR glial fibrillary acidic protein; NF-H, 200 kDa neurofilament; NF-L, 68 kDa neurofilament; TUJ1, monoclonal antibody for neuron-specific [3-tubulin; S-100, S 100 protein; Vim, vimentin; CG, chromogranin; CGRP, calcitonin gene-related peptide; SS, somatostatin; SE substance P; Syn, synaptophysin; GRP, gastrin releasing peptide; CRF. corticotropin-releasing factor; AADC, aromatic L-amino acid decarboxylase; TH, tyrosine hydroxylase; 6H7, monoclonal antibody for a neural cell adhesion molecule produced by us
put in a 60-ram plastic dish (Falcon Primaria, Becton Dickinson, Calif.) containing RPMI 1640 (Nissui Pharmaceutical Co., Toyko, Japan) with 10 % fetal calf serum (Flow Lab., Lot No. 29101954, North Ryde, Australia). A cell line consisting of one cell type was obtained within 15 months and designated as the KK-2 cell line. Cells (1 • 108) were inoculated subcutaneously into the backs of 5-8 week-old BALB/c female nude mice (nu/nu genotype; Clea Japan Inc., Japan). The karyotype of the KK-2 cells was determined by using the Giemsa-trypsin banding method [29]. Specimens of the original tumor and xenografts fixed in 10 % formalin were stained with hematoxylin and eosin (H&E) and immunostained by the avidin-biotin-complex (ABC) method [I5]. For indirect immunofluorescence, the cells were cytcentrifuged at 100 rpm for 10 min and then fixed in cold acteone at - 2 0 ~ for 20 rain for subsequent use.Western blotting was conducted on the cell line [31]. Cultured cells were lysed in Laemmli's sample buffer by brief sonication and diluted to a concentration of 1 mg protein/ml in the same buffer. The cell lysate was then heated in boiling water for 3 min. SDS-PAGE was carried out on a 4 O~/o-12 O concave gradient polyacrylamide gel. The proteins were then transferred to polyvinylidene difluoride membranes (Immobilon PVDF Transfer Membrane; Millipore, Bedford, Mass.) and immunostained by the ABC method. The antibodies and antisera shown in Table 1 were used for phenotypic characterization of the cell line. For transmission electron microscopy (TEM), the specimens were fixed in 2.5 % glutaraldehyde, and then 1% osmium tetroxide, dehydrated in a graded series of alcohol and embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate, and examined with a TEM (Hitachi H-600). Human y-enolase was assayed in a sandwich enzyme immunoassay system by Dr. K, Kato, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi [16].
Results H i s t o l o g i c a l e x a m i n a t i o n (Fig. 1) s h o w e d t h a t t h e original t u m o r c o n s i s t e d o f s m a l l , r o u n d n u c l e i s u r r o u n d e d
Fig, 1, H&E-stained section of the original tumor. The tumor consists of undifferentiated small, round cells with chromatin-rich nuclei. The cells in the small area form Homer Wright rosettes (arrows). x 400
54
by a minimal rim of cytoplasm. The nuclei were rich in chromatin and contained small single or double nucleoli. The cells formed Homer Wright rosettes in small areas. Otherwise, there were neither fibrillary areas nor ganglionic cells. Mitotic figures were conspicuous and numerous. Immunohistochemistry of the paraffin sections indicated neuron-specific enolase (NSE) to be weakly and diffusely positive, whereas glial fibrillary acidic protein (GFAP), S-100 protein (S-100), and neurofilament proteins (NF) were negative in tumor cells. Electron microscopy showed the original tumor to be comprised of undifferentiated cells with large nuclei, free ribosomes to be abundant, and elements of rough endoplasmic reticulum and Golgi complex to be scant. Cilia and basal bodies were occasionally present, and parallel microtubules and synapsis elements were absent. No definite morphological differentiation to mature neuronal or glial cells could be detected in the tumor cells, and a final diagnosis of PNET was thus made. Our KK-2 cell line has been growing well as suspension cell clusters or spheroids for the past 3 years by repeated subculturing. The cell clusters measured 50 to 1800 ~tm in diameter. The population doubling time of the KK-2 cells is 96 h. After being transplanted into nude mice, they elicited tumor growth of about
1 0 m m • 1 5 m m • 10mm in size within 3months. Chromosomes of the KK-2 cells were examined I year after the primary culture. The modal number of chromosomes was 46XX; 6q-, 10q- and 15q- were observed in all cells, and 11q-, 12p-, 22q-, 6p+ in some cells. Light microscopy showed that the KK-2 cells possess scanty cytoplasm, round and oval nuclei and that mitosis rarely occurs. The center of a large cell cluster was necrotic. In TEM, the cultured cells had little cytoplasm and chromatin-rich nuclei (Fig. 2a). Cytoplasmic processes were seen to connect to each other by junctional apparatus (Fig. 2b). Very rarely, these processes contained microtubules and a few dense-cored granules (Fig. 2c). In other respects, the cultured cells and those of the transplants were morphologically quite similar to those of the original tumor. By immunohistochemistry, only NSE and protein gene product (PGP) 9.5 were detected in paraffin sections of original tumor and xenografts fixed with formalin. However, in cultured cells, PGP 9.5 (27 kDa), NSE (48kDa), a monoclonal antibody specific for neuron-associated class III ~-tubulin isotype (TUJ1; 54 kDa), and vimentin (Vim; 57 kDa) were detected by Western blotting (Fig. 3), and NSE, PGP 9.5,Vim and a monoclonal antibody to a neural cell adhesion molecule (NCAM) (6H7) were positive in nearly all cells as
Fig. 2a-c. Transmission electron microscopy of the cultured cells. a The cultured cells possess scanty cytoplasm and chromatin-rich nuclei, b High magnification of the cultured cells. Cytoplasmic processes are connected to each other by a cell junction (arrow)and
there are some dense-cored granules (arrowheads) in cytoplasmic processes, e Cytoplasmic processes contain parallel microtubules (arrowheads) interspersed with a few cored granules (arrow). a • 5000; b,e x 30000
55 indicated by immunofluorescence (Fig. 4). GFAP, S100, NF, synaptophysin (Syn), and neuropeptides such as calcitonin gene-related peptide (CGRP), gastrin releasing peptide (GRP), corticotropin-releasing factor (CRF), substance P (SP) and somatostatin (SS), and neuroendocrine markers such as chromogranin (CG), aromatic L-amino acid decarboxylase ( A A D C ) and tyrosine hydrolase (TH) were all negative (Table 2). NSE content detected by immunoassay of KK-2 cells 20 months following the establishment of the line was 820 ng/mg protein.
Discussion
Fig. 3. Western blots of the cultured cells. Protein gene product (PGP 9.5; 27 kDa; lane A), neuron-specific enolase (NSE; 48 kDa; lane B), Vimentin (Vim; 57 kDa; lane C), and TUJ1 (54 kDa; lane D) are detected
Fig. 4a-d. Indirect immunofluorescence on cytocentrifuged preparations. KK-2 cells are immunostained with PGP 9.5 (a), NSE (b), Vim (c) and 6H7 (d) antibodies. PGP 9.5, NSE and Vim are
A continuous cell line of supratentorial P N E T without definite glial, ependymal or neuronal differentiation, as far as could be determined by light and electron microscopy, was established. Some cell lines from medulloblastoma [6, 7, 18] and neuroblastoma [1, 10, 27, 30, 32] have been established. In medulloblastoma cell lines, TE-671 [18] and D 283 Med [6] show indication of
positive in scanty cytoplasm, but 6H7 stains the cell surface only. • 400
56 Table 2. Comparison of immunohistochemical results on original tumor, transplant, and KK-2 cell linea
Original tumor fixed by formalinb Immunofluorescence of KK-2 fixed by a c e t o n e T r a n s p l a n t o f KK-2 f i x e d by f o r m a l i n b W e s t e r n b l o t t i n g o f KK-2
NSE PGP9.5
Vim
6H7
NF-L NF-H
+
+
-
ND
.
+
++
+
+
.
+
+
-
ND
.
+
++
+
ND
-
.
TUJ1 .
. .
. .
GFAP
.
+
. .
. .
.
TH
.
.
.
AADC
.
.
. -
S-100
.
.
. .
Syn
CGRP
GRP
CRF
ND
-
-
-
-
-
-
ND
ND
ND
. ND
.
.
SP
SS
CG
.
ND ND ND
a+, positive; + + , strongly positive; - , negative; ND, not done b I m m u n o s t a i n e d by A B C m e t h o d
neuronal differentiation. However, to our knowledge, the present cell line is the first continuous cell line of supratentorial PNET without definite glial, ependymal, or neuronal differentiation. The population doubling time of KK-2 was 96 h; this is somewhat longer than that of other cell lines (44.5 to 57.6 h [6, 27,321. Immunohistochemically, NSE was present in KK-2 cells. In an immunochemical assay, the NSE content of KK-2 cells was shown to be 820 ng/mg protein, much less than in a series of neuroblastoma and ganglioneuroblastoma (2060 + 890 ng/mg protein, n = 7) [16]. In the neoplastic state, the specificity of NSE as indication of neuronal origin or differentiation cannot be assumed in all cases [3] since immunopositivity for NSE has been demonstrated in glioblastoma multiforme [33] and numerous non-neural cells [28]. However, the antibody against NSE has proved useful as a marker for developing embryonal neuronal cells and for the recognition of peripheral neuroblastomas, melanomas, amine precursor uptake and decarboxylation (APUD) cells, neuroendocrine cells and their tumors [3]. PGP 9.5 was the most strongly positive in all cells in the present tumor. PGP 9.5 is a 27 kDa soluble protein that has been isolated from the human brain and is found in neurons, nerve axons and all neuroendocrine cells in normal adult human tissues and immature neural tissues at early embryonic stages [5]. The distribution of immunoreactive PGP 9.5 in tissues is virutally the same as that of NSE [34]. PGP 9.5 has been found to serve reliably as a marker in PNET [22]. Positive staining for PGP 9.5 was evident in 16/21 tumors, and the results of staining with antibody to PGP 9.5 were clear-cut and without background staining [12]. No NF proteins could be detected using two monoclonal antibodies (anti-68 and 200 kDa NF) by immunoblotting or immunofiuorescence. In their study of a less differentiated human medulloblastoma cell line (D341 Med), Friedman et al. [7] detected NF proteins of high and middle molecular weight by both immunoblotting and immunohistochemistry. Differences in sensitivity of detection cannot be compared for the two studies, since the antibodies used were different. However, most tumors of PNET and continuous cell lines of medulloblastoma express NF proteins [6, 7, 11], our cell line may possibly belong to the least differentiated neuroectodermal tumor. Gould et al. [11] detected NF in surgical specimens from 16 out of 22 PNET tumors. Their
diagnosis of tumors was made based on the 1985 W H O classification [24], and their PNET included medulloblastoma and neuroblastoma. The epitope recognized by TUJ1, specific for neuronassociated class III ~-tubulin isotype, was detected in KK-2 by Western blotting analysis but not immunostaining. Class III ~-tubulin is expressed by neurons and not by glia. TUJ1 stains neuronal cell bodies, dendrites, axons and axonal terminations [9, 14]. Primitive neuroepithelial structures resembling neural tubes (medulloepithelial rosettes) contain single or small groups of cells which react with TUJ1. Immature neuroblasts and maturing polar neurons also show immunoreactivity with TUJ1, whereas reactivity for microtubule-associated protein 2, tau, the 200 kDa isoform of NF, NSE and Syn is restricted for the most part ot maturing neurons [4]. Our cell line contained vimentin, the first intermediate filament (IF) protein to appear in embryologic development, regardless of cell type. IF protein is later replaced by several IF proteins specific for each particular cell type [3]. The presence of vimentin has been confirmed in early differentiating neurons in one report [2], which indicated that vimentin-positive structures first appear in the embryo on day 11, but that the neural tube is still be negative at this stage. On day 12, vimentin-positive fibers appear throughout the central nervous system. Vimentin and NF proteins continue to be present together in neurons during a limited period of embryonal development. On the other hand, GFAP and NF, IF proteins specific for glial and neuron, respectively, were not detected in KK-2 cells. The cells may, thus, be analogous to undifferentiated cells at the primitive stage of embryonic neuronal development. A monoclonal antibody, 6H7, was produced by the immunization of small cell carcinoma of the lung and characterized at our laboratory [20]. The cell surface antigen recognized by this antibody, belonging to the cluster-1 monoclonal series, was found to correspond to NCAM [8, 21]. NCAM are expressed in various tissues at an early stage of embryogenesis, but its expression becomes restricted to tissues of neuroectodermal origin as development progresses. NCAM on cell membranes are believed involved in cell-cell interactions and may possibly be essential to neural embryogenesis and development. This pan-neuroectodermal antigen is expressed in most neuroectodermal small, round cell
57
tumors including neuroblastoma, retinoblastoma, ependymoblastoma and small cell carcinoma of the lung [211. The expression of neuropeptides, such as SS and SP has been reported in central PNET [11]. SS, SP, CGRP, G R P and C R F were negative in KK-2 cells by i m m u n o histochemistry. KK-2 was also e x a m i n e d for the expression of CG, A A D C and T H , all d e t e c t e d f r e q u e n t l y or occasionally in A P U D cells and t u m o r s including small cell lung c a n c e r [17, 19]. T h e s e m a r k e r s were all negative. B a s e d o n the findings o f positivity for N C A M antigen, v i m e n t i n , N S E , T U J 1 and P G P 9.5, and negative results for N F p r o t e i n s and GFAP, t h e K K - 2 m a y be c o n s i d e r e d a v e r y primitive n e u r o e c t o d e r m a l t u m o r cell line. I n c o n s i d e r a t i o n o f the original m e a n i n g o f P N E T , the diagnosis of " P N E T " s h o u l d be m a d e only for t u m o r s p r e s e n t i n g no indication of m o r p h o l o g i c a l l y n e u r o n a l or glial differentiation, as o b s e r v e d for the tumor. To o u r k n o w l e d g e , t h e r e is no o t h e r c o n t i n u o u s s u p r a t e n t o r i a l P N E T cell line which fulfills these criteria. U s e o f this cell line m a y facilitate clarification of the origin o f P N E T as well as the p r o p e r t i e s o f h u m a n n e u r o e c t o d e r m a l t u m o r s and n o r m a l n e u r o n a l developments.
Acknowledgements. The authors thank Dr. A. Frankfurter for generously providing the TUJ1 used in this study.We are grateful to Mrs Kuniko Kadoya of the Morphology Division at our institute for her technical assistance. The members of the Electron Microscope Laboratory Center of the Kitasato University School of Medicine are thanked for preparing electron micrographs.
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