Establishment and Characterization of a Malignant Melanoma Cell Line (YP-MEL) Derived from a Patient with Neurocutaneous Melanosis

Path. Res. Pract. 190, 178-185 (1994)

Establishment and Characterization of a Malignant Melanoma Cell Line (YP-MEL) Derived from a Patient with Neurocutaneous Melanosis y. Nagashimal, Y. Miyagp,4, I. Aokil, T. Funabiki 2 , K. Ikuta2 , M. Umeda3, Y. Kuchino4 and K. Misugi1 1Departments of Pathology and 2Pediatrics, Yokohama City University School of Medicine, Yokohama, 3Cell Biology Division, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan, 4Biophysics Division, National Cancer Center Research Institute, Tokyo, Japan

SUMMARY

A cell line, YP-MEL, was established from an intracranial malignant melanoma occurring in a'neurocutaneous melanosis (NCMsis) patient. The established cell line was successfully cultured in serum-free medium with a doubling time of 41 h. The cells were refractile and small in size, with occasional pigmented giant cells. Histochemical and immunohistochemical features were compatible with common malignant melanoma and its cell line. Chromosome analysis revealed many supernumerary chromosomes and marker chromosomes including double minutes (DMs). When transplanted into nude mice, YP-MEL formed tumors histologically consistent with the original tumor. Addition of sera to the medium caused cellular spreading and elongation of cytoplasmic processes with an increase of melanin contents and tyrosinase activity. Because there was no melanoma eel/line derived from a NCMsis patient, YP-MEL might be a beneficial tool for study on NCMsis.

Introduction Malignant melanoma is a neoplasm derived from melanocytes. In spite of comprehensive therapeutic regimens, its prognosis is still unfavorable due to its aggressive behavior and unresponsiveness to chemotherapy and therapeutic irradiation l2 • A clearer understanding of the pathogenesis of malignant melanoma is important not only for improving the prognosis but also forpreventing its development. Neurocutaneous melanosis (NCMsis) is a syndrome characterized by generalized pigmented nevi, involving not only the skin but also the viscera and central nervous system. Affected patients are predisposed to malignant transformation of the pigmented nevi l 7. Malignant melanoma of the skin, viscera and central nervous system 0344-0338/94/0190-0178$3.50/0

occurs before adolescence, and the patients die due to cancer progression 17 • Although the basic genetic abnormality responsible is not known2, some melanoma-related gene(s) is considered to playa role in the syndrome, similar to the APC gene in familial adenomatous polyposis of the colon l8 . In this paper, we report the establishment and characterization of a cell line derived from malignant melanoma occurring in a NCMsis patient. This cell line has an amelanotic phenotype, and has been cultivated serially in serum-free medium; its melanogenesis is accelerated by addition of sera to the medium. Immunohistochemical and histochemical features were similar to common malignant melanoma. This cell line might be a beneficial tool for study for NCMsis. © 1994 by Gustav Fischer Verlag, Stuttgart

Melanoma Cell Line Derived from NCMsis . 179

Material and Methods Case report. A 4-year-old boy was referred to Yokohama City University Hospital with complaint of abrupt visual disturbance in February, 1987. The patient had had multiple pigmented nevi since birth, but they had not shown obvious enlargement. There was no familial history of phacomatosis including NCMsis, or of malignant melanoma. Computerized tomography on admission revealed hydrocephalus. Because of the presence of multiple pigmented nevi (Fig. 1) and hydrocephalus, the patient was diagnosed as having NCMsis. Consecutive decompression operations failed to improve his poor condition. On the operation, the cerebral surface was discolored black. The biopsied specimen obtained from the pia mater showed abnormal proliferation of melanocytes (Fig. 2a), which were positively stained with Fontana-Masson staining (Table 1). Two months after admission, progression of multiple brain tumors became evident. A second decompression surgery was performed, and the biopsied specimen showed obvious neoplastic features, that is, malignant melanoma (Fig. 2b). Finally, 6 months after admission, the patient died of respiratory failure due to compression of the brainstem by the intracranial tumors. Autopsy was not permitted.

Table 1. Reagents utilized to examine in vitro differentiation Reagents

Tested concentrations

Reference

L-histidinol Retinoid TPAl DMS02 alpha MSH3 beta MSH3 Sodium butyrate dbcAMp4

1 mM, 2 mM 10- 7 M, 10- 6 M, 10- 5 M 10 nM, 20 nM, 30 nM 1 %,3 %,5% 10- 8 M ] 0- 7 M 10- 6 M 10- 8 M', 10- 7 M', 10- 6 M 0.1 mM, 1.0 mM 0.1 mM, 1.0 mM

18 ]9,20,2] 22,23 22, 24

25 25

24 26,27

112-0-tetradecanoylphorbol 13-acetate, 2Dimethylsulfoxide, 3Melanocyte-stimulating hormone, 4Dibutyric cyclic AMP.

Fig. 2. Light micrographs of the first biopsied specimen (a), culture source (b) and xenotransplanted tumor (c). - a: The specimen of the first biopsy. The pia mater contained considerable numbers of melanin-laden cells, which showed no neoplastic feature. - b: The culture source. There were malign?nt melanoma cells forming epithelioid architecture. Occasionally, melanin pigment was noted. c: Nude mouse xenotransplant of YP-MEL. The formed tumor showed similar appearance with the culture source. Bar represents 20 ~m.

Fig. 1. Clinical features of the patient. The patient had multifocal pigmented nevi in the trunk and extremities.

180 . Y. Nagashima et al.

Cell culture. The culture source was a small tumor tissue fragment

biopsied from the brain (Fig. 2b). It was minced with surgical blades, washed several times with culture medium and placed in a culture flask. The medium used was RPMI 1640 (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10 % fetal calf .serum (FCS; HyCione Lab., Logan, UT). Passage was performed using 0.25. % trypsin. On fifth passage generation, the culture medium was replaced by serum-free ASF 103 medium (Ajinomoto Co., Tokyo, Japan). All subsequent studies were carried out using this serum-free medium. To examine the growth characteristics, 1 x 104 YP-MEL cells were seeded into 24-well culture plates (Coster, New York, NY), and cultured. Cell numbers were counted with a Coulter Counter (Co ulter Electronics Ltd., London, England) . For evaluation of plating efficiency, 100 cells were seeded into 60-mm culture dishes. Colony-forming ability on soft aga r plates was examined by culturing 100 or 1000 cells on 0.5 % soft agar medium 2 !. Monitoring of mycoplasma was performed according to the method reported by Chen 3 . Briefly, VERO cells cultured in YP-MEL-conditioned medium were stained with Hoechst 33258 and examined for mycoplasma fl uo rescence in the cytoplasm.

Table 2. Reagents utilized to examine in vitro differentiation Reagents

Tested concentra tions

Reference

L-histidinol Retinoid TPA I DMS0 2 alpha MSH l beta MSH3 Sodium butyrate dbcAMp4

1 mM, 2 mM 10- 7 M, 10- 6 M, 10- 5 M 10 nM, 20 nM, 30 nM

20 23,25,26 24, 29 24,28 21 21 28 22, 27

1 %,3 %,5 % lO- g M 10- 7 M 10- 6 M 10- 8 M', 10- 7 M', 10- 6 M

0.1 mM, 1.0 mM 0.1 mM, 1.0 mM

112-0 -tetradecanoylphorbol 13-acetate, 2Dimethylsulfoxide, 3Melanocy te-stimulating hormone, 4Di butyric cyclic AMP. Tokyo, Japan). The formed tumors were then examined histopathologically, along with the biopsied specimens and cultured cells.

In Vitro Differentiation Morphological and Chromosomal Examinations Morphological observations were performed on the in vitro cells using a phase-contrast microscope. For electron microscopy. YP-MEL cells cultured on plastic dishes were fixed with 3 % glutaraldehyde, post-fixed with 1 % osmium tetroxide, dehydrated with ethanol and embedded in expoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed using a Hitachi H-600 transmission electron microscope. _ For histochemical and immunohistochemical studies, the biopsied samples, cultured cells were stained with PAS, Fontan aMasson stainings, and antibodies listed in Table 1. Cytogenetic analysis was performed on YP-MEL cells of fifth passage generation before rransfer to serum-free culture condition. Chromosome slides were prepared after 4 h of colcemid treatment (final concentration 0.005 JAg/ml) by the standard air-drying method. Chromosome numbers were counted on 100 metaphases. Karyotyping by the G-banding method was performed according to Dutrillax and Lejeune5 .

DNA and RNA Analyses Genomic DNA was prepared from YP-MEL cells according to the standard method 26 • Ten micrograms of DNA was completely digested with EeoRI, PstI or HindIII, separated electrophoretically, and then transferred to a nylon membrane. Total RNA from exponentially growing YP-MEL cells was prepared by the guanidine-cold phenol method4, and poly(A)+ RNA was selected with Oligotex-dT30 (Takara Shuzo, Kyoto, Japan). Ten micrograms of poly(A)+ RNA was separated on agarose gel containing 1.6 M formamide, and then transferred to a nylon membrane. A genomic 2.1 -kbp BglIII-EcoRI fragment containing human Nmyc exon 3 was prepared from a plasmid pN-myc2238 (supplied by the Japanese Cancer Research Resources Bank), and labelled with (- 32P)dCTP as a hybridization probe by nick translation. After hybridization, the membranes were then exposed to Kodak XAR-OMAT autoradiography film using an intensifying screen.

Heterotransplantation to N ude Mice To examine the tumo rigenicity of YP-MEL cells cells in nude mice, 1 x 107 cells were inoculated subcutaneously into the lumbodorsal area of BALB/c athymic nude mice (Clea Japan,

Twenty thousand of YP-MEL cells suspended in 2 ml of serum-free ASF 103 medium, were seeded onto 35-mm plastic dishes, and after overnight culture, the test reagents shown in Ta ble 2 were added. After one week exposure to the reagents, the pigmentation of the cells was monitored with phase-contrast microscopy. Melanin contents and tyrosinase activity were assayed as previously reported7,14 . As the positive control, B1 6 murine melanoma cell line (obtai ned fro m Japanese Cell Bank), was employed. Protein contents we re measured according to Bradford! for normalization of melanin contents and tyrosinase acti vity. Synthetic melanin (Sigma Chern. Co., St. Louis, MO) and mushroom tyrosinase (Sigma Chern. Co.) were employed as the standard materials.

Results The biopsied specimen taken at the first decompression surgery showed thickened pia mater, with proliferation of moderate-sized cells (Fig. 2a). The cells showed moderate nucleocytoplasmic ratio, without atypism. The nuclei were round to oval in shape, and contained fine chromatin. The nucleoli were not prominent. The cytoplasm was clear or contained brown pigment granules. Fontana-Masson stain showed positive reaction (Table 1). In contrast, the second biopsied specimen showed neoplastic melanocytes with solid arrangements (Fig. 2b). The nucleocytoplasmic ratio was high. The nuclei were large, and contained coarse chromati'n with prominent nucleoli. The cytoplasm was generally clear, but occasionally melanin pigment was noticed (Table 1). FontanaMasson staining also showed positivity. A part of this biopsied tissue was cultured in vitro. After 2 weeks of primary culture, the tumor cells formed a monolayer sheet composed of small refractile cells with bipolar cytoplasmic processes. Usually, melanin pigment was not visible. 'O ccasionally, pigmented giant cells were noted. We named the cell line YP-MEL; it showed good consistant growth and could be passaged serially. The cells were also found to grow in serum-free ASF 103 medium. Since the 5th passage, YP-MEL has been maintained in ASF 103

Melanoma CeJJ Line Derived from NCMsis . 181

Fig. 3. Phase-contrast micrographs of YP-MEL cells. - a: YPMEL cells cultured in ASF 103 medium. The cells were small in size and refractile. Occasionally, large multinucleated cells were noted (Arrow). - b: YP-MEL cells cultured under the condition of FCS addition at 20 %. The cells were spread and flat in shape. Note intracytoplasmic granules. The bar represents 50 flm.

medium, for more than 100 passage generations (Fig. 3a). The population doubling time was 41 h (Fig. 4), and plating efficiency was 8.8 % . Colony-forming ability on 0.5 % soft agar medium plates was less than 1 %.

Cell Density 2 (Cells/em i

1 x

loST I

1

1 x 10 4

I

I

Hoechsr 33258 staining showed no fluorescence in the cytoplasm of Vero cells cultured in YP-MEL-conditioned medium, confirming that the YP-MEL cells were free from mycoplasma contamination. Electron microscopic examination revealed well developed rough endoplasmic reticulum and mito<;:hondria. Immature melanosomes were observed, and none were fully matured (Fig. Sa, arrowheads). The results of special and immunohistochemical stainings were summerized in Table 1, which were almost identical to those of the other melanoma celllines 28 . Chromosome analysis was performed at the fifth passage. The chromosome number ranged from 73 to 154 with a mode of 82 (Table 3). Ten metaphases were analyzed for karyotyping. Figure 6 shows a representative karyotype with 82 chromosomes including many supernumerary chromosomes and markers. Because of the presence of double minutes (DMs), we examined possible abnormalities of the N-myc gene, which is frequently amplified in tumors with DMs27. No amplification or increased transcription was revealed by Southern and Northern blotting analyses (Fig. 7). When transplanted inro athymic nuce mice, YP-MEL cells formed melanotic tumors measuring 1-2 cm in diameter, after a I-month latency period. Histologically, the tumors showed rep resentative features of malignant Table 3. Frequency of chromosome number in YP-MEL

2

Culture Days. Fig. 4. Growth curve of YP-MEL cells in serum-free condition . The population doubling time was 41 h.

Chromosome number 73 74 75 76 77 78 79 80 81 82 83 84 85 86 154 Frequency 1

3

6

5

9 14 12 15 16 10

5

3

1

1

182 . Y. Nagashima et al.

melanoma, consisting of large polygonal cells with solid cell sheets (Fig. 2c). The tumor cells had eosinophilic cytoplasm and large oval or round nuclei with prominent nucleoli. Some of them contained melanin pigment. These features were similar to those of the original tumor (Fig.2b). The results of histochemical and immunohistochemical examinations were summerized in Table 1, being representative as a malignant melanoma. We attempted to induce melanogenesis of YP-MEL cells by the in vitro treatment with reagents reported previously as differentiation- or melanogenesis-inducing factors for cultured melanoma cells. Although none of these reagents

induced melanin production by YP-MEL, the monolayer turned black macroscopically when more than 1 % of FCS was added to the medium. The cells changed to a spread form and became flat. Intracytoplasmic granules were evident (Fig. 3b, compare with Fig. 3a). These morphological changes were observed 24 h after serum supplementation, and were reversible when the cells were returned to serum-free medium. Calf serum also showed a similar effect, but a higher concentration (more than 10 %) was required to induce melanogenesis. Growth characteristics including plating efficiency and colony-forming ability on soft agar plates were not influenced. Ultrastructurally,

Fig. 5. Transmission electron micrographs of YP-MEL cells. - a: YP-MEL cells cultured in serumfree ASF 103 medium. There were immature melanosomes (arrowheads) with low electron density. - b: YP-MEL cells cultured in ASF 103 supplemented with 20 % FCS. The melanosomes showed increased electron density. The bar represents 0.2 Ilm.

Melanoma Cell Line Derived from N CMsis . 183

7

i.

14

x

ii,OMs

mar

FCS cone.

PDT! (hrs)

Saturation density (cells/cm 2)

Melanin content (flg/107 cells)

0 1 5 10 20

41 41 41 40 41

3.0 3.0 2.5 2.3 2.3

54 67 93 96 103

x 105 x 10 5 x 10 5 X 105 x 105

18

22

21

~'A

*'

11

17

16

15

20

19

Table 4. Effects of the addition of FCS in a serum-free culture medium on the growth characteristics, melanin production and tyrosinase activity of YP-MEL cells

(%)

10

9

8

13

6

",

Fig. 6. A representative karyotype of YP-MEL cell with 84 chromosomes. There were excess chromosomes including marker chro mosomes (lp+, Ip +, 5q- , 10q-, and 13p+, arrowheads). DMs were also 2 and 17 were noted. apparently deleted.

*'

4

3

2

± ± ± ± ±

8.1 3.5 11.0 13 .6 9.3 2

(A)

± ± ± ± ±

~~

kb

0.5 0.2 0.6 2 0.5 2 0.7 2

9 .4 6.6 4.4 2 .3 2.0 -

The values in the Table are the averages of 3 repeated experiments and standard deviations in duplicate cultures. - 1Population doubling time; 2p < 0.05; comparing to the data under tile serum-free condition.

Fig. 7. Results of Southern (A) and Northern (B) analyses for N-myc oncogene. - A: Ten micrograms of PstI, HindIII or EcoRI-digested DNA from human placen ta and YP-MEL cells were hybridized with 32P-Iabelled human N-myc exon 3 probe. Lanes 1, 2, 3: human placental DNAs digested with PstI, HindIII or EcoRI, respectively. Lanes 4,5,6: YP-MEL DNAs digested with PstI, HindIII, EcoRI, respectively . - B: Ten micrograms of poly(A) + RNA from YP-MEL cells were hybridized with rue same probe used in Southern hybridization. The agarose gel was stained by ehtidium bromide fo r sample quantitation, showing that extracted RNA was not degraded and was good for Northern analysis (beside).

YP- MEL

;;;:;; c::-b 0 Q.'" ~ ~CJ

Tyrosinase activity (mU/mg protein) 3.0 4.1 5.7 6.0 6.7

placenta

(8)

N-myc -285 -18S

fi-a ctin

Y

184 . Y. Nagashima et al.

maturation of melanosomes was observed (Fig. Sb, compare with Fig. Sa). Melanin contents were increased upon addition of serum, and tyrosinase activity was also elevated (Table 4). Discussion NCMsis is a hereditary syndrome characterized by congenital multifocal pigmented nevi and mental retardation. The nevi occur in the viscera and meninges, and malignant transformation to melanoma is observed frequently, not only in the skin but also in the viscera and central nervous system. In spite of extensive studies, the basic mechanism of its pathogenesis is still obscure. A previous cytogenetic study on melanoma-prone families 2 was unable to reveal any specific karyotypic abnormality. However, the investigators speculated that some form of genetic instability was present in members of melanomaprone families. Recently, a transgenic animal model of generalized melanosis was established using simian virus 40 large T antigen tagged with the tyrosinase promoter l 9 . In this experimental system, the transgenic mice had multifocal and heterotopic melanotic nevi in the gastrointestinal tract, respiratory tract and central nervous system. Such mice produce malignant melanomas and died due to tumor spreading. In this paper, we have described a newly established cell line derived from malignant melanoma which developed in an NCMsis patient. In this case, histological specimens were obtained both from nevus on the pia mater and from intracranial malignant melanoma. Unfortunately, cytogenetic study on somatic or tumor cells of the patient failed to be performed. The cell line established from the intracranial melanoma, named YP-MEL, was adapted to serum-free conditions and grew vigorously. A previous study on the karyotype of malignant melanoma showed frequent involvement of chromosomes 1, 6, 7 and 9, followed by chromosomes Sand 10 (reviewed in reference 16 ). Abnormalities of chromosomes 1 and 6 in malignant melanoma were considered to be associated with the Rh and HLADR loci, respectivelylO. Another study failed to reveal any specific chromosome abnormality in malignant melanoma/dysplastic nevus syndrome9 . In our cell line, many types of marker chromosomes including DMs were observed, which were not necessarily identical to the reported findings. Considerable ones of these chromosomal abnormalities seemed to have occurred in the primary tumor, because the chromosomal examination was carried out with the cells of the fifth passage generation. DMs had not been reported in any previous cytogenetic studies of malignant melanoma. We considered that they might bear some abnormally amplified oncogene, such as N-myc in neuroblastoma 27 . Therefore, we examined the N-myc gene by Southern and Northern blotting, but no amplification or increased transcription of the oncogene was evident. Some other oncogene might be amplified in OMs, but no further study to investigate this possibility was conducted.

When transplanted into athymic nude mice, the YPMEL cell line generated melanotic tumors after a I-month latency period. Histologically, the tumor showed representative features of malignant melanoma with melanin production. Light microscopy including histochemistry and immunohistochemistry revealed identical features with the culture source. By contrast, the xenotransplant was apparently different from the melanocytes on the pia mater noted in the first biopsy specimen. The tumor cells formed solid and epithelioid architectures in the culture source and xenotransplant, whereas melanocytes on the first biopsy specimen were distributed individually. The nuclei of the formers contained coarse chromatin with prominent nucleoli, but those of the latters showed fine chromatin. Histochemistry and immunohistochemistry features of them were identical. Unfortunately, YP-MEL cells failed to shown a specific feature comparing the other melanoma cell lines, although it occurred in a patient with NCMsis. Because this cell line could be cultured under serum-free condition, we attempted to examine the melanogenesisinducing effect of reagents previously reported to cause differentiation of melanoma cells in vitro (Table 2). In spite of repeated experiments, no morphological change in YP-MEL cells was observed. However, when FCS was added to the growth medium at concentrations of more than 1 %, YP-MEL cells turned black with a flat, polygonal and spread morphology, and elaborated an intracytoplasmic granular substance. These findings were consistent with the induced differentiation reported in previous papers. The amount of melanin and tyrosinase activity were increased, indicating accelerated melanogenesis. Electron microscopy also revealed maturation of melanosomes in the FCS-supplemented cultures. Thus, there should be some melanogenesis-inducing activity in sera other than the reagents listed in Table 1, including melanocyte-stimulating hormone (MSH). The growth characteristics of the cells were not altered when serum was added to the medium. Such phenomenon has already been reported in the induction of differentiation by L-histidino[25, although most of differentiation-inducing factors cause growth retardation 6, 8, 11 , ]3, 15,20,22,23, 24. We have thus described the characteristics of a malignant melanoma cell line, YP-MEL. Because there was no reported malignant melanoma cell line derived from NCM patients, this cell line might be valuable for the study of malignant melanoma and NCMsis.

Acknowledgement The authors wish to thank Mr. Makoto Iwamoto, Ms. Michiko Ehara, Ms. Hiromi Fujisawa, and Ms. Tomoko Watanabe for their technical and secretarial assistance. This research was supported in part by Grant-in-aids from the Japanese Ministry of Education, Science and Culture, and from Kihara Memorial Yokohama Biological Research Foundation.

Melanoma Cell Line Derived from NCMsis . 185

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Received June 24, 1993 . Accepted in revised form October 7, 1993

Key words: Malignant melanoma - Neurocutaneous melanosis (NCMsis) - Cel/line - YP-MEL - Serum-free culture Serum - Melanogenesis Y. Nagashima, Departments of Pathology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-kll, Yokohama 236, Japan, Tel. 045-787-2587, Fax 045 -7 86-0191