Presence and Regulation of Tyrosinase Activity in Human Neuroblastoma Cell Variants in Vitro1

[CANCER RESEARCH 45, 1628-1632, April 1985]

Robert A. Ross2 and June L. Biedler

Department ol Biological Sciences, Fordham University, Bronx, New York 10458 [R.A.R.], and Laboratory of Cellular and Biochemical Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [J.L.B.]

ABSTRACT the catecholamine-biosynthetic enzymes. We postulated that this second, variant cell type could represent either a melanocyte The human neuroblastoma cell line SK-N-SH comprises cells or a neurilemmal (Schwann) cell, 2 types of cells that are within that undergo morphological and biochemical interconversion be the repertoire of the neural crest and have a flat, substrate- tween a primitive sympathoblast and a variant, epithelial-like cell adherent appearance in tissue culture (11, 12). To ascertain type which does not express the neuronal characteristics of the whether the epithelial-like cells of SK-N-SH are expressing either SK-N-SH cell line. Since neural crest cells, from which neuro- of these cellular phenotypes, biochemical studies were under blastomas are presumed to arise, can undergo transdifferentia- taken. tion in culture from a neuronal phenotype into other cellular phenotypes, particularly into neurilemmal cells and melanocytes, the present study was undertaken to determine whether this capacity is preserved in malignant cells of the peripheral nervous MATERIALS AND METHODS system. Activities for tyrosinase, a melanocyte marker enzyme, and 2':3'-cyclic nucleotide phosphohydrolase, a Schwann-cell Materials. Cell culture constituents (medium, fetal bovine serum, and marker enzyme, were measured in clones of the two cell types. antibiotics) were obtained from Grand Island Biological Co. (Grand Island, While no significant differences in 2':3'-cyclic nucleotide phos NY). L-["C]Tyrosine and S-[14C]adenosylmethionine were purchased phohydrolase activity were measurable, tyrosinase activity was from New England Nuclear (Boston, MA). Drugs and hormones were obtained from Sigma Chemical Co. (St. Louis, MO). detectable only in the flattened neuroblastoma variant cell lines Cell Culture. All cell lines were cultured in a 1:1 mixture of Eagle's and was comparable to that in some human melanoma cell lines. minimum essential medium and Ham's F12 nutrient medium and supple The tyrosinase activity in neuroblastoma cell variants increased mented with 15% (v/v) heat-inactivated fetal bovine serum, 100 Â»Â»gof with cell density and was significantly elevated by melanocyte- streptomycin per ml, and 100 ID of penicillin per ml. Cells were refed stimulating hormone and 8-bromo-cyclic adenosine monophos- every 2 to 3 days. phate, similar to that seen in melanoma cells in culture. Thus, The clones and subclones from the human neuroblastoma cell line our findings show that human neuroblastoma cells can undergo SK-N-SH were established at Memorial Sloan-Kettering Cancer Center. bidirectional transdifferentiation in vitro between a neuronal and HA-A and HA-L, 2 melanoma cell lines, were obtained from Dr. J. Trent a melanocyte phenotype, possibly reflecting a process which (32). The melanoma cells lines SK-MEL (10) were provided by Dr. L. J. occurs in the patient. Old. F-ECH and F-ALF, 2 human fibroblast cell lines, were described previously (6, 8, 9). Enzyme Assays. Cells were collected for enzyme analysis as de INTRODUCTION scribed previously (23). Pellets were homogenized in 5 mw phosphate buffer (pH 7.4) containing Triton X-100 and an aliquot taken for protein in vivo, human neuroblastomas express multiple cellular phe determination (18) using bovine serum albumin as standard. After cen notypes. In addition to cells with neuronal characteristics, cells trifugar ion at 6000 x g for 10 min, the supernatant was decanted, and aliquots were taken for enzyme assay. with chondrocytic, Schwannian, and melanocytic phenotypes CNP3 activity was assayed by the spectrophotometric method of have been distinguished (4, 14, 27, 30). In vitro, while human Prohaska (19, 21) using 5 to 10 /tg of protein per assay. Specific activity neuroblastomas typically express neuronal properties (3, 23, 26, was expressed as ^mol of phosphate released/h/mg protein. 28, 29, 36), evidence for morphological and biochemical hetero Tyrosinase activity was assayed by the method of Hearing and Ekel geneity has been shown (2, 5,25,34). For example, recent work (16) whereby ["CJtyrosine is converted to [14C]melanin, and this product on the SK-N-SH cell line (25) has demonstrated 2 distinct types is subsequently trapped on filter paper, washed extensively, and counted of cells with a common ancestral origin, only one of which by liquid scintillation spectrometry. Specific activity is expressed as pmol expresses neuronal properties. The 2 cell types can undergo of melanin formed per h per mg protein. This assay procedure enables bidirectional coordinate morphological and biochemical intercon distinction between tyrosinase activity and activity for tyrosine hydrox- version in culture as confirmed by karyotype analysis (25). Clones ylase, the rate-limiting enzyme in norepinephrine biosynthesis, which is of these 2 cell types have been isolated. One type resembles a found in most neuroblastoma cell lines (23). The 2 primary enzymes involved in the synthesis of the neurotrans- primitive sympathoblast and contains the requisite enzymes for mitter norepinephrine, tyrosine hydroxylase and dopamine 0-hydroxyl- synthesis of norepinephrine (8, 23). The other, variant cell type ase, were assayed as described previously (23, 24). Specific enzyme is flat, is substrate adherent, and does not express activities for activity was expressed as pmol or nmol of product formed per h per mg 1Supported by Grant NS-17738 from the National Institute of Neurological and protein. Communicative Disorders and Stroke and Grants CA-31553 and CA-08748 from the National Cancer Institute and by the Kteberg Foundation. 3The abbreviations used are: CNP, 2' :3'-cyclic nudeotkte 3'-phosphohydrolase; : To whom requests for reprints should be addressed. MSH, melanocyte-stimulating hormone; DBH, dopamine-fi-hydroxylase; cAMP, Received 10/9/84; accepted 1/9/85. cyclic adenosine 3':5'-monophosphate.

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RESULTS activity. The tyrosinase activity expressed in these cells is lower but of the same order of magnitude as that found in some of the CNP Activity. To determine whether the flat, variant human human melanoma cell lines. Tyrosinase activity is not detectable neuroblastoma cells are of a Schwann Å“il lineage, a biochemical in subclones with a neuroblastic morphology (SH-SY5Y, SH- marker of this cell type, CNP, was assayed. High levels of CNP EP5, and SH-EP17). Conversely, tyrosine hydroxylase activity is activity are found in human and rat Schwann cells and rat glioma expressed by the neuroblastic cells but not by the melanoma cells grown in vitro (22, 31, 38). As seen in Table 1, clones and cells nor by the variant neuroblastoma cell clones. Neither tyro subclones of SK-N-SH expressing either a neuroblastic or a sinase nor tyrosine hydroxylase activity is detectable in the variant morphology have low levels of CNP activity, similar to fibroblast controls. that seen in human fibroblasts and muscle cells and approxi Regulation by Cell Density. To determine whether tyrosinase mately 10% of that measured in C-6 rat glioma cells or human activity in the variant neuroblastoma cells is regulated with cell Schwann cells in vitro (19, 22, 38). density, enzyme activity was measured in SH-EP1 cells at differ Tyrosinase Activity. To determine whether the neuroblas ent stages of the growth cycle. Chart 1 shows that tyrosinase toma cell variants are melanocytes, the activity of tyrosinase, an activity decreases rapidly with cell dispersal (to approximately enzyme which synthesizes melanin from L-tyrosine and is local 20% of control) and then increases during logarithmic growth ized to melanocytes and melanoma cells, was measured. Activity phase to reach stable levels of activity as the cells enter station was assayed in clones and subclones of both neuroblastoma ary growth phase on Day 14. cell types and compared with that in human melanoma cells and Regulation by MSH. Exogenous MSH increases tyrosinase fibroblasts. Tyrosine hydroxylase activity was assayed simulta activity in melanocytes and melanoma cells in culture (15, 35, neously as a marker of the noradrenergic neuronal phenotype 37). To determine whether tyrosinase activity in the melanocyte- (23). As shown in Table 2, human neuroblastoma clones with an like human neuroblastoma cells can be increased by this hor epithelial-like morphology (SH-EP1 and SH-FE) have tyrosinase mone, a-MSH in varying concentrations (0.1 to 1.0 U.M)was added to stationary cultures of SH-EP1 cells. After 3 days, cells Tabtel were collected and tyrosinase activity determined. The addition CNP activity in neuroblastomaand glioma clones and subclones of Â«-MSHcaused a dose-dependent increase in enzyme activity CNP activity in SH-EP1 cells (Chart 2). While no increase was seen with 0.1 Cell line" Phenotype 0/mol/h/mg of protein) U.Ma-MSH, 0.5 and 1.0 UM caused a 2- to 4-fold increase in 2.53 Â±0.736 SH-SY5Y Neuroblast tyrosinase activity, respectively. MSH had no effect on either the SH-EP17 Neuroblast 3.32 Â±0.49 SH-EP1 Variant 2.89 Â±0.14 cell morphology or tyrosine hydroxylase activity of neuroblast SH-FE Variant 3.37 Â±0.13 (SH-SY5Y) cells (data not shown). C-6 Glioma 34.05 Â±5.10 Regulation by cAMP. Cyclic nucleotides are mediators of the * The SH-SY5Y and SH-FE cell lines were cloned from SK-N-SH.The SH-EP1 and SH-EP17cell lines are subclones of SH-EP,a flattened, epithelial-likecell type also cloned from SK-N-SH. Note that whereas SH-EP1 retains the phenotype of 14 r- the SH-EP variant, the SH-EP17 now expresses a neuroblast phenotype. C-6 is a rat glioma cell line. Mean Â±SEin 3 cultures. 12 Table 2 Tyrosinehydroxylase and tyrosinase activities in human neuroblastoma, melanoma,and fibroblast cell lines (pmol/h/mg)Cell Enzymeactivity _ 10

type*NeuroblastomaSH-SY5Y hydroxylase6.8

(n)6SH-EP5 Â±0.1Â°1.9 (n)SH-EP17(n)SH-EP1 Â±0.40.8 Â±0.1NDNDNDNDNDNDNDNDNDND'I 6 (v)SH-FE Â±7.48.0 (v)MelanomaHA-AHA-LSK-MEL-5SK-MEL-13SK-MEL-26SK-MEL-28FibroblastF-ECHF-ALFTyrosinaseNONOND23.8Â±0.982.8

Â±10.9287.4 Â±32.3308.6 Â±36.474.6 Â±8.9ND526.9

Â±72.78NDNDTyrosine

' SH-SY5Y and SH-FE clones were derived from SK-N-SH. The SH-EP sub- 4 8 12 16 clones (SH-EP5,SH-EP17,and SH-EP1)were cloned from SH-EP,an epithelialcell Time in culture (days) variant. While SH-EP1retains the phenotype of the parent, SH-EP5and SH-EP17 have undergoneconversion to the neuroblastic phenotype. Chart 1. Time course of changes in tyrosinase activity in SH-EP1 cells. Each n, neuroblast; v, variant; ND, not detectable. value represents the mean of 3 to 4 cultures, except Day 16 which is the mean of c Mean Â±SEof 3 to 6 cultures. 2 cultures. Cells were confluent by Day 12 after plating. Bars. SE.

CANCER RESEARCH VOL. 45 APRIL 1985 1629

45 45

40- 40

35 g* l 30 IÂ» Â«t l* 3t H oÂ» r 10 10 -t

0Â«- Control Control 0.1 0.5 1.0 0.1 0.5 1.0 [oMSH] MM [CAMP]mM Chart 2. Effect of a-MSH on tyrosinase activity in SH-EP1 cells. Each value Chart 3. Effect of 8-bromo-cAMP on tyrosinase activity in SH-EP1 cells. Each represents the mean of 3 separate cultures. Bars, SE. ", P < 0.001 compared to value represents the mean of 3 to 4 cultures. **, P < 0.001 compared to untreated untreated cultures. control cultures. Bars, SE. intracellular effects of exogenous hormones. To determine 10 50 whether tyrosinase activity in the variant human neuroblastoma cells is regulated by cAMP, SH-EP1 cells were incubated for 3 days in the presence of various concentrations of 8-bromo- cAMP. Chart 3 shows that, while 0.1 mw had no effect on Ã• 8 40 tyrosinase activity, 0.5 and 1.0 mw cAMP caused a significant 3- to 5-fold increase in enzyme activity, respectively. To examine the differential effects of cAMP on cell type in SK- Â£â€¢6 N-SH clones, 1.0 mw 8-bromo-cAMP was added to cultures with 30 either neuroblast (SH-SY5Y) or melanocyte (SH-EP1 ) phenotype and tyrosinase and DBH activities measured after 3 days. Chart 4 shows that the same concentration of drug caused a 5-fold increase in tyrosinase activity in SH-EP1 cells, whereas it caused a 3-fold increase in DBH activity in SH-SY5Y cells. DBH and tyrosinase activities were not detectable in SH-EP1 or SH-SY5Y cultures, respectively, before or after treatment with cAMP. 10

DISCUSSION

Previous studies have shown that the human neuroblastoma Control l.OmMcAMP Control 1.0 mM cAMP cell line SK-N-SH comprises 2 cell types which undergo coordi SH-SY5Y SH-EP1

nate morphological and biochemical interconversion in culture Chart 4. Differential responses of SK-N-SH subctones to 8-bromo-cAMP. Each (25). One cell type resembles a primitive sympathoblast. The value represent the mean of 3 to 4 cultures. **, P < 0.001 compared to untreated present findings support the hypothesis that the second cell controls. Bars. SE. type, which is flattened and strongly substrate adherent, is a melanocyte. Not only do these variant neuroblastoma cells con (15,35), tyrosinase activity in SH-EP1 and SH-FE neuroblastoma tain activity for tyrosinase, an enzyme unique to melanocytes cell variants is regulated by cell density and cAMP. From these and melanoma cells, but the enzyme activity is increased by and previous biochemical studies, in conjunction with recent addition of MSH to the culture medium. As in melanoma cells electron microscopic studies showing the presence of melano-

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. MELANOCYTE PHENOTYPE IN HUMAN NEUROBLASTOMA CELLS somes in SH-EP1 cells (33), we conclude that, within the SK-N- REFERENCES SH cell line, there is spontaneous and bidirectional transdifferentiation between neuroblast and melanocyte cell types. 1. Anderson, B., and Robertson, D. M. Melanin containing neurofibroma: case report with evidenceof Schwann cell origin of melanin.Can. J. Neural. Sci., 6: While the expression of a melanocyte phenotype and/or 139-143,1979. melanogenesis has been reported in Schwann cells and gangli- 2. Aubert, C., Janiaud,P., Rouge, F., Hansson,C., Rorsman, H., and Rosengren, E. Melanogenesis in cultured human neuroblastomas. Ann. Clin. Res., 72: oneuroblastoma in vivo and in primary cultures of avian neural 288-294, 1980. crest and human neuroblastoma tumors (1,2,4,11,14,20), the 3. Barnes,E. N., Biedler,J. L., Spengler,B. A., and Lyser, K. M. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. Presence and Regulation of Tyrosinase Activity in Human Neuroblastoma Cell Variants in Vitro

Robert A. Ross and June L. Biedler

Cancer Res 1985;45:1628-1632.

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