phenotypic diversification in human neuroblastoma cells ... · arises from the neural crest....

[CANCER RESEARCH 49. 219-225. January 1. 1989]

Phenotypic Diversification in Human Neuroblastoma Cells: Expression of DistinctNeural Crest Lineages1

Valentina Ciccarone,2 Barbara A. Spengler, Marian B. Meyers, June L. Biedler, and Robert A. Ross3

Laboratory of Neurobiology, Department of Biological Sciences, Fordham University, Bronx, New York 10458 Â¡V.C., R. A. R.Â¡,and Laboratory of Cellular andBiochemical Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [B. A. S., M. B. M., J. L. B.J

ABSTRACT

Previous studies of the human neuroblastoma cell line SK-N-SH haddemonstrated the presence of and phenotypic interconversion (transdif-

ferentiation) between two morphologically and biochemically distinct celltypes: N (neuroblastic) cells with properties of noradrenergic neuronsand S (substrate-adherent) cells with properties of melanocytes. Current

studies have sought to test the generality of these findings among othercultured human neuroblastoma cell lines and to define further the S-cell

phenotype and that of a newly identified, morphologically intermediate,I-type cell. Morphologically homogeneous populations (clonal sublines

or subpopulations) of N, S, and I cells were isolated from five additionalneuroblastoma cell lines and analyzed biochemically for neuronal, glial,and melanocytic marker enzyme activities and norepinephrine uptake.Immunoblot techniques were used to detect intermediate filament proteins(neurofilament protein, vimentin, glial fibrillar}' acidic protein) and fibro-

nectin. All N-type cells exhibited neuronal marker enzyme activities,

specific uptake of norepinephrine, and presence of one or more neurofilament proteins. S-type cells generally lacked neuronal characteristics but

contained, instead, tyrosinase activity (a melanocytic marker enzyme),vimentin, and fibronectin. This combination of attributes is suggestive ofa multipoli-m embryonal precursor cell of the neural crest. I-type cellsdifferentially expressed both S- and N-cell properties and could represent

either a stem cell or an intermediate in the transdifferentiation process.Studies of the biological significance of human neuroblastoma cell trans-

differentiation and the molecular mechanisms underlying this processmay be of relevance to the biological and clinical behavior of this tumorin the patient.

INTRODUCTION

Human neuroblastoma is a cancer of early childhood andarises from the neural crest. Histopathological examination ofpatients' tumors has revealed the presence in neuroblastomas

of a variety of neural crest cell types, ranging from neuroblaststo melanocytes, glial cells, and chondrocytes (1-3). Cell linesestablished from several neuroblastoma tumors have shown asimilar diversity in cell morphology (4-6). From one such cellline, SK-N-SH, clonal sublines comprising one or the other oftwo distinct cell types have been isolated and extensively characterized (5, 7-12). One cell type, termed "N," is neuroblastic

in appearance, with a small, rounded, loosely adherent cell bodyand numerous neurite-like processes, and contains noradrenergic biosynthetic enzymes as well as an uptake mechanism fornorepinephrine. N cells also express opioid, muscarinic, andnerve growth factor receptors (10, 13). The second cell type,termed "S," is larger and flattened, resembling epithelial orfibroblast cells, and is highly substrate-adherent. The latter cells

Received 5/25/88; revised 9/13/88; accepted 9/29/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grants NS-17738 (NINCDS) and CA-31553 and CA-

08748 (NCI) from the National Institutes of Health and by the Kleberg Foundation.

2A Shering-Plough Fellow. Present address: Laboratory of Immunoregulation,

Biological Response Modifiers Program, Frederick Cancer Research Facility,National Cancer Institute. Frederick, MD 21701.

3To whom requests for reprints should be addressed, al Department of

Biological Sciences, Fordham University. Bathgate and Fordham Road. Bronx.NY 10458.

do not exhibit neuronal properties (neurotransmitter biosynthetic enzyme activities, catecholamine uptake, or receptorproteins) but have properties of melanocytic, Schwannian, and/or meningea! cell types, e.g., tyrosinase activity specific formelanocytes (8, 14), stromal (types I and III) collagens indicative of glial or Schwann cells (11, 15-18), and cell surface

antigen expression resembling that of meningeal cells (10).Cytogenetic and cloning studies have shown that both morphological cell types are descended from a common precursor celland both are capable of spontaneous interconversion to theother cell type (7). This bidirectional conversion between twowell-defined, differentiation lineages of the neural crest we havetermed "transdifferentiation."

Studies of clones of SK-N-SH identified the process of trans-

differentiation (7, 8, 19). The present study seeks to determinethe generality of this phenotypic diversity among human neuroblastoma cell lines in culture. The variety of attributes reportedso far for the S-type cells (10-12, 16, 17) underscores thedesirability of obtaining S-cell populations from additionalneuroblastoma cell lines to better determine their differentiation phenotype. Therefore, in the present study, N- and S-typeclones or populations were isolated from five different neuroblastoma cell lines. A newly identified, third cell type, termed"I," with a morphology intermediate between that of N and S

cells, was also cloned from two of these lines and from SK-N-SH. All sublines were characterized biochemically for markerenzymes specific for neuronal, melanocytic, and Schwann/glialcells: tyrosine hydroxylase and dopamine-^-hydroxylase (neuronal), tyrosinase (melanocytic), and CNP4 (Schwann or glial

cells) (14, 20-22). In addition, we measured the ability of cellsto specifically take up norepinephrine.

Other indicators of differentiation lineage are intermediatefilament and extracellular matrix proteins. Intermediate filament proteins are cell- and tissue-specific components of thecytoskeleton which appear early in development, are usuallyretained by cells in culture, and change in both type and amountduring cell differentiation (23-26). Neuronal cells (neurons andneuroblasts) contain neurofilaments composed of three relatedpolypeptides: a core protein (M, 68,000) and proteins (M,150,000 and 200,000) which attach to the core protein andarise later in development (27). By contrast, melanocytes,Schwann cells, and mesenchymal cells (such as fibroblasts andimmature glial cells) contain the intermediate filament proteinvimentin (25, 28), whereas mature astrocytes and glial cellsspecifically express GFAP (29, 30). Assay for production ofextracellular matrix proteins, particularly by S cells, was suggested both by the flatness and adhesiveness of S cells and byresults of DeClerck et al. (11), demonstrating production oflarge amounts of stromal collagens (isotypes I and III) by S-type cells, and of Alitalo et al. (31) and Scarpa et al. (18), whoshowed that uncloned neuroblastoma cell lines synthesize fibronectin in culture. Thus, we assayed representative N, S, and Ipopulations immunochemically for presence of tissue-specific

4 The abbreviations used are: CNP, 2'.3'-cyclic nucleotide 3'-phosphohydro-

lase: GFAP. glial fibrillar) acidic protein: PBS. phosphate buffered saline.

219

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PHENOTYPIC DIVERSIFICATION IN HUMAN NEUROBLASTOMA

intermediate filament proteins and of the extracellular matrixprotein, fibronectin, in an attempt to understand more clearlythe nature of both S- and I-type cells. Our results have demonstrated that transdifferentiation is a prevalent phenomenonamong human neuroblastoma cell lines in culture.

MATERIALS AND METHODS

Cells and Culture Methods. Human neuroblastoma cell lines weregrown in plastic tissue culture flasks or dishes in a 1:1 mixture ofEagle's minimum essential medium with nonessential amino acids andHam's nutrient mixture F-12 supplemented with 15% heat-inactivated

fetal bovine serum. Cells were removed from the substrate by incubationwith either divalent cation-free PBS or 0.02% EDTA, 0.12% trypsin inPBS.

Clones were isolated in 96-well cluster dishes by a limiting dilutionmethod. Since there is a naturally occurring interconversion betweenN- and S-cell types (7) and since S-cells from some lines had a lowplating efficiency and limited growth capacity in vitro, serial subcloningand/or differential culturing techniques were used in maintenance orselection of the homogeneous cell populations imperative for this typeof study. Culturing techniques for N-cell populations included growthto high cell densities, split ratios of 1:50 or greater, and removal fromthe substrate with PBS alone or with 0.12% trypsin in PBS. Populationsof S-type cells were isolated and maintained by selective removal of lessadherent N-type cells from the cultures with strong agitation or tryp-sinization. The tightly adherent S cells were subcultured with 0.02%EDTA, 0.12% trypsin in PBS. Clones and purified populations used inthese studies are listed in Table 1.

Enzyme Analysis. Cells for enzyme assay were collected as previouslydescribed (22) and frozen until assay. Pellets were homogenized in 5mM potassium phosphate buffer (pH 7.4) containing 0.2% Triton X-100. After removal of an aliquot for protein determination (32) andcentrifugation at 6000 x g for 10 min, the supernatant was decantedfor assay. Tyrosine hydroxylase was assayed by the method of Coyle(33), dopamine-/3-hydroxylase by the method of Molinoff et al. (34),lyresinase by the procedure of Hearing and Ekel (14), and CNP by themethod of Prohaska et al. (21).

Norepinephrine Uptake. The specific cellular uptake of norepineph-rine was determined by the method of Silberstein et al. (35). Briefly,neuroblastoma cells in late exponential growth phase in 35-mm culturedishes were incubated with 0.1 nmol of [3H]norepinephrine for l h at37Â°C.After three washes with Hanks' balanced salt solution, cells were

homogenized and counted by liquid scintillation spectrometry. Uptakeis expressed as cpm/mg protein.

Intermediate Filament Protein and Fibronectin Analysis. Cells in lateexponential growth phase in 100-nim plastic tissue culture dishes wererinsed twice with cold PBS and scraped from the substrate. Fractionsenriched for cytoskeletal proteins were prepared as described by Hynesand Destree (36) and stored at -70Â°C. For fibronectin analysis, each

cell culture with its extracellular matrix was harvested nonenzymati-cally. Conditioned medium was collected from N or S cells grown inserum-free medium for 24 h and proteins precipitated with 10% tri-chloroacetic acid. Protein samples were separated on 7.5% polyacryl-amide gels (37) and transferred to nitrocellulose (38). The electropho-retic blots were blocked for l h in 5% powdered nonfat dry milk (39)in 100 mM Tris/HCl, pH 7.4, then incubated overnight or for 2 h withantibodies directed against specific intermediate filaments or fibronectin. Bound antibody was detected with peroxidase-labeled secondaryantibody (Cappell) and 4-chloro-l-naphthol (40). Antibodies used were:monoclonal antibody NPB to human M, 200,000 and 150,000 neuro-filament subunits and NP7 to human GFAP obtained from Dr. P.Davies, rabbit polyconal antibody 70-C to the rat M, 68,000 neurofil-ament subunit from Dr. F. C. ChiÃ¹,and monoclonal antibody to humanvimentin from Boehringer Mannheim Co., and monoclonal antibodyMICIO to fibronectin from Dr. K. Lloyd.

RESULTS

Prevalence of Divergent Morphologies. In a panel of 14 human neuroblastoma cell lines, neuroblastic (N-type) cells con

stituted the predominant cell type; however, cells with a flattened, S-type morphology were detectable in 10 cell lines [SK-N-SH, SK-N-BE(l), SK-N-BE(2), LA-N-1, IMR-32, SMS-SAN, CHP-234, SMS-KCN, SMS-KCNR, and NAP] established from eight patients and have been isolated from six ofthese lines (Table 1). No S-type cells were seen in the otherfour human neuroblastoma cell lines (SMS-MSN, SMS-KAN,SMS-KANR, and LA-N-2). In four lines [SK-N-SH, LA-N-1,SMS-KCN, and SK-N-BE(2)], S cells constituted a major portion of early-passage cultures and could be readily isolated. Intwo other lines, NAP and SK-N-BE(l), S-type cells were lessapparent and relatively few in number. A third type (I) cell wasalso isolated from the SK-N-SH, LA-N-1, and SK-N-BE(2)lines. For all but the clonal sublines of SK-N-SH and LA-N-1,S-type cell populations ceased to proliferate or grew extremelyslowly after only a few subcultivations. By contrast, all clonallyderived N- or I-type cells were immortal. All clones werekaryotyped with the exception of three S cell sublines whereadequate number of mitotic cells were not obtained. Presenceof consistent chromosome markers indicated origin from acommon precursor cell of N, S, and I cells cloned from any oneneuroblastoma cell line.5

In all instances, designation of clones as N, S, or I was basedsolely on morphology. Two criteria were used to assign clonesto morphological groups: (a) cell size, nuclear/cytoplasmic volume ratio, and adhesiveness, and (/)) presence, number, andlength of cellular (presumably neuritic) processes. N cells typically are small and loosely adherent, with a large nucleus/cytoplasm ratio and multiple, moderately long cell processes. Scells are larger and strongly adherent, with abundant cytoplasmand no processes (Fig. 1). Small, flattened, moderately adherentcells with relatively scant cytoplasm and a variable display ofcell processes are classed as I cells.

N-type clones showed morphological similarity in culture

Table 1 Derivation of human neuroblastoma sublines

Parental cell SublinelinedesignationSK-N-SH

SH-SY5YSH-EPSH-EP1SH-EP1ESH-INLA-N-1

LAl-15nLAl-19nLAl-21nLAI

-5sLAI-6sLAl-22n/iSK-N-BE(2)

BE(2)-M17BE(2)-M17VBE(2)-M17FBE(2)-M17MBE(2)-7sBE(2)-CSK-N-BE(l)

SK-N-BE(l)nSK-N-BE(l)sSMS-KCN

KCN-62nKCN-65nKCN-71nKCN-83nKCN-9SNAP

NAP(H)nNAP(H)s"

Homogeneous population derivedCell

typeNSSSINNNSSN/INNSISINSNNNNSNSby

culturingDescriptionThrice

clonedClonalsublineTwiceclonedThriceclonedClonalsublineClonal

sublineClonalsublineClonalsublineClonalsublineClonalsublineClonalsublineTwice

clonedThriceclonedThriceclonedThriceclonedClonalsublineClonalsublinePurified

population"Purifiedpopulation"Clonal

sublineClonalsublineClonalsublineClonalsublineClonalsublinePurified

populationPurifiedpopulationtechniques.

See "Materialsand Methods."

' B. A. Spengler and J. L. Biedler, unpublished results.

220




Fig. I. Phase contrast photomicrographsof N and S-cells. A, KCN-65n; fi, SK-N-BE(l)n; C, NAP(H)n; D, KCN-9s; E, SK-N-BE(l)s; and F, LA-N-ls. Dense cultures of N-cells generally contained large aggregates ofcells with radiating neurite-like processes(pseudoganglia) (A and C), whereas S cellsgrew as monolayers (D-F). Quite variable inshape and size. S-cells also occasionally exhibited extensive formation of stress fibers (F).Magnification, 250x.

Fig. 2. Phase contrast photonof I cells. A, SH-IN; B, BE(2)-C; Ci. Magnification, 250x.

(Fig. 1,/i-C), differing mainly in length and number of neurite-like processes and degree of adhesiveness to the surface (rangingfrom none to slight). S cells showed a greater difference in cellmorphology among clones (Fig. 1, D-F). These cells, by definition highly substrate adhesive, ranged in appearance from

fibroblast-like, growing in swirling arrays, to epithelioid withno apparent directional orientation. As a group, the moderatelyadherent I-type cells were likewise heterogeneous, with noprocesses (SH-IN), occasional long processes [BE(2)-C], ornumerous long cell processes (LAl-22n/i) (Fig. 2).

221




Table 2 Biochemical characteristics of human neuroblastoma cell variants

CelltypeN1SGliomaMelanomaÂ°

Abbreviations: THClone

orlinetestedLAl-15nLAl-19nLAl-21nKCN-62nKCN-65nKCN-71nKCN-83nSH-SY5YBE(2)-M17NAP(H)nSH-INBE(2)-CLAl-22n/iLAI

-5sLAI-6sKCN-9SSH-EP

1BE(2)-7sNAP(H)sC-6MeWo,

tyrosine hydroxylase:TH"

(pmol/h/mg)322

Â±31'17

+534Â±9257

Â±10331Â±36483

Â±85184Â±197Â±

12331Â±414760

Â±3456+41673Â±29500000000DÃŸH

(nmol/NEuptakeh/mg)(cpm/mg xIO3)0.614.411.245.4

+0.31.6Â±0.25.0

Â±0.210.6+1.812.8Â±0.50.9

Â±0.30000012.1

Â±1.525.3+0.521.6+3.942.0

Â±1.5234.2

+36.4324.7Â±6.622.5Â±1.834.5

Â±3.08.2+2.904.3

Â±0.31.9Â±0.4003.80.9Tyrosinase(pmol/h/mg)tf0000000001.4

Â±0.501.0

Â±0.61.2+0.66.5016.6

Â±1.2CNP

Cimol,h/mg)6.2

Â±1.88.9+2.49.3

Â±0.27.4

Â±1.45.9+1.55.9

+1.54.5Â±0.65.7

Â±0.96.7Â±2.47.8+2.12.8Â±0.55.7

Â±0.17.1Â±0.91.8

+0.614.534.0

Â±3.616.2Â±1.21D0H.

dopamine-/3-hydroxylase; NE,norepinephrine.*Values, mean + SE of three to eight cultures or the average of twocultures.'0, value less than two timesblank.Phenotype

NMrkD200-116-97-66-SINS"1 N S1â€”Brain PhenotypeMrkD200-118-97-^

66-N

S 1N S 1-

.N

S1â€”

â€¢ â€”BrainÃŽ"

45- 45-

Lane 1 6 7 8 9 10 Lane 1 8 10

Fig. 3. Immunoblot analysis of expression of the M, 68,000 neurofilamentprotein subunit by N, S, and I clones derived from three neuroblastoma cell lines.Lanes and cell lines: /, SH-SY5Y; 2, SH-EP; 3, SH-IN; 4, LAl-19n; 5, LAl-5s;6, LAl-22n/i; 7, BE(2)-M 17V; 8. BE(2)M 17F; 9. BE(2)-M17M; 10, human braincontrol.

Marker Enzyme Analysis. All of the N-type and I-type clonescontained activity for tyrosine hydroxylase and/or dopamine-0-hydroxylase and showed specific uptake of norepinephrine(Table 2). By contrast, neither of these characteristics waspresent in the S-type clones.

Marker enzymes for melanocytic and glial cell types weremeasured in N, I, and S clones from four human neuroblastomacell lines and compared to those in rat glioma (C-6) and humanmelanoma cells (MeWo) (Table 2). While tyrosinase activitywas undetectable in N- and I-type clones, four of the five S-type clones assayed contained low levels of this enzyme whencompared to melanoma cells. There were no significant differences in the levels of CNP activity in N, I, and S clones. Allbut one S clone had low levels of CNP activity, less than 20%of that found in rat C-6 glioma cells. One clone, BE(2)-7s, hadan average CNP activity of 14.5 ^mol/h/mg protein, similar tothat measured for the human melanoma cell line, MeWo.

Intermediate Filament Proteins. We examined cytoskeleton-enriched fractions from N, I, and S human neuroblastoma cell

Fig. 4. Immunoblot analysis of expression of the M, 150,000 and 200.000neurofilament protein subunits. Cell lines and lanes are identical to those in Fig.3.

Table 3 Intermediate filament expression in human neuroblastoma clones

CelltypeNISCloneor line

testedSH-SY5YSK-N-BE(l)nBE(2)-M17LAl-19nNAP(H)nKCN-71nIMR-32SH-INBE(2)-CLAl-22n/iSH-EPSK-N-BE(l)sBE(2)-M17FLAI

-5sNAP(H)sKCN-9SNeurofilament

protein(M,)68,000

150,000 200,000 VimentinGFAP+

+ Â±--+

+ +Â±++ â€” â€”â€”-+ Â±-++

+++ â€”+++ â€”++-1- â€”+__ _ +_+

â€”__ _ +___ _ +_-

+--

clones for presence of proteins of three intermediate filaments:neurofilament proteins (M, 200,000, 150,000 and 68,000), vi-mentin, and GFAP.

Immunoblot analysis of the M, 68,000 core neurofilament222




PhenotypeMrkD

200-

116-

97-

66-

45-

I N N

Lane 1 8Fig. 5. Vimentin synthesis in N, S, and I clones analyzed by immunoblolting.

Lanes and cell lines: /, SH-SY5Y; 2, SH-EP; 3, SH-IN; 4, LAl-19n; 5. LAl-Ss;6, LAl-22n/i; 7, BE(2)-M17V; 8, BE(2)-MI7F; 9, BE(2)-MI7M.

Table 4 Fibronectin synthesis in human neuroblastoma cells

CelltypeNSSublineSH-SY5YSK-N-BE(l)nLAl-15nNAP(H)nSH-EPSK-N-BE(I)sLAI

-5sNAP(H)sFibronectinCell/matrix

Mediumâ€”â€”--â€”â€”â€”+

+++

++

stemcell

Fig. 6. Models of human neuroblastoma cell transdifferentiation. Transdiffer-entiation occurs by (A) a highly coordinated switch from an N to an S program(or vice versa) or (B) a multistep, loosely coordinated programatic switch whichproduces I-type cells as transient intermediates. Alternatively (( '). I cells are stem

cell precursors from which N and S cells arise. Interconversion may occur directlybetween N and S. It may also occur via the stem cell (not shown). Both N and Scells may be capable of further differentiation to mature states associated withloss of proliferative capacity.

protein (Fig. 3) revealed that this protein is present in three ofthe four N-type clones and in all three of the I-type clonesexamined. Similarly, all of the N and I clones contained the M,150,000 neurofilament protein (Fig. 4). However, only three ofthe seven N and I clones expressed detectable M, 200,000

neurofilament protein. None of these proteins was expressed inS-type clones (Figs. 3 and 4 and Table 3).

Vimentin was examined by immunoblot analysis in clonesfrom six human neuroblastoma cell lines (Table 3). As shownin Fig. 5. S-type clones expressed large amounts of vimentin,whereas their N-type counterparts contained none or very littleof this protein. I-type cells also expressed vimentin. Indirectimmunofluorescence of vimentin in S-type cells revealed anextensive network of cytoplasmic and perinuclear fibrils. Incontrast, in those N cells which contained small amounts ofvimentin, reactivity was localized only to the perinuclear region(data not shown).

GFAP is an intermediate filament protein of unknown function but specific to mature glial cells. GFAP content wasassessed in N and S clones from five different human neuroblastoma cell lines and was present in neither cell type (Table3) when compared to a positive glioma cell line (U251 astro-cytoma) (data not shown).

Fibronectin. We next sought to determine whether there isdifferential expression of fibronectin in S- and N-cell types inhuman neuroblastoma cells. The amount of cell-associated fibronectin (in the cell and extracellular matrix) and fibronectinsecreted into the medium was determined by immunoblot analysis of four human neuroblastoma N and S pairs. Whereas thisprotein was undetectable in cultures of N-type clones, it wasdetectable in both compartments of S-type clones (Table 4).

DISCUSSION

Human neuroblastoma, one of the most common childhoodsolid tumors, is thought to arise from neural crest cells duringembryonic development. In general, this tumor has proven veryrefractory to conventional radiation and chemotherapy, possibly related to the biological plasticity of the cells comprisingthis tumor (1-3, 10, 41,42). Conversely, in some instances, thetumors may also spontaneously regress, maturing into benignganglioneuromas (2, 43). An important question is whethercellular plasticity plays a crucial role in the regression of suchtumors in the patient.

In the present study, cells cloned or purified from six differenthuman neuroblastoma cell lines initially showed one of threedistinct morphologies: neuroblastic (N), flat and substrate-adherent (S), or morphologically (and biochemically) intermediate(I). Occurrence of these clearly identifiable morphological subtypes was prevalent among the independently derived humanneuroblastoma cell lines studied here. Each distinct morphologyalso had a distinct, characteristic biochemical phenotype. Two-dimensional gel electrophoresis of metabolically labeled proteins from N and S cells revealed cell-specific protein patternsindicative of an underlying similarity among cells displayingsimilar differentiation phenotypes (9).6 Thus, an importantfinding of this study is that three different cell types, each withdistinct morphological and biochemical features, may arise inneuroblastoma cell lines in culture.

All 11 newly derived N-type clones have properties of nor-adrenergic neuroblasts similar to those observed in SK-N-SHsublines (7, 12). In addition to containing the biosyntheticenzymes and an uptake mechanism for norepinephrine, all ofthe N-type clones assayed (six out of seven) contained the coreM, 68,000 and the M, 150,000 neurofilament proteins. Sincethe larger neurofilament subunits arise later in embryonic development (44-46), differential expression of the M, 200,000

* M. B. Meyers and J. L. Biedler, unpublished results.

223




neurofilament protein suggests that different neuroblastomasmay arise at different stages of embryonic or perinatal development.

Identification of the S-cell phenotype was a major focus ofthe studies reported here. These cells were found to containproteins specific to melanocyte and glial cell lineages of neuralcrest: S cells synthesize fibronectin, a property of glial,Schwann, and melanocytic cells (18,47,48) and S cells producevimentin but not GFAP. Immature, motile glial cells producevimentin whereas mature glial cells cease to express this intermediate filament and begin to produce GFAP (28, 29). Thus,the presence of vimentin and the absence of GFAP in S cells isconsistent with a proposed embryonic glial phenotype.

Other studies of S-type cell populations, derived from manyof the same cell lines studied here, have placed S cells in variousother (Schwannian, meningea!) neural crest lineages. For example, studies by Rettig et al. (10) demonstrated that S cellsexpress cell surface proteins unique to embryonic meningealcells. On the other hand, DeClerck et al. (11) showed that S-type clones of SK-N-SH and LA-N-1 synthesize stremai colla-gens (types I and III) similar to Schwann cells. These seeminglydiscordant findings can be resolved by suggesting that the S-type cell represents an embryonic neural crest precursor cellwhich is multipotent and capable of some degree of differentiation towards a glial, meningeal, or melanocytic phenotype (49,50). Studies of the avian neural crest and of sensory ganglia inculture have demonstrated their ability to give rise to eitherglial or melanocytic cells, and such studies led Weston andcoworkers to suggest that glia and melanocytes arise from acommon, partially restricted, progenitor cell (49-52). It remains to be determined whether S cells from human neuroblastoma can be induced to undergo further maturation alongeither a glial or a melanocytic pathway or whether they arerestricted to an embryonic stage of differentiation owing totheir transformed nature.

In light of the above considerations, the occurrence of I cellsin neuroblastoma cell cultures is of considerable interest. Thesecells have characteristics of both N and S cells. I cells aremoderately substrate adherent and contain vimentin as do S-cells, but also frequently have small numbers of neurite-likeprocesses and contain neurotransmitter enzymes and neurofil-ament proteins as do N cells. A similar, but transient, expression of both neuroblastic and melanocytic properties has beenobserved in individual avian neural crest cells grown on fibro-blast-derived extracellular matrix (51), suggesting that extrinsicfactors can influence phenotypic conversion of neural crest-derived cells. Thus, I-type cells may represent a cellular intermediate in the N- and S-cell transdifferentiation process inwhich expression of multiple phenotypes may be extrinsicallyas well as intrinsically regulated (Fig. 6B). Alternatively, the Icell could represent a stem cell from which both N and S cellsarise (Fig. 6C). The consequent N and S cells could then becapable of direct interconversion (Fig. 6, A and C). Additionalexperiments will be necessary to characterize the role of the I-type cell in transdifferentiation.

Our various findings suggest that N- and S- (and possibly I)-cell types remain multipotent and capable of differentiating intoone of the other two cell types and that this is a highly regulatedand coordinated process. However, the limited growth potentialof many of the S-cell populations also suggests that S-cells, atleast, may undergo terminal differentiation. In support of thispossibility are recent observations that S-type cells will neithergrow in soft agar nor form tumors in nude mice (53). Thebiological implications of the phenotypic interconversion phe

nomenon and the molecular mechanisms underlying transdifferentiation are subjects of current investigation. The transdifferentiation process that we have described here could be involved in both tumor regression and relapse (progression)observed in patients with neuroblastoma. Further study ofmechanisms and consequences of transdifferentiation of humanneuroblastoma cells in culture will prove useful in understanding the enigmatic nature of this childhood cancer.

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phenotypic diversification in human neuroblastoma cells ... · arises from the neural crest....

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