multiple neurotransmitter synthesis by human neuroblastoma … · multiple neurotransmitter...

[CANCER RESEARCH 38, 3751-3757, November 1978]0008-5472/78/0038-0000$02.00

Multiple Neurotransmitter Synthesis by Human Neuroblastoma Cell

Lines and ClonesJune L. Biedler1, Suzanne Roffler-Tarlov,2 Melitta Schachner,3 and Lewis S. Freedman

Laboratory of Cellular and Biochemical Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [J. L. B.Â¡;Department ofNeuroscience, Children's Hospital Medical Center, Boston, Massachusetts 02115 [S. R.-T., U. S.Â¡;and Department of Neurology, Division of Behavioral

Neurology, New York University Medical Center, New York, New York 10016[L. S. F.Â¡

ABSTRACT

Continuously cultured human neuroblastoma cell linesSK-N-SH, SK-N-BE(1), SK-N-BE(2), and SK-N-MC, as well

as several clonally derived sublines, were assessed fortheir neurotransmitter characteristics. Two different methods were used: measurement of cholinergic and adrener-

gic enzyme activities and detection of neurotransmitterssynthesized from radioactive precursors. Dopamine-/3-hy-

droxylase (EC 1.14.2.1), choline acetyltransferase (EC2.3.1.6), acetylcholinesterase (EC 3.1.1.7), and butyryl-

cholinesterase (EC 3.1.1.8) levels were compared to thoseof various normal and neuronal cell controls. The capacityof neuroblastoma lines and clones to convert radioactivetyrosine, choline, and glutamate to transmitter productswas determined. SK-N-SH, SK-N-BE(1), and SK-N-BE(2)cells had moderately high levels of dopamine-0-hydroxyl-ase activity. Whereas neuroblast-like clones of SK-N-SHhad similar levels of this enzyme, epithelial-like cloneshad no detectable activity. SK-N-BE(2) cells showed the

greatest capacity to convert tyrosine to dopamine. Although the SK-N-SH line itself was not tested, both neu-roblast and epithelial-like clones, shown by karyotype

analysis to have a common cell precursor, convertedtyrosine to dopamine. In contrast, the SK-N-MC line and

its clones appeared devoid of adrenergic properties. Allcells tested except human fibroblast-like cells of normalorigin were able to convert radioactive choline to acetyl-

choline. While all the cell lines had measurable cholineacetyltransferase activity, levels were substantiallyhigher (at least 4- to 12-fold) for SK-N-MC cells and itsclonally derived subline MC-IXC. All cells tested converted glutamate to y-aminobutyric acid. Results indicatethat these human neuroblastoma cell lines have predominating neurotransmitter characteristics that are sometimes, but not always, expressed in clones derived fromthem. Since the SK-N-BE(1) and SK-N-BE(2) lines were

isolated 5 months apart from the same patient and havesimilar activity levels of dopamine-ÃŸ-hydroxylase, these

characteristics may be stable and representative of thetumor cells in vivo. Furthermore, results obtained by thetwo different methodological approaches suggest that,whereas SK-N-MC cells show cholinergic and not adrenergic traits, SK-N-SH and possibly SK-N-BE(2) may be mul-

tipotential with regard to neuronal enzyme expression.

1 Recipient of NIH Grants CA-08748 and CA-18856. To whom requests for

reprints should be addressed.2 Recipient of NIH Grants NS-09704 and NS-12200.3 Recipient of NIH Grant NS-12659-O1A1. Present address:: Department of

Neurobiology, Heidelberg University, Im Neuenheimer Feld 347, 69 Heidelberg, GFR.

Received February 16,1978; accepted August 18,1978.

INTRODUCTION

In recent years continuous lines of human neuroblastomacells in culture have become available for studies relatingto tumor cell growth and neuronal cell biology. A remarkable feature of the established neuroblastoma lines described thus far is the expression of biochemical propertiescharacteristic of normal neuronal cells in vivo. For example,enzyme activities associated with neurotransmitter biosynthesis have been detected in approximately 10 independently derived cell lines (3, 7, 8, 10, 18, 19, 22). Among themany unresolved questions concerning neuroblastoma arewhether and to what extent an individual tumor consists ofa mixture of functionally differing neuronal phenotypes andwhether and to what extent a single neuroblastoma cellmay have the potential for synthesis of more than onetransmitter. Results of several combined clinical and biochemical studies suggest that the cells established in culture reflect the characteristics of the tumor from which theywere derived (4, 21). Although karyotype analysis of thehuman cell lines has revealed the presence of consistentmarker chromosomes (4, 6, 7, 19, 21) suggestive of clonalorigin of individual neuroblastomas, the possibility of functional diversity and multipotentiality of the tumor cells invivo cannot be explored by studying the tumor cell population as a whole. To obtain evidence regarding functionalproperties as well as phenotypic stability of human neuroblastoma cells, we isolated clones from the neuroblastomalines SK-N-SH and SK-N-MC and began their characterization. These 2 parental lines were compared to a hithertouncharacterized set of neuroblastoma lines, SK-N-BE(1)and SK-N-BE(2), isolated at different times from the samepatient and possessing several biological and biochemicalfeatures in common. In this report we describe findingsobtained by measurement of cholinergic and adrenergicenzyme activities and by detection of neurotransmitterssynthesized from radioactive precursors.

MATERIALS AND METHODS

Neuroblastoma Cell Lines and Clones. Cell line SK-N-SH, established in culture in December 1970 from a bonemarrow biopsy, was previously described (4). The linecomprises 2 morphologically distinct cell types, one neuro-blastic and the other epithelial-like. Clones representingboth types were recently isolated and are described in Table1. Cell line SK-N-BE(1) was established in vitro in June 1972from a bone marrow biopsy. A subsequent marrow biopsyin November 1972, from the same patient, yielded a secondcontinuous line designated SK-N-BE(2). The latter has beendescribed briefly (7). Cell line SK-N-MC, established in

NOVEMBER 1978 3751

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J. L. Biedler et al.

September 1971 from a metastatic tumor mass, and thetwice cloned derivatives MC-IIE and MC-IXC have beendescribed (4). The source of the human fibroblast-like cellpopulations, F-ECH and F-LSO, has been given (4). F-ALF

was established more recently from histologically normalendometrial tissue and is also fibroblast-like in appearance.

All of the human neuroblastoma and nonneuronal controlcell lines used in these studies are listed in Table 1.

Cell lines were grown in Eagle's minimum essential medium supplemented with nonessential amino acids (Eagle's

formulation), 15% fetal bovine serum, penicillin (100 ID/ml),and streptomycin (100 Â¿ig/ml). Cell lines were maintainedroutinely in 25-sq cm tissue culture flasks (Falcon Plastics

Co., Oxnard, Calif.). For isolation of clones, 2 methodswere used. By the first method 100-mm plastic tissue

culture dishes (Falcon) were seeded with 200 cells/dish.Colonies were isolated with the use of metal cylinders.These populations were subcloned as soon as feasible andgave rise to the lines SH-SY5, SH-SY5Y, MC-IIE, and MC-IXC. The other clonal lines, SH-IN, SH-EP, and SH-FE, were

isolated as colonies after seeding microscopically verifiedsingle cells in individual 0.4-ml wells in Micro Test II Plates

(Falcon).Population-doubling Time, Saturation Density, and Het-

erotransplantability Determinations. Estimates of growthrate/'n vitro and maximum cell density attained in stationary

growth phase were based on cell counts of replicate cellcultures. Plastic tissue culture dishes (60 mm; Falcon) wereseeded with 0.2 x 106 or 4 x 106 cells in 6 ml of growth

medium per plate. If cells were adequately adherent, celllayers were rinsed once with PBS.4 Cells were removedfrom the substrate with 1 ml of a trypsin (0.12%)-EDTA(0.02%) solution, resuspended in 9 ml of serum-containinggrowth medium, and pipetted to obtain single-cell suspen

sions as viewed microscopically. If cells were loosely adherent or floating, the supernatant medium was removed andcentrifuged at 1000 rpm. The pelleted cells were resuspended and pooled with those removed from the dish byEDTA-trypsin. For these experiments as well as for cloning,cells were incubated in a water-jacketed C02 incubator

(National Appliance Co., Portland, Oreg.) in an atmosphereof approximately 5% CO2 in air at 37Â°.Medium was replaced

twice per week. In each experiment, duplicate plates werecounted 3 to 5 times/week with the aid of a Model F CoulterCounter (threshold, 13.5; aperture current, 32; attenuation,2; Coulter Electronics Inc., Hialeah, Fla.).

For determination of tumor-producing capacity, cellswere inoculated into both cheek pouches of 19- to 22-day-

old female, weanling golden Syrian hamsters. Animals weregiven s.c. injections of 2.5 mg of cortisone acetate at thetime of cell inoculation and 2 times/week thereafter. Cheekpouches were examined once per week and tumors weremeasured. Criteria for "positive" tumor growth have been

given in a previous report (4).Preparation of Cells for Enzyme Analysis. Stationary-

growth-phase cultures of cells in 25-sq cm flasks were

rinsed once with cold PBS lacking calcium and magnesium

and harvested by a 2- to 3-min exposure to 0.005% trypsin

at room temperature. Cells were resuspended in cold PBSsupplemented with 2% serum, washed twice with cold PBS,and pelleted at 4Â°.Cell pellets frozen at -20Â° were provided

for enzyme assays. Estimation of growth phase was basedon cell counts of replicate cultures. Population-doubling

times and saturation densities have been determined for allparental cell lines and clonal lines included in this study.Cultures maintained in 25-sq cm flasks were refed 3 times/

week by partial replacement of growth medium. Estimatesof cell number were reproducible, for each cell line, andgrowth curves of cells grown in flasks duplicated thoseobtained with tissue culture dishes (Chart 1 ; Ref. 4).

Enzyme Assays. Dopamine-/3-hydroxylase activity wasmeasured by a modified, previously described radioenzy-

matic method (2, 11), utilizing tyramine as substrate for theenzyme. Choline acetyltransferase activity was measured

Table 1Derivation of human neuroblastoma and control cell lines

Cells DescriptionSK-N-SH Uncloned neuroblastoma line cultured December

1970SH-SY Neuroblast-like clonal subline of SK-N-SHSH-SY5 Subcloned from SH-SYSH-SY5Y Subcloned from SH-SY5SH-IN Neuroblast-like clone of SK-N-SHSH-EP Epithelial-like clone of SK-N-SHSH-FE Epithelial-like clone of SK-N-SH

SK-N-BE(1) Uncloned neuroblastoma line cultured June 1972SK-N-BE(2) Uncloned neuroblastoma line cultured November

1972

SK-N-MC Uncloned neuroblastoma line cultured September1971

MC-IIE Twice-cloned subline of SK-N-MCMX-IXC Twice-cloned subline of SK-N-MC

F-ECH Fibroblast-like line cultured from normal tissueF-LSO Fibroblast-like line cultured from normal tissueF-ALF Fibroblast-like line cultured from normal tissue

64.0

32.0-

16.0-

Â«O

? 8-Â°~

t 4.0-

iJ2 2.0S

0.5

0.25

10 15 20 25Day

4 The abbreviations used are: PBS, calcium- and magnesium-free phosphate-buffered salts solution (8.0 g NaCI, 0.20 g KCI. 2.16 g Na2HPO4-7H2O,and 0.20 g KH2PO, per liter of water, pH 7.3); GABA, -y-aminobutyric acid.

Chart 1. Determinations of cell number during exponential and stationarygrowth phases. Each point represents the mean of 2 values. O, SK-N-BE(1),single experiment; â€¢¿�,SK-N-BE(2), 3 experiments.

3752 CANCER RESEARCH VOL. 38



Neuretransmitters in Human Neuroblastoma Cells

according to the method of Giller and Schwartz (9). Measurements of acetylcholinesterase and pseudocholinester-ase were made as described by Hall (12).

Determinations of Neurotransmitter Synthesis. Cellswere scraped from the surface of 25-sq cm plastic flasks,pipetted, and counted. In the first of 2 experiments, 5 x 103to 3 x 106 cells of each of the test cell lines were pelletedand resuspended in 100 /Â¿\of specially formulated L-15culture medium (Microbiological Associates, Bethesda,Md.), pH 7.2, to which fetal calf serum, bovine serumalbumin, dextrose, and imidazole had been added (15) andpreincubated at 37Â°for 30 min. The L-15 medium containedno glutamate, glutamine, tyrosine, phenylalanine, or cho-line. In this first experiment, glutamate and glutamine wereadded to the preincubation mixture. Radioactive precursorsof transmitter compounds (20 /J.Mi_-[3,5-3H]tyrosine, 60.3Ci/mmol, and 0.3 mw [mef/?y/-14C]choline chloride, 30 mCi/

mmol) from New England Nuclear, Boston, Mass., werethen added. The radiochemical purity of the precursors wastested before use (13). For preservation of the catechol-amines and the acetylcholine that might be formed, 0.1 mMpargyline and eserine, 30 /Â¿g/ml,were added to the incubation mixture to inhibit monoamine oxidase and acetylcholinesterase, respectively. Cells were incubated withgentle shaking at 37Â°for 2 hr, pelleted, and extracted by

alternate freezing and thawing in a mixture of 90 Â¿tlof pH 2buffer (0.47 M formic acid-1.4 M acetic acid), and 15 Â¿ilofsodium dodecyl sulfate (1% in pH 2 buffer) according topreviously described procedures (13, 15). Samples of cellextract, supernatant medium, and medium incubated inabsence of cells were applied to separate lanes of WhatmanNo. 3MM chromatography paper. Standard, nonradioactivesolutions of the relevant precursors and products wereadded to each of the lanes. Precursors and their productswere separated by high-voltage electrophoresis at pH 2, asdescribed by Hildebrand ef al. (13). The paper strips wereexposed to iodine vapors for development of choline andacetylcholine spots and were then dipped into a ninhydrinsolution containing cadmium acetate to visualize the positions of all other compounds. Strips were cut into 1-cmsections and eluted with 0.5 ml of 0.1 N HCI for 30 min (15).Four ml of Aguasol (New England Nuclear) were added toeach vial, and radioactivity was counted in a liquid scintillation counter (Packard Instrument Co., Inc., DownersGrove, III.).

In the second experiment, after 30 min preincubation inthe L-15 medium, cells were incubated in medium containing 20 /aw [3-3H]glutamate (16.2 Ci/mmol) in addition to theother radioactive precursors. Use of [14C]choline ratherthan the tritiated compound allowed easy separation of theacetylcholine and GABA formed by the tissue (see Fig. 3,Ref. 16). Radioactive glutamate was boiled in 2 N HCI for 1hr just prior to use to remove a contaminant that can beconverted to GABA in the absence of glutamic acid decar-boxylase (15). Cell preparation and incubation procedureswere as described for Experiment 1 except that enzymeinhibitors were not included. Extraction, separation, andquantification of radioactive products were carried out asdescribed. Protein was estimated by the method of Lowryet al. (14).

RESULTS

Morphology and Growth Characteristics of Cell Linesand Clones. The 2 independently derived human neuroblastoma lines, SK-N-SH and SK-N-MC, have been described in detail (4). As noted in the earlier report, clonallyderived populations of either neuroblast- or epithelial-likeSK-N-SH cells sometimes do not retain morphological homogeneity after several months of culture. SH-SY wasobserved to contain a small proportion of epithelial-likecells and was therefore subcloned. The twice-cloned SH-SY5 and the thrice-cloned SH-SY5Y were apparently homogeneous neuroblast-like cell populations when studiedfor transmitter properties. Examples of the 2 morphologicalvarieties of clones are shown in Figs. 1 and 2.

The SK-N-BE(1) and SK-N-BE(2) lines were established invitro from bone marrow biopsy specimens obtained in Juneand November 1972, from a 2-year-old male with disseminated neuroblastoma (3). The lines are similar in morphologyand consist primarily of small, retractile cells with short,neurite-like cell processes (Figs. 3 and 4). Cells tend to growin tight aggregates. The average population-doubling timeof SK-N-BE(2) cells was 27 hr, and they attain high saturation densities ranging from 137 to 186 x 10" cells/sq cm(Chart 1). In a single determination, the population-doubling time of SK-N-BE(1) cells was approximately 4 days(Chart 1); this line is difficult to culture. Both lines require aplastic rather than glass surface for attachment, but eventhen cell-substrate adhesiveness of SK-N-BE(1) cells isrelatively low. Both cell populations, SK-N-BE(1) cellsespecially, are unusually sensitive to treatment with trypsinor hypotonie solutions and to mechanical manipulationssuch as pipeting. Karyotype analysis revealed the presenceof several consistent marker chromosomes that are sharedby the 2 lines and indicate their common heritage.5 Themodal chromosome number is 46 for SK-N-BE(1) cells and44 for SK-N-BE(2) cells (6); these lines, like the SK-N-SHand SK-N-MC lines (6), are near diploid.

In heterotransplantation tests, 107 SK-N-BE(1) cells wereinoculated into 2 groups of cortisonized hamsters. Notumors were produced in the 24 pouches inoculated. In aseries of 5 experiments with the SK-N-BE(2) line, an inoculum of 107cells produced tumors in 8 of a total of 45 cheek

pouches (average frequency, 18%). In comparison withtumors produced by SK-N-SH and SK-N-MC cells (4), SK-N-BE(2) tumors had longer latent periods. However, the tumor-producing capacity as well as the morphological attributes of SK-N-BE(2) cells were consistent with a malignantneuronal cell origin. Even though SK-N-BE(1) cells were nottumorigenic in this heterotransplantation system, their similarity to SK-N-BE(2) cells with respect to morphology andkaryotype indicates that the SK-N-BE(1) line also originatedfrom metastatic neuroblastoma.

Synthetic and Degradative Neurotransmitter Enzymes.Neuroblastoma cell lines SK-N-SH, SK-N-MC, SK-N-BE(2),and cloned sublines were assayed for dopamine-/3-hydrox-ylase, choline acetyltransferase, acetylcholinesterase, andbutyrylcholinesterase (pseudocholinesterase). Specific activities (nmol/hr/mg protein) are listed in Table 2. SK-N-SH

5 J. L. Biedler and B. A. Spengler, manuscript in preparation.

NOVEMBER 1978 3753




Table 2

Specific activities of neuronal enzymes in human neuroblastomacell lines and controls

Replicate cultures of each of the human cell lines were harvestedwith 0.005% trypsin, pelleted, and washed twice with cold Dul-becco's phosphate-buffered saline. Growth phase was verified by

cell counts. Cells were stored as pellets at -20Â°. Replicate cell

pellets were used for assay of all 4 enzyme activities, except asnoted. Activity values are based on determinations at 2 differentpassages of the cell lines, with exceptions noted.

nmol/hr/mgproteinCellsSK-N-SHSH-SYSH-SY5SH-SY5YSH-INSH-EPSH-FESK-N-BEd)SK-N-BE(2)SK-N-MCMC-IIEMC-IXCF-ECHF-LSOF-ALFMS-ISC'1NIE-115GPMouse

brainC-1300tumorSuperior

cervicalganglion,ratDopamine-

ÃŸ-hydrox-

ylase7.16Â°11.473.84"3.47''7.19NDrND1.202.26NDNDNDNDND0.545.325-8Choline

acetyl-transfer-

ase0.371

.91*1.341.440.900.258.46"1.0022.911.070.440.66"41.0Acetyl-

cholines-

terase170180*32040089016090d701901108010''6010810Butyryl-

cholines-

terase7020"701090710200"16031017040130"30

" Values of 5.88, 6.76, 8.14, and 7.86 (mean, 7.16) were obtained

for 4 independent cultures, at different passage levels, that werenot used for assay of the other enzymes.

" Only 1 set of cell cultures was assayed.r ND, not detectable.'' Mean of values obtained at 3 different culture passages.e Cholinergic and adrenergic mouse neuroblastoma C-1300

clones (1) were provided by Dr. Marshall Nirenberg to Dr. MenekGoldstein.

and its several neuroblast-like clones, SH-SY, SH-SY5, SH-SY5Y, and SH-IN, had moderate levels of dopamine-/3-hy-droxylase activity, whereas the epithelial-like clones, SH-EPand SH-FE, had no detectable activity. SK-N-SH cells had

consistently higher activity in the stationary phase of growthas compared to the exponential growth phase (8). Thedopamine-/3-hydroxylase activity values for SK-N-SH andthe neuroblast-like clones were similar in magnitude to those

obtained for superior cervical rat ganglia, which containadrenergic postganglionic cell bodies (Table 2). Cholineacetyltransferase activities of SK-N-SH cells and clones

were negligible and approximated those exhibited by thehuman fibroblast controls, F-ECH, F-LSO, and F-ALF. Do-pamine-/3-hydroxylase activity was detected in SK-N-BE(1)and SK-N-BE(2) cells also but only in stationary-phasecultures. Choline acetyltransferase activity of SK-N-BE(2)cells was at control level. For comparison, the dopamine-ÃŸ-hydroxylase activities of cholinergic and adrenergicclones (1) of mouse neuroblastoma C-1300 are included inTable 1. The SK-N-MC line and its subclones were negativefor dopamine-/3-hydroxylase. The choline acetyltransferaseactivity of SK-N-MC cells, however, was at least 4 times

higher than that measured in the other neuroblastoma linesand controls, whereas clonal MC-IXC cells had at least 12-fold greater activity. In contrast, the acetylcholine-synthe-sizing enzyme activity of the MC-IIE clone was similar to

that of controls.Acetylcholinesterase activity was low in all cell lines

compared to that of mouse brain (Table 1). Pseudocholin-esterase activity was higher than acetylcholinesterase activity in the SK-N-BE(2) line and the SK-N-MC line and clones.

Conversion of Radioactive Precursors to Neurotrans-

mitter Compounds. Several of the human neuroblastomalines and clones that were assayed for neurotransmitterenzyme activity were also tested for their capacity to synthesize and accumulate putative neurotransmitters from radio-

labeled precursor compounds. Results of the first of 2experiments are given in Table 3. Three clones derived fromthe SK-N-SH line as well as SK-N-BE(2) cells convertedtyrosine to dopamine. Of the 3 clones, SH-SY was especiallyactive whereas, of all lines tested, SK-N-BE(2) cells madethe most dopamine. Clonal MC-IXC cells did not converttyrosine to dopamine. None of the lines produced norepi-

nephrine. This product was not detectable either in cells or

Table 3Conversion of radioactive precursors to candidate neurotransmitter compounds

(Experiment 1)Cells were incubated for 2 hr in 100 Â¿tlof L-15 medium containing [3H]tyrosine (20 Â¿IM),

[14C]choline (0.3 mw), pargyline (0.1 mw), and eserine (30 Â¿ig/ml). No radioactive

products were detected in medium incubated in absence of cells.Radioactive product(dpm/108DopamineCell

lineSH-SY

SH-INSH-EPSK-N-BE(2)MC-IXCCells18,467

6,0251,152

126,230NDMedium1,853

NDND

9,016NDNorepinephrineCellsND"

NDNDNDNDMediumND

NDNDND

NDcells)AcetylcholineCells4,354

12,260421

15,9948,787Medium1,553

4,380ND

1,4003,333

" ND, not detectable.




Neurotransmitters in Human Neuroblastoma Cells

in the incubation medium (Table 3). All of the cells testedconverted choline to acetylcholine.

The second experiment included the MC-IIE subclonederived from SK-N-MC along with the cell lines used inExperiment 1. The synthetic capacity of these cells wascompared to that of fibroblast-like F-ECH cells and small (1to 2 mg) pieces of freshly dissected mouse cerebellar cortexand hypothalamus. All of the cells and tissues were testedfor their capacity to convert tyrosine and choline to neuro-transmitters, as in Experiment 1, and in addition to convertglutamate to the transmitter GABA. Results of this experiment (Table 4) agreed with those obtained in Experiment 1(Table 3). SK-N-BE(2) cells were again the most active inthe conversion of tyrosine to dopamine, even when compared to adrenergically innervated mouse hypothalamus.Again, SH-SY was the most active of the 3 SK-N-SH clones.In the clones derived from SK-N-MC, normal fibroblast-likeF-ECH cells, and mouse cerebellar cortex, no dopaminewas detected. None of the tumor cells showed detectableaccumulation of norepinephrine. Under the conditions usedin this test, conversion of tyrosine to norepinephrine doesoccur since it is detectable with mouse hypothalamus(Table 4). As before, all of the tumor cells were able to formacetylcholine from choline, comparable to the result obtained with mouse cerebellar cortex. Fibroblast controlcells did not synthesize acetylcholine. All cell lines testedconverted glutamate to GABA. The 2 neuroblastic clones ofSK-N-SH were the most active and the fibroblast-like cellsthe least able to synthesize GABA. However, the capacity ofthe neuroblastoma cells to form GABA was far less thanthat exhibited by cerebellar cortex which contains GABa-minergic neurons (Table 4).

DISCUSSION

In order to assess neuronal-like properties of humanneuroblastoma lines established in continuous culture, wehave made quantitative measurements of the activities of

Table 4Conversion of radioactive precursors to candidate

neurotransmitter compounds (Experiment 2)Cells were incubated for 2 hr in 100 /*lof L-15 medium containing

[3H]tyrosine (18 Ã•J.M),[14C]choline (0.3 mw), and [3H]glutamate (20

fiM). The enzyme inhibitors pargyline and eserine were not used inthis experiment. No radioactive products were detected in mediumincubated in absence of cells. The values represent radioactivitymeasured in both cell extracts and supernatant medium.

Radioactive product (dpm/mg protein)

Cell line Dopamine

Norepi- Acetyl-

nephrine choline GABA

SH-SYSH-INSH-EPSK-N-BE(2)

MC-IIEMC-IXCF-ECHMouse

cerebellarcortexMouse

hypothalamus4,6171,93186527,458

NDNDNDND5,740ND"NDNDNDNDNDNDND3,2802,5417,1792,1658722,0035,338ND1,78975,55950,81139,77015,06310,45524,3995,3301,086,246

" ND, not detectable.

several neurotransmitter-related enzymes in cell extractsunder optimal conditions for enzyme activity. We have alsotested the intact, living cells for their ability to synthesizeand store neurotransmitters by presenting to them transmitter precursors without additional cofactors. This methodtests the presence of the transmitter-synthesizing enzymeand also the capacity of the cells to synthesize and storeneurotransmitters under the culture conditions of the experiment. Results obtained by these 2 approaches indicatethat all 4 cell lines, SK-N-SH, SK-N-BE(1), SK-N-BE(2), andSK-N-MC, have biochemical properties of neuronal cells.We hesitate to classify human neuroblastoma lines as eitheradrenergic or cholinergic but rather describe them in termsof their most prominent neurotransmitter characteristics. Infact, results obtained by 2 different methods suggest thatSK-N-SH and possibly SK-N-BE(2) cells are multipotentialwith regard to neuronal enzyme expression. SK-N-MC cellsand clones, however, showed no adrenergic traits.

Adrenergic characteristics of the cells were assessed bymeasurement of the activity of dopamine-/3-hydroxylase,the enzyme catalyzing the conversion of dopamine to norepinephrine, and by detection of catecholaminergic products in the cells after incubation with the precursor aminoacid tyrosine. The SK-N-SH, SK-N-BE(1), and SK-N-BE(2)lines had moderately high activities of dopamine-/3-hydrox-ylase, similar to those obtained for the highly adrenergicN1E-115 C-1300 clone described by Amano et al. (1) and forsympathetic cervical ganglia (Table 2). These cells or theirclones were also able to convert tyrosine to dopamine. Thefinding that SK-N-BE(2) cells efficiently convert tyrosine todopamine suggests that these cells may have high tyrosinehydroxylase activity, and recent tests have shown this to beso (17). None of the cells that converted tyrosine to dopa-mine/3-hydroxylated the dopamine to form norepinephrineeven though substantial in vitro dopamine-/3-hydroxylaseactivity was present in several of the cell lines. A possibleexplanation is that some part of the machinery of a normalnoradrenergic cell is missing, since, under the conditionsof these experiments, adrenergic cells do make norepinephrine as shown for hypothalamic tissue (Table 4) andfor cultured superior cervical ganglion (15). Perhaps thedopamine-/3-hydroxylase is not localized in storage vesicles. Dopamine that is converted to norepinephrine but notstored may be rapidly destroyed by monoamine oxidase.Another possible explanation is that a cofactor may beabsent. We do know that the putative factor is not ascorbicacid since its addition did not change the results (notshown).

The SK-N-MC line and the MC-IXC clone, showing noadrenergic traits, had particularly high choline acetyltrans-ferase activities. There are, however, quantitative discrepancies between the activity of this enzyme and the amountof choline converted to acetylcholine. For example, although MC-IXC or SK-N-MC cells had the highest activity(23 nmol/hr/mg protein), SK-N-BE(2) and SH-IN cells madesomewhat more acetylcholine (Table 3). The basis for thesediscrepancies is not known; they may reflect the methodological differences discussed earlier and/or may indicatethat the precursor-product conversion method is a reliableindex of the functional capacities of neuroblastoma cells.

NOVEMBER 1978 3755




All of the cell lines tested converted glutamate to GABA.It is clear that production of GABA by the test cells was farless than that measured for cerebellar tissue in whichseveral types of GABA-utilizing neurons are present. In astudy of various human neuroblastoma lines including SK-N-SH and SK-N-MC, West ef al. (22) found very low activitylevels of glutamic acid decarboxylase, the enzyme responsible for the conversion of glutamate to GABA. Schubert efal. (20), in a study of neuronal and glial cell lines derivedfrom chemically induced tumors of the rat, found GABA inall lines examined. These results and the observed capacityof all of the human cell lines to make GABA from glutamate(Table 4) suggest that this capacity may be present commonly at least in cells of neural origin and thus may notserve to discriminate between malignant neuronal cells andother types of cells.

Investigation of transmitter characteristics of the clonesshowed that they either reflect the biochemical propertiesof parental lines or express those properties to a considerably lower degree. The neuroblast-like clones of SK-N-SHhave similar or higher dopamine-/3-hydroxylase activitieswhereas the epithelial-like clones have no measurable activity. Results of karyotype analysis indicate that the 2 sets ofclones have identical marker chromosomes and are, therefore, derived from a common precursor cell (J. L. Biedlerand B. A. Spengler, unpublished results). The basis for themorphological and biochemical divergence of the 2 celltypes is unknown. All clones, like SK-N-SH cells, convertedcholine to acetylcholine. The SK-N-MC-derived clones havecholinergic and not adrenergic characteristics; the MC-IIEclone has comparatively low choline acetyltransferase activity. Thus, these human neuroblastoma lines do not appear to comprise mixtures of adrenergic and cholinergiccells. SK-N-MC cells have only cholinergic propertieswhereas the predominantly adrenergic SK-N-SH cells havemultiple neurochemical characteristics.

Phenotypic stability of human neuroblastoma cells in vivoand in vitro is suggested from observations of the SK-N-BE(1) and SK-N-BE(2) cell lines established in culture frombiopsy material obtained 5 months apart from a patient withdisseminated disease. Morphological and biochemical similarities, as documented in this report, and karyotypic identity with respect to several marker chromosomes,5 demon

strated the constancy of certain cellular properties andsupport previous observations (4, 21) that human neuroblastoma lines in continuous culture may indeed maintainthe neurochemical attributes of the patient's tumor. Neuro

blastoma, the second most common solid tumor of youngchildren, remains refractory to therapeutic control. Availability of a variety of continuous lines of human neuroblastoma cells that exhibit the specialized, neuronal propertiesof the tumor of origin permits a rational approach toexperimental evaluation of potentially useful chemothera-peutic agents. In preliminary experiments (5) we observedthat several neuroblastoma cell lines and clones weredifferentially sensitive to vincristine, in comparison to otheralkaloids and to some widely used chemotherapeutic

agents, in keeping with its recognized clinical efficacy inthe management of this form of cancer.

ACKNOWLEDGMENTS

Thanks are due trom Dr. Schachner to Dr. J. H. Schwartz for the use ofhis facilities.

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Neurotransmitters in Human Neuroblastoma Cells

Fig. 1. Clonal SH-SY subline derived from SK-N-SH; culture in late exponential growth phase. Cells have long, neurite-like processes and typically grown intight aggregates separated by a distance of 2 to 5 times the diameter of an aggregate. Phase contrast, â€¢¿�450.

Fig. 2. Epithelial-like SH-EP cells cloned from SK-N-SH; culture in exponential growth phase. Cells are flattened, are tightly adherent to plastic substrate,and do not exhibit neurite-like processes. The epithelial-like cells apparently cease to multiply at about the time they become confluent. Phase contrast, x450.

Fig. 3. SK-N-BE(1) line in exponential growth phase. Cells are small, are poorly attached to substrate, and have occasional processes. Phase contrast, x450.

Fig. 4. SK-N-BEI2) line in exponential growth phase. Cells resemble those of the SK-N-BEO ) line but are somewhat more adherent. Single, large nucleoliare a prominent feature. Phase contrast, x 450.

NOVEMBER 1978 3757



1978;38:3751-3757. Cancer Res June L. Biedler, Suzanne Roffler-Tarlov, Melitta Schachner, et al. Cell Lines and ClonesMultiple Neurotransmitter Synthesis by Human Neuroblastoma

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