[CANCER RESEARCH 38, 3751-3757,
0008-5472/78/0038-0000$02.00
November
1978]
Multiple Neurotransmitter Synthesis by Human Neuroblastoma Cell
Lines and Clones
June 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.¡;Department of
Neuroscience, Children's Hospital Medical Center, Boston, Massachusetts 02115 [S. R.-T., U. S.¡;and Department of Neurology, Division of Behavioral
Neurology, New York University Medical Center, New York, New York 10016 [L. S. F.¡
ABSTRACT
INTRODUCTION
Continuously cultured human neuroblastoma cell lines
SK-N-SH, SK-N-BE(1), SK-N-BE(2), and SK-N-MC, as well
In recent years continuous lines of human neuroblastoma
cells in culture have become available for studies relating
to tumor cell growth and neuronal cell biology. A remarka
ble feature of the established neuroblastoma lines de
scribed thus far is the expression of biochemical properties
characteristic of normal neuronal cells in vivo. For example,
enzyme activities associated with neurotransmitter biosyn
thesis have been detected in approximately 10 independ
ently derived cell lines (3, 7, 8, 10, 18, 19, 22). Among the
many unresolved questions concerning neuroblastoma are
whether and to what extent an individual tumor consists of
a mixture of functionally differing neuronal phenotypes and
whether and to what extent a single neuroblastoma cell
may have the potential for synthesis of more than one
transmitter. Results of several combined clinical and bio
chemical studies suggest that the cells established in cul
ture reflect the characteristics of the tumor from which they
were derived (4, 21). Although karyotype analysis of the
human cell lines has revealed the presence of consistent
marker chromosomes (4, 6, 7, 19, 21) suggestive of clonal
origin of individual neuroblastomas, the possibility of func
tional diversity and multipotentiality of the tumor cells in
vivo cannot be explored by studying the tumor cell popula
tion as a whole. To obtain evidence regarding functional
properties as well as phenotypic stability of human neuro
blastoma cells, we isolated clones from the neuroblastoma
lines SK-N-SH and SK-N-MC and began their characteriza
tion. These 2 parental lines were compared to a hitherto
uncharacterized set of neuroblastoma lines, SK-N-BE(1)
and SK-N-BE(2), isolated at different times from the same
patient and possessing several biological and biochemical
features in common. In this report we describe findings
obtained by measurement of cholinergic and adrenergic
enzyme activities and by detection of neurotransmitters
synthesized from radioactive precursors.
as several clonally derived sublines, were assessed for
their neurotransmitter characteristics. Two different meth
ods were used: measurement of cholinergic and adrenergic enzyme activities and detection of neurotransmitters
synthesized from radioactive precursors. Dopamine-/3-hydroxylase (EC 1.14.2.1), choline acetyltransferase
(EC
2.3.1.6), acetylcholinesterase
(EC 3.1.1.7), and butyrylcholinesterase (EC 3.1.1.8) levels were compared to those
of various normal and neuronal cell controls. The capacity
of neuroblastoma lines and clones to convert radioactive
tyrosine, choline, and glutamate to transmitter products
was determined. SK-N-SH, SK-N-BE(1), and SK-N-BE(2)
cells had moderately high levels of dopamine-0-hydroxylase activity. Whereas neuroblast-like clones of SK-N-SH
had similar levels of this enzyme, epithelial-like clones
had no detectable activity. SK-N-BE(2) cells showed the
greatest capacity to convert tyrosine to dopamine. Al
though the SK-N-SH line itself was not tested, both neuroblast and epithelial-like
clones, shown by karyotype
analysis to have a common cell precursor, converted
tyrosine to dopamine. In contrast, the SK-N-MC line and
its clones appeared devoid of adrenergic properties. All
cells tested except human fibroblast-like cells of normal
origin were able to convert radioactive choline to acetylcholine. While all the cell lines had measurable choline
acetyltransferase
activity,
levels were substantially
higher (at least 4- to 12-fold) for SK-N-MC cells and its
clonally derived subline MC-IXC. All cells tested con
verted glutamate to y-aminobutyric acid. Results indicate
that these human neuroblastoma cell lines have predom
inating neurotransmitter
characteristics that are some
times, but not always, expressed in clones derived from
them. Since the SK-N-BE(1) and SK-N-BE(2) lines were
isolated 5 months apart from the same patient and have
similar activity levels of dopamine-ß-hydroxylase, these
characteristics may be stable and representative of the
tumor cells in vivo. Furthermore, results obtained by the
two different methodological approaches suggest that,
whereas SK-N-MC cells show cholinergic and not adrener
gic traits, SK-N-SH and possibly SK-N-BE(2) may be multipotential 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 Heidel
berg, GFR.
Received February 16,1978; accepted August 18,1978.
NOVEMBER
MATERIALS
AND METHODS
Neuroblastoma Cell Lines and Clones. Cell line SK-NSH, established in culture in December 1970 from a bone
marrow biopsy, was previously described (4). The line
comprises 2 morphologically distinct cell types, one neuroblastic and the other epithelial-like. Clones representing
both types were recently isolated and are described in Table
1. Cell line SK-N-BE(1) was established in vitro in June 1972
from a bone marrow biopsy. A subsequent marrow biopsy
in November 1972, from the same patient, yielded a second
continuous line designated SK-N-BE(2). The latter has been
described briefly (7). Cell line SK-N-MC, established in
1978
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3751
J. L. Biedler et al.
September 1971 from a metastatic tumor mass, and the
twice cloned derivatives MC-IIE and MC-IXC have been
described (4). The source of the human fibroblast-like
cell
populations,
F-ECH and F-LSO, has been given (4). F-ALF
and harvested by a 2- to 3-min exposure to 0.005% trypsin
at room temperature. Cells were resuspended in cold PBS
supplemented with 2% serum, washed twice with cold PBS,
and pelleted at 4°.Cell pellets frozen at -20° were provided
was established more recently from histologically
normal
endometrial tissue and is also fibroblast-like
in appearance.
All of the human neuroblastoma
and nonneuronal control
cell lines used in these studies are listed in Table 1.
Cell lines were grown in Eagle's minimum essential me
dium supplemented with nonessential amino acids (Eagle's
for enzyme assays. Estimation of growth phase was based
on cell counts of replicate cultures. Population-doubling
times and saturation densities have been determined for all
parental cell lines and clonal lines included in this study.
Cultures maintained in 25-sq cm flasks were refed 3 times/
formulation),
15% fetal bovine serum, penicillin (100 ID/ml),
and streptomycin
(100 ¿ig/ml). Cell lines were maintained
routinely in 25-sq cm tissue culture flasks (Falcon Plastics
Co., Oxnard, Calif.). For isolation of clones, 2 methods
were 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 and
gave rise to the lines SH-SY5, SH-SY5Y, MC-IIE, and MCIXC. The other clonal lines, SH-IN, SH-EP, and SH-FE, were
isolated as colonies after seeding microscopically
verified
single cells in individual 0.4-ml wells in Micro Test II Plates
(Falcon).
Population-doubling Time, Saturation Density, and Heterotransplantability
Determinations.
Estimates of growth
rate/'n vitro and maximum cell density attained in stationary
growth phase were based on cell counts of replicate cell
cultures. Plastic tissue culture dishes (60 mm; Falcon) were
seeded with 0.2 x 106 or 4 x 106 cells in 6 ml of growth
medium per plate. If cells were adequately adherent, cell
layers were rinsed once with PBS.4 Cells were removed
from the substrate with 1 ml of a trypsin (0.12%)-EDTA
(0.02%) solution, resuspended in 9 ml of serum-containing
growth medium, and pipetted to obtain single-cell suspen
sions as viewed microscopically.
If cells were loosely adher
ent or floating, the supernatant medium was removed and
centrifuged
at 1000 rpm. The pelleted cells were resus
pended and pooled with those removed from the dish by
EDTA-trypsin. For these experiments as well as for cloning,
cells were incubated in a water-jacketed
C02 incubator
week by partial replacement of growth medium. Estimates
of cell number were reproducible,
for each cell line, and
growth curves of cells grown in flasks duplicated those
obtained with tissue culture dishes (Chart 1 ; Ref. 4).
Enzyme Assays. Dopamine-/3-hydroxylase activity was
measured by a modified, previously described radioenzymatic method (2, 11), utilizing tyramine as substrate for the
enzyme. Choline acetyltransferase
activity was measured
Derivation
Cells
SK-N-SH
SH-SY
SH-SY5
SH-SY5Y
SH-IN
SH-EP
SH-FE
old female, weanling golden Syrian hamsters. Animals were
given s.c. injections of 2.5 mg of cortisone acetate at the
time of cell inoculation and 2 times/week thereafter. Cheek
pouches were examined once per week and tumors were
measured. Criteria for "positive" tumor growth have been
and control
cell lines
Description
Uncloned neuroblastoma line cultured December
1970
Neuroblast-like clonal subline of SK-N-SH
Subcloned from SH-SY
Subcloned from SH-SY5
Neuroblast-like clone of SK-N-SH
Epithelial-like clone of SK-N-SH
Epithelial-like clone of SK-N-SH
SK-N-BE(1)
SK-N-BE(2)
Uncloned neuroblastoma line cultured June 1972
Uncloned neuroblastoma line cultured November
1972
SK-N-MC
MC-IIE
MX-IXC
Uncloned neuroblastoma line cultured September
1971
Twice-cloned subline of SK-N-MC
Twice-cloned subline of SK-N-MC
F-ECH
F-LSO
F-ALF
Fibroblast-like line cultured from normal tissue
Fibroblast-like line cultured from normal tissue
Fibroblast-like line cultured from normal tissue
(National Appliance Co., Portland, Oreg.) in an atmosphere
of approximately 5% CO2 in air at 37°.Medium was replaced
twice per week. In each experiment, duplicate plates were
counted 3 to 5 times/week with the aid of a Model F Coulter
Counter (threshold, 13.5; aperture current, 32; attenuation,
2; Coulter Electronics Inc., Hialeah, Fla.).
For determination
of tumor-producing
capacity, cells
were inoculated into both cheek pouches of 19- to 22-day-
Table 1
of human neuroblastoma
64.0
32.016.0«O
?
8-°~
t
4.0-
i
J2 2.0
S
0.5
given in a previous report (4).
Preparation of Cells for Enzyme Analysis. Stationarygrowth-phase
cultures of cells in 25-sq cm flasks were
rinsed once with cold PBS lacking calcium and magnesium
0.25
15
10
20
25
Day
4 The abbreviations
used are: PBS, calcium- and magnesium-free
phos
phate-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.
3752
Chart 1. Determinations
of cell number during exponential and stationary
growth phases. Each point represents the mean of 2 values. O, SK-N-BE(1),
single experiment; •¿,
SK-N-BE(2), 3 experiments.
CANCER
RESEARCH
VOL. 38
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Neu retransmitters in Human Neuroblastoma Cells
according to the method of Giller and Schwartz (9). Meas RESULTS
urements of acetylcholinesterase and pseudocholinesterMorphology and Growth Characteristics of Cell Lines
ase were made as described by Hall (12).
and
Clones. The 2 independently derived human neuro
Determinations of Neurotransmitter Synthesis. Cells blastoma
lines, SK-N-SH and SK-N-MC, have been de
were scraped from the surface of 25-sq cm plastic flasks,
scribed
in
detail (4). As noted in the earlier report, clonally
pipetted, and counted. In the first of 2 experiments, 5 x 103
derived
populations
of either neuroblast- or epithelial-like
to 3 x 106 cells of each of the test cell lines were pelleted
SK-N-SH
cells
sometimes
do not retain morphological ho
and resuspended in 100 /¿\of specially formulated L-15
mogeneity after several months of culture. SH-SY was
culture medium (Microbiological Associates, Bethesda,
observed to contain a small proportion of epithelial-like
Md.), pH 7.2, to which fetal calf serum, bovine serum
cells and was therefore subcloned. The twice-cloned SHalbumin, dextrose, and imidazole had been added (15) and
SY5 and the thrice-cloned SH-SY5Y were apparently ho
preincubated at 37°for 30 min. The L-15 medium contained
no glutamate, glutamine, tyrosine, phenylalanine, or cho- mogeneous neuroblast-like cell populations when studied
line. In this first experiment, glutamate and glutamine were for transmitter properties. Examples of the 2 morphological
varieties of clones are shown in Figs. 1 and 2.
added to the preincubation mixture. Radioactive precursors
The SK-N-BE(1) and SK-N-BE(2) lines were established in
of transmitter compounds (20 /J.Mi_-[3,5-3H]tyrosine, 60.3
vitro from bone marrow biopsy specimens obtained in June
Ci/mmol, and 0.3 mw [mef/?y/-14C]choline chloride, 30 mCi/
and November 1972, from a 2-year-old male with dissemi
mmol) from New England Nuclear, Boston, Mass., were
then added. The radiochemical purity of the precursors was nated neuroblastoma (3). The lines are similar in morphology
and consist primarily of small, retractile cells with short,
tested before use (13). For preservation of the catecholneurite-like cell processes (Figs. 3 and 4). Cells tend to grow
amines and the acetylcholine that might be formed, 0.1 mM
in tight aggregates. The average population-doubling time
pargyline and eserine, 30 /¿g/ml,were added to the incu
of SK-N-BE(2) cells was 27 hr, and they attain high satura
bation mixture to inhibit monoamine oxidase and acetyl
tion densities ranging from 137 to 186 x 10" cells/sq cm
cholinesterase, respectively. Cells were incubated with
gentle shaking at 37°for 2 hr, pelleted, and extracted by (Chart 1). In a single determination, the population-dou
bling time of SK-N-BE(1) cells was approximately 4 days
alternate freezing and thawing in a mixture of 90 ¿tl
of pH 2
buffer (0.47 M formic acid-1.4 M acetic acid), and 15 ¿ilof (Chart 1); this line is difficult to culture. Both lines require a
sodium dodecyl sulfate (1% in pH 2 buffer) according to plastic rather than glass surface for attachment, but even
then cell-substrate adhesiveness of SK-N-BE(1) cells is
previously described procedures (13, 15). Samples of cell
relatively low. Both cell populations, SK-N-BE(1) cells
extract, supernatant medium, and medium incubated in
especially,
are unusually sensitive to treatment with trypsin
absence of cells were applied to separate lanes of Whatman
or
hypotonie
solutions and to mechanical manipulations
No. 3MM chromatography paper. Standard, nonradioactive
such
as
pipeting.
Karyotype analysis revealed the presence
solutions of the relevant precursors and products were
of
several
consistent
marker chromosomes that are shared
added to each of the lanes. Precursors and their products
by the 2 lines and indicate their common heritage.5 The
were separated by high-voltage electrophoresis at pH 2, as
modal chromosome number is 46 for SK-N-BE(1) cells and
described by Hildebrand ef al. (13). The paper strips were
44 for SK-N-BE(2) cells (6); these lines, like the SK-N-SH
exposed to iodine vapors for development of choline and
and SK-N-MC lines (6), are near diploid.
acetylcholine spots and were then dipped into a ninhydrin
In heterotransplantation tests, 107 SK-N-BE(1) cells were
solution containing cadmium acetate to visualize the posi
tions of all other compounds. Strips were cut into 1-cm inoculated into 2 groups of cortisonized hamsters. No
sections and eluted with 0.5 ml of 0.1 N HCI for 30 min (15). tumors were produced in the 24 pouches inoculated. In a
series of 5 experiments with the SK-N-BE(2) line, an inocu
Four ml of Aguasol (New England Nuclear) were added to lum of 107cells produced tumors in 8 of a total of 45 cheek
each vial, and radioactivity was counted in a liquid scintil
pouches (average frequency, 18%). In comparison with
lation counter (Packard Instrument Co., Inc., Downers
tumors produced by SK-N-SH and SK-N-MC cells (4), SK-NGrove, III.).
In the second experiment, after 30 min preincubation in BE(2) tumors had longer latent periods. However, the tu
mor-producing capacity as well as the morphological attri
the L-15 medium, cells were incubated in medium contain
ing 20 /aw [3-3H]glutamate (16.2 Ci/mmol) in addition to the butes of SK-N-BE(2) cells were consistent with a malignant
other radioactive precursors. Use of [14C]choline rather neuronal cell origin. Even though SK-N-BE(1) cells were not
than the tritiated compound allowed easy separation of the tumorigenic in this heterotransplantation system, their sim
ilarity to SK-N-BE(2) cells with respect to morphology and
acetylcholine and GABA formed by the tissue (see Fig. 3,
karyotype indicates that the SK-N-BE(1) line also originated
Ref. 16). Radioactive glutamate was boiled in 2 N HCI for 1
hr just prior to use to remove a contaminant that can be from metastatic neuroblastoma.
Synthetic and Degradative Neurotransmitter Enzymes.
converted to GABA in the absence of glutamic acid decarNeuroblastoma cell lines SK-N-SH, SK-N-MC, SK-N-BE(2),
boxylase (15). Cell preparation and incubation procedures
and cloned sublines were assayed for dopamine-/3-hydroxwere as described for Experiment 1 except that enzyme
inhibitors were not included. Extraction, separation, and ylase, choline acetyltransferase, acetylcholinesterase, and
quantification of radioactive products were carried out as butyrylcholinesterase (pseudocholinesterase). Specific ac
tivities (nmol/hr/mg protein) are listed in Table 2. SK-N-SH
described. Protein was estimated by the method of Lowry
5 J. L. Biedler and B. A. Spengler, manuscript in preparation.
et al. (14).
NOVEMBER
1978
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3753
J. L. Biedler et al.
Table 2
Specific activities of neuronal enzymes in human neuroblastoma
cell lines and controls
Replicate cultures of each of the human cell lines were harvested
with 0.005% trypsin, pelleted, and washed twice with cold Dulbecco's phosphate-buffered
saline. Growth phase was verified by
cell counts. Cells were stored as pellets at -20°. Replicate cell
pellets were used for assay of all 4 enzyme activities, except as
noted. Activity values are based on determinations
at 2 different
passages of the cell lines, with exceptions noted.
and its several neuroblast-like
clones, SH-SY, SH-SY5, SHSY5Y, and SH-IN, had moderate levels of dopamine-/3-hydroxylase activity, whereas the epithelial-like clones, SH-EP
and SH-FE, had no detectable activity. SK-N-SH cells had
consistently higher activity in the stationary phase of growth
as compared to the exponential
growth phase (8). The
dopamine-/3-hydroxylase
activity values for SK-N-SH and
the neuroblast-like clones were similar in magnitude to those
obtained for superior cervical rat ganglia, which contain
adrenergic postganglionic
cell bodies (Table 2). Choline
proteinCellsSK-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
nmol/hr/mg
acetyltransferase
activities of SK-N-SH cells and clones
were negligible and approximated
those exhibited by the
acetylhuman fibroblast controls, F-ECH, F-LSO, and F-ALF. Doß-hydroxtransfercholinescholinespamine-/3-hydroxylase
activity was detected in SK-N-BE(1)
ylase7.16°11.473.84"3.47''7.19NDrND1.202.26NDNDNDNDND0.545.325-8Choline
terase7020"701090710200"16031017040130"30
ase0.371 terase170180*32040089016090d701901108010''6010810Butyryland SK-N-BE(2) cells also but only in stationary-phase
cultures. Choline acetyltransferase
activity of SK-N-BE(2)
*1.341.440.900.258.46"1.0022.911.070.440.66"41.0Acetyl.91
cells was at control level. For comparison, the dopamineß-hydroxylase activities
of cholinergic
and adrenergic
clones (1) of mouse neuroblastoma
C-1300 are included in
Table 1. The SK-N-MC line and its subclones were negative
for dopamine-/3-hydroxylase.
The choline acetyltransferase
activity of SK-N-MC cells, however, was at least 4 times
higher than that measured in the other neuroblastoma lines
and controls, whereas clonal MC-IXC cells had at least 12fold greater activity. In contrast, the acetylcholine-synthesizing 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). Pseudocholinesterase activity was higher than acetylcholinesterase
activ
ity in the SK-N-BE(2) line and the SK-N-MC line and clones.
Conversion of Radioactive Precursors to NeurotransbrainC-1
mitter Compounds. Several of the human neuroblastoma
tumorSuperior
300
lines and clones that were assayed for neurotransmitter
cervi
enzyme activity were also tested for their capacity to synthe
cal
ganglion,ratDopaminesize and accumulate putative neurotransmitters
from radio" 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 were
not 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
Goldstein.
by Dr. Marshall
Nirenberg
to Dr. Menek
labeled precursor compounds.
Results of the first of 2
experiments are given in Table 3. Three clones derived from
the SK-N-SH line as well as SK-N-BE(2) cells converted
tyrosine to dopamine. Of the 3 clones, SH-SY was especially
active whereas, of all lines tested, SK-N-BE(2) cells made
the most dopamine. Clonal MC-IXC cells did not convert
tyrosine to dopamine. None of the lines produced norepinephrine. This product was not detectable either in cells or
Table 3
Conversion of radioactive precursors to candidate neurotransmitter compounds
(Experiment 1)
Cells were incubated for 2 hr in 100 ¿tlof L-15 medium containing [3H]tyrosine (20 ¿IM),
[14C]choline (0.3 mw), pargyline
(0.1 mw), and eserine (30 ¿ig/ml). No radioactive
products were detected in medium
(dpm/108DopamineCell
incubated in absence of cells.
Radioactive product
lineSH-SY
SH-IN
SH-EP
SK-N-BE(2)
MC-IXCCells18,467
12,260
4,380
6,025
ND
ND
NDNDND
421
ND
1,152
ND
ND
15,994
1,400
9,016
126,230
NDNDMediumND
8,787Medium1,553
NDMedium1,853
NDNorepinephrineCellsND"NDcells)AcetylcholineCells4,354
3,333
" ND, not detectable.
3754
CANCER
RESEARCH
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VOL. 38
Neurotransmitters in Human Neuroblastoma Cells
several neurotransmitter-related enzymes in cell extracts
under optimal conditions for enzyme activity. We have also
tested the intact, living cells for their ability to synthesize
and store neurotransmitters by presenting to them transmit
ter precursors without additional cofactors. This method
tests the presence of the transmitter-synthesizing enzyme
and also the capacity of the cells to synthesize and store
neurotransmitters under the culture conditions of the ex
periment. Results obtained by these 2 approaches indicate
that all 4 cell lines, SK-N-SH, SK-N-BE(1), SK-N-BE(2), and
SK-N-MC, have biochemical properties of neuronal cells.
We hesitate to classify human neuroblastoma lines as either
adrenergic or cholinergic but rather describe them in terms
of their most prominent neurotransmitter characteristics. In
fact, results obtained by 2 different methods suggest that
SK-N-SH and possibly SK-N-BE(2) cells are multipotential
with regard to neuronal enzyme expression. SK-N-MC cells
and clones, however, showed no adrenergic traits.
Adrenergic characteristics of the cells were assessed by
measurement of the activity of dopamine-/3-hydroxylase,
the enzyme catalyzing the conversion of dopamine to nor
epinephrine, and by detection of catecholaminergic prod
ucts in the cells after incubation with the precursor amino
acid tyrosine. The SK-N-SH, SK-N-BE(1), and SK-N-BE(2)
lines had moderately high activities of dopamine-/3-hydroxylase, similar to those obtained for the highly adrenergic
N1E-115 C-1300 clone described by Amano et al. (1) and for
sympathetic cervical ganglia (Table 2). These cells or their
clones were also able to convert tyrosine to dopamine. The
finding that SK-N-BE(2) cells efficiently convert tyrosine to
dopamine suggests that these cells may have high tyrosine
hydroxylase activity, and recent tests have shown this to be
so (17). None of the cells that converted tyrosine to dopamine/3-hydroxylated the dopamine to form norepinephrine
DISCUSSION
even though substantial in vitro dopamine-/3-hydroxylase
In order to assess neuronal-like properties of human
activity was present in several of the cell lines. A possible
neuroblastoma lines established in continuous culture, we
explanation is that some part of the machinery of a normal
have made quantitative measurements of the activities of
noradrenergic cell is missing, since, under the conditions
of these experiments, adrenergic cells do make norepi
Table 4
Conversion of radioactive precursors to candidate
nephrine as shown for hypothalamic tissue (Table 4) and
neurotransmitter
compounds (Experiment 2)
for cultured superior cervical ganglion (15). Perhaps the
Cells were incubated for 2 hr in 100 /*lof L-15 medium containing
dopamine-/3-hydroxylase is not localized in storage vesi
[3H]tyrosine (18 ÕJ.M),[14C]choline (0.3 mw), and [3H]glutamate (20
cles. Dopamine that is converted to norepinephrine but not
fiM). The enzyme inhibitors pargyline and eserine were not used in
stored may be rapidly destroyed by monoamine oxidase.
this experiment. No radioactive products were detected in medium
incubated in absence of cells. The values represent radioactivity
Another possible explanation is that a cofactor may be
measured in both cell extracts and supernatant medium.
absent. We do know that the putative factor is not ascorbic
acid since its addition did not change the results (not
Radioactive product (dpm/mg protein)
shown).
NorepiAcetylThe SK-N-MC line and the MC-IXC clone, showing no
Cell line
Dopamine
nephrine
choline
GABA
adrenergic traits, had particularly high choline acetyltransSH-SYSH-INSH-EPSK-N-BE(2)
ferase activities. There are, however, quantitative discrep
ancies between the activity of this enzyme and the amount
of choline converted to acetylcholine. For example, al
MC-IIEMC-IXCF-ECHMouseNDNDNDND5,740ND"NDNDND
though MC-IXC or SK-N-MC cells had the highest activity
2,0035,338ND1,78975,55950,81139,77015,063
10,45524,3995,3301,086,246
NDNDNDND3,2802,5417,1792,165872
(23 nmol/hr/mg protein), SK-N-BE(2) and SH-IN cells made
somewhat
more acetylcholine (Table 3). The basis for these
cerebellarcortexMouse
discrepancies is not known; they may reflect the methodo
logical differences discussed earlier and/or may indicate
hypothal
amus4,6171,93186527,458
that the precursor-product conversion method is a reliable
" ND, not detectable.
index of the functional capacities of neuroblastoma cells.
in the incubation medium (Table 3). All of the cells tested
converted choline to acetylcholine.
The second experiment included the MC-IIE subclone
derived from SK-N-MC along with the cell lines used in
Experiment 1. The synthetic capacity of these cells was
compared to that of fibroblast-like F-ECH cells and small (1
to 2 mg) pieces of freshly dissected mouse cerebellar cortex
and hypothalamus. All of the cells and tissues were tested
for their capacity to convert tyrosine and choline to neurotransmitters, as in Experiment 1, and in addition to convert
glutamate to the transmitter GABA. Results of this experi
ment (Table 4) agreed with those obtained in Experiment 1
(Table 3). SK-N-BE(2) cells were again the most active in
the conversion of tyrosine to dopamine, even when com
pared 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-like
F-ECH cells, and mouse cerebellar cortex, no dopamine
was detected. None of the tumor cells showed detectable
accumulation of norepinephrine. Under the conditions used
in this test, conversion of tyrosine to norepinephrine does
occur since it is detectable with mouse hypothalamus
(Table 4). As before, all of the tumor cells were able to form
acetylcholine from choline, comparable to the result ob
tained with mouse cerebellar cortex. Fibroblast control
cells did not synthesize acetylcholine. All cell lines tested
converted glutamate to GABA. The 2 neuroblastic clones of
SK-N-SH were the most active and the fibroblast-like cells
the least able to synthesize GABA. However, the capacity of
the neuroblastoma cells to form GABA was far less than
that exhibited by cerebellar cortex which contains GABaminergic neurons (Table 4).
NOVEMBER
1978
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3755
J. L. Biedler et al.
All of the cell lines tested converted glutamate to GABA. agents, in keeping with its recognized clinical efficacy in
It is clear that production of GABA by the test cells was far the management of this form of cancer.
less than that measured for cerebellar tissue in which
several types of GABA-utilizing neurons are present. In a ACKNOWLEDGMENTS
study of various human neuroblastoma lines including SKThanks are due trom Dr. Schachner to Dr. J. H. Schwartz for the use of
N-SH and SK-N-MC, West ef al. (22) found very low activity
his facilities.
levels of glutamic acid decarboxylase, the enzyme respon
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CANCER
RESEARCH
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VOL. 38
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 in
tight aggregates separated by a distance of 2 to 5 times the diameter of an aggregate. Phase contrast, •¿
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, x
450.
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, x
450.
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 nucleoli
are a prominent feature. Phase contrast, x 450.
NOVEMBER
1978
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3757
Multiple Neurotransmitter Synthesis by Human Neuroblastoma
Cell Lines and Clones
June L. Biedler, Suzanne Roffler-Tarlov, Melitta Schachner, et al.
Cancer Res 1978;38:3751-3757.
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