[CANCER RESEARCH 37, 1372-1376, May 1977]

SUMMARY

Cultured human neuroblastoma cell lines were assayedfor biochemical characteristics of neuronal function. Celllines studied included LA-N-i , LA-N-2, IMR-32, SK-N-SH,and SK-N-MC. Veratridine-dependent uptake of 22Na@implied the presence of the action potential Na@ionophore inLA-N-i , LA-N-2, IMR-32, and SK-N-SH. The time course of22Na@uptake and inhibition of uptake by tetrodotoxin supported this. SK-N-MC had no veratridine-dependent 22Na@uptake. Tyrosine hydroxylase (EC 1.14.10.), glutamic aciddecarboxylase (EC 4.1 .1.15), and acetyicholine contents inneuroblastoma cells were compared to those in brain. LA-N-1 and IMR-32 contained 15 and 5 times as much tyrosinehydroxylase, respectively, whereas LA-N-2 , SK-N-SH , andSK-N-MC contained only 0.5 to 5% of that in brain. Acetylcholine was present in LA-N-2 in 15- to 20-fold greaterquantities than in brain; other lines had only 10 to 50% ofthatinbrain.None ofthecelllinescontainedglutamicaciddecarboxylase. Thus, continuously propogated human neuroblastoma cell lines may have the action potential Na@ionophore and may be adrenergic (LA-N-i and IMR-32),cholinergic (LA-N-2), or inactive (SK-N-SH and SK-N-MC).This is the first demonstration of the action potential Na@ionophore and of acetylcholine production in human neuroblastoma cell lines.

INTRODUCTION

Established neuroblastoma cell lines provide models forstudying both neoplasia and normal differentiation andfunction of neurons (1i). Murine Ci300 neuroblastoma cellsgrowing in mice have been used as a laboratory model todevelop chemotherapy for human neuroblastoma (9). Invitro Ci300 cells synthesize neurotransmitters (3), have the

I In this investigation, adrenergic and cholinergic cells were defined as

those that had tyrosine hydroxylase and acetylcholine, respectively. Cellsdefined as “inactive―lacked these markers; this is an operational definitionsince such cells may have other adrenergic or cholinergic markers such asdopamine p hydroxylase or cholineacetyl transferase (4) and thus may nottruly be inactive.

2 This study was supported by the University of California Cancer Re

search Coordinating Committee, the California Institute for Cancer Research, and National Science Foundation Grant BMS 75-10093.@

3 Recipient of Contract E (04-1) GEN-12 from the Energy Research and

Development Administration.4 Recipient of National Cancer Institute Research Career Development

Award 1 K04 CA 00069. To whom requests for reprints should be addressed,at the Department of Pediatrics, UCLA School of Medicine, Los Angeles,Calif. 90024.

Received December 8, 1976; accepted January 24, 1977.

action potential Na@ionophore (7), and can have brainspecific cell-surface antigens (1, 2). Furthermore, morphological differentiation of these cells in vitro is accompaniedby alterations in neurochemical, electrophysiological, andcell-surface properties. These studies suggest that understanding of human neuroblastoma and possibly of neuronaldifferentiation and function could be advanced if a numberof well-characterized human neuroblastoma cell lines wereavailable for analysis of both tumor-associated and organspecific characteristics.

Two new human neuroblastoma cell lines were recentlyestablished in 1 of our laboratories (22). These lines alongwith 3 previously established lines (4, 24) have been exammed for biochemical properties of neural tissues. We reportthat continuous lines of human neuroblastoma cells possess the action potential Na@ionophore and can be adrenergic, inactive, or cholinergic.

MATERIALS AND METHODS

Reagentsand Supplies.Reagentsandtheirsourcesare:L-[3,5-3H]tyrosine (1.0 Ci/mmole), 22NaCI(carrier free), andDL-[i-14C]glutamic acid (23 mCi/mmole) from Amersham/Searle, Arlington Heights, Ill.; Liquifluor from New EnglandNuclear, Boston, Mass.; tetrodotoxin (purified by Sankyo),catalase, pyridoxal phosphate, and DL-6-methyl-5,6,7,8-tetrahydropterine dihydrate from Calbiochem, La Jolla, Calif.;hydroxide of Hyamine iO-X from Packard Instrument Co.,Downers Grove, Ill. ; tyrosine and ouabain from SigmaChemical Co. , St. Louis, Mo. ; Hi-Pure nitrogen from LiquidCarbonic Corp., Los Angeles, Calif.; CGC-24i and Dowex50-2X from J. T. Baker Chemical Co., Phillipsburg, N. J.;

and veratridine from Aldrich Chemical Co. , Milwaukee, Wis.All other reagents were reagent grade. Plastic wells (“centerwells―)were obtained from Kontes Glass Co., Vineland, N.J.

Cell Culture. Cell culture is described in detail in thepreceding paper (22). Briefly, LA-N-i was derived from bonemarrow metastases of a neuroblastoma in a 2-year-oldmale. LA-N-2 was derived from a biopsy specimen of theprimary abdominal neuroblastoma of a 3-year-old female(21). Both lines were grown in Waymouth's medium contaming 30% heat-inactivated fetal calf serum, glutamine(100 mM), and gentamycin (50 @g/ml).SK-N-SH (4), 5K-N-MC (4), 1MR-32 (24), T24 (6), MT (14), and J82 (18) weregrown in Eagle's minimal essential medium containing 15%heat-inactivated fetal calf serum , glutamine (100 mM), andgentamycin (50 @g/ml).

1372 CANCERRESEARCHVOL. 37

Adrenergic,1 Cholinergic, and Inactive Human NeuroblastomaCell Lines with the Action-Potential Na@lonophore2

Gregory J. West, Jiro Uki, Harvey R. Herschman,3 and Robert C. Seeger@

Department of Biological Chemistry and Laboratory of Nuclear Medicine and Radiation BiologyfG. J. W., J. U., H. A. H.J, and the Departmentof Pediatrics@R.C. 5.1, UCLA School of Medicine, Los Angeles, California 90024

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Neuroblastoma Cells with Na@Ionophore

test tube. The tube was sealed with a sleeve stopper fromwhich a plastic well containing 0.2 ml Hyamine hydroxidewas suspended to collect “CO2.Oxygen was flushed fromthe tube with a stream of nitrogen entering through a hypodermic needle and exiting through a spinal needle. Thereaction was started by injecting 1 ml of enzyme sample.The tubes were incubated at 37°for 60 mm in a shakingwater bath.. At the end of this time, 0.1 ml of 8 N H@ZSO4wasinjected and shaking was continued for another 30 mm. Atthe end of the experiment, the plastic well was placed in 10ml of Liquifluor:toluene (42:1000) for counting.

Tyrosine Hydroxylase. Tyrosine hydroxylase was measured by a modification ofthe method of Nagatsu eta!. (16).L-[3,5-3H]Tyrosine was purified by absorption to and elutionfrom Dowex 50-X2. L-[3,5-3H]Tyrosine (250 @I)was acidifiedwith 0.2 ml of 0.17 N acetic acid and absorbed to the resin.The column was washed with 100 ml of distilled water andthen with 10 ml of 0.5 N HCI. The radioactive tyrosine waseluted with 20 ml of 3 N HCI. The HCI was evaporated with arotary evaporator, and the sample was resuspended andstored in absolute ethanol.

Cells (two 100-mm Petri plates) were harvested as follows.Medium was removed from the plates. Cells were washed 3times with cold 0.9% NaCI solution. To each plate wasadded 0.1 ml of 1 mM potassium phosphate buffer, pH 6.2.Cells were scraped from the dish with a rubber policemanand transferred to a test tube with a Pasteur pipet. Cellhomogenates were prepared by freezing and thawing 3times. The supernatant from a i000 x g, 10-mm centrifugation was used for protein and tyrosine hydroxylase determination.

For assay of enzyme activity, 3 x i0@ cpm of L-[3,5-3H]tyrosine (for each assay tube) were evaporated in astream of nitrogen and resuspended in 10 @lof 1 m@ Ltyrosine per assay tube. The reaction mixture, which waspreincubated for 2 mm at 37°,contained 0.1 M potassiumphosphate buffer (pH 6.2), 2000 units of catalase, 1 mM DL6-methyl-5,6,7,8-tetrahydropterine dihydrate, 0.1 mM mercaptoethanol, and enzyme solution in a final volume of 100

@l.The reaction was started by the addition of 10 @lof thetyrosine solution. After 20 mm at 37°,200@ of 0.17 M aceticacid were used to stop the reaction. A boiled enzyme solution was used to determine the blank. Tritiated water wasisolated by chromatography over Dowex 50-X2 and countedin 10 ml of Bray'ssolution(5).Allassayswere done induplicate. We found the monomethyl derivative of 5,6,7,8-tetrahydropterine to give 2.5 times the tyrosine hydroxylaseactivity as that observed with the 6,7-dimethyl derivative.

RESULTS

Veratridine-dependent Na@Uptake. Veratridine-dependent Na@uptake implies the presence of the action potentialNa@ionophore (7). Neuroblastoma cell lines LA-N-i , LA-N-2,SK-N-SH, and IMR-32 each have a large veratridine-dependent Na@uptake (Table 1). In contrast, line SK-N-MC consistently had small positive values of veratridine-dependent Na@uptake which were not significantly different from controlhuman cell lines T24, derived from a bladder carcinoma,and MT, derived from an osteogenic sarcoma.

Veratridine-dependentNaF Uptake. Cell lineswere assayed for veratridine-dependent Na@uptake according tothe procedure of Catterall and Nirenberg (7). Cells grown toa confluent monolayer in 60-mm culture dishes were preincubated at 37°for 2 to 3 mm in a shallow water bath with 2ml of assay medium (50 mM N-2-hyd roxyethylpiperazi ne-N'-2-ethanesulfonic acid, i30 mM NaCI, 5.4 mM KCI, 1.8 mMCaCl2, 0.8 mM MgSO4, 5.5 mM glucose, and 1.0 mMNaH2PO4adjusted to pH 7.4). The medium was then aspirated and replaced with assay medium containing 5 mMouabain, 0.2 mM veratridine (omitted in controls), and 0.5

@Ci22NaCIper ml. The incubation was resumed for 15 mmor for a variable time in the case of the time-course study.The assay medium was aspirated , and the plate was washedrapidly 4 times with wash medium (164 mM NaCI, 5.4 mMKCI, 1.8 mM CaCI2, 0.8 mrvi MgSO4, and 5.0 mM NaH2PO4adjusted to pH 7.4). The cell monolayer was dissolved with.2ml of 0.4 N NaOH. One ml was counted in a gamma coUnter,and 0.2 ml was used to measure protein concentration (13).The assay medium and wash medium were modified slightlyin the case of the time-course studies by the omission ofphosphate. In the wash medium it was replaced by 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid . Thisdoes not affect the rate of 22Na@uptake. For further characterization of the nature of the observed Na@influx, cellswere also assayed in the presence of 0.001 , 0.1 , and 10 @Mtetrodotoxin. All Na@ uptake determinations were performed on triplicate culture dishes.

Acetylcholine. Cells were grown to confluence in 100-mmtissue culture dishes, washed 3 times with 0.14 M NaCI, 0.15mM CaCl2, 0.50 mM MgSO4, and 0.01 M 2-amino-2-hydroxymethyl-i ,2 propanediol base adjusted to pH 7.45 with HCI,scraped off the plate with a rubber policeman, and collectedby centrifugation (20 mm, 27,000 x g, and 4°).The pelletwas weighed by difference and stored at —70°until assay.Acetylcholine was assayed in the laboratory of Dr. DonaldJenden according to the method of Jenden et al. (12) asmodified by Freeman et a!. (10). Briefly, the cells werehomogenized in 2 ml of formic acid:acetone (15:85). Particulate matter was removed by centrifugation. After 2 etherwashes, acetylcholine was extracted with dipicrylamine.Acetylcholine was then demethylated to the tertiary amineby treatment with benzene thiolate. The sample was injected into a gas chromatograph, and the effluent wasmonitored by continuous mass spectrometry. The output ofthe mass spectrometer was analyzed by a computer forquantitation of the acetyicholine.

Glutamic Acid Decarboxylase. Glutamic acid decarboxylase was assayed by the method of Wu and Roberts (26)with the exception that the substrate solution was 0.2 M inK2HPO4.Cells were grown to confluence and washed asdescribed in the previous section. The cell monolayer waswashed once more with 0.5 ml of the standard buffer of Wuand Roberts (26) and scraped off in a total of 2.5 ml ofstandard buffer. The cell suspension was homogenized by25 strokes of a “B―pestle in aDounce homogenizer. Particulate matter was removed by centrifugation at 27,000 x gfor 10 mm. [i-'4C]Glutamic acid was purified by chromatography on CGC-24i by the method of Moore and Stein (15).The radioactive substrate (0.1 ml; 0.2 @tCi)was placed in a

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Veratridine-de

Cell linependent

Na@uptake (nmoles Na@/

mg protein)Stimulation ratioTetrodotoxin inhibition(@&M)LA-N-i1160

± 90°5.8 ±1.00.03LA-N-2830± 103.4 ±0.40.05SK-N-MC47± 181.1 ±0.02NT―SK-N-SH1500±1505.5 ±0.20.08IMR-321040±1304.2 ±0.40.8T240± 201.0 ±0.1NTMT—40± 370.9 ±0.1NT

G. J. West et a!.

Table 1

Effect of veratridine and tetrodotoxin on 22NA@uptake in humancell lines

Replicateplates of confluent cells were incubated 15 mm at 37°in the presence of the indicated effectors plus 5 mM ouabain and22NaCI(0.5 MCi/mI). “Veratridine-dependentNa@uptake―is equalto the Na@uptake in plates with 0.2 mM veratridine minus theuptakein thosewithout. The“stimulationratio―is the ratio of these2 values.“Tetrodotoxininhibition―is the concentration of tetrodotoxin necessary to inhibit veratridine-dependent Na@ uptake by

Tetrodotoxin was used to characterize further the natureof the veratridine-dependent Na@uptake . Veratridine-dependent Na@uptake was measured in the presence of 0.001,0.1 , and 10 MMtetrodotoxin (Table 1). Uptake of Na@by LAN-i , LA-N-2, SK-N-SH, and IMR-32 was inhibited by tetrodotoxin. The concentration resulting in 50% inhibition rangedfrom 0.03 to 0.8 @Mfor the various cell lines.

If veratridine-dependent Na@uptake represents (a) passive diffusion of Na@through the open Na@ionophore with(b) subsaturating Na@concentration, then the time coursefor uptake of Na@should be given by a single exponentialand should fit the equation (7):

(22Na@In (@N@)@ _ (2:Na+)( kt

where (22Na@fl@,is the concentration of radioactivity insidethe cell at equilibrium and (22Na@)@@is the concentration attime t. If the left side of the equation is plotted against time,a straight line with slope k is the result. SK-N-SH was chosen as a representative cell line and assayed (Chart 1). It isapparent from the straight-line plot that Equation A is satisfied, consistent with both assumptions.

Neurotransmitters.Cultured neuroblastomacell lineswere assayed for tyrosine hydroxylase, glutamic acid decarboxylase, and acetyicholine (Table 2). Activities in cell lineswere compared to activities found in freshly preparedmouse or rat brain. LA-N-i and IMR-32 contained 15 and 5times as much tyrosine hydroxylase, respectively, as brainextracts. In contrast, LA-N-2, SK-N-SH, and SK-N-MC contamed only 0.5 to 5% of the tyrosine hydroxylase activity ofbrain. Acetyicholmne was present in LA-N-2 in 20-fold greaterquantities than in brain; other lines had only 10 to 50% thatin brain. None of the cell lines had high specific activities ofglutamic acid decarboxylase when compared to brain ornonneuronal cell lines.

ClinicalCorrelations.All tumorswerediagnosedpathologically as neuroblastomas; however, not all produced catecholamines. The patient from whom LA-N-i was derivedhad elevated levels of vanillylmandelic and homovanillicacid in his urine, and the cell line had high levels of tyrosinehydroxylase. LA-N-2 was derived from a child who did notexcrete catecholamines, and the cell line lacked tyrosinehydroxylase. However, a freshly frozen sample of the tumorfrom which LA-N-2 was derived contained acetylcholine asdid the cell line.5 Line SN-N-MC lacked tyrosine hydroxylase; the patient did not excrete catecholamines (4). Urinarycatecholamines were not reported for the patient fromwhom IMR-32 was derived. The only discrepancy was withSK-N-SH. In this case the patient excreted vanillylmandelicacid , but tyrosine hydroxylase was not present in the cellline. However, Biedler et a!. (4) reported that SK-N-SH cellshave dopamine-fJ-hydroxylase, an enzyme characteristic ofadrenergiccells.

DISCUSSION

Human neuroblastoma cells in continuous culture werefound to retain membrane transport and neurotransmitter

(A)

aMean±S.D.b NT, not tested.

Minutes

Chart 1. Time course of °°Na@uptake for cell line SK-N-SH. 0, no veratridine; •,plus 0.2 mM veratridine. Incubation conditions as in Table 1. Top,plotted directly; bottom , plotted according to Equation A (see text). (Na@)r,=1650 nmoles Na@per mg protein.

functions characteristic of nervous tissue. Using thesefunctions, the cell lines were classified as positive or negative for the action potential Na@ionophore and as adrenergic, inactive, or cholinergic.

Veratridine-dependent Na@uptake implies that a cell linehas the action potential Na@ionophore and thus is likely tobe electrophysiologically active. Human neuroblastoma celllines have not been examined previously for this property.Four of the 5 lines tested , LA-N-i , LA-N-2, SK-N-SH, andIMR-32, had veratridine-dependent Na@uptake.

This observation was extended by using tetrodotoxin, aspecific inhibitor of the action potential Na@ionophore (17),to inhibit veratridine-dependent Na@uptake. The equilibrium constant for binding of tetrodotoxin to nerve prepara

a R. C. Seeger, G. J. West, H. lsaacs, and N. Shore, manuscript in prepara

tion.

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Neuroblastoma Cells with Na@Ionophore

Table2Neurotransmitters and enzymes of neurotransmitter biosynthesis in

human cell linesSpecific activities of tyrosine hydroxylase and glutamic acid de

carboxylaseare reported. One unit of tyrosine hydroxylaseactivityis equal to 1 pmole of product per mm. One unit of glutamic aciddecarboxylaseactivity is equal to 1 pmole of product per mm.

of neurons commited to that transmitter will account foronly a fraction of the mass of brain.

Cell lines LA-N-i and IMR-32 contained large amounts oftyrosine hydroxylase relative to brain. We therefore condude they are adrenergic. In contrast, LA-N-2 had largeamounts of acetylcholine, and we conclude it is cholinergic.SK-N-SH and SK-N-MC lacked tyrosine hydroxylase andacetylcholine, and by these criteria we classify them asinactive. However, SK-N-SH has been reported to have dopamine fj hydroxylase, an adrenergic marker, and SK-N-MChas cholineacetyl transferase, a cholinergic marker (Ref. 4;J. Biedler, personal communication). The discordance between these latter markers and those we utilized suggeststhat neuroblastoma cells can express markers of a givendifferentiation pathway to variable degrees. This is supported by our observation that LA-N-i cells but not those ofthe other adrenergic cell lines have multiple cytoplasmicdense cores (22). Our findings, together with those of Biedler et a!. (4), are consistent with the clinical histories suggesting that the established cell lines are representative ofthe tumors from which they were derived.

Our finding that IMR-32 cells have elevated levels of tyrosine hydroxylase differs from the report of Prasad et a!. (19)who found that IMR-32 cells had no detectable tyrosinehydroxylase activity. However, they did induce 9 pmoles/mm/mg protein by 5-bromodeoxyuridine treatment. Asidefrom possible differences in culture conditions or divergence of cell lines, we cannot account for this difference.

LA-N-2 is the 1st human neuroblastoma cell line demonstrated to produce acetyicholine. A cholinergic clone ofmurine neuroblastoma C1300 has been reported (3); however, LA-N-2, even before cloning, appears to be synthesizing acetyicholmne more actively. Three additional humanneuroblastoma cell lines recently have been reported tohave choline acetyltransferase, another marker for cholinergic activity (20); however, 2 lines also had tyrosine hydroxylase, leaving one that expressed only the cholinergicmarker. None of these lines appear to be as active as LA-N-2since choline acetyltransferase levels ranged from 200 to495 pmoles/min/mg protein. Control values for choline acetyltransferase in brain and nonneural tissues were not included in this report; however, Wilson et a!. (25), using thesame method, reported mouse brain to have 1500 pmoles/mm/mg protein. The discovery that LA-N-2 produced onlyacetylcholine and that the biopsy specimen from which LAN-2 was derived also had acetylcholine may have significantclinical implications, both for differential diagnosis of neuroblastoma-like tumors that do not produce catecholaminesand for estimating prognosis.

LA-N-i is the ist human neuroblastoma cell line demonstrated to have the action potential NaF ionophore, tyrosinehydroxylase, and cytoplasmic dense cores (22) in combination, suggesting that these cells have the capability of synthesizing and packaging catecholamines. If this is true, andif the cells can be induced to release catecholamines, LA-N-i may provide a good human-derived model for many studies of adrenergic neurons.

LA-N-2, together with LA-N-i , provides a valuable modelfor comparing cholinergic and adrenergic cells. It has notbeen possible to obtain completely pure populations of

Glutamicaciddecarboxyl

Tyrosine hydrox Acetylcholinease (mullylase (units/mg(nmoles/g tus units/mg pro

Celllineprotein)sue)tein)LA-N-i688

±17―10.8 ± 7.80.2 ±0.1LA-N-20.0―504±2090.0 ±0.05SK-N-MC0.5

± 0.67 .8 ± 4.90.9 ±0.4SK-N-SH5.3± 0.74.2 ± 3.50.1 ±0.2IMR-32219±122.5 ± 1.00.1 ±0.04T240.7± 1.51 .4 ± 1 .00.0 ±0.01MT0.3± 0.60.9 ± 1.00.0 ±0.05J822.1± 0.21 .5 ± 0.50.1 ±0.1Brain41

.2 ± 1.1“22C5.3 ±1.5―

a Mean ± S.D.

b Values less than blank.C Rat.

d Mouse.

tions is 3 to 5 nM in frog and rabbit (8). Catterall and Nirenberg (7) observed 50% inhibition of veratridine-dependent Na@uptake with 0.01 @Mtetrodotoxin in mouse Ci300clone Ni8 cells, an electrically excitable line. We also haveobtained this result with Ni8 cells. Stallcup and Cohn (23)report 50% inhibition of veratridine-dependent Na@uptakein rat neuronal cell lines with 1 @tMtetrodotoxin. These linesare all electrically excitable. Thus, the human neuroblastoma cell lines we have studied are sensitive to tetrodotoxinat concentrations well within the range observed for electrically excitable cells (Table 1). IMR-32 approaches the upperend of the range, but the others are in fact very low. Weconclude, therefore, that tetrodotoxin acts specifically inthese cases to block the action potential Na@ionophore andthat the veratridine-dependent Na@uptake we have observed results from the action of this ionophore. The timecourse of veratridine-dependent Na@uptake is consistentwith the view that this represents passive diffusion throughthe action potential Na@ionophore locked in the open configuration.

Cell line SK-N-MC did not show significant veratridinedependent Na@uptake and apparently does not contain theaction potential Na@ionophore. However, absence of veratridine-dependent Na@uptake does not mean that a cell lineis electrophysiologically inactive. Ions other than Na@maybe involved in action potential generation. Some cases havebeen noted of veratridine-insensitive but electrophysiologically active cell lines (D. Schubert, personal communication).

We classified cell lines as adrenergic, cholmnergic, gabaergic, or inactive by testing for the presence of tyrosinehydroxylase, acetyicholine, and glutamic acid decarboxylase, respectively. To be considered representative of neurons committed to production of a given neurotransmitter,we believea cell lineshouldhavea specificactivityof thattransmitter, or enzymes involved in its synthesis, significantly greater than that for whole brain since the population

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G. J. West et a!.

neurons, much less pure samples of a particular type ofneuron. These cell lines, which originated from singleclones (22) and which express either adrenergic or cholinergic properties, offer the opportunity to perform ultrastructural, electrophysiobogical, and biochemical comparisonson homogeneous populations of cells with neuronal characteristics. In this respect, they complement the variousmurine clones (3) and the recently described functional ratneural tumors (20). Finally, they also may be uniquely valuable as experimental models for developing new and improved therapies of child hood neuroblastoma.

ACKNOWLEDGMENTS

We thank Sylvia Rayner and Hilary Rosenblatt for excellent technicalassistance and Dr. Eugene Roberts for helping with the glutamic acid decarboxylase assay. We are grateful to Dr. Donald Jenden and his staff forperforming the acetylcholine assays. Cell lines were provided by Dr. June L.Biedler, Sloan-Kettering Institute for Cancer Research (SK-N-MC, SK-N-SH),Dr. Robert McAllister, Children's Hospital of Los Angeles (MT) and Dr. CarolO'Toole (T-24, J-82).

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1376 CANCERRESEARCHVOL. 37

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1977;37:1372-1376. Cancer Res   Gregory J. West, Jiro Uki, Harvey R. Herschman, et al.  

Ionophore+Cell Lines with the Action-Potential NaAdrenergic, Cholinergic, and Inactive Human Neuroblastoma

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