voltage-operated calcium channels in small cell lung...

[CANCER RESEARCH 50. 3892-3896, July 1. 1990]

Voltage-operated Calcium Channels in Small Cell Lung Carcinoma Cell Lines:Pharmacological, Functional, and Immunological Properties1

Emanuele Sher,2 Atanasio Pandiella,3 and Francesco Clementi

CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milano, via Vantitela 32, 20129 Milano, Italy

ABSTRACT

Different subtypes of voltage-operated calcium channels (VOCCs) areexpressed in different tissues and can be distinguished by functional andpharmacological criteria. One type of high voltage-activated calciumchannel, specifically recognized by the peptide neurotoxin u-conotoxin(wCTx), is expressed only in neurons.

Seven different human small cell lung carcinoma (SCC) cell lines werealso found to bind 125I-o>CTx.The binding was specific, saturable, and of

high affinity."5I-wCTx binding was not antagonized by the calcium channel ligands

verapamil, nitrendipine, and diltiazem. There was a correlation betweenthe amount of toxin binding and the detection of depolarization-inducedcalcium fluxes studied with the fluorimetrie probe Fura2. Fura2 experiments also demonstrated that, in addition to wCTx-sensitive calciumchannels, SCC cell lines also expressed wCTx-insensitive calcium channels, which were antagonized by nitrendipine and verapamil.

'"I-uCTx-labeled VOCCs from SCC cells were, furthermore, precip

itated by anti-VOCC autoantibodies obtained from patients affected bythe Lambert-Eaton myasthÃ©niesyndrome, a neuromuscular disease oftenassociated with SCC. The present findings further indicate the presenceof neuronal molecules with important biological function on SCC plasmamembrane and add new insights into the pathogenetic mechanism ofautoimmune neurological paraneoplastic diseases, like Lambert-EatonmyasthÃ©niesyndrome.

INTRODUCTION

In vitro growing human SCC4 cell lines have provided in last

few years a unique model for studying, at the molecular level,the biological properties of this very aggressive lung tumor.Although there is still controversy about the histogenesis ofSCCs (1), it is known that they express a number of neuronalmarkers (2).

VOCCs are key molecules in the regulation of Ca2+ influxinto neuronal cells and in particular in the control of Ca2+-

dependent release of neurotransmitters and peptide hormones(3) that are known to play a fundamental role in SCC physiology(4). VOCCs are heterogeneous molecules, present both in peripheral tissues and in the nervous system. Although differentsubtypes of VOCCs are known to exist, their classification,based on electrophysiological and/or pharmacological properties, is still a matter of debate (5, 6).

Drugs like the dihydropyridines, phenylalkylamine and ben-zothiazepine, are known to bind preferentially to a type of highvoltage-activated calcium channel (also called L type) expressedboth at the periphery and in the nervous system (7). oj-Cono-toxin, on the other hand, is known to bind selectively to a high

Received 11/7/89; revised 2/16/90.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.

1This work was partially supported by a CNR Grant: Special Project "Chimicafine."

2To whom requests for reprints should be addressed.'Supported by a CNR-CSIC Italian-Spanish Cooperative Treat (Grant 14.1-

1989). Present address: Sloan Kettering Institute for Cancer Research, New York,NY 10021.

4The abbreviations used are: SCC, small cell carcinoma; LES, Lambert-EatonmyasthÃ©niesyndrome; VOCC, voltage-operated calcium channel; mCTx = u-conotoxin; EGTA, ethyleneglycol bis(/3-aminoethyl ether)-JV,A',A'',A''-tetraaceticacid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

voltage-activated calcium channel (also called N type) expressedonly in neurons (8-11).

The characterization of the types of VOCCs expressed bySCC would give more insights into the histogenesis of thistumor, the mechanism of hormone release by these cells, andthe pathogenesis of the Lambert-Eaton myasthÃ©niesyndrome,a paraneoplastic neuromuscular disease often associated withSCC (12,13) and known to be caused by autoantibodies directedagainst the neuronal, Ã¼jCTx-sensitive,calcium channel (14).

It has been reported that VOCCs are indeed expressed inSCC cells. McCann et al. (15) first provided electrophysiological evidence that VOCCs were responsible for Ca2+ spike elec-

trogenesis in SCC cells in culture. Furthermore, more recently,Roberts et al. (16) and De Aizpurua et al. (17) studied depolarization-induced 45Ca2+fluxes in SCC cell lines and found that

they could be antagonized by some LES autoantibodies (16,17),adenosine and wCTx (17). However, a careful characterizationof Ca2* channel densities and of their pharmacology in SCC

cells is still lacking. We studied VOCC properties using different tools: binding assays with l25I-ojCTx to quantitate channel

density, Fura2 for studying the functional and pharmacologicalcharacteristics of VOCCs, and immunoprecipitation assay tostudy the immunological similarities between SCC and neuronal VOCCs.

The data reported here indicate that SCCs express a complement of VOCCs which is similar to the one expressed byneurons and further stress the role of tumor VOCCs as thetriggering antigen for the autoimmune pathogenesis of theLambert-Eaton myasthÃ©niesyndrome. A preliminary accountof this work has already been published (18).

MATERIALS AND METHODS

Cell Culture. All SCC lines were kindly provided by Prof. G. Caudinoand Dr. A. Mondino from the Department of Biomedicai Sciences andOncology, University of Torino Medical School, Torino,Italy, with theexception of the NCI-H-146 and a second batch of NCI-N-592 whichwere a kind gift of Dr. C. Soranza from the National Cancer Instituteof Milano, Italy. IMR32, HeLa subclones, HRT-18, KB, and A-172were obtained from American Type Culture Collection (Rockville,MD). SK-N-BE were kindly provided by Dr. G. Melino (Roma, Italy),SK-N-SH, VERO, and PGK by Prof. C. De Giuli Morghen (Universityof Milano, Milano, Italy), SH-SY5Y by Dr. M. Toselli (Pavia, Italy),TE671 by Dr. D. Sophianos (Athens, Greece), NIE-115 by Dr. M.Oortgiesen (Utrecht, The Netherlands), NG-108-15 by Dr. M. Parenti(University of Milano, Milano, Italy), PCI2 and A431 by Prof. J.Meldolesi (University of Milano, Milano, Italy), HepG2 by Dr. P. Rosa(our laboratory), and A-549 by Dr. A. Ciomei (Farmitalia-Carlo Erba,Milano, Italy).

SCC cell lines were all grown in RPMI-1640 medium, supplementedwith 10% fetal calf serum, 100 lU/ml penicillin, and 100 Mg/ml streptomycin in 10-cm diameter Falcon tissue culture dishes. The samemedium was used for SK-N-BE, SK-N-SH, SH-SY5Y, and PC 12 cells,while all other cell lines were grown in minimum essential medium,supplemented as above. All the cells were kept at 37Â°Cin a humidified

atmosphere of 5% CO2 in air.'"'-uCTx-Binding Assays. Binding studies and saturation, associa

tion, dissociation, and competition experiments were performed essen-

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CALCIUM CHANNELS IN SMALL CELL LUNG CARCINOMA

tially as described in Ref. 19, utilizing crude cell homogenates. Theincubation buffer consisted of 0.3 M sucrose, 5 ITIMHepes/Tris, pH7.4, supplemented with 0.01 mg/ml lysozyme and 1 mg/ml bovineserum albumin.

For the screening of the different cell lines, a unique concentrationof '25I-o)CTx(100 pM) was used and nonspecific binding determined in

parallel tubes, in the presence of an excess (1 MM)of cold wCTx. Thesame concentration of '25I-o>CTxwas used for association and dissocia

tion experiments, as well as in competition assays. In the latter, thecompeting drugs were added to the binding mixture, at the indicatedconcentrations, 10 min before the labeled toxin.

Saturation binding curves were obtained by adding increasingamounts of 125I-uiCTx(5-250 pM) to a fixed amount of cell homogenate.

For each concentration, nonspecific binding was determined as described above. The binding reaction was terminated at the indicatedtimes by washing the samples three times with an ice-cold washingbuffer consisting of 160 mM choline chloride, 1.5 mM CaCl2, 5 mMHepes/Tris, 1 mg/ml bovine serum albumin, pH 7. Radioactivity in thepellets was determined in a Beckman 4000 gamma-counter.

|Ca2+l,-Measurements with Fura2. SCC cells were recovered from the

dishes and passed three times through a sterile syringe to obtain asingle cell suspension. After centrifugation (5 min, 800 rpm), the cellswere resuspended in a Krebs-Ringer-Hepes solution consisting of 150mM NaCl, 5 mM KC1, 1.2 mM MgSO4and KH2PO4, 2 mM CaCl2, 6mM glucose, and 25 mM Hepes-NaOH (pH 7.4).

Loading with Fura2 and fluorescence measurements were performedas previously described (19).

Immunoprecipitation of '"I-uCTx-labeled VOCCs. Immunoprecipi-

tation experiments were performed by a method we recently described(14). Briefly, IMR32 or GLC-8 cells were homogenized and a saturatingconcentration (100 pM) of '"I-uCTx was added to the preparation in

order to label all VOCCs present. After 90 min at room temperature,when the binding reached equilibrium, a final concentration of 1%Triton XI00 was added to the homogenate. After 2 h at 4Â°C,the

samples were centrifuged for 30 min at 15000 rpm in a SORVALLRC-5B centrifuge, and the supernatant was used as the source of labeled

antigen for the radioimmunoassay.Increasing concentrations (0.5-5 M') of LES or control serum were

added to a fixed amount of antigen (40-50 pM), in the presence ofcontrol "carrier" serum, to achieve a constant amount of human IgG,in a total volume of 200 /il. After an overnight incubation at 4Â°C,50 jjl

of goat anti-human IgG was added to each tube and left 2 h at roomtemperature. The samples were then centrifuged and washed threetimes. The radioactivity in the pellets was then determined in thegamma-counter. LES serum was obtained from patients already shownto synthesize anti-VOCC autoantibodies (14).

RESULTS

125I-u)CTx-binding sites were expressed in all seven different

human SCC cell lines that we have studied (Table 1), regardlessof whether they were of the "classic" or "variant" type, the

latter known to be less differentiated and usually lacking someneuronal markers. The number of binding sites ranged from0.18 to 5.12 fmoles/mg of protein, as determined by using asingle concentration of l25I-coCTx(100 pM). 125I-u)CTx-binding

sites were also present in a number of human neuronal celllines but were undetectable in human tumor cells originatingfrom other tissues (Table 1). Since NCI-H-69 and GLC-8 werethe SCC lines more useful in terms of number of binding sitesand growth rate, they were chosen for the more detailed experiments described below.

Fig. 1 shows a saturation curve for 125I-u)CTxbinding toGLC-8 cells. The binding was specific (<30% nonspecific binding near the ATd,in the presence of 1.6 UMcold cuCTx), saturable,and of high affinity. From the Scatchard plot shown in theinset, a Bma%of 3.24 fmoles/mg of protein and a Ka of 4.2 x10"" M could be calculated. These values would correspond to

Table 1 "'I-aCTx-binding sites on different cultured cell linesThe binding assay was performed using a saturating concentration of 12*I-

0,'CTx (100 pM) as described in "Materials and Methods.

'"1-u.CTx bound

Cell line (fmoles/mg of protein)

Small cell lung carcinomaNCI-H-69 (human, classic type) 0.18 Â±0.1 'NCI-N-592 (human, classic type) 0.22 Â±0.2NCI-H-209 (human, classic type) 0.52 Â±0.2NCI-H-345 (human, classic type) 0.55 Â±0.1NCI-H-146 (human, classic type) 5.20 Â±0.5GLC-8 (human, classic type) 1.45 Â±0.9GLC-1 (human, variant type) 0.28 Â±0.1

NeuronalIMR32 (human neuroblastoma) 5.0 3.1SK-N-BE (human neuroblastoma) 4.5 2.0SK-N-SH (human neuroblastoma) 9.0 3.5SH-SY5Y (human neuroblastoma) 11.5 6.0NIE-115 (mouse neuroblastoma) 1.1 0.2NG-108-15 (mouse neuroblastoma x rat glioma) 3.4 1.9PCI2 (rat pheochromocytoma) 7.4 5.4

OthersA-549 (human lung adenocarcinoma) ND*

A431 (human epidermoid carcinoma) NDHepG2 (human hepatoma) NDTE-671 (human rhabdomyosarcoma) NDPGK (human squamous cell carcinoma) NDHeLa (human epitheloid carcinoma) NDHeLa-229 (human epitheloid carcinoma) NDHeLa-53 (human epitheloid carcinoma) NDVERO (monkey kidney) NDHRT-18 (human rectal tumor) NDKB (human epidermoid carcinoma) NDA172 (human glioblastoma) ND

a Values represent the mean Â±SD of different experiments (from 3 to 25 withthe different cell lines).

*ND, no binding detectable.

50 200100 150Free 125l-ooCtx [pM]

Fig. 1. Equilibrium binding of '2!I-u>CTx to GLC-8 cell homogenate. Each

value of this representative experiment is the mean of triplicate samples. Fromthe Scatchard plot shown in the inset a Ka of 4.2 x 10~" M and a Ã„mâ€žof 3.24

fmol/mg of protein can be calculated.

the expression of ~800 l25I-u)CTx-binding sites/cell. Similarbinding properties were found in the NCI-H-69 cell line (notshown).

At 37Â°C,125I-o)CTxbinding reached equilibrium in less than

15 min (Fig. 2A) and the binding was almost irreversible. Atboth 37 and 4Â°C,only a small component of specific binding

(10-20%) was lost after 6-8 h (Fig. 2Ã„), more likely due toproteolytic degradation than to true dissociation.

We found that nitrendipine, verapamil, and diltiazem, threedrugs known to bind to L-type calcium channels, were unableto compete for '"I-coCTx binding, in GLC-8 and NCI-H-69

cells at concentrations up to 10 /UM(not shown). The samebinding specificity has been reported for the IMR32 and thePC 12 neuronal cell lines (19).

Detection of Functional VOCCs with Fura2. The results of3893



Fig. 2. Association ill and dissociation(fi) kinetics of '"I-uCTx binding to GLC-8

cells. Values represent specific binding withthe value after 2 h taken as 100%. Dissociationwas started with addition of an excess of un-labeled toxins and sample dilution (arrow) followed by incubation at 37'C (â€¢)or 4Â°C(O).


Itoo.,

câ€¢0

5 0-1

30 60 90 120 30 60 90 120

'"I-u)CTx binding to SCC cell lines strongly suggested the

presence of VOCCs in these cells. In order to directly demonstrate the presence of functioning VOCCs, suspensions of SCCcells were loaded with the fluorescent Ca2+ indicator Fura2,and changes in [Ca2*]Â¡were continuously monitored.

Basal [Ca2+]Â¡in GLC-8 cells was found to be 121 Â±6.7 nM(mean Â±SD, n = 3) (Fig. 3), a value similar to that describedin other cultured SCC cell lines (20). Exposure of GLC-8 cellsto a depolarizing concentration of K.C1(50 mivi) resulted in afast (peak in Â»10s) increase in [Ca2+]Â¡,reaching a maximum of

182Â±2.6nM(/i = 3)(Fig. 3a).The initial phase of [Ca2*]Â¡increase was found to be of

transient nature, being followed by a slowly declining plateauphase ([Ca2+]Â¡= 140 Â±10 nM, n = 6). Addition of an excess of

EGTA (3 ITIM)during the plateau phase resulted in a rapiddecline of [Ca2+]Â¡toward basal, prestimulated values (Fig. 3a).

On the other hand, addition of EGTA before depolarizationcompletely prevented the KCl-induced increase in [Ca2+]Â¡(Fig.

30).

180 â€”

120-

b)

KCI KCI

160_

120-

I" "O- ?" H)c) l L Â» d)

^MÃ‚ ^^ Â»alH. HX*N* W "'- Â«MJ

KCI KCI

2 min.Fig. 3. Fura2 measurements of depolarization-induced calcium influx in GLC-

8 cells. KCI (50 HIM) induces a rapid increase in [Ca1*], (a). [Ca2*)Â¡increase was

due to influx since it was antagonized (a) and prevented (In by the addition of 3HIM EGTA to the cell suspension. Preincubation of the cells with 1.6 >iM njCTxfor 30 min at 37"C (c) reduced but did not block completely calcium influx. Theopened channels were blocked by Verapamil ( Vp) completely in uCTx-pretreatedcells (f ) and partially in uiCTx-untreated cells (</).

Minutes

Preincubation of the cells with 1.6 pM u>CTx for 30-60 minat 37Â°Chad no significant effect on basal [Ca2+]Â¡(117 Â±8.7

nM, n = 3). u)CTx reduced significantly (peak of 163.7 Â±7.6nM, n = 3, â€”25%),but did not abolish, depolarization-inducedCa2+ influx (Fig. 3c). When verapamil (10 /*M, Fig. 3c) or

nitrendipine (100 nM, not shown) were given to the cells, theywere able to completely antagonize the VOCCs "spared" bycoCTx, with a return of [Ca2+]Â¡to basal levels. On the otherhand, when verapamil was given to cells which were not prein-cubated with uCTx, it blocked a fraction of VOCCs (Fig. 3d)but, often, not completely.

Taken together, these data indicate, on one hand, thatVOCCs of SCC cells are functionally very similar to thosepresent in cells of neuronal lineage and, on the other hand,suggest the presence of at least two different subtypes of VOCCsin SCC cells, one "uCTx sensitive" and the another "u>CTxinsensitive" but verapamil sensitive.

Immunoprecipitation of l25I-u>CTx-labeled VOCCs by LESAutoantibodies. l25I-o)CTx-labeled VOCCs were solubilizedfrom both IMR32 human neuroblastoma cells and GLC-8human SCC cells, as described in the "Materials and Methods."

In both cell lines LES autoantibodies immunoprecipitatedthis o)CTx-labeled antigen, in a dose-dependent manner (Fig.4, closed symbols). In contrast, same dilutions of control serumdid not precipitate labeled VOCCs from the two cell types (Fig.4, open symbols). Analogous immunological cross-reactivitybetween neuronal and SCC VOCCs was found using anotherSCC cell line (NCI-H-59) and autoantibodies from differentpatients with LES (not shown).

DISCUSSION

Roberts et al. (16) and De Aizpurua et al. (17) using the45Ca2+flux technique have recently provided elegant evidence

for the presence of VOCCs in SCC cells. Furthermore, in theirstudies, it was clearly demonstrated that LES autoantibodieswere able to reduce depolarization-induced 45Ca2+influx into

SCC cells. However, the number and the characteristics of theVOCC subtypes present were not clearly determined. We addressed this last point utilizing different techniques, like thebinding of l25I-u>CTxto "neuronal" type VOCCs and Fura2 for

functional studies. We also performed VOCC immunoprecipi-tation experiments, by using a new radioimmunological assayspecific for neuronal type VOCCs (14).

We found l25I-o>CTx-binding sites in all seven SCC cell lines

used in this study, as well as in all neuronal cell lines tested.The number of 125I-o>CTx-binding sites was, however, usually

3894




2 ,

nIo

-Oa

1.5 .

u0)

Eo.u

2345

jul of serumFig. 4. Immunoprecipitation of '"I-ojCTx-labeled VOCCs from IMR32 and

GLC-8 cells. Increasing concentration of LES (closed symbols) or control (opensymbols) serum were reacted with 12*I-uCTx-labeled VOCCs solubili/ed fromIMR32 (triangles) or GLC-8 (circles) cells. This is a representative experiment offour with the same results.

lower in SCC than in neuronal cells. We were unable to detect'2$I-u)CTx-binding sites in a number of human cell lines origi

nating from other tissues and, particularly interesting, was theirabsence from a non-SCC lung tumor cell line (A-549). Ourdata, together with those of De Aizpurua et al. on other differentSCC cell lines, indicate that the presence of VOCCs is a generalfeature of SCC. Furthermore, it is noteworthy that all SCClines express not just "unclassified" VOCCs but a subtype of

VOCC (labeled by u>CTx), which was thought to be presentonly in neurons (6-11). Therefore, this molecule can be addedto the growing list of neuronal markers expressed by SCC cells.

However, it is known that in a single cell, in particular in asingle neuron, different subtypes of VOCCs may coexist whichcan be identified by their functional and pharmacological properties (6-11).

In competition-binding experiments we found that wCTx anddrugs affecting the L-type VOCC, like nitrendipine, verapamil,and diltiazem, did not compete for the same binding site. Thisseems to represent a general feature of the binding propertiesof these ligands to neuronal tissues (9-11, 21-26).

The experiments with the Fura2 technique demonstrate, furthermore, that SCC cells express both oiCTx-sensitive VOCCsand u>CTx-insensitive VOCCs. The latter are probably identifiable as L-type VOCCs since they are antagonized by verapamiland nitrendipine.

Therefore, previous pharmacological results from De Aizpurua et al. (17) and our own present data would indicate thepresence of at least two types of VOCCs in SCC cells. Thisevidence is important in trying to define which VOCC subtype(if any) is indeed involved in the control of hormone secretionfrom SCC cells.

As already mentioned, the presence of neuronal type VOCCson SCC cells is not only interesting for determining the biological properties of this tumor but has also clinical implicationfor the pathogenesis of LES. LES patients synthesize autoanti-bodies directed against neuronal wCTx-labeled VOCCs (14, 27,

28).

We here demonstrate that neuronal type calcium channels ofSCC cells are not only pharmacologically and functionallysimilar to their neuronal counterparts but they share commonantigenic sites. In fact, LES autoantibodies were able to im-munoprecipitate with the same potency '25I-o)CTx-labeled

VOCCs from both neuronal and SCC cells.Therefore, our data are in agreement with the immunological

recognition of SCC VOCCs by LES autoantibodies, claimedon the basis of functional experiments (16, 17).

The epitopes recognized by LES autoantibodies on theVOCCs of neuronal and SCC cells are mainly external to theu)CTx-binding site since in our immunoprecipitation assay thisis already occupied by the toxin. This is not a surprise, since,until now, LES autoantibodies directly acting as antagonists onthe VOCCs have not been described; LES autoantibodies arethought to exert their pathogenetic effect by inducing VOCCantigenic modulation (14, 29, 30).

The strong similarities, at the pharmacological and immunological level, between VOCCs in SCC and VOCCs in neurons, further support the idea that in LES tumor antigens maytrigger an autoimmune response affecting also the VOCCsexpressed on nerve terminals. The absence of neuronal VOCCsin tumor cell lines different from SCC further clarify the biological basis for the stronger association of LES with SCC thanwith other cancers. Still unknown is the triggering factor forthe production of anti-VOCC autoantibodies in those patients(~30%) who do not have an underlying SCC.

ACKNOWLEDGMENTS

We thank all the colleagues who provided us with the different celllines. We thank also E. Biancardi, M. Passafaro, and N. Hukovic whohelped in some experiments and P. Tinelli for technical support. Weare indebted to Profs. E. Carbone and G. Caudino (University ofTorino) for their suggestions and critical reading of the manuscript.

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1990;50:3892-3896. Cancer Res Emanuele Sher, Atanasio Pandiella and Francesco Clementi Immunological PropertiesCarcinoma Cell Lines: Pharmacological, Functional, and Voltage-operated Calcium Channels in Small Cell Lung

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