FEBS 11695 Volume 312, number 1, 66-70 0 1992 Federation of European Biochemical So&tics 00145793/9265,00
Neuronal-type
Paola Tarroni”, “CNR Center
November
nicotinic receptors in human neuroblastoma lung carcinoma ceil lines Francesca
and small-cell
Rubboli”, Bite Chini”, Ruud Zwartb, Marga Oortgiesen”, and Francesco Clementi”
of C}~topharrr~~cofeg~,Deportmwt
of Tosicologv,
of Medical Phawracology, Utliversity of Milan, Mih, Universitv of Utrecht, Utrecht, The Neri~erlands
Received 4 September
1992; revised version received 17 September
1992
Emanuele
Sher”
Italy and bResearr/r Institute
1992
subunit of the neuronal nicotinic receptor, sharing 88% homology with the ratbd subunit, has been cloned from a human neltroblastoma cell line. The gene encoding the human p4 subunit isexpresscd in association with the a3 gene in neuroblastoma and small-cclt lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional ncuronal nicotinic receptors. We suggest that these rcccptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosccrctory human tumors.
A,6
Nicotine; Receptor; Cloning; Neuroblastoma: Small-cell lung carcinoma
1, INTRODUCTION Neuronal nicotinic receptors are present in the censystem (CNS), the peripheral ganglia and the adrenal medulla. In these tissues the activation of nicotinic receptors causes a localized depolarization of the plasma membrane which may also lead to ncurotransmitter release [1,2]. Different gene products (al-a,; &&), identified in the rodent and avian systems, participate in the formation of neuronal nicotinic receptors [3], which have distinct localization [3] and pharmacological profiles [4]. Very little information is available on the molecular properties of human neuronal nicotinic receptors. We took advantage of previously choracterized human cell lines with neuronal properties. Neutoblastoma (NB) is a tumor of the peripheral nervous system which is known to release amines and peptide hormones into the circulation [S]. Over recent years, in vitro-stabilized NB cell lines have proved to be invaluable tools for the characterization of different types of neuronal receptors [6--S].On the other hand, small-cell lung carcinoma (SCC) cell iines derived from epithelial lung tumors consist of secretory cells which also express a number of neuronal traits [9]. Binding studies have tral mxvcus
Correporrderzce address: P. Tarroni, CNR Center of Cytopharmacology, via Vanvitrlli 32. 20129 Milano. Italy. Fax: (39) (2) 749 0574. The hS, nucleotide sequence has beeendeposited in the EMBL data library under accession number x68275. AC~mWims: SCC, small-ccl: lung carcinoma; NE, neuroblasioma; Ach, acetylcholine; Cch, carbachol; TPMP’, triphcnylmcthylphosphonium: nt, nucleotidcs.
66
previously shown that SCC cell lines express muscarinic cholinergic receptors [ 101,but recent evidence suggests that they may also express cholinergic receptors of the nicotinic type [ 11,123. We here report (i) the cloning of the human p4 nicotinic subunit, (ii) the selective expression of a5 and p4 nicotinic subunits in human NB and SCC cell lines, and (iii) the presence in the same cells of functional nicotinic receptors. 2. MATERIALS AND METHODS A @IO EDNA library from the lMR32 human NB cell line (kindly provided by H. Soreq) was screened under low stringency conditions (hybridization was performed in 43% v/v formamide, 5x SSC, IX Denhnrdt’s, 20 mM sodium phosphate butler, pH 6.8.0.18 w/v SDS. and 20~g/ml denatured fish sperm DNA at 37°C. Filters were washed at a final stringency of 2x SSC, 0. I% SDS at 6G”C.I) USillg probe PC%49 (kindly provided by S. Heinemann) which encodes the rat pz nicotinic subunit [13]. Two positiveoverloppingclones.ilh2l (nt221-1,372)and Al122 (nt R81-1,360), were isolated (numbers in parentheses indicate the nucleotide residues or the cDNA carried by the clone). DNA scqucncing was perfamed in M13mp18 and M13mpl9 using the Scqucnasc Version 2.0 kit (USB). The two clones were round to contain fragments of’ the human fl,, subunit cDNA. Further inrormetion (nt I-34G) was obtained by sequencing the PCR amplification products from 1MR32 mRNA described below. 2.2, PCIi ulllp/t~cdotr First strand cDNAn were synthesized from poly(A) RNA using the first strand cDNA synthesis kit (Stratagene) and the supplied random henamcr primer. I/lOth or the first strand reaction was amplified as previously described [ 141,The ,9.,5’ sense primer, S’rfl,-S (CAA CAA CCT GAT CCG CCC AGC; nt 1’77l97J, was drawn on thepd rat seqence [ 153.The primers for human,& (this paper), human aj [lb] and p-uctin [II] arc as follows: 3’l@,,-AS(GAA GGG AAA GTA CTT
Volume 312, number 1
FEES LETTERS
CAC CTC; r-113X-346);S’ha,-S (CGA CAT CAA GTA CAA CT; nt 558-574); 3’ha,-AS (TAG AGC TTC TCG TCA CC; nr 1,161I, 177); S;B-act-S(CGT GGG GCG CCC CAG GCA CCA GGG; nt 143-167): 3’p-act-AS (CGG TTG GCC TTG GGG TTC AGG GGG G; nt 364-388) The ampification products were analyzed by Southern blot using the full-length a, and the 2112I insert as probes, and human @actin was detected by ethidium bromide staining. The identity ofp, PCR products was confirmed by DNA sequencing.
November 1992
human Pr, rot
P4
human rat
3.1. Hutnan p4rlicotinic receptor suburlit Fig. 1 shows an alignment of the amino acid sequences of the human and rat /$ subunits. The deduced protein sequence of the human pd subunit (451 amino acids) was iacomplete at its N-terminal but revealed the canonic primary strucutre of a nicotinic receptor subunit, which was characterized by four hydrophobic putative membrane-spanning regions and a large cytoplasmic loop. At the amino acid level, the homology between human and rat subunits was 88%. The most divergent region was represented by the cytoplasmic domain (77% homology), where the human subunit also had a deletion of four amino acids. 3.2. kpression
of ha3 and h/3., in nerrrortal und nonrwtrronul ceN titles
It is known that rat and chick a, and /X,subunits are
DO
p4 DE~~TfHVLPLKPEH~YRL~SSRYE~ILRIPN~NA llllllllllI III1 IIIIIIIIIIIII IIIIIIIIlll EPTMTTSIWLL(QSWTDYRLhWIISSCIR~NILRlPAnRYLILPDIVLIHNA
74
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0
100 0.
t
DtTYEVSVYTNLI’.‘XSMG?iVLHLPPAIYKSACK~EVKYFPFDQQ2?ZTLKF IIllllllIll Illllll IIlllIIlllllllll llllllll DGTYEVSWT~I~SUGS~OWLPPAt7KShncIeVKliFPFDPQBEAVKF
124 II
ASWTYDHTEfDMVTMTPThSMDDFTPSGeWDIVALPGRR~NPQDPS~ III1 IIllllllI III llllllllIIlllllllIIIIIIIIllllI RSHTSDHTNI~MVLKSCTA:MDDFTPSGEnDIVA~~~NPQDP5~
humn
3. RESULTS AND DlSCUSSIGN
24
0
Electropbysiological experiments on SK-N-BE cells were carried out using the standard whole-cell patch-clump technique [I 81.Resting potential was t?om -50 to -G5 mV. Patch electrode resistance was 3-4 MR, and 70-758 of the series resistance was compensated for. Membrane potential was held at -SO mV. Mcmbranc currents were lowpass filtered (8 pole Bessel; -3 dB at 1 kHz), digitized (12 bits: 1,024 points/record) and stored on magnetic discs for off-lint computer analysis. The external solution contained (in mM): NaCl 135, KCI 5.5, C&I, I& MgC& 0.8, HEPES 20, glucose 25. and sucrose 3G.5(pH 7.3.340 mOsm). The pipette solution contoined (in mM): KCI 100. KF 50. HEPES IO, sucrose 50, and EGTA IO (pH 7.2, 340 mOsm). For whole-cell superRation, a continuous flow (I .5 mllmin) or external solution was applied kIlTI a I mm diamctcr capillary positioned within 0.5 mm of the cell. The cells were superllrsed at room temperature (RT) with known concentrations ofthc agonist and/or antagonists. To improve recovery Tram dcsensilizdtion, cells were washed with cxternal solution for 4 min between each response. [“ti]TPMP+ binding assays were performed as in [20]. with some modifications. The dirrerent human call lines were obtained and grown as described [ 191.The binding assay was pcrformcd at RT on cell homogenates in a bulTer (in mM: N&I IGO, KCI 5, CaCI, 2, phenylmethylsulronyI fluoride 0.1, and HEPES IO. pH 7.4) containing 0.5 mdml BSA, [‘H]TPMP+ 50 nM (46.1 Ci/mnol; DuPont-NEN) and different concentrations ofcarbnchol (Cch) (Sigma). Non-specific binding was measured in the presence of 5 mM unlabeled TPMP’. Alter incubation, tlic samples were resuspended in 3 ml of bulTer containing BSA (I mg/ml), and filtered on GFIB filters (Whatman). The filters were washed 3 times with 3 ml buffer and radioactivity counted.
. . .TSSSPLISIK~LS~LISVHBR llllllllI I Ill llllllll RLAN~KLMWLLNKTRYNNLIRPAT~~L~E~RLELSLSQLXSVN~R
rat
P4
hurcx, rat
150
174 200
YTYDFIIKRKPLFYTINLIIP~rPLUILYFILPBOCGE~KISYL IIIIlllllIIIII llllllllI I lIIIfIIIIIIIII!IlIIIIII VTYC?fIKRKPLPII~SECCEKKT.lUS%la
224
IALTFFLLLISWIVPPTSLDVPL~GKYX&FTl4VLVTFSIV-CSYCVLNVlIH IllllllllIIIIIIIIIII lllllll lllllllIIIII IlllllII mTSLDfPLIGK7pH
274
RSPCTIITMhPrNKRCFLILFTFLF~~GPDSSPARE IlIIlIIll III IIIIIIIIIIIIIIII I I II RSPSTHI~SSWVKECFLHKLPTFLF~~RPCLEVSLVRVPHPSOLHLATAD
224
ATATS----‘IiPSNFYGNSMYFV?iPASARSNSPAGSTPVAXPRDFWLRSS IIIII II IIIIIII Ilil I III IllI I I I TAATSALCPTSPSNLYOSSMYFVHPVPAAPKSAVSSHTAGLPRDRRLRSS
170
b4 GRFIIODVPEAIECVSF~AQHP?nlDDEDQS~DWKWh Illi I llIIIIIIIIIII II III1 III1 /lIIIIIlllIII CRFREDLQ~:AtEGVSFIAPHLESDDRUQIVIEDHKWII*IWDRISL)IWW
p4 ~~CVLGTVGLFLPPLFQTHAUECPYAAQP3 III III IIIllIIlI II I ’ QIHAPSKDS nrop p4 m t
stop
2bO
200
210
400
420 450
451 475
Fig. I. Alignment of human and rat /?, subunit amino acid sequences. The one-letter amino acid notation is used. Putative transmembranc regions are underlined. Asterisks (*) indicate tbc cysteines conserved in all ligand-gatcd ion channels. Circles (‘) indicate glycosylation sites.
highly expressed in the peripheral nervous system and that , when expressed in Xenopus occytes, they yield functional nicotinic receptors with a ganglionic pharmacology [21]. IMR32 and SK-N-BE human NB cells have already been shown to express ali [ 161and a5 nicotinic subunits, but not ccl [123. By means of Northern blot analysis (Fig. 2), we now show that both IMR32 and SK-N-BE express a single b., transcript of about 3.1 kb. Given that NB originates from ganglionic peripheral neurons, this findings in human cells is in line with previous reports on rat. Furthermore, PCR amplification (Fig. 3) showed that the a3 and pA nicotinic subunits were expressed in two different SCC cell lines (NCW-I-69; NCI-N-592 not shown) although they were not expressed in either a lung non-SCC (A549) or a rhabdomyosarcoma (TE671) ceii iine. Thus the expression of the c+ and & nicotinic subunits is not confined to the nervous system but also appears in cells of the neuroendocrine type.
Volume 312, number 1
FEBS LETTERS
November : 992
Fig, 3. PCRnnalysisof tq and fi,, RlUAin different human cell lines. Human os and @,amplification prwducts from SK-N-RR, NWH-69, A 549 and TE 671 cells were analyzed by Southern blot with specific cDNAprobes. The PCR products of ha, and the t&were only present in SK-N-BE and NCI-H-69 cells, The same results wcrc obtained in NCI-N-592 cells (not shown). The @actin control (B-act) was detectable by ethidium bromide staining in all of the lanes. Fig. 2. Northern blot analysis of RNAfrom human neuroblastoma cells. Analysis of poly(A) R.NA from SK-N-BE and fMR32cells(LO mg each) was performed under high stringency conditions (final wash stringency: 0. lx SCC, 0.1% SDS, SOOC)using the Ah21 clone insert as probe. Autoradiography was at -7OOC for 2 days, 3.1 indicates the length of the transcript in lib.
NB C&Y
Superfusion with 1 mM acetylcholine (Ach) resulted in transient inward currents in most of the SK-N-BE (n = 13 cells tested) (Fig. 4) with peak amplitudes ranging from 0.2%3,50 nA. The Ach-induced inward current was completely and reversibly blocked by both 10 ~LM d-tubocurarine (Fig, 4a) and I yM TPMP’ (Fig. 4b), thus confirming the nicotinic nature of the Ach-induced currents. SK-N-BE and lMR32 are the first human neuronal cell lines of peripheral origin found to express the a3 and/I4 nicotinic subunits which might be involved in the formation of the functional nicotinic receptors here described, However, we cannot exclude the possibility that other a and /3 nicotinic subunits may be expressed in these cells and contribute towards the formation of different functional nicotinic channels. Several attempts were made to patch-clamp SCC cells, but the recordings were insufficiently stable to permit the characterization of the acetylcholine-induced currents. We therefore used the following approach to obtain indirect proof of the presence of functional nicotinic receptors, 68
3.4. [3JI]TPMP’ hex
birtdhtg lo humutt NB attd SCC cc/l
TPMP” binding to the nicotinic receptor is allosteritally modulated by cholinergic effecters [20]. This channel blocker binds to residues located inside the pore of the channel, which become available after receptor activation, The blocking effects of TPMP’ on the muscular nicotinic receptor have been previously characterized [22j, and we have shown here that it also blocks neuronal nicotiaic currents in SK-N-BE cells (see above). As expected [3H]TPMP+ binding in SK-N-BE cells was positively modulated, in a dose-dependent manner, by Cch (Fig. 5B>.The specific binding of i;HjT’PMPi was maximally stimu!ated by lo-” M Cch. Cch also stimulated the specific binding of [jHjTfMP+ in NCI-H-69 (Fig. SC) and NCI-N-592 (not shown) SCC cell lines which, like NB, express the cc3and ,& nicotinic receptor subunits. On the other hand, in the lung non-SCC cell line (A549), which does not express either a3 of /?., subunits, the binding of [‘H]TPMP’ was not stimulated by Cch (Fig. SD). As a control, an allosteric modulation of [3H]TPMP+ binding was also shown in TE671 cells, which are known to express muscle-type nicotinic receptors and the a, nicotinic subunit (Fig. SA). The expression of functional neuronal nicotinic receptors in SCC cell lines is a novel and intriguing tinding. We suggest that these receptors should be considered as new members of the growing list of neuronal markers selectively expressed by this type of lung tumor. Neuronal nicotinic receptors are known to mediate the secretagogue effects of endogenous acetylcholine
Volume 312, number
1
November 1992
a
b
ACh _- .---._.-_ -+
____L
13 mh wash
Pig. 4. Electrophysizlogical recordings in SK-N-BE cells. (a) inward control currents induced by 1 mM Ach (left) are completely blocked after 3 min of superfusion with lO,uM d-tubocurarinc (center). Recovery from tile block is observed al’tc-r4 min ofwashin8with control external medium (right). (b) Inward control currents induced by I mM Ach (left) are completely blocked after 7 min ofsuperfusion with 1NM TPMP’ {center). Recovery from the block is observed after 13 min of washing with control external medium (ri8ht). Superfusion periods are indicated by bars.
and exogenous nicotine in a number of tissues, and we have also demonstrated the secretagogue effects of nicotine on SCC cell lines (E. Sher, manuscript in preparation). The effects of nicotine on cell proliferation have
previously been described [i 1,23,24], but no information is available on the molecular nature of nicotine targets. The present characterization of neuronal nicotinic receptors in SSC, a subtype of human lung cancer, the association of which cigarette smoking is well demonstrated [25], offers new insights into the possible mechanisms by which nicotine might affect tumor cell physiology and proliferation. Ac~/to~lll~~~r~,z~~~~,~~ We thank N. BorSesc and C. Gotti for their discussions and extensive comments on the manuscript; J. Mahsco for oiigonucleotidc synth&s; S. Heinemann for the sift of the/& cDNA probe; H. Soreq for the lMR32 EDNA library; G. Melino, G. Gaudino. 5. Tzartos and A. Ciomei for cell lines; and K. Smart and I. RutToni for manuscript revision. The paper was supported by CNR tar8eted projects ‘Prevcnrion and Control of Disease Factors’ and ‘AginS’.
REFERENCES
-_
J
CCh-lctg CM3 Fig. 5. Dose-response curves of the cB’ectsof Cch on [‘H]TPMP+ binding. Cch-induced increases in [‘H]TDMP+bindinp were cvzluatcd in TE 671 (A). SK-N-BE (9, NCi-Ii-69 (C; and A549 CD; cd!s. Specific [‘HJTPMP’ binding in the absence of carbachol was considcred as 100%.Yalues represent the averuge of 3-8 experiments, each performed in triplicate.
[I] Wannacott. S., Irons, J., Lunt. 0.0.. Rapier. CM. and Albuquerque. E.X. (1988) in: Nicotinic Acctylcholine Receptors in the Nervous System (Clementi, F., Got& C. and Shcr. E. eds.) NATO ASI Series H: Cell Biology vol. 25. pp. 41-60. Springer* Vcrlag, Berlin. [2] Roll, L.W. and Perlman, R.L. (1983) Neuroscicnoc 10,979-985. [31 Dencris. ES.. Connolly, J., Rogers. S.W. andDuvoisin. R.(i991) Trends Eharmacol. Sri. 12. 34-40. [4] Luetjc, C.W. and Patrick, 3. (1991) .I. Neurosci. it, 537-845. [S] H&o, R.J.. SeeSer, RX., Yu, AL. and O’Connor, D.T. (1990) J. Clin. Invest. 85, 1555-l 559. {6] Clementi, F.. Cabrini. D.. Gotti, C. and Sher. E. (1986) 1. NCUFOthem. 47, 29l-297. [7] Gotti. C., Esparis 08ando. A. and Clcmcnti, F. (1989) Ncurossicnee 32, 759-767. 69
Volume
312, number
I
FEN
[S] Abbracchio. M.P.. Cattabcni, F., Clementi, F. and Sher, A. (1989) Neuroscience 30. 519-825. [9] Teeli& M.E. and Carney, D.N. (1985) in: Biology of Lung Cancer: Diagnosis and Treatment Biocbcmical Markers of Lung Cancer (Rosen, ST., Mulshine. J., Cutitta. F. and Abram% P.C. cds.) pp. 43-58, Marccl Dckkcr, New York. [IO] Cunningham, J., Lennon, Y., Lamben, E. and Scheithauer, B. (1985) 1. Neurochem. 45. 159-167. [I I] Maneckjee, R. and Minna, J.D. (1990) Proc. Natl. Acad. Sci. USA 87, 3294-3298. [I 21 Chini, B.. Clementi, F.. Hukovic. N. and Sher. E. (1992) Proc. Natl. Acad. Sci. USA S9, 1572-1576. [I31 Dcneris. ES.. Connolly, J., Boulter, J., Wada, E., Wada, K., Swanson, L.W.. Patrick, J. and Heinemann, S. (1988) Neuron I. 45-54. 1141 Frohman. M.A., Dush, MR. and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 5995-9002. [15] Duvoisin, R.M., Deneris, ES., Patrick, J. and Hcinemann, S. (1989) Neuron 3,487-496. [16] Fornasari, D., Chini, B., Tarroni, P. and Clementi, F. (1990) Neurosci. Lett. I I I. 351-356.
LETTERS
November
1992
[l7] Ponte, P., Ng:, S-Y,, Engee),J., Gunning, P. and Kedes, L. (1984) Nucleic Acid Res. 12. 1687-1696. [18] Hamill, OP., Marty,.A., Nchcr, E., Sakmann, B. and SiBworth, F.J. (1981) Pflllgcrs Arch. 391, 85-100. [I91 Shcr, E., Pandiella, A. and Clementi, F. (1990) Cancer Res. 5, 3892-3896. WI Lauffer, L, and Hucho, F. (1982) Proc. Natl. Acad. Sci. USA 79, 24062409. [2ll Couturier, S., Erkmun, L., Valera, S., Rungger, D., Bertrand, S., Boulter. J.. Ballivet. M. and Bertrand, D. (1990) J. Biol. Chcm. 2G5, 17560-l 7567. Spivak, C.E. and Albuquerque, E.X. (1984) Mol. Pharmacol. 27, 246-255. Shuller, H.M. (1989) Biochem. Pharmacol. 38, 34393442. Tominaua. K.. Kinoshita. Y.. Hato. F.. Masuda. A. and Matsuy&a, M’. (1989) Celi. Mol. Biol: 35’679-686. . lannuzzi, MC. and Scoggin, C.H. (1986) Am. Rev. Rasp. Dis. 134, 593-608.
Neuronal-type
Paola Tarroni”, “CNR Center
November
nicotinic receptors in human neuroblastoma lung carcinoma ceil lines Francesca
and small-cell
Rubboli”, Bite Chini”, Ruud Zwartb, Marga Oortgiesen”, and Francesco Clementi”
of C}~topharrr~~cofeg~,Deportmwt
of Tosicologv,
of Medical Phawracology, Utliversity of Milan, Mih, Universitv of Utrecht, Utrecht, The Neri~erlands
Received 4 September
1992; revised version received 17 September
1992
Emanuele
Sher”
Italy and bResearr/r Institute
1992
subunit of the neuronal nicotinic receptor, sharing 88% homology with the ratbd subunit, has been cloned from a human neltroblastoma cell line. The gene encoding the human p4 subunit isexpresscd in association with the a3 gene in neuroblastoma and small-cclt lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional ncuronal nicotinic receptors. We suggest that these rcccptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosccrctory human tumors.
A,6
Nicotine; Receptor; Cloning; Neuroblastoma: Small-cell lung carcinoma
1, INTRODUCTION Neuronal nicotinic receptors are present in the censystem (CNS), the peripheral ganglia and the adrenal medulla. In these tissues the activation of nicotinic receptors causes a localized depolarization of the plasma membrane which may also lead to ncurotransmitter release [1,2]. Different gene products (al-a,; &&), identified in the rodent and avian systems, participate in the formation of neuronal nicotinic receptors [3], which have distinct localization [3] and pharmacological profiles [4]. Very little information is available on the molecular properties of human neuronal nicotinic receptors. We took advantage of previously choracterized human cell lines with neuronal properties. Neutoblastoma (NB) is a tumor of the peripheral nervous system which is known to release amines and peptide hormones into the circulation [S]. Over recent years, in vitro-stabilized NB cell lines have proved to be invaluable tools for the characterization of different types of neuronal receptors [6--S].On the other hand, small-cell lung carcinoma (SCC) cell iines derived from epithelial lung tumors consist of secretory cells which also express a number of neuronal traits [9]. Binding studies have tral mxvcus
Correporrderzce address: P. Tarroni, CNR Center of Cytopharmacology, via Vanvitrlli 32. 20129 Milano. Italy. Fax: (39) (2) 749 0574. The hS, nucleotide sequence has beeendeposited in the EMBL data library under accession number x68275. AC~mWims: SCC, small-ccl: lung carcinoma; NE, neuroblasioma; Ach, acetylcholine; Cch, carbachol; TPMP’, triphcnylmcthylphosphonium: nt, nucleotidcs.
66
previously shown that SCC cell lines express muscarinic cholinergic receptors [ 101,but recent evidence suggests that they may also express cholinergic receptors of the nicotinic type [ 11,123. We here report (i) the cloning of the human p4 nicotinic subunit, (ii) the selective expression of a5 and p4 nicotinic subunits in human NB and SCC cell lines, and (iii) the presence in the same cells of functional nicotinic receptors. 2. MATERIALS AND METHODS A @IO EDNA library from the lMR32 human NB cell line (kindly provided by H. Soreq) was screened under low stringency conditions (hybridization was performed in 43% v/v formamide, 5x SSC, IX Denhnrdt’s, 20 mM sodium phosphate butler, pH 6.8.0.18 w/v SDS. and 20~g/ml denatured fish sperm DNA at 37°C. Filters were washed at a final stringency of 2x SSC, 0. I% SDS at 6G”C.I) USillg probe PC%49 (kindly provided by S. Heinemann) which encodes the rat pz nicotinic subunit [13]. Two positiveoverloppingclones.ilh2l (nt221-1,372)and Al122 (nt R81-1,360), were isolated (numbers in parentheses indicate the nucleotide residues or the cDNA carried by the clone). DNA scqucncing was perfamed in M13mp18 and M13mpl9 using the Scqucnasc Version 2.0 kit (USB). The two clones were round to contain fragments of’ the human fl,, subunit cDNA. Further inrormetion (nt I-34G) was obtained by sequencing the PCR amplification products from 1MR32 mRNA described below. 2.2, PCIi ulllp/t~cdotr First strand cDNAn were synthesized from poly(A) RNA using the first strand cDNA synthesis kit (Stratagene) and the supplied random henamcr primer. I/lOth or the first strand reaction was amplified as previously described [ 141,The ,9.,5’ sense primer, S’rfl,-S (CAA CAA CCT GAT CCG CCC AGC; nt 1’77l97J, was drawn on thepd rat seqence [ 153.The primers for human,& (this paper), human aj [lb] and p-uctin [II] arc as follows: 3’l@,,-AS(GAA GGG AAA GTA CTT
Volume 312, number 1
FEES LETTERS
CAC CTC; r-113X-346);S’ha,-S (CGA CAT CAA GTA CAA CT; nt 558-574); 3’ha,-AS (TAG AGC TTC TCG TCA CC; nr 1,161I, 177); S;B-act-S(CGT GGG GCG CCC CAG GCA CCA GGG; nt 143-167): 3’p-act-AS (CGG TTG GCC TTG GGG TTC AGG GGG G; nt 364-388) The ampification products were analyzed by Southern blot using the full-length a, and the 2112I insert as probes, and human @actin was detected by ethidium bromide staining. The identity ofp, PCR products was confirmed by DNA sequencing.
November 1992
human Pr, rot
P4
human rat
3.1. Hutnan p4rlicotinic receptor suburlit Fig. 1 shows an alignment of the amino acid sequences of the human and rat /$ subunits. The deduced protein sequence of the human pd subunit (451 amino acids) was iacomplete at its N-terminal but revealed the canonic primary strucutre of a nicotinic receptor subunit, which was characterized by four hydrophobic putative membrane-spanning regions and a large cytoplasmic loop. At the amino acid level, the homology between human and rat subunits was 88%. The most divergent region was represented by the cytoplasmic domain (77% homology), where the human subunit also had a deletion of four amino acids. 3.2. kpression
of ha3 and h/3., in nerrrortal und nonrwtrronul ceN titles
It is known that rat and chick a, and /X,subunits are
DO
p4 DE~~TfHVLPLKPEH~YRL~SSRYE~ILRIPN~NA llllllllllI III1 IIIIIIIIIIIII IIIIIIIIlll EPTMTTSIWLL(QSWTDYRLhWIISSCIR~NILRlPAnRYLILPDIVLIHNA
74
De
0
100 0.
t
DtTYEVSVYTNLI’.‘XSMG?iVLHLPPAIYKSACK~EVKYFPFDQQ2?ZTLKF IIllllllIll Illllll IIlllIIlllllllll llllllll DGTYEVSWT~I~SUGS~OWLPPAt7KShncIeVKliFPFDPQBEAVKF
124 II
ASWTYDHTEfDMVTMTPThSMDDFTPSGeWDIVALPGRR~NPQDPS~ III1 IIllllllI III llllllllIIlllllllIIIIIIIIllllI RSHTSDHTNI~MVLKSCTA:MDDFTPSGEnDIVA~~~NPQDP5~
humn
3. RESULTS AND DlSCUSSIGN
24
0
Electropbysiological experiments on SK-N-BE cells were carried out using the standard whole-cell patch-clump technique [I 81.Resting potential was t?om -50 to -G5 mV. Patch electrode resistance was 3-4 MR, and 70-758 of the series resistance was compensated for. Membrane potential was held at -SO mV. Mcmbranc currents were lowpass filtered (8 pole Bessel; -3 dB at 1 kHz), digitized (12 bits: 1,024 points/record) and stored on magnetic discs for off-lint computer analysis. The external solution contained (in mM): NaCl 135, KCI 5.5, C&I, I& MgC& 0.8, HEPES 20, glucose 25. and sucrose 3G.5(pH 7.3.340 mOsm). The pipette solution contoined (in mM): KCI 100. KF 50. HEPES IO, sucrose 50, and EGTA IO (pH 7.2, 340 mOsm). For whole-cell superRation, a continuous flow (I .5 mllmin) or external solution was applied kIlTI a I mm diamctcr capillary positioned within 0.5 mm of the cell. The cells were superllrsed at room temperature (RT) with known concentrations ofthc agonist and/or antagonists. To improve recovery Tram dcsensilizdtion, cells were washed with cxternal solution for 4 min between each response. [“ti]TPMP+ binding assays were performed as in [20]. with some modifications. The dirrerent human call lines were obtained and grown as described [ 191.The binding assay was pcrformcd at RT on cell homogenates in a bulTer (in mM: N&I IGO, KCI 5, CaCI, 2, phenylmethylsulronyI fluoride 0.1, and HEPES IO. pH 7.4) containing 0.5 mdml BSA, [‘H]TPMP+ 50 nM (46.1 Ci/mnol; DuPont-NEN) and different concentrations ofcarbnchol (Cch) (Sigma). Non-specific binding was measured in the presence of 5 mM unlabeled TPMP’. Alter incubation, tlic samples were resuspended in 3 ml of bulTer containing BSA (I mg/ml), and filtered on GFIB filters (Whatman). The filters were washed 3 times with 3 ml buffer and radioactivity counted.
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210
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Fig. I. Alignment of human and rat /?, subunit amino acid sequences. The one-letter amino acid notation is used. Putative transmembranc regions are underlined. Asterisks (*) indicate tbc cysteines conserved in all ligand-gatcd ion channels. Circles (‘) indicate glycosylation sites.
highly expressed in the peripheral nervous system and that , when expressed in Xenopus occytes, they yield functional nicotinic receptors with a ganglionic pharmacology [21]. IMR32 and SK-N-BE human NB cells have already been shown to express ali [ 161and a5 nicotinic subunits, but not ccl [123. By means of Northern blot analysis (Fig. 2), we now show that both IMR32 and SK-N-BE express a single b., transcript of about 3.1 kb. Given that NB originates from ganglionic peripheral neurons, this findings in human cells is in line with previous reports on rat. Furthermore, PCR amplification (Fig. 3) showed that the a3 and pA nicotinic subunits were expressed in two different SCC cell lines (NCW-I-69; NCI-N-592 not shown) although they were not expressed in either a lung non-SCC (A549) or a rhabdomyosarcoma (TE671) ceii iine. Thus the expression of the c+ and & nicotinic subunits is not confined to the nervous system but also appears in cells of the neuroendocrine type.
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Fig, 3. PCRnnalysisof tq and fi,, RlUAin different human cell lines. Human os and @,amplification prwducts from SK-N-RR, NWH-69, A 549 and TE 671 cells were analyzed by Southern blot with specific cDNAprobes. The PCR products of ha, and the t&were only present in SK-N-BE and NCI-H-69 cells, The same results wcrc obtained in NCI-N-592 cells (not shown). The @actin control (B-act) was detectable by ethidium bromide staining in all of the lanes. Fig. 2. Northern blot analysis of RNAfrom human neuroblastoma cells. Analysis of poly(A) R.NA from SK-N-BE and fMR32cells(LO mg each) was performed under high stringency conditions (final wash stringency: 0. lx SCC, 0.1% SDS, SOOC)using the Ah21 clone insert as probe. Autoradiography was at -7OOC for 2 days, 3.1 indicates the length of the transcript in lib.
NB C&Y
Superfusion with 1 mM acetylcholine (Ach) resulted in transient inward currents in most of the SK-N-BE (n = 13 cells tested) (Fig. 4) with peak amplitudes ranging from 0.2%3,50 nA. The Ach-induced inward current was completely and reversibly blocked by both 10 ~LM d-tubocurarine (Fig, 4a) and I yM TPMP’ (Fig. 4b), thus confirming the nicotinic nature of the Ach-induced currents. SK-N-BE and lMR32 are the first human neuronal cell lines of peripheral origin found to express the a3 and/I4 nicotinic subunits which might be involved in the formation of the functional nicotinic receptors here described, However, we cannot exclude the possibility that other a and /3 nicotinic subunits may be expressed in these cells and contribute towards the formation of different functional nicotinic channels. Several attempts were made to patch-clamp SCC cells, but the recordings were insufficiently stable to permit the characterization of the acetylcholine-induced currents. We therefore used the following approach to obtain indirect proof of the presence of functional nicotinic receptors, 68
3.4. [3JI]TPMP’ hex
birtdhtg lo humutt NB attd SCC cc/l
TPMP” binding to the nicotinic receptor is allosteritally modulated by cholinergic effecters [20]. This channel blocker binds to residues located inside the pore of the channel, which become available after receptor activation, The blocking effects of TPMP’ on the muscular nicotinic receptor have been previously characterized [22j, and we have shown here that it also blocks neuronal nicotiaic currents in SK-N-BE cells (see above). As expected [3H]TPMP+ binding in SK-N-BE cells was positively modulated, in a dose-dependent manner, by Cch (Fig. 5B>.The specific binding of i;HjT’PMPi was maximally stimu!ated by lo-” M Cch. Cch also stimulated the specific binding of [jHjTfMP+ in NCI-H-69 (Fig. SC) and NCI-N-592 (not shown) SCC cell lines which, like NB, express the cc3and ,& nicotinic receptor subunits. On the other hand, in the lung non-SCC cell line (A549), which does not express either a3 of /?., subunits, the binding of [‘H]TPMP’ was not stimulated by Cch (Fig. SD). As a control, an allosteric modulation of [3H]TPMP+ binding was also shown in TE671 cells, which are known to express muscle-type nicotinic receptors and the a, nicotinic subunit (Fig. SA). The expression of functional neuronal nicotinic receptors in SCC cell lines is a novel and intriguing tinding. We suggest that these receptors should be considered as new members of the growing list of neuronal markers selectively expressed by this type of lung tumor. Neuronal nicotinic receptors are known to mediate the secretagogue effects of endogenous acetylcholine
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a
b
ACh _- .---._.-_ -+
____L
13 mh wash
Pig. 4. Electrophysizlogical recordings in SK-N-BE cells. (a) inward control currents induced by 1 mM Ach (left) are completely blocked after 3 min of superfusion with lO,uM d-tubocurarinc (center). Recovery from tile block is observed al’tc-r4 min ofwashin8with control external medium (right). (b) Inward control currents induced by I mM Ach (left) are completely blocked after 7 min ofsuperfusion with 1NM TPMP’ {center). Recovery from the block is observed after 13 min of washing with control external medium (ri8ht). Superfusion periods are indicated by bars.
and exogenous nicotine in a number of tissues, and we have also demonstrated the secretagogue effects of nicotine on SCC cell lines (E. Sher, manuscript in preparation). The effects of nicotine on cell proliferation have
previously been described [i 1,23,24], but no information is available on the molecular nature of nicotine targets. The present characterization of neuronal nicotinic receptors in SSC, a subtype of human lung cancer, the association of which cigarette smoking is well demonstrated [25], offers new insights into the possible mechanisms by which nicotine might affect tumor cell physiology and proliferation. Ac~/to~lll~~~r~,z~~~~,~~ We thank N. BorSesc and C. Gotti for their discussions and extensive comments on the manuscript; J. Mahsco for oiigonucleotidc synth&s; S. Heinemann for the sift of the/& cDNA probe; H. Soreq for the lMR32 EDNA library; G. Melino, G. Gaudino. 5. Tzartos and A. Ciomei for cell lines; and K. Smart and I. RutToni for manuscript revision. The paper was supported by CNR tar8eted projects ‘Prevcnrion and Control of Disease Factors’ and ‘AginS’.
REFERENCES
-_
J
CCh-lctg CM3 Fig. 5. Dose-response curves of the cB’ectsof Cch on [‘H]TPMP+ binding. Cch-induced increases in [‘H]TDMP+bindinp were cvzluatcd in TE 671 (A). SK-N-BE (9, NCi-Ii-69 (C; and A549 CD; cd!s. Specific [‘HJTPMP’ binding in the absence of carbachol was considcred as 100%.Yalues represent the averuge of 3-8 experiments, each performed in triplicate.
[I] Wannacott. S., Irons, J., Lunt. 0.0.. Rapier. CM. and Albuquerque. E.X. (1988) in: Nicotinic Acctylcholine Receptors in the Nervous System (Clementi, F., Got& C. and Shcr. E. eds.) NATO ASI Series H: Cell Biology vol. 25. pp. 41-60. Springer* Vcrlag, Berlin. [2] Roll, L.W. and Perlman, R.L. (1983) Neuroscicnoc 10,979-985. [31 Dencris. ES.. Connolly, J., Rogers. S.W. andDuvoisin. R.(i991) Trends Eharmacol. Sri. 12. 34-40. [4] Luetjc, C.W. and Patrick, 3. (1991) .I. Neurosci. it, 537-845. [S] H&o, R.J.. SeeSer, RX., Yu, AL. and O’Connor, D.T. (1990) J. Clin. Invest. 85, 1555-l 559. {6] Clementi, F.. Cabrini. D.. Gotti, C. and Sher. E. (1986) 1. NCUFOthem. 47, 29l-297. [7] Gotti. C., Esparis 08ando. A. and Clcmcnti, F. (1989) Ncurossicnee 32, 759-767. 69
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[S] Abbracchio. M.P.. Cattabcni, F., Clementi, F. and Sher, A. (1989) Neuroscience 30. 519-825. [9] Teeli& M.E. and Carney, D.N. (1985) in: Biology of Lung Cancer: Diagnosis and Treatment Biocbcmical Markers of Lung Cancer (Rosen, ST., Mulshine. J., Cutitta. F. and Abram% P.C. cds.) pp. 43-58, Marccl Dckkcr, New York. [IO] Cunningham, J., Lennon, Y., Lamben, E. and Scheithauer, B. (1985) 1. Neurochem. 45. 159-167. [I I] Maneckjee, R. and Minna, J.D. (1990) Proc. Natl. Acad. Sci. USA 87, 3294-3298. [I 21 Chini, B.. Clementi, F.. Hukovic. N. and Sher. E. (1992) Proc. Natl. Acad. Sci. USA S9, 1572-1576. [I31 Dcneris. ES.. Connolly, J., Boulter, J., Wada, E., Wada, K., Swanson, L.W.. Patrick, J. and Heinemann, S. (1988) Neuron I. 45-54. 1141 Frohman. M.A., Dush, MR. and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 5995-9002. [15] Duvoisin, R.M., Deneris, ES., Patrick, J. and Hcinemann, S. (1989) Neuron 3,487-496. [16] Fornasari, D., Chini, B., Tarroni, P. and Clementi, F. (1990) Neurosci. Lett. I I I. 351-356.
LETTERS
November
1992
[l7] Ponte, P., Ng:, S-Y,, Engee),J., Gunning, P. and Kedes, L. (1984) Nucleic Acid Res. 12. 1687-1696. [18] Hamill, OP., Marty,.A., Nchcr, E., Sakmann, B. and SiBworth, F.J. (1981) Pflllgcrs Arch. 391, 85-100. [I91 Shcr, E., Pandiella, A. and Clementi, F. (1990) Cancer Res. 5, 3892-3896. WI Lauffer, L, and Hucho, F. (1982) Proc. Natl. Acad. Sci. USA 79, 24062409. [2ll Couturier, S., Erkmun, L., Valera, S., Rungger, D., Bertrand, S., Boulter. J.. Ballivet. M. and Bertrand, D. (1990) J. Biol. Chcm. 2G5, 17560-l 7567. Spivak, C.E. and Albuquerque, E.X. (1984) Mol. Pharmacol. 27, 246-255. Shuller, H.M. (1989) Biochem. Pharmacol. 38, 34393442. Tominaua. K.. Kinoshita. Y.. Hato. F.. Masuda. A. and Matsuy&a, M’. (1989) Celi. Mol. Biol: 35’679-686. . lannuzzi, MC. and Scoggin, C.H. (1986) Am. Rev. Rasp. Dis. 134, 593-608.
