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Home , Bradykinin, Galanin, Neurotensin, Neurotensin receptor

[ RESEARCH 56. 5758-5764, December 15, 1996] , , and Rapidly Stimulate Activation of Mitogen-activated Kinase in Small Cell Cancer Cells

Thomas Seufferlein and Enrique Rozengurt'

Imperial Cancer Research Fund, P. 0. Box 123, 44 Lincoln ‘sinnFields, London WC2A 3PX, United Kingdom

ABSTRACT and p44me@@k,aredirectly activated by on specific tyrosine and threonine residues by the dual-specificity MEKs, of Addition of phorbol 12,13-dibutyrate (PDB) to H 69, H 345, and H 510 which at least two isofonus, MEK-1 and MEK-2, have been identified small cell lung cancer (SCLC) cells led to a rapid concentration- and in mammalian cells (12—14).Several pathways leading to MEK acti time-dependent increase in p42―@ activity. PD 098059 [2-(2'-amino 3'-methoxyphenyl)-oxanaphthalen-4-one], a selective inhibitor of mitogen vation have been described. Tyrosine kinase receptors induce p42r@a@@@( activated protein kinase (MAPK) kinase 1, prevented activation of via a son of sevenless (SOS)-mediated accumulation of p2l@-GTP, p42maPk by PDB in SCLC cells. PDB also stimulated the activation of which then activates a kinase cascade comprising p74@', MEK, and p90r$k, a major downstream target of p42―@. The effect of PDB on both p42Jp44maPk (10, 11). Activation of seven transmembrane domain p42―@and p90rsk activation could be prevented by down-regulation of receptors also leads to p42@'@ activation, but the mechanisms in protein kinase C (PKC) by prolonged pretreatment with 800 aM PDB or volved are less clear, although both p2l@- and PKC-dependent treatment of SCLC cells with the PKC inhibitor bisindolylmaleinside (GF pathways have been implicated (15—20).Activated MAPKs directly 109203X), demonstrating the involvement ofphorbol ester-sensitive PKCS phosphorylate and activate various , e.g., p90@ (21, 22), and In the signaling pathway leading to p42 activation. Various neuropep factors (23) and have been strongly implicated in the tides, such as , , , neurotensin, and gala control of cell proliferation (24—27).However, the role of p42@@a@@k Ida, which promote clonal growth in SCLC cells, also induced activation of p42―@'Inthese cells. In particular, galanin and neurotensin stimulated and @44mapkinSCLC cell growth remains unknown. @ activation In SCLC cells by a pathway that was dependent on the Here we report that PDB potently stimulates and p9@JrSk activity of PKC. Furthermore, galanin-stimulated clonal growth of SCLC activity in the SCLC cell lines H 69, H 345, and H 510 through a cells in semisolidmediumcouldbe preventedby the PKCInhibitorGF PKC-dependent pathway. Furthermore, that promote 109203X and by PD 098059. Thus, our results suggest that activation of clonal growth in SCLC cells, such as bombesin, bradykinin, vaso p42―@ plays an important role in -induced growth of pressin, galanin, and neurotensin, also induce p42m@ activation in SCLC. these cells.

MATERIALS AND METHODS INTRODUCTION Cell CUltUre.SCLCcell linesH 69, H 345, andH 510 weregenerously SCLC2 constitutes 25% of all pulmonary and is character donated by Dr. A. Gazdar (Southwestern Medical Center, University of Texas, ized by a very low 2-year survival despite initial sensitivity to radio Dallas) and purchased from the American Type Culture Collection. Stocks therapy and chemotherapy (1). Thus, novel therapeutic strategies are were maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine needed, and these could arise from defining the factors and signaling serum (heat inactivated at 57°Cfor1 h) in a humidified atmosphere of 10% pathways that stimulate the proliferation of SCLC. C02:90% air at 37°C.Theywere passaged every 7 days. For experimental A variety of neuropeptides, including bombesin or -releasing purposes, the cells were grown in RPM! 1640 with HITESA (10 nM hydra , bradykinin, vasopressin, galanin, and neurotensin, promote cortisone, S ,xg/ml , 10 @xg/mitransferrin, 10 nM , 30 nt@i clonal growth of certain SCLC cell lines and have been proposed to selenium, and 0.25% BSA). act as autocrine and paracrine growth factors for these cells (2—5). Immune Complex Kinase Assay for p42 and p9O'@ Activation. SCLC cells culturedin HITESAfor 3-5 days were washed twice in RPMI 1640 Neuropeptides bind to seven transmembrane domain G protein-linked and disaggregated by gentle pipetting. Aliquots of 3 X l0@SCLC cells in receptors and induce polyphosphoinositide hydrolysis to generate the single-cellsuspensionwere incubatedin 5 ml of RPMI 1640for 30 mm at 37°C. two second messengers, inositol-l,4,5-trisphosphate and diacylglyc The cells were then treated with factors as described in the figure legends and erol, which mobilize Ca2@ from intracellular stores and activate PKC, subsequentlylysed at 4°Cin1 ml of a solutioncontaining 10 m@iTns-HCI(pH respectively (6, 7). SCLC cells express multiple diacylglycerol- and 7.6), 5 mrs@EDTA,50 mMNaCI,30 mrsisodiuminorganicpyrophosphate,50mM phorbol ester-sensitive PKCs, including a, @,@,and€(8)?Activation NsF, 2 mM Na3VO4, 1% Triton X-l00, 10 @xg/mlapmtinin, 10 pg/mi , of PKCs by phorbol esters has been demonstrated to stimulate growth and 1 mMphenylmethylsulfonylfluoride(lysis buffer).Lysates were clarifiedby and transcription of gastrin-releasing peptide mRNA in SCLC cells centrifugationat 15,000 X g for 10 mm at 4°C.Immunoprecipitationwas per (9). However, the downstream signaling pathways stimulated by PKC fonned using a polyclonal anti-p42'°@ antibody or a polyclonal anti@p9@Y@k in SCLC cells remain poorly characterized. antibody, incubatingthe samples on a mtating wheel for 2 h. Protein A-agarose beads (40 @x1,1:1slurry) were added for the second h. Immune complexes were The MAPKS constitute a family of highly conserved -threo collected by centrifugation and washed twice in lysis buffer and twice in kinase nine kinases that are activated in response to a wide range of extra buffer [15 mM Tns-HCI (pH 7.4) and 15 mM MgCl@]. The kinase reaction was cellular signals (10, 11). The two best characterized isoforms, p42maPk performed by resuspending the pellet in 25 p1 of kinase assay mixture containing kinase buffer, 100 @xMATP,100 MCi/mi [‘y-32P]ATP,200@iMmicracystinlescine Received 5/28/96; accepted 10/15/96. analogue (LR) and 1 mg/mI of either myelin basic pmtein peptide The costs of publication of this article were defrayed in part by the payment of page (APRTPGGRR) or S6 peptide (RRRLSSLRA) for the assays of p42°@and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. @,nn@sk respectively. Incubations were perfonned for 10 mm (linear assay condi I To whom requests for reprints should be addressed. Phone: 44-171-269-3455; Fax: tions) at 30°C and terminated by spotting 20 @.dof the supernatant onto P81 44-171-269-3417. chromatographypaper (Whitman). Filters were washed four times for 5 rain in 2 The abbreviations used are: SCLC, small cell lung cancer; G protein, guanine 0.5% o-phosphoric acid, immersed in acetone, and dried before Cerenkov count nucleotide-binding protein; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PDB, phorbol 12,13-dibutyrate;PKC, protein kinase C; GF l09203X, bisindolyl ing. The average radioactivity of two blank samples containing no immune maleimide; PD 098059, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one. complex was subtractedfrom the result of each sample. The specific activity of 3 Unpublished observations. [y-32P)ATP used was 900-1200 cpm/pmol. 5758

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1996 American Association for Cancer Research. STIMULATIONOFMrroGEN-ACTIvATEDPROTEINKINASEIN SCLC

A 100 .100 80@60 H —3 60.20 II[Inn @ .@a. C_ 101520 E 0 40 • PD098059(JLM) c5@e

0—I' @ U 10 100 Time (mm) PDB (nM)

B 100 @8O 80@ —3 @ 60 i!40 40=<20°@―@'

PDB PDB PDBpre - + - - + - + + — GF - - + - - + - - + - - +

Fig. 1. Effect of PDB on p42―°―@'andp9O@°'@inH 69 SCLC cells. A, cultures of H 69 cells were treated with 400 nst PDB for various times (Ieftpanet) or with various concentrations of PDB for 5 olin (right panel) as indicated. Cells were lysed, and p42@@aPkimmunecomplex kinase assays or mobility shift assays (left panel. inset) were performed as described in “MaterialsandMethods.―Theresults shown in each case are representative of three independent experiments. The positions of nonphosphorylated p42'°@and the slower-migrating phosphorylated form of p42'°°@inthe mobility shift assay are indicated. Results of the kinase assays are the means of duplicates and are expressed as percentages of the maximum PDB-stimulated p42'°@activation (6,000—9,000cpm/3X l0@cells at 5 mm). Right panel, inset, cultures of H 69 cells were treated with various concentrations of PD 098059 for 3 h (open columns). Control cells received an equivalent amount of solvent (filled column). Cells were then stimulated with 400 nsi PDB for 5 mm and subsequently lysed, and immune complexkinaseassaysforp42°'°@activitywereperformedasdescribedin“MaterialsandMethods.―Resultsofthekinaseassaysarethemeansoftwoindependentexperiments,each perfonned in duplicate, and are expressed as percentages of the maximum PDB-stimulated p42'°°@activation(6,000—9,000 cpm/3 X l0@cells at 5 mm). B, cultures of H 69 cells were pretreated with 800 nsi PDB for 48 h (PDB pre, +, dark hatched columns) or 3.5 @xsiGFl09203X for 1 h (GF, +, light hatched columns). Control cells received an equivalent amount of solvent (—,filled columns). Cells were subsequently stimulated with 400 nsi PDB for 5 mm, and p42@@a@l@andp90@@kimmunecomplex kinase assays (left and right panels, respectively) were performed as described in “Materialsand Methods.―Results are the means of three independent experiments, each performed in duplicate, and are expressed as percentages of the maximum PDB-stimulated activation (6,000—9,000cpm/3X 106cells at 5 mm for p42'°@and55,000—110,000cpm/3X l0@cells at 5 mm for p9(1―).Bars,SE. The specific activity of [y@2PJATPusedin all experiments was 900—1,200cpm/pmol.

p42 Mobffity Shift Assays. Activation ofp42'°@ can be determined by copy. Cell number was determinedusing a Coulter Counter, and l0'@cellswere the appearance of slower-migrating forms in SDS-PAGE gels, which results from mixed with HITESA containing 0.3% agamse and galanin in the presence or the phosphorylation of specific threonine and tyrosine residues within its subdo absence of PD 098059, at the concentrations indicated, and layered over a solid main Vifi (28). SCLC cells were treated as described above and subsequently base of 03% agarose in H1TESA with galanin in the presence or absence of PD incubated with factors as indicated. The cells were then lysed in 2X SDS-PAGE 098059 at the same concentrationin 33-mm dishes.The cultures were incubated sample buffer and analyzed by SDS-PAGE. were subsequentlytram in humidified 10% C02:90% air at 37°Cfor21 days and then stained with the vital ferredto Immobion membranes,whichwere blockedusing 3% nonfatdried milk stain nitro-blue tetrazolium. Colonies of >120 @unin diameter (16 cells) were in PBS (pH 7.2)and incubated for 1 h at 22°Cwitha polyclonal p42°@ antiserum counted using a microscope. (1:1000) in PBS containing 3% nonfat dried milk Immunoreactivebands were Materials. The PKC inhibitor GF l09203X and microcystin LR were oh visualized using WI-labeled protein A, followed by autoradiography. Uquid CUltUreAssay.SCLCcells,3-5 daysafterpassage,werewashed mined from Calbiochem-Novabiochem,Ltd. (Nottingham. United Kingdom). and resuspended in HITESA. Cells were then aliquoted in 24-well Falcon -derivedgrowth factor (BB homodimer), ‘@l-labeledproteinA (30 mCi/ plates at a density of 5 X l0@cells in 1 ml of HITESA in the presence or mg), and [@y-32P]ATP(10rnCihnl) were from Amersham (Buckinghamshire, absence of PD 098059. At various times, cell number was determined from a United Kingdom).The MEK-l inhibitor PD 098059 was a generous gift from minimum of 3 wells per condition using a Coulter Counter, after cell clumps Alan R. Saltiel (Department of Signal Transduction, Parke Davis Research Divi were disaggregated by passing the cell suspension five times through 19- and, sion, Ann Arbor, MI). The polyclonal anti-p42°'' antibody raised against a subsequently, 21-gauge needles. COOH-terminal peptide (EETARFQPGYRS) and the polyclonal antibody against Clonogenic Assay. SCLC cells, 3-5 dayS after passage, were washed and p90rSk @jagainst a COOH-terminal peptide (IESSILAQRRVRKLPSTI'L) resuspended in H1TESA. Cells were then disaggregated by two posses through a were generous gifts from Dr. J. Van Lint (Katholieke Universiteit, Leuven, 19-gaugeneedle into an essentially single-cellsuspensionas judged by micros Belgium).All other reagents were of the purest grade available. 5759

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1996 American Association for Cancer Research. STIMULATION OF MFF0GEN-ACnvATED PROTEIN IUNASE IN SCLC A H345 kinase assays for @2mapkactivity.As shown in Fig. 1A, left panel, PDB induced a marked activation of p42―°@,whichpeaked after 5 mm of incubation and remained elevated at 50% of maximum after 60 mm of incubation. Similar results were obtained in @42mapkand > @mapk mobility shift assays (Fig. 1A, left panel, inset; and data not 0 shown). PDB induced p42@k activation in a concentration-depen Ia dent manner. Half-maximum and maximum stimulation of p42m@@( c@J activity were achieved at 25 and 100 nt@tPDB, respectively (Fig. IA, ‘a II right panel). Recently, a novel compound, PD 098059, has been characterized as a selective inhibitor of MEK-l activity in vitro and in vivo (29, 30). PDB — PDB Pretreatment of H 69 cells with various concentrations of PD 098059 PDBpre - + - + - + prevented PDB-stimulated p42@@k activation in a concentration-dc pendent manner. A maximum 85% inhibition of PDB-induced B H510 p42°'@ activation was achieved at 15 p.MPD 098059 (Fig. 1A, right panel, inset). Thus, the activation of @,42mapkbyPDB involves MEK-1 io4 as an upstream regulator in H 69 cells. To substantiate the role of PKC in PDB-induced p42'°°@activa :@° 801 J80@ @ >@E@rI I tion, H 69 cells were treated either with 800 nisi PDB for 48 h to fl60I@ 160h,r down-regulate phorbol ester-sensitive PKCs (31) or for 1 h with the tm I selective PKC inhibitor OF 109203X (32) and subsequently chal @j.@°t• 140c@@lenged with 400 mMPDB for 5 mm. As shown in Fig. lB, left panel, down-regulation of PKC as well as treatment with OF 109203X 20@ j20 completely prevented PDB-induced @42mapk activation in H 69 cells. 0I.@.______. .______10 Indeed, down-regulation of PKC by PDB pretreatment reduced — PDB — PDB @i2map@@activityin H 69 cells to levels below that obtained in the PDBpre - + - + - + - + control cells. PDB also inducedstrikingactivationof p90@, a majordownstream Fig. 2. PDB activates p42ma@@andp90@ in H 345 and H 510 SCLC cells. A, Cultures of H 345 cells were pretreated with 800 nsi PDB for 48 h (PDB pre, +, hatched columns). target of @42mapk(21,22). Down-regulation of PKC or treatment of Control cells received an equivalent amount of solvent (—,filled columns). Cells were the cells with the PKC inhibitor OF 109203X abolished p9(Y°―acti subsequently stimulated with 400 flMPDB for 5 mm, and p42―°@'andp9(Y°―immune vation in response to PDB in H 69 cells (Fig. 1B, right panel). complex kinase assays (left and right panels. respectively) were performed as described in “MaterialsandMethods.―Resultsare the means of three independent experiments, each To further substantiate the role of PKC in the activation of @42mapk performed in duplicate, and are expressed as percentages of the maximum PDB-stimulated and p90@ in SCLC cells, we examined whether PDB could also activation (3,000—6,000 cpm/3 X I0@cells at 5 miss for p42―°'@'and20,000—25,000 cpm/3 X l0@cellsat 5 mm for p9@,y%k).Bars,SE. B, cultures of H 510 cells were pretreated activate @42mapkinother SCLC cell lines. As shown in Fig. 2, PDB with 800 nM PDB for 48 h (PDB pre, +. hatched columns). Control cells received an induced a marked increase in p42m@@activity and p9(Y@kactivity in equivalent amount of solvent (—.filledcolumns). Cells were subsequently stimulated with both H 345 (Fig. 2A) and H 510 (Fig. 2B) SCLC cells. PDB-induced 400 nM PDB for 5 mm, and p42m@@and @9(yskimmune complex kinase assays (left and right panels, respectively) were performed as described in “Materialsand Methods.― activation of both kinases was prevented when PKC was down Results are the means of three independent experiments, each performed in duplicate, and regulated by chronic pretreatment with PDB (Fig. 2, A and B). Thus, are expressed as percentages of the maximum PDB-stimulated activation in each exper iment (3,000—6,000 cpm/3 x 106 cells at 5 mm for p42@@@Pkand60,000—80,000 phorbol ester-sensitive PKCs are major activators of the MAPK cpm/3 X lO@cellsat 5 mm for p9O@). Bars, SE. cascade in SCLC cell lines. Effect of Neuropeptides on p42―@@Activationin SCLC Cells. Multiple neuropeptides can stimulate the growth of SCLC cells (4, 5). RESULTS Because activation of p42m@ 1@oneof the major pathways leading to PDB Induces @42mapkandp9@FSkActivation in H 69, H 345, and mitogenesis in a variety of cell lines (24—27), we next examined H 510 SCLC Cell Lines. To determine whether phorbol esters could whether neuropeptides could activate p42Ifl@Pkin SCLC cells. As activate @42mapkinSCLC cells, lysates of H 69 cells stimulated with shown in Fig. 3, left panel, treatment of H 69 cells with bradykiin, 400 nM PDB for various times were analyzed by immune complex neurotensin, and galanin each at 100 nM caused 2.0-, 2.0-, and 2.6-fold

H69 H345 H510

Fig. 3. Multiple neuropeptides stimulate @,42maPkactivation in SCLC cell lines. Cultures of H 69, H 345, and H 5 10 cells were treated with bradykinin (BK), neurotensin (Ni), galanin (Gal). vaso pressin (VP), or bombesin (Bom) as indicated, each at 100 flMfor 5 0—S' @ mm. Cells were subsequently lysed, and immune complex kinase assays were performed as described in “Materialsand Meth ods.―Results are the means of at least three independent experiments, each performed in duplicate, and are expressed as cpm/3 X 106 cells. Bars, SE.

— BK NT Gal — BK NT Born VP — BK VP Gal

5760

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STIMULATION OF MITOGEN-ACflVATED PROTEIN KINASE IN 5CLC

A H345 H69

.@% 1000

@__% 800 a 600 @:2.

200

Fig. 4. Role ofphorbol ester-sensitive PKCs in p42'°@ 0 activation in response to neurotensin and galanin. A. cal []@J— Neurotensln lures of H 345 cells (leftpanel) or H 69 cells (right panel) were pretreated with 800 nat PDB for 48 h (PDB pre, +, PDB pre + — + — + — + hatched columns). Control cells received an equivalent amount of solvent (—,filled columns). Cells were subse B H510 H69 quently stimulated with 100 nat neurotensin for 5 mm, and p42'°@immunecomplex kinase assays were performed as described in “MaterialsandMethods.―Results are the means of three independent experiments ±SE each per formed in duplicate and are expressed as cpm/3 X l0@ cells.Bars,SE.B,leftpanels,culturesofH 510cellswere > pretreated with 800 nMPDB for48 h(PDBpre, +, hatched 0-@'@ columns). Control cells received an equivalent amount of solvent (—,filled columns). Cells were subsequently stim ulated with 100 nsi galanin for 5 mm, and p42°'@ and @ p90r$k complex kinase assays (top and bottom panels, respectively) were performed as described in “Ma terials and Methods.―Resultsare the means of three mdc pendent experiments, each performed in duplicate, and are expressed as c@3 X l0@cells. Bars, SE. B, right panels, cultures ofH 69 cells were pretreated with 800 nsi PDB for 48 h (PDB pre, +, hatched columns). Control cells re ceived an equivalent amount of solvent (—,filledcolumns). Galanln Cells were subsequently stimulated with 100nsi galanin for — + — + 5 rain, and p42'°°@'andp9(Y°―immunecomplex kinase assays (top and bottom panels. respectively) were per formed as described in “MaterialsandMethods.―Results are the means of three independent experiments, each per formed in duplicate, and are expressed as cpm/3 X l0@ 50000 cells. Bars, SE. —-@

30000

I 0000 0 T I Galanln PDBpre — + — + — +

@ increases in @2mapkactivity, respectively. Treatment of H 345 cells PDB. This treatment prevented neurotensin-induced activa with 100 mM bradykinin, neurotensin, bombesin, and vasopressin tion in H 345 cells (Fig. 4A, left panel). The stimulation of p42―@ induced 2-, 1.9-, 4-, and 3.2-fold increases in p42m@@@activity,re activity induced by neurotensin in H 345 SCLC cells was also mark spectively (Fig. 3, middle panel). In H 510 SCLC cells, 100 nti edly attenuated by treatment of the cells with the PKC inhibitor OF bradykinin, vasopressin, and galanin stimulated p42'°°@activationby l09203X (from a 1.9-fold increase above control to control levels). 3-, 5.2-, and 4.2- fold, respectively (Fig. 3, right panel). Thus, mul Down-regulation of PKCs also prevented neurotensin-induced tiple neuropeptides that act as growth factors for SCLC cell lines can p42ma@@@cactivation in H 69 SCLC cells, substantiating the role of activate p42@@as@@kinthese cells. Consequently, we examined the sig phorbol ester-sensitive PKCs in neurotensin-induced p42―@ activa naling mechanisms by which these neuropeptides induce p42―@'@ tion (Fig. 4A, right panel). In contrast, the release of Ca2@ from activation in SCLC cells. intracellular stores in response to neurotensin was not affected by Role of PKC in $2maPk Activation in Response to Neurotensin PDB pretreatment of parallel cultures of H 345 or H69 SCLC cells and Galanin. Neurotensin stimulates the release of Ca2@ from intra (data not shown). cellular stores in SCLC cells (5), suggesting that the neurotensin We have previously demonstrated that activation of the galanin mediates phospholipase C-dependent PKC activation in these receptor in H 510 and H 69 cells stimulates phospholipase C activity, cells. To examine the role of PKC in neurotensin-induced @>42mapk which leads to Ca2@ mobilization (33) and activation of enzymes of activation, phorbol ester-sensitive PKC isoforms were down-regulated the PKC family (7). Consequently, we examined whether PKCs could in H 345 SCLC cells by prolonged treatment of the cells with 800 flM be upstream regulators of galanin-induced @,42mapkactivation in 5761

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STIMULATION OF MITOGEN-ACI'IVATED PROTEIN KINASE IN 5CLC A and H 69 cells were pretreated with pertussis toxin. As shown in Fig. H 510 H 69 5A,thistreatmentonlyslightlyreducedthebasalandgalanin-induced @ activation in H 510 and H 69 cells, respectively (Fig. 5A, left and right panels, respectively). As a control, we verified that pretreat ment of H 510 cells with the same preparation and concentration of pertussis toxin inhibited @42mapkactivation in response to lysophos phatidic acid, which binds to a 0, protein-coupled receptor, by 90% in parallel experiments (data not shown). PDB stimulates @42mapkactivationby a MEK-l-dependent path C@10 way in SCLC cells (Fig. lA). To examine whether MEK-l is also the main upstream regulator of galanin-induced @,42mapkactivityin SCLC cells, H 510 and H 69 cells were treated with the selective MEK-1

— Galanin inhibitor PD 098059. PD 098059 at 20 @.tM completely prevented — Galanin

PTX — + — + — + — +

B H510 H69 A H69 auw -@-- - 2001

@ 2000 I@ 100@0ii:1j1______g @ C@1‘a iooo 0@n n@__ fl — PDB Galanin P0098059 — + — + — + — Galanin — Galanin H 510 PD098059 — + — + — + — +

Fig. 5. Role of pertussis toxin-sensitive G proteins and MEK-l in galanin-induced p42'°°―activation.A. cultures of H 510 cells (left panel) or H 69 cells (right panel) were Il) pretreated for 3 h with 30 ng/ml pertussis toxin (PTX, +, hatched columns). Control cells •0 received an equivalent amount of solvent (—,filled columns). Cells were subsequently Ia stimulated with 100 n@igalanin for 5 mm, and p42ma1@kimmune complex kinase assays C were performed as described in “MaterialsandMethods.―Resultsare the means of three 0 independent experiments, each performed in duplicate, and are expressed as percentages 0 C.) of the maximum galanin-stimulated p42'°°@―activation(1900—2100cpm/3 X 106 cells at 5 mm and I 100—1300cpm/3 X lO'@cells at 5 mm, respectively). Bars, SE. B, cultures of H 510 cells (left panel) or H 69 cells (right panel) were treated with 20 pxi PD 098059 for 3 h (+, open columns). Control cells received an equivalent amount of solvent (—, Galanln filled columns). Cells were subsequently stimulated with 100 nsi galanin for 5 mm, and PD 098059 + + p42°'°@immunecomplex kinase assays were perfonned as described in “Materialsand Methods.―Results are the means of three independent experiments, each performed in B H510 duplicate, and are expressed as cpm/3 X 106 cells. Bars, SE. i ‘aft'

SCLC cells. Down-regulation of phorbol ester-sensitive PKC iso forms by prolonged treatment with PDB prevented activation of : @2m@@pkbygalanin in H 510 cells (Fig. 4B, top left panel) and in g 400 H 69 cells (4B, top right panel). In addition, galanin potently 0 C-) stimulated p9O'@ activity in H 5 10 cells and H 69 cells, and this effect was also prevented by down-regulation of phorbol ester — Galanln sensitive PKCs (Fig. 4B, bottom left and bottom right panels, GF109203X — + — + respectively). The stimulation of p42mat5@@activityinduced by ga lanin was also markedly attenuated in H 510 SCLC cells treated Fig. 6. Effect of PD 098059 on colony formation in response to PDB and galanin in SCLC cells. A, top panel, a single-cell suspension of H 69 cells was plated in agarose with OF 109203X (from a 1.6-fold increase above control to medium containing HITESA at a density of 1 X l0@cells/dish in the absence or presence control levels). Thus, galanin-induced p42maP@@activation is de of 400 OMPDB or 100 nsi galanin and/or 20 @MPD098059 (+, open columns) and incubated for 3 weeks as described in “Materialsand Methods.―Bottom panel, a single pendent on PKC in H 510 and H 69 cells. In contrast, the increase cell suspension of H 510 cells was plated in agarose medium containing HITESA at a in intracellular concentration induced by galanin was not density of 1 x l0'@cells/dishin the absence or presence of 100 nsi galanin and/or 20 pxi affected by prolonged pretreatment of H 510 or H 69 SCLC cells PD 098059 (+, open columns) and incubated for 3 weeks as described in “Materialsand Methods.―insets,H 69 cells (top panel) and H 510 SCLC cells (bottom panel) were with PDB (data not shown). aliquoted in 24-well plates in HITESA in the presence or absence of 20 @xMPD098059 Role of Pertussis Toxin-sensitive G Proteins and MEK-1 in (PD, +). Cell number was determined from a minimum of 3 wells per condition when Galanin-induced p42m@ Activation in SCLC Cells. Several re cells were growing exponentially at days 7 and 8 using a Coulter Counter. B, a single cell suspension of H 510 cells was plated in agarose medium containing HITESA at a density ports indicate that the expressed by pancreatic j3-cells of I X 10―cells/dishin the absence or presence of 100 nsi galanin and/or 3.5 psi GF and pituitary cell lines is coupled to a pertussis toxin-sensitive het 1O92O3X (+, open columns) and incubated for 3 weeks as described in “Materialsand Methods.―Columns,mean number of colonies formed on five separate dishes; bars, SE. @ erotrimeric 0 protein of the subfamily (34). To examine In all cases, a representative of three independent experiments is shown. Where no error whether a G@protein mediates p42m@@@@activationby galanin, H 510 bar is shown, it lies within the dimensions of the symbol. 5762

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galanin-induced p42ma@@activityin immune complex kinase assays in a pertussis toxin-insensitive mechanism (33). In the present article, we H 510 and H 69 cells, respectively (Fig. SB, left and right panels, show that galanin induces activation of p42m@@@(inH 510 and H 69 respectively), demonstrating the involvement of MEK-l in the sig SCLC cells. This effect is also independent of a pertussis toxin naling pathway leading to MAPK activation by galanin in SCLC cells. sensitive 0 protein but requires functional PKC. Because neuropep Effect of PD 098059 on PDB- and Galanin-induced Colony tide receptors are frequently expressed in multiple molecular forms Formation in H 510and H 69 SCLC Cells. Severalreportsindicate (39), we propose the existence of a different molecular subtype of the that activation of the MAPK cascade plays a role in mediating galanin receptor in SCLC cells, which is coupled to phospholipase C mitogenesis in various cell lines (24—27). Oalanin and PDB are and mediates activation of @,42mapkbya pathway involving PKC and known to stimulate colony formation of SCLC cells in semisolid MEK-1 but independently of a pertussis toxin-sensitive 0 protein. media (9, 33). Consequently, we determined whether inhibition of the Oalanin markedly stimulates the clonal growth of H 510 and H 69 MAPK cascade by PD 098059 could also prevent PDB and galanin cells in semisolid medium (33). The specific MEK-l inhibitor PD from stimulating clonal growth in H 510 and H 69 SCLC cells. As 098059 prevented @2mapkactivationby galarnn and strikingly inhib shown in Fig. 6A, 20 @MPD098059 prevented the increase in colony ited galanin-induced colony formation in these cell lines. This mdi @ formation of H 69 SCLC cells in response to either PDB or galanin cates that activation of p42°'@ necessary for galanin-induced (Fig. 6A, top panel). This MEK-l inhibitor also caused a profound colony formation in H 510 and H 69 cells. Thus, our findings dem decrease of colony growth induced by galanin in H 510 SCLC cells onstrate that the MAPK cascade is an important signal transduction (Fig. 6A, bottom panel). Because PKCs are major upstream regulators pathway that mediates colony growth in SCLC cell lines. of the MAPK cascade in SCLC cell lines, it could be expected that inhibition of these enzymes should also prevent colony formation. ACKNOWLEDGMENTS Fig. 6B shows that OF 109203X markedly inhibited colony growth in response to galanin in H 510 SCLC cells. We also verified that PD We thank Alan R. Saltiel for the generous supply of PD 098059 and Dr. Jo @ 098059 reduced the proliferation of the H 69 and H 5 10 SCLC cell Van Lint for and anti-p90@ antibodies. lines in liquid culture by 43 and 40%, respectively (Fig. 6A, top and bottom panels, insets). REFERENCES

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Thomas Seufferlein and Enrique Rozengurt

Cancer Res 1996;56:5758-5764.

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