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[CANCER RESEARCH 56. 5758-5764, December 15, 1996] Galanin, Neurotensin, and Phorbol Esters Rapidly Stimulate Activation of Mitogen-activated Protein Kinase in Small Cell Lung 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 phosphorylation 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 enzymes, e.g., p90@ (21, 22), and In the signaling pathway leading to p42 activation. Various neuropep transcription factors (23) and have been strongly implicated in the tides, such as bradykinin, vasopressin, bombesin, 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, neuropeptides 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 neuropeptide-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 cancers 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 gastrin-releasing purposes, the cells were grown in RPM! 1640 with HITESA (10 nM hydra peptide, bradykinin, vasopressin, galanin, and neurotensin, promote cortisone, S ,xg/ml insulin, 10 @xg/mitransferrin, 10 nM estradiol, 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 leupeptin, 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 serine-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. arginine 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.

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