(CANCERRESEARCH52, 6031-6035, November1, 19921 Gastrin Stimulates Ca2' Mobilization and Clonal Growth in Small Cell Lung Cancer Cells Tariq Sethi and Enrique Rozengurt' Imperial Cancer Research Fund@P. 0. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, England ABSTRACT tion. Nevertheless, compelling evidence that gastrin acts as a cellular growth factor or as an autocrine factor in tumors has Gastnin has been postulated to be a physiological growth factor, but been difficult to document in clonal cell populations. Indeed, compelling in vitro evidence of this has been difficult to obtain. In the studies using receptor antagonists and colon carcinoma cell present study we investigated whether small cell lung carcinoma cell lines could provide a useful model system to study the effects of gastrin lines resulted in controversial results (6—8).The lack of a con on signal transduction and cell proliferation in vitro. We found that the venient model system has impeded the elucidation of the pos addition of gastrin to small cell lung cancer cells loaded with the fluo sible action of gastrin as a direct growth factor in vitro. rescent Ca2 indicator fura 2-tetraacetoxymethylester causes a rapid Cell lines established from SCLC2 provide a useful model and transient increase In the intracellular concentration of Ca2@ system to study the effects of hormonal peptide agonists and (lCa2i@)followedby homologousdesensitization. The tCa2@I1response antagonists on early signaling events and on cell proliferation was especially prominent in the small cell lung carcinoma cell line (9—13).The SCLC cell line H345 has been shown to express H51O. In this cell line, gastnin I, gastrin H, cholecystokinin residues receptors for gastrin (14, 15), but the effect of this peptide on 26-33 (CCK-8), and unsulfated CCK-8 increased lCa2i1 in a concen H345 growth was not determined. In the present study we tratlon-dependent fashion with half-maximum effects at 7, 23, 3, and 5 report that gastrin, at nanomolar concentrations, stimulates the flM, respectively. The Ca2-mobilizing effects of gastrin and CCK-8 were prevented clonal growth of the SCLC cell line H510, identified as an by proglumide, benzotnlpt, and the specific gastrln/CCKB receptor excellent model system for studying the effects of gastrin in antagonist L365260. Gastrin stimulated the clonal growth of H510 cells vitro. The results demonstrate that gastrin acts as a direct in semisolid (agarose-containing) medium, increasing both the number growth factor and show, for the first time, that this hormone and the size of the colonies. Gastrin and CCX agonists were equally can stimulate the proliferation of cells outside the gastrointes effectivein promoting clonal growth. The broad-spectrum neuropeptide tinal tract. antagonists It-Arg',n-Phe@-Trp7'9,Leu―l substance P and IArg6,n- Trp7'9,MePhe@@substance P (6-11) markedly inhibited gastnin-stimu lated Ca2 mobilization and clonal growth. These results show that MATERIALS AND METHODS gastnin acts as a direct growth factor through gastnin/CCKB receptors Cell Culture. SCLC cell line H5l0 was purchased from the Amen and demonstrate, for the first time, that these peptides can stimulate the can Type Culture Collection. Stocks were maintained in RPMI 1640 proliferation of ceUsoutside the gastrointestinal tract. supplemented with 10% (v/v) fetal bovine serum (heat inactivated at 57'C for I h) in a humidified atmosphere of 10% CO2/90% air at 37'C. INTRODUCTION They were passaged every 7 days. For experimental purposes, the cells were grown in HITESA which consists of RPMI 1640 supplemented The possibility that the gastrointestinal peptide gastrin could with 10nMhydrocortisone, 5 Mg/mlinsulin, 10 @ig/mltransfernin,10nM act as a hormonal growth factor has attracted considerable in estradiol, 30 flM selenium, and 0.25% bovine serum albumin (16). terest. A considerable body of evidence has demonstrated that Determination of Intracellular Ca2@Concentration. Aliquots of 4—5 the administration of gastrin induces growth-promoting effects x 106SCLC cells cultured in HITESA for 3—5dayswere washedand in the digestive tract and exocrine pancreas (1). In particular, an incubated for 2 h at 37C in 10 ml fresh HITESA medium. Then 1 @M increase in the circulating levels of gastrin has been related to fura-2-tetraacetoxymethylester AME from a stock of 1 m@tindimethyl hyperplasia of the gastric enterochromaffin-like cells (2). Fur sulfoxide was added, and the cells were incubated for a further 5 mm. thermore, a decrease in circulating gastrin induced by antrec The cell suspension was centrifuged at 2000 rpm for 15 s, and the cells were resuspended in 2 ml of electrolyte solution [140 m@iNaC1,5 mr@i tomy resulted in atrophy of the colonic mucosa in the rat, an KC1,0.9 mMMgCl2, 1.8 mr@iCaCl,25 mviiglucose,16 mv@t4(-2-hydrox effect reversed by the administration of pentagastrin (3). Gas yethyl)-piperazineethanesulfonic acid, 16 mv@iTris, and a mixture of tin also appears to be a growth-promoting hormone for ma amino acids at pH 7.2], transferred to a quartz cuvette, and stirred lignant cells grown as xenografts in nude mice (4, 5). While continuously at 31T. Fluorescence was recorded continuously in a these observations strongly suggest that gastrin acts as a growth Perkin-Elmer LS5 luminescence spectrometer with an excitation wave factor, it is difficult to obtain unambiguous evidence for a direct length of 336 nm and an emission wavelength of 510 nm. ICa2i1 was growth-promoting effect of gastrin in vivo because the admin calculated as previously described (13). istration of this peptide could stimulate the release of other Clonogenic Assay. Cultures of the SCLC cell line H5l0, 3—5days biologically active peptides or growth factors which could act as postpassage in HITESA, were washed and resuspended in the same the proximal effectors of the action of gastrin. medium. Cells were then disaggregated into a single cell suspension by two passes through a 19-gaugeneedle and then through 20-tim nylon Cultured cells have provided useful experimental systems for gauze. Viabilitywas determined byTrypan blue exclusion on a standard elucidating the extracellular factors that promote cell growth hemocytometer. Approximately 10'@viablecells/mI, measured using a without the many complexities of whole animal experimenta Coulter counter, were suspended in HITESA containing 0.3% agarose. Received 4/30/92; accepted 8/24/92. 2 The abbreviations used are: SCLC, small cell lung cancer, GRP, gastnin-re Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentof leasing peptide; HITESA, 10 n,.i hydrocortisone, 5 big/mI insulin, 10 ,@g/mltrans page charges. This article must therefore be hereby marked advertLcement in accord ferrin, 10 nM estradiol, 30 nM selenium, and 0.25% bovine serum albumin; AME, ancewith 18 U.S.C. Section 1734solelyto indicatethis fact. tetraacetoxymethylester, [Ca2'J@,intracellular concentration of Ca2@;CCK, chole I To whom requests for reprints should be addressed. cystokinin;CCK-8,cholecystokininresidues26—33. 6031 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. GASTRIN STIMULATION OF CLONAL GROWTH Then, 1 ml of this mixture was plated in five replicate 35-mm plastic Gastrin-IGastrin-Il dishes containing a 2-mI base layer of 0.5% agarose in HITESA that had hardened. The two layers contained neuropeptide at the same con 150 centration. Cultures were incubated at 37T in a humidified atmosphere at 10% C02/90% air for 21 days and then stained with the vital stain 100 nitroblue tetrazolium. Colonies of >1 20 @mdiameter (16 cells) were counted under a microscope, and, using a x 4 lens, the image is relayed via a TV camera and analyzed on a Macintosh IIcx computer running 50 the digital image processing and analysis program Image. C Materials. Gastrin 17-I (unsulfated), gastrin 17-Il (sulfated on po 0 sition 6 from the COOH terminus), CCK residues 26—33sulfated on + / C@,1 the 7th position from the COOH terminus (CCK-8), desulfated CCK-8 Ides(S03)CCK-81,and CCK 10—27werepurchased from Sigma Chem C) @ ical Co. (St. Louis, MO); (i-Arg',n-Phe5,o-Trp7@9,Le& substance P 150 and @Arg@,t@-Trp7'9,MePhe8lsubstanceP (6—11)were purchased from Peninsula Laboratories (Belmont, CA); L364,7l8 and L365,260 werea 100 kind gift from John Walsh (University ofCalifornia, Los Angeles, CA); fura-2-AME was from Calbiochem Corporation (La Jolla, CA); and 50 agarose was from SeaKem (Rockland, ME). All of the other reagents were of the highest grade commercially available. 0 RESULTS [Peptide] nM Gastrin Stimulates Ca2@ Mobilization. The addition of gas Fig. 2. Dose-dependent effect of gastrin-I, gastnin-lI, CCK-8, and des(S03)- trin I or CCK-8 to either H69 or H345 SCLC lines loaded with CCK-8on ICa2ii. H5l0 cellswerepreloadedwith fura 2/AME and fluorescence the fluorescent Ca2@indicator fura-2 AME caused a rapid and was monitored as previously described. desCCK-8, des(S03)CCK-8. @ICa2@1j(i.e., transient increase in [Ca211. These findings (Fig. 1) are in peak (Ca2'J@—basalICa2@1i)wascalculatedat each concentration. Points, mean agreement with other reports that demonstrated that gastrin of 3—5independent experiments bars, SEM. induces Ca2@mobilization in the SCLC cell line H345 (14, 15). However, the increase of[Ca2'j1 by gastnin or CCK-8 (i@[Ca2@11,including bradykinin, vasopressin, or galanin (@[Ca2i1, 100, 20—30nM)was considerably smaller than that induced by other 70, and 50 nM, respectively). Identical results were obtained peptides including bradykinin (H69) or GRP (H345) (@[Ca2@11, when CCK-8 was added instead of gastrin I (Fig. 1). The in 80—100and 85—120nM, respectively). crease in [Ca2@11induced by gastrin I in H510 cells results from The salient feature shown in Fig. I is that gastrin I and Ca2@ mobilization from internal stores, since it still occurred CCK-8 induced a prominent Ca2@ mobilization in the SCLC after the addition of ethyleneglycol-bis-(fl-aminoethyl ether)- cell line H510.