Gastrin Stimulates Ca2' Mobilization and Clonal Growth in Small Cell Lung Cancer Cells

Home , Bradykinin

(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 (6â€”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 (9â€”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,Leuâ€•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 4â€”5 the administration of gastrin induces growth-promoting effects x 106SCLC cells cultured in HITESA for 3â€”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, 3â€”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,cholecystokininresidues26â€”33. 6031

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 26â€”33sulfated on + / C@,1 the 7th position from the COOH terminus (CCK-8), desulfated CCK-8 Ides(S03)CCK-81,and CCK 10â€”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 (6â€”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@â€”basalICa2@1i)wascalculatedat each concentration. Points, mean agreement with other reports that demonstrated that gastrin of 3â€”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, 20â€”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 80â€”100and 85â€”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. The magnitude of the ICa2i1 response induced N,N,IV',N-tetraacetic acid to chelate extracellular Ca2@, just by gastrin I in this cell line (i@[Ca2it, 150 nM) was greater than prior to the addition of gastrin I (results not shown). In view of the responses induced by other Ca2@-mobilizing neuropeptides the findings depicted in Fig. 1, the H510 SCLC line has been used in additional experiments to characterize the Ca2@-mobi H69 â€”leo H345 lizing effects of gastrin I and CCK-8.

206- Repeated additions of gastrin I caused the homologous de sensitization of Ca2@ mobilization. Furthermore, the addition of gastrin I attenuated the increase in [Ca2@]1induced by 1u-@ CCK-8, and reciprocally, brief exposure to CCK-8 prevented 127â€” the [Ca2@J1response induced by gastrin I. Neither gastrin I nor â€”110 120-p CCK-8 prevented the increase in [Ca2@J1inducedthrough a @ Gastrini cc K-S Gastrin-1 ccK-8 BK C 267â€” distinct neuropeptide receptor such as bradykinin (data not @:; H510 shown). These results suggest that gastrin I and CCK-8 induced % 275â€” Ca2@mobilization in H510 through a common receptor.

208 J\ BK 200â€” Gastrin I, gastrin II, des(S03)CCK-8, and CCK-8 increased -183 â€”161 the peak level of [Ca2'j1 in a concentration-dependent fashion -156 (Fig. 2). The concentrations required to induce half-maximum -.156 stimulation by these agonists were 7, 2.5, 5, and 2.5 n@, respec @ 110i@@@+vp â€”110 100â€”@@ -100 tively. Thus, the receptors expressed by H510 cells recognize @ Gsstrin-I I Mm CC@8BK tVP C gastrin and CCK agonists with approximately equal apparent Fig. I . Effect of gastrin-I and CCK-8 on [Ca2@J1in comparison to other neu affinities. ropeptides in various SCLC cell lines. SCLC cell lines H69 (lop left), H345 (top Effect ofVarious CCK/Gastrin Antagonists. In order to gain right), and H5l0 (lower) were cultured in HITESA for 3â€”5days.Aliquots of 4â€”5 x l0@cellswerewashedandincubatedin10ml freshHITESAmediumfor2 h insight into the receptor that mediates the [Ca2@]1response to at 37C. Then, I @LMfura-2/AMEwas added for 5 mm. The cells were washed and gastrin and CCK-8 in H510 cells we tested the effect of various resuspended in 2 ml of electrolyte solution the composition of which is described in â€œMaterialsand Methods.â€•This cell suspension was placed in a quartz cuvette receptor antagonists. Proglumide, a derivative of glutaramic and wascontinuouslystirred. Fluorescencewasmonitored in a Perkin-ElmerLs5 acid, and benzotript, a tryptophan derivative, block the binding luminescence spectrophotometer, and basal and peak [Ca2'J@values were deter and biological effects of both gastnin and CCK analogues mined. Gastrin-I, CCK-8, cholecystokinin 23â€”26;BK,bradykinin; GRP, gastnin releasing peptide; VP, vasopressin; Gal, galanin. All peptides were used at a final (reviewed in Ref. 17). Accordingly, the Ca2@-mobilizing effects concentration of 100 flM. of either gastrin II or CCK-8 were prevented by the addition of 6032

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. GASTRIN STIMULATION OF CLONAL GROWTH either proglumide or benzotnpt (Fig. 3). These antagonists, 216 however, do not discriminate between different types of CCK/ 141 â€” gastrin receptors. Recently, a CCK receptor antagonist has been identified that is selective for gastrin/CCK@ receptors as com C pared to pancreatic CCKA receptors (18, 19). Fig. 4 shows that the addition of the specific gastrin/CCK@ receptor antagonist c@,I L-365260 (15 nM) completely blocked the increase in [Ca211 C.) induced by 5 nM gastrin II. The antagonist inhibited the [Ca2@j1 81-h â€˜U response by either gastrin II or CCK-8 in a concentration 108 1 Mm dependent manner; 50% inhibition concentration values were 4 tCCK.8 and 1 nM, respectively. In contrast, the CCKA preferring antag Ga1trin4l -.135 onist L-364,718 (15 nM) had little effect on the increase in 120 [Ca2@j1inducedby 5 flM gastrin II (data not shown). The fact that gastrin and CCK agonists were able to induce Ca2@mobi lization with comparable potencies (Fig. 2) and that L-365260 80 f -90 was a potent inhibitor of these responses (Fig. 4) indicates that 105 :@ L3L6OCC@C@ K-8+BK Gal H5l0 cells express a gastrin/CCKB-type receptor. L365,260 @strin@ii@BK Gal Gastrin Stimulates Clonal Growth in SCLC. The preceding findings indicate that the H510 SCLC cell line can provide a 100 useful model system for determining whether gastrin can act as C 80 a direct growth factor in vitro. Consequently, we attempted to determine whether gastrin can stimulate colony formation by N 60 this SCLC cell line in agarose-containing medium. Fig. 5 shows @@40

216@ <20

â€”180 â€”154 0 [L365,260] nM â€”125 â€”114 Fig. 4. Effect ofantagonist L365,260 on gastrin-induced Ca2@mobilization in the SCLC cell line H5l0. Top, H510 cells preloaded with fura-2/AME; fluores Proglu C cence was monitored as previously described. The following final concentrations were used: Gastrin-!!, gastrin II (5 nM); BK, bradykinin (10 nM) L365,260 (15 nM); gastrin-JI+, gastnin II (100 nM). Bottom, dose-dependent inhibition of Ca2' mobilization induced by 5 ElMgastrin II and 5 n@ CCK-8 by L365,260. C) that gastrin I and CCK-8 caused a striking increase in the ability ::@ of these cells to form colonies in semisolid medium. Gastrin Benzf t tCCK-8+tBK increased both the number and the size of colonies, in a dose CCK-8 dependent manner (Fig. 6). This increase in clonal growth was U' 1 Mm comparable to that induced by other growth factors. Gastrin I, gastrin II, des(SO3)CCK-8, and CCK-8 stimulated colony for mation at comparable concentrations (Fig. 7). In contrast, 100 CCK-1020, which lacks the COOH-terminal sequence critical for receptor binding, neither increased [Ca2'j1 nor stimulated colony formation. The ability of gastrin to induce colony growth was attenuated by the addition of the specific gastrin/ c,,1 CCKn receptor antagonist L-365260 (results not shown). 4' 0 The peptides [D-Arg1,o-Phe5,o-Trp7'9,Leut IJ substance P and @121 [Arg6,o-Trp7'9,MePhe8j substance P (6â€”11)have been shown to inhibit signal transduction and cell proliferation by multi 0 1000 1 10 ple neuropeptides that induce Ca2@mobilization in SCLC cells [Benzotript] MM [Proglumide] mM (1 1, 13, 20). These broad-spectrum neuropeptide antagonists Fig. 3. Effect of gastrin II and CCK-8 and their antagonists proglumide and inhibited both the [Ca2']1 response and the stimulation of col benzotript on (Ca2@J,in the H510 SCLC cell line. Top, H510 cells, cultured, ony formation induced by gastrin (data not shown). washed, and loaded with fura-2/AME, were resuspendedin electrolytesolution and placed in a quartz cuvette. Fluorescence was monitored, and basal and peak ICa2i@ was calculated as described previously. The following abbreviations and DISCUSSION finalconcentrationswere used: gastrin-Il, gastrin 11(5 nM);CCK-8, CCK-8 (5 nM); Proglu, proglumide (10 mM); gastrin-1J+, gastrin II (100 nM);BK, bradykinin (10 nM); Benz, benzotript (I mM); Gal, galanin (25 nM); CCK-8+, cholecystokinin 8 In the present study we demonstrate that the addition of (100 nM). Bottom, dose-dependent inhibition of Ca2@mobilization induced by 5 gastrin at nanomolar concentrations to the SCLC line H510 nM gastrin II by benzotript and proglumide. A@Ca2@l, (i.e., peak [Ca2@J@â€”basal (Ca2'j@)was calculated at each antagonist concentration. @[Ca2'ltinduced by 5 nti causes a rapid mobilization of Ca2@ from intracellular stores. gastninII is taken as 100%. The magnitude ofthe ICa2i1 signal evoked by gastrin was more 6033

Gastrin-I

Fig. 5. Effect of gastrin I and CCK-8 on colony formation in H5l0 SCLC cells. Cul tures, 3â€”5dayspostpassage in HITESA, were washed, and 10@viable cells/mI were plated in HITESA mediumcontaining0.3% agaroseon top of a base of 0.5% agarose in culture me dium as described in â€œMaterialsandMeth ods.â€•Both layers contained either gastrin I or CCK-8atvariousconcentrations,asindicated. Cultures were incubated at 37T in a humidi fled atmosphere at 10% C02/90% air for 21 days and then stained with nitrotetrazolium CCK-8 blue. Colonies on a control dish and from dishes with 10, 50, and 200 [email protected] I and CCK-8 are shown. 10 50 200 (Peptide] nM

Gastrin-I Gastrin-Il ,j 600

I- 400 .0 E .C 200 z U) >5 L 0 .@ 600 desCCK-8 CCK-8 0 (U) @400[ - 100 10 [Gastrin-IJnM 20O@jII@r-. Area(mm2) 0.1

Fig. 6. Effect of dose-dependent stimulation ofgastrin I on colony number and 0 10 50100 0 10 50100 size in H5l0 SCLC cells. Cultures, 3â€”5days postpassage, were washed and resuspended in HITESA. Single cells (l0@) in 0.3% agarose were layered on top [Peptide] nM of 0.5% agarose, both layerscontaining gastninI at the concentration indicated. Fig. 7. Gastrin I, gastrin II, CCK-8, and des-(S03)CCK-8 stimulate colony Colonies were incubated and stained as described. Colonies represent aggregates formation in H510 SCLC cells. Cultures, 3â€”5dayspostpassage, were washed and ofcells >0.01 mm2 visualized by a TV camera and analyzed using the Macintosh resuspended in HITESA. Single cells (l0@) in 0.3% agarose were layered on top Image program. of 0.5% agarose; both layers contained peptide at the same concentration as indicated. Colonies were incubated and stained as described in â€œMaterialsand Methods.â€•Colonies represent aggregates of cells >16 cells (l20-@im diameter) counted under a microscope.Columns, mean of 3â€”4independentexperiments pronounced than that elicited by other Ca2@-mobilizing pep (each with 5 replicates Â±SEM). tides in this cell line. Thus, the SCLC cell line H510 has been identified as a useful model system for studying the effects of Gastrin has been postulated as a cellular growth factor, but gastrin in vitro. compelling in vitro evidence of this has been difficult to obtain. Gastnin and CCK share a common COOH-terminal pen SCLC cell lines, including H510, are able to form colonies in tapeptide and consequently bind to common cell surface recep semisolid medium, and their clonogenic ability is markedly in tors. The CCKA receptors have a 500-fold higher affinity for creased by multiple neuropeptides (21). The SCLC cell line CCK-8 than for gastrin. In contrast, CCKB receptors bind ei H510 provides an excellent model system in which to deter ther CCK-8 or gastrin with approximately equal apparent af mine whether gastrin can act as a direct growth factor. Conse finities (17). Here we demonstrate that gastrin I, gastrin II, quently, we determined the effect of gastrin on the ability of CCK-8, and des-(SO3)CCK-8inducerapid Ca2@mobilization these cells to form colonies in agarose-containing medium. in SCLC H510 at comparable concentrations. Furthermore, Here we demonstrate that gastrin markedly stimulates the the selective gastrin/CCK@ antagonist L360265 (18, 19) clonal growth of H510 cells. Gastrin I, gastrin II, des-(SO3)- blocked the increase in [Ca2@]1induced by either gastrin or CCK-8, and CCK-8 induce colony formation at comparable CCK-8. Thus, the effects of gastrin and CCK in SCLC H510 concentrations, in agreement with the effects obtained on Ca2@ are mediated by gastrin/CCK@ receptors. mobilization. [o-Arg1,o-Phe5,o-Trp7'9,Leu' â€˜@SubstanceP and 6034

[Arg6,o-Trp7-9,MePhe8] substance P (6â€”11), which have been 9. Carney, D. N., Gazdar, A. F., and Minna, J. D. Positive correlation between histological tumor involvement and generation of tumor cell colonies in identified as broad-spectrum neuropeptide antagonists (20), agarose in specimens taken directly from patients with small cell carcinoma prevented the Ca2@mobilization induced by gastrin and strik of the lung. Cancer Res., 40: 1820â€”1823,1980. ingly inhibited basal and gastrin-stimulated colony formation. 10. Heikkila, R., Trepel, J. B., Cuttitta, F., Neckers, L. M., and Sausville E. A. These results demonstrate that gastrmn acts as a direct growth Bombesin-relatedpeptidesinducecalciummobilizationina subsetofhuman small cell lung cancer cell lines. J. Biol. Chem., 262: 16456â€”16460,1987. factor in vitro and show, for the first time, that this hormonal 11. Woll, P. J., and Rozengurt, E. A neuropeptide antagonist that inhibits the peptide can stimulate the proliferation of cells outside the gas growth of small cell lung cancer in vitro. Cancer Res., 50: 3968â€”3973, 1990. trointestinal tract. 12. Woll, P. J., and Rozengurt, E. Multiple neuropeptides mobilise calcium in small cell lung cancer: effects of vasopressin, bradykinin, cholecystokinin, Lung cancer remains the commonest fatal malignancy in galanin and neurotensin. Biochem. Biophys. Res. Commun., 164: 66â€”73, the developed world. SCLC constitutes 25% of the total and 1989. follows an aggressive clinical course, despite initial chemo 13. Sethi, T., and Rozengurt, E. Galanin stimulates Ca2' mobilization, inositol phosphate accumulation and clonal growth in small cell lung cancer cells. sensitivity (22). Identification of the factors that stimulate Cancer Res., 51: 1674â€”1679,1991. the proliferation of SCLC cells will be important in the design 14. Staley,J., Jensen, R. T., and Moody, T. W. CCK antagonists interact with of alternative and more effective therapeutic strategies. SCLC CCK-Breceptorsonhumansmallcelllungcancercells.Peptides(NY),11: is characterised by its ability to secrete many hormones and 1033â€”1036,1990. 15. Lin, S. W., Holladay, M. W., Barrett, R. W., et al. Distinct requirements for neuropeptides including GRP, neurotensin, and vasopressin activation at CCK-A and CCK-B/gastrin receptors: studies with a C-terminal (23â€”25).Results from our laboratory show that, at optimal hydrazide analogue of cholecystokinin tetrapeptide (30â€”33).Mol. Pharma concentrations, neurotensin, vasopressin, GRP, galanin, and col., 36: 881â€”886,1989. 16. Simms, E., Gazdar, A. F., Abrams, P. G., and Minna, J. D. Growth of human bradykinin induce comparable increases ofSCLC clonal growth small cell (oat cell) carcinoma of the lung in serum-free growth factor-sup in responsive cell lines (21). Consequently, it has been hypoth plemented medium. Cancer Res., 40: 4356â€”4363,1980. esized that SCLC growth is regulated by multiple autocrine 17. Jensen, R. T., Wank, S. A., Rowley, W. H., Sato, S., Gardner, J. D. Inter action of CCK with pancreatic acinar cells. Trends. Pharmacol. Sci., 10: and/or paracrine circuits involving Ca2@-mobilizing neuropep 418â€”423,1989. tides (12, 21, 26). Interestingly, SCLC cells have been shown to 18. Lotti, V. J., and Chang, R. L. A new potent and selective nonpeptide gastrin express gastrin and CCK peptides (27, 28). Thus, the findings antagonist and brain cholecystokinin receptor ligand 365,260. Eur. J. Phar macol., 162: 273â€”280,1984. presented here demonstrating that gastrin and CCK-8 can act 19. Bock,M. G., DiPardo, R. M., Evans,B. E., et al. Benzodiazepinegastninand as direct growth factors for the SCLC cell line H510 further brain cholecystokinin receptor ligands: L-365,260. J. Med. Chem., 32: 13â€” extend the hypothesis that SCLC growth may be regulated by 16, 1989. 20. Woll, P. J., and Rozengurt, E. (DArg',DPhe5,DTrP7'9,LeU' â€˜IsubstanceP, a multiple autocnine and paracrine loops involving neuropep potent bombesin antagonist in Swiss 3T3 cells, inhibits the growth of human tides. small cell lung cancer cells in vitro. Proc. NatI. Acad. Sci. USA, 85: 1859â€” 1863, 1988. REFERENCES 21. Sethi, T., and Rozengurt, E. Multiple neuropeptides stimulate clonal growth ofsmall cell lung cancer: effects ofbradykinin, vasopressin, cholecystokinin, I. Johnson, L. R. Trophic effects ofgastnin on the colon. In: S. R. Wolman and galanin and neurotensin. Cancer Res., 51: 3621â€”3623,1991. A. J. Mastromarmno(eds.),Progress in Cancer Research and Therapy, Vol. 22. Smyth, J. F., Fowlie, S. M., Gregor, A., Crompton, G. K., Busuttil, A., 29, pp. 327â€”336.New York: Raven Press, 1984. Leonard, R. C., and Grant, I. W. The impact of chemotherapy on small cell 2. Ryberg, B., Axelson, J., Hakanson, R., Sundler, F., and Mattson, H. Trophic carcinoma of the bronchus. Quant. J. Med., 61: 969â€”973,1986. effects of continuous infusion of @Lys'5J.gastrin-17 in the rat. Gastroenter 23. Cuttitta, F., Carney, D. N., Muishine, J., et al. Bombesin-like peptides can ology, 98: 33â€”40,1990. function as autocrine growth factors in human small-cell lung cancer. Nature 3. Johnson, L. R., and Guthrie, P. D. Proglumide inhibition oftrophic action of (Lond.), 316: 823â€”826,1985. pentagastnn. Am. J. Physiol., 246: G62-G66, 1984. 24. Goedert, M., Reeve, 1. G., Emson, P. C., and Bleehen, N. M. Neurotensin in 4. Singh, P., Walker, J. P., Townsend, C. M., and Thompson, J. C. Role of human small cell lung carcinoma. Br. J. Cancer, 50: 179â€”183,1984. gastrin and gastrin receptors on the growth of a transplantable mouse colon 25. Sausville, E., Carney, D., and Battey, J. The human vasopressin gene is linked carcinoma (MC-26) in Balb/c mice. Cancer Res., 46: 1612â€”1616,1986. to the oxytocingeneand is selectivelyexpressedin a cultured lungcancercell 5. Watson, S., Durrant, L., and Morris, D. Gastrin: growth enhancing effects on line. J. Biol. Chem., 260: 10236â€”10241,1985. human gastric and colonic tumour cells. Br. J. Cancer., 59: 554â€”558,1989. 6. Hoosein,N.M.,Kiener,P.A.,Curry,R.C.,Rovati,L C.,McGilbra,D.K., 26. Sethi, T., Langdon, S., Smyth, J., and Rozengurt, E. Growth of small cell and Brattain, M. G. Antiproliferative effects of gastnin receptor antagonists lung cancer cells: stimulation by multiple neuropeptides and inhibition by and antibodies to gastrin on human colon carcinoma cell lines. Cancer Res., broad spectrum antagonists in vitro and in vivo. Cancer Res., 52: 2737s- 48:7179â€”7183,1988. 2742s, 1992. 7. Hoosein, N. M., Kiener, P. A., Curry, R. C., and Brattain, M. G. Evidence for 27. Rehfeld, J. F., Bardrum, L., and Hilsted, L. Gastrin in human bronchogenic autocnine growth stimulation of cultured colon tumor cells by a gastrin/ carcinomas: constant expression but variable processing of progastrin. cholecystokinin-like peptide. Exp. Cell Res., 186: 15â€”21,1990. Cancer Res., 49: 2840â€”2843,1989. 8. Thumwood, C. M., Hong, J., and Baldwin,G. S. Inhibition of cell prolifer 28. Geijer, T., Folkesson, R., Rehfeld, J. F., and Monstein, H-J. Expression of ation by the cholecystokinin antagonist L-364,718. Exp. Cell Res., 192: the cholecystokinin gene in a human (small-cell) lung carcinoma cell line. 189â€”192,1991. FEBS Lett., 270: 30â€”32,1990.

6035

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. Gastrin Stimulates Ca2+ Mobilization and Clonal Growth in Small Cell Lung Cancer Cells

Tariq Sethi and Enrique Rozengurt

Cancer Res 1992;52:6031-6035.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/21/6031

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/52/21/6031. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.