ICANCER RESEARCH 54. 6143-6147, December 1. 19941

ABSTRACT

Serwn stimulates both Ca2@mobilization and colony growth of manysmall cell lung cancer (SCLC) cell lines, but the factors involved remainunknown. We demonstrate that 1-oleoyl-Iysophosphatidicacid (LPA), likeserum, induced a dose-dependent increase in intracellular Ca2@ in the

11-510,H-345, and H-69 SCLC cell lines with halfmaximal concentrationsof 18 flM,22 flM,and 20 nM,respectively. Two lines of evidence revealedthat LPA was the major factor in serum responsible for mobilizing Ca2@in these SCLC cell lines: (a) both LPA and serum exhibited cross desensitization in the @2+mobilization assay; and (b) phospholipase B pretreatment of either LPA or serum prevented the ability of these agents tostimulate Ca2@mobilization. In marked contrast, LPA at concentrationsbetween 2 BM and 20 @M,unlike serum, failed to stimulate colony formadon. Furthermore, phospholipase B treatment of serum did not inhibitserum-induced colony formation. We therefore searched for growth factors which could induce colony growth through a Ca2@-independentpathway. We found that both human recombinant hepatocyte growthfactor and stem cell growth factor Increased colony growth, but failed tostimulate an increase in intracellular Ca2@in the H-510, H-345, and H-69SCLC cell lines. Our results indicate that LPA-depletedserum, hepatocytegrowth factor, and stem cell growth factor stimulate colony formation inSCLC cells through a Ca2'-independent pathway.

INTRODUCTION

Lung cancer is the most common fatal malignancy in the developedworld. SCLC2 constitutes 25% of all pulmonary cancers and followsan aggressive clinical course. Despite initial sensitivity to radio andchemotherapy, the 2-year survival of patients with SCLC remainsvery low (1). Thus, novel therapeutic strategies are urgently required,and these will most likely arise from a better understanding of thefactors and signaling pathways that stimulate the proliferation ofSCLC.

SCLC is characterized by the ability to secrete a variety of hormonal neuropeptides including gastrin-releasing peptide, vasopressin,cholecystokinin, and neurotensin (2—7).Among these, gaStrin-releasing peptide has been shown to act as an autocrine growth factor forcertain SCLC cell lines (8—10).Furthermore, a variety of neuropeptides, including those secreted by SCLC, induce rapid mobilization ofCa2@ from internal stores of SCLC cell lines (1 1—13)and promoteclonal growth of these cells in semisolid medium (13—15).Consequently, the emerging view is that SCLC growth appears to beregulated by multiple autocrine and paracrine circuits involving Ca2@mobilizing neuropeptides. Another class of growth factor, thepolypeptide growth factors, has been implicated in the proliferation of

Received 6/27/94; accepted 9/30/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

@ To whom requests for reprints should be addressed.2 The abbreviations used are: SCLC, small cell lung cancer; [Ca2]1, intracellular

calcium; FBS, fetal bovine serum; HITESA, 10 nsi hydrocortisone-5 @tg/mlinsulin-lO@tg/mltransferrin-lO nsi estradiol-30 n@.iselenium-0.25% bovine serum albumin; HGF,

recombinant human hepatocyte growth factor; PIP2-PLC, phosphatidyl inositol-specificphospholipase C; LPA, l-oleoyl-lysophosphatidic acid; SCF, recombinant human stemcell factor; EC5,@,50% effective concentration.

several cancers (16). Indeed, receptors for the polypeptide factorsHGF and SCF (17—21)have been demonstrated on many differentSCLC cell lines, but their role as growth promoting agents for SCLChas not been defined.

While the response of individual SCLC cell lines to a range ofneuropeptides is heterogenous, serum has been shown to stimulate themobilization of Ca2@ and colony growth of most SCLC cell linesexamined (9, 22). The role of Ca2@ mobilization and the identity ofgrowth factor(s) in serum which induce SCLC growth remain unknown. Recently, the bioactive lipid LPA has been shown to be oneof the major lysophospholipids in serum to stimulate growth and topromote differentiation in a variety of cell lines (reviewed in Ref. 23).LPA binds to a putative receptor which activates heterotrimeric Gproteins leading to the stimulation of PIP2-PLC. This results in therapid hydrolysis of phosphotidylinositol 4,5-bisphosphate into inositol1,4,5-trisphosphate and diacylglycerol, which cause Ca2@ mobilization and protein kinase C activation, respectively (23, 24). It remainsunknown whether LPA is also responsible for serum-induced Ca2@mobilization and growth in SCLC.

In the present study we demonstrate that LPA accounts for theability of serum to mobilize calcium in SCLC cell lines. However,LPA neither stimulated colony formation nor mediated serum-inducedcolony growth of SCLC cells. This suggested that rapid Ca2@ mobilization was not required for serum-induced growth of SCLC. Wetherefore searched for growth factors that could stimulate colonygrowth without inducing Ca2@ mobilization. Here we report that thepolypeptide growth factors HGF and SCF stimulate colony growth ofSCLC cell lines through a Ca2tindependent pathway.

MATERIALS AND METhODS

Cell Culture. SCLC cell lines H-510, H-69, and H-345 were generouslydonated by Dr. A. Gazdar (Bethesda, MD) and purchased from the AmericanType Culture Collection. Stocks were maintained in RPMI 1640 supplemented

with 10% (v/v) fetal bovine serum (heat inactivated at 57°C for 1 h) in a

humidified atmosphere of 10% C02/90% air at 37°C. They were passaged

every 7 days. For experimental purposes, the cells were grown in HITESA,which consists of RPMI 1640 supplemented with 10 n@i hydrocortisone-5p@g/mlinsulin-lO @xWmltransferrin-lO nM estradiol-30 nM selenium-0.25%bovine serum albumin.

Measurement of Intracellular Calcium. [Ca2@ 1 was measured with theflourescent Ca2@indicator fura-2 using a modification of the procedure described previously (25). Aliquots of 4—5X 106 SCLC cells in HITESA for 3—5

days were washed and incubated for 2 h at 37°Cin 10 ml fresh HITESAmedium. Then fura-2 tetraacetoxymethyl ester was added to a final concen

tration of 1 p@Mand the incubation continued for a further 10 mm. After a 30-scentrifugation at 1500 rpm the cells were resuspended in 2 ml of electrolytesolution at 37°Ccontaining 120 mM NaCl-5 mM KC1-1.8 mM CaCl2-0.9 mMMgCI2-25m@iglucose-16 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid-6 mM Tris/HCI and an amino acid mixture equivalent to Dulbecco's

modified Eagle's medium (jH 7.2) and transferred to a quartz cuvette. Thesuspension was stirred continuously and maintained at 37°C.Fluorescence wasmonitored in a Perkin Elmer Cetus IS-S luminescence spectrophotometer withan excitation wavelength of 336 nm and emission wavelength of 510 nm.

6143

Lysophosphatidic Acid-depleted Serum, Hepatocyte Growth Factor and Stem Cell

Growth Factor Stimulate Colony Growth of Small Cell Lung Cancer Cellsthrough a Calcium-independent Pathway

Michael J. Secki, Thomas SeufTerlein, and Enrique Rozengurt'

imperial Cancer Research Fund, P.O. Box 123. 44 Lincoln ‘sinn Fields. London WC2A 3PX. United Kingdom

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POLYPEPTIDEGROWTH FACTORS AND SCLC COLONY GROWTH

Various additions were made as indicated in the figure legends after a 1-mmstabilization period. [Ca2@J@was calculated using the formula:

/F— F.[Ca2@inM KI\Fmax

where F is the fluorescence at the unknown [Ca2@]1,Fmax @5the fluorescenceafter addition of 0.02% Triton X-100, and Fmjn is the fluorescence after theCa2@in the solution is chelated with 10 m@i[ethylenebis(oxyethylenenitnlo)]tetraacetic acid. The value of K was 220 flM for fura-2 (25).

Phospholipase B Treatment ofLPA and Serum. LPA and lysophosphatidates bound to albumin in serum are inactivated by phospholipase B (26, 27).Here, 100 @tlof a 500-g.@MLPA stock solution dissolved in phosphate-bufferedsaline/0.01% bovine serum albumin (w/v) or 100 pA of FBS were incubatedwith or without 100 IU of phospholipase B for 2 h at 37°C. The phospholipase

B-treated LPA or FBS were used immediately in the Ca2@ mobilization orcolony assays as indicated in the figures. In control experiments the activity of

phospholipase B was destroyed by heating to 75°C for 1 h prior to the 2-h

incubation with LPA or serum.

Growth Assay. SCLC cells 3—5days postpassage were washed and resuspended in HITESA. Cells were then dissaggregated into an essentially singlecell suspension by two passes through a 19-gauge needle and then through a20-sm nylon gauze. Cell number was determined using a Coulter Counter, andio@viable cells were mixed with HITESA containing 0.3% agarose andagonist at the concentrations indicated and layered over a solid base of 0.5%agarose in HITESA with agonist at the same concentration in 33-mm plasticdishes. The cultures were incubated in humidified 10% C02/90% air at 37°Cfor 21 days and then stained with the vital stain nitroblue tetrazolium. Coloniesof >120 @xmdiameter (16 cells) were counted using a microscope.

Materials. LPA and phospholipase B (Vibrio, EC 3.1.1.4) were obtainedfrom Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum was purchasedfrom GIBCO-BRL and diluted with HITESA to the amounts indicated. Recombinant human HGF was generously provided by Dr. E. Gherardi;

recombinant human SCF was purchased from Biotechnology Products Ltd.(Abingdon, UK). Fura-2-tetracetoxy methyl ester was purchasedfrom Calbiochem Corp. (La Jolla, CA) and agarose was from SeaKem(Rockland, ME). All other reagents were of the highest grade available.

RESULTS

Serum and LPA Stimulate Ca2@ Mobilization in SCLC Cell

Lines: Cross-desensitization and Effect of Phospholipa.se B Pretreatment. Fresh serum induces rapid Ca2@ mobilization in multipleSCLC cell lines. As shown in Fig. IA, addition of increasing amountsof FBS caused a dose-dependent increase in [Ca2@]1in the H-510,H-345, and H-69 cells with an EC50 of 0.03, 0.02, and 0.05% (v/v),respectively. The maximum increase in [Ca2@]1 was achieved byaddition of 0.5% FBS in H-510 and H-69 cell lines and 1% in theH-345 cell line. Since LPA has been identified as one of the majorcomponents in serum capable of mobilizing Ca2@ in rat glioma C6cells (27), we reasoned that LPA may also stimulate Ca2@ mobilization in SCLC cell lines. As shown in Fig. 1B, LPA-like serumstimulated a dose-dependent increase in [Ca2@]1in the H-510, H-345,and H-69 cells with an EC50 of 18, 22, and 20 nM, respectively. LPAinduced a maximum increase in [@2±] at a concentration of 200 flMin all SCLC cell lines. Furthermore, the maximum amount of Co2@mobilization achieved with LPA was almost identical to the amountreleased by serum.

If LPA was the major factor in serum responsible for mobilizingCo2@thenbothLPAandserumshouldexhibitcross-desensitizationin the Ca2@mobilization assay. Accordingly, addition of 200 nM LPAto H-510, H-345, and H-69 cells, a concentration which inducedmaximum Ca2@ mobilization, markedly attenuated the subsequentresponse to 1% serum (Fig. 24). Similarly, when cells were maximally stimulated with FBS the subsequent Ca2@response to LPA wasinhibited (Fig. 2B). Importantly, treatment with either LPA and/orFBS did not block the subsequent response of cells to neuropeptide,

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indicating that the mobilizable Ca2@ pool was not depleted after theinitial stimulation with either LPA or FBS (data not shown). Theseresults clearly demonstrate heterologous desensitization between LPAand serum in SCLC cell lines and suggest that LPA mediates theCa2tmobilizing effects of serum in these cells.

To substantiate the notion that LPA was the major factor in serumto stimulate Co2@ mobilization in the SCLC cell lines, we nextexamined the effect of phospholipase B pretreatment on either LPA or

6144

Fig. 1. Dose-response curves for the effects of FBS and LPA on Ca2@ mobilization inthe SCLC cell lines H-Sb, H-345, and H-69. (A) FBS induces a dose-dependent increasein [Ca2@]1.The H-Sb, H-MS. and H-69 SCLC cell lines were cultured in H1'FESA for3—5days. Aliquots of 4—5x 106 cells were washed and incubated in 10 ml fresh HITESAmedium for 2 h at 37°Cand then loaded with 1 @sifura-2 tetraacetoxymethyl ester for 5mm. The cells were washed and resuspended in 2 ml of electroyte solution in a quartzcuvette, and fluorescence was monitored in a spectrophotometer as described in “Materials and Methods.―Basal and peak [Ca2@] were detennined at each FBS amount addedto calculate the il [Ca2']1. (B) LPA induces a dose-dependent increase in ICa2@I1.TheH-Sb, H-345, and H-69 SCLC cell lines were preloaded with fura-2 tetraacetoxymethylester, and fluorescence was monitored as described previously. Basal and peak [Ca2@Jwere determined at each LPA concentration added to calculate the@ [Ca2']. Points, atleast 3 independent experiments.

AH510 H345 H69

E.—3

0

B

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Fig. 2. Cross-desensitization between LPA and FCS in Ca2@ mobilization assays in theSCLC cell lines H-Sb, H-345, and H-69. (A) Treatment with LPA prevents subsequentFBS-stimulated Ca24 mobilization. The H-Sb, H-345, and H-69 SCLC cell lines werestimulated initially with 200 nsi LPA. Following a 2-mm recovery period (arrow), thecells were challenged with 1% FBS. (B) Treatment with FBS prevents subsequentLPA-stimulated Ca24 mobilization. The H-Sb, H-345, and H-69 SCLC cell lines werestimulated initially with 1% FBS. Following a 2-mm recovery period (arrow), the cellswere challenged with 200 nsi LPA. [Ca2@] values were determined as described in“Materialsand Methods.―Columns, at least 3 independent experiments. Values areexpressed as a percentage of the maximum increase in 1Ca21 induced by the firststimulating agent compared to the second agent.

FBS LPA FBS LPA FBS LPA- _- _

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300

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Table I Effect of LPA on the colony growth of the SCLC cell lines H-Sb,H-345. andH-69A

single cell suspension was plated in agarose medium containing HITESA at adensity of 1 X@ cells/dish in the absence (—)or presence of 25 flMor 2 @MLPA or 10%FBS (v/v with HITESA) and incubated for 3 weeks as described in “MaterialsandMethods.―Each number represents the mean number of colonies formed on 5 separatedishes ±SE. In all cases, a representative of 3 independent experiments isshown.Colonies/dishAddition

H-Sb H-345H-69—

110±14105±1398±15LPA(25niw) 105±12 95±18 118±12LPA(2@u.i) 118±14 144±20 123±18FBS(10%) 620±23 714±26 770±32

0...1 I 10

POLYPEFFIDEGROWTHFACTORSAND5CLCCOLONYGROWTH

serum. Phospholipase B has been shown to inactivate LPA by hydrolyzing the ester bond-linking fatty acid to the 1-position of theglycerol backbone of lysophospholipids (26, 27). LPA or serum werepretreated in the presence or absence of 100 IU/ml of phospholipaseB for 2 h (26, 27). The results shown in Fig. 3A revealed that theability of LPA to induce Ca2@ mobilization could be inactivated bypretreating LPA with phospholipase B. We verified that this enzymealso inactivated LPA in assays of Ca2@ mobilization and tyrosinephosphorylation using Swiss 3T3 cells (results not shown). Crucially,incubation of serum with phospholipase B markedly reduced theability of the phospholipase B-treated serum to induce @2+mobilization in the H-510, H-345, and H-69 SCLC cell lines (Fig. 3B).Treatment of serum or LPA with heat-inactivated phospholipase B didnot affect the ability of either serum or LPA to induce Co2@mobilization. In addition, phospholipase B pretreatment of various neuropeptides did not inhibit their ability to subsequently stimulate Ca2@mobilization in these SCLC lines (data not shown). These resultsindicated that LPA was likely to be the dominant factor in serumcontributing to Ca2@ mobilization in the SCLC cell lines examined.

Differenfial Stimulation of Colony Formation by Serum andLPA. It has been shown previously that neuropeptides which stimulate Co2@ mobilization also induce colony formation in SCLC celllines (13.-is). Fig. 4A shows that serum stimulated the growth of theH-510, H-345, and H-69 small cell lines in a dose-dependent fashionwith an EC50 of 0.6, 1.5, and 3% FBS, respectively. In markedcontrast, LPA at either 25 tIM,a concentration similar to that requiredto produce half-maximum Ca2@ mobilization, or 2 p.M, a concentration 10-fold higher than that needed to induce maximum Ca2@ mobilization, failed to either stimulate or inhibit colony formation in allthree of the SCLC cell lines examined (Table 1). In contrast, FBScaused a marked increase in colony formation of parallel cultures.

Furthermore, incubation of serum with phospholipase B, underconditions that markedly reduced its ability to mobilize Ca2@ frominternal stores, failed to reduce colony formation stimulated by serum.In fact, phospholipase B-pretreated serum enhanced colony growth2-fold as compared to untreated serum. We verified that both

@ 200

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200

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Fig. 3. Effect of phospholipase B treatment on LPA- (A) or serum- (B) induced Ca2@mobilization in the SCLC cell lines H-Sb, H-345, and H-69. [Ca2@11values weredetermined as described in “Materialsand Methods.―(A) H-Sb, H-345, and H-69 cellswere stimulated with either 200 nat LPA or 200 ns LPA which had been treated with 100LUphospholipase B. (B) The same cell lines were stimulated with either 1% FBS or 1%FBS which had been treated with 100 IU phospholipase B. Basal and peak [Ca2]1 weredetermined for 200 nsi LPA or 1% FBS with and without phospholipase B pretreatmentto calculate the@ [Ca2']1. Columns, representative experiments of 3 independentexperiments.

Fig. 4. Effect of FBS on colony growth in the SCLC cell lines H-Sb, H-345, and H-69.Left, a single cell suspension was plated in agarose medium containing HITESA at adensity of 1 X io@cells/dish in the absence or presence of increasing percentages of FBS(v/v with HITESA) and incubated for 3 weeks as described in “Materialsand Methods.―Points, mean number of colonies formed on 5 separate dishes, bars, SE. Right, cellsprepared in an identical way were incubated in the absence (0) or presence (P) of 100IU/ml of phospholipase B (P), 1% FBS, or 1% PBS pretreated with phospholipase B(FBS + P) for 3 weeks as described in “Materialsand Methods.―Columns,mean numberof colonies formed on 5 separate dishes, bars, SE. In all cases, a representative of 3independent experiments is shown. Where no error bar is visible, it lies within thesymbol.

A H 510

phospholipase B pretreatment of LPA and phospholipase B itself didnot stimulate colony growth (Fig. 4B). In addition, pretreatment ofneuropeptide with phospholipase B did not affect the subsequentability of neuropeptide to stimulate colony growth (data not shown).Taken together, the results demonstrated that serum-induced colonyformation in SCLC was not mediated by LPA and occurred through asignaling pathway independent of rapid mobilization of Ca2@ frominternal stores.

HGF and SCF Stimulate Colony Growth But Do Not Mobilize@2+ The preceding results prompted us to search for defined

polypeptide growth factors that could substitute for serum in promoting colony growth through a Ca2@-independent pathway. The recaptors for the polypeptide growth factors, HGF and SCF, have beenshown to be expressed by many different SCLC cell lines (17—21),buttheir effects on SCLC [Ca2@]1 and colony growth have not beenexplored. We initially examined the ability of these growth factors toinduce Co2@mobilization. Fig. 5 shows that 10 ng/ml of HGF or SCF,a concentration known to be biologically active in other cell systems,failed to stimulate an increase in [Ca2@], in any of the 3 cell lines

6145

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POLYPEPTIDE GROWTH FACTORS AND SCLC COLONY GROWTH

examined. In contrast, addition of LPA after HGF or SCF caused theexpected large increase in [Ca2@],, demonstrating that these factorsneither induce nor interfere with Ca2@ mobilization in SCLC celllines.

We next tested the ability of HGF and SCF to induce colony growthin the SCLC lines. Fig. 6 shows that HOF induced a dose-dependentincrease in colony formation of the H-510, H-345, and H-69 small celllines with an EC50 of 1.8, 1.3, and 1 ng/ml, respectively. Maximumcolony formation was achieved at a concentration of 10 ng/ml of HGFin all 3 cell lines. Similarly we found that SCF could induce adose-dependent increase in colony formation in the H-510 small cellline with an EC50 of 0.9 ng/ml (Fig. 7). SCF induced maximumcolony formation at a concentration of 3 ng/ml. This effect was notconfmed to the H-510 cell line, because SCF also stimulated colonyformation in both the H-345 and H-69 SCLC cell lines (Fig. 7,bottom). Thus, the polypeptide growth factors HGF and SCF stimulate SCLC colony growth without inducing a rapid increase in [Ca2@]1in the H-510, H-345, and H-69 SCLC cell lines.

H-345

270-

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‘Mi.Fig. 5. Effect of HGF and SCF on Ca2@mobilization in the SCLC cell lines H-Sb,

H-345, and H-69. H-Sb, H-MS. and H-69 cells were stimulated consecutively with 10ng/ml HGF, 10 ng/ml SCF, diluent, and 200 nsi LPA as indicated. The tracings shown arerepresentative of 2 independent experiments. [Ca2'], values were determined as describedin “Materialsand Methods.―

HGF(nglml)

Fig. 6. Effect of HGF on colony growth in the SCLC cell lines H-5b0, H-MS. andH-69. A single cell suspension was plated in agarose medium containing Hfl'ESA at adensity of 1 X io@cells/dish in the absence or presence of increasing concentrations ofHGF and incubated for 3 weeks as described in “Materialsand Methods.―Points, meannumber of colonies formed on 5 separate dishes; bars, SE. In all cases, a representativeof 3 independent experiments is shown. Where no error bar is visible, it lies within thesymbol.

H-510 1469

‘,-0 10 0 10SCF (nglml)

Fig. 7. SCF stimulates colony growth in the SCLC cell lines H-Sb, H-345, and H-69.Top, a single cell suspension of H-Sb cells was plated in agarose medium containingHITESA at a density of 1 X 10@cells/dish in the absence or presence of increasingconcentrations of SCF and incubated for 3 weeks. Bottom, a single cell suspension ofH-345 or H-69 was plated in agarose medium containing HITESA at a density of 1 X 10―cells/dish in the absence or presence of 10 ng/ml SCF and incubated for 3 weeks. Pointsand columns, mean number of colonies formed on 5 separate dishes, bars, SE. In all cases,a representative of 3 independent experiments is shown. Where no error bar is visible, itlies within the symbol.

DISCUSSION

Serum is known to stimulate both Ca2@ mobilization and colonygrowth in many SCLC cell lines, but the factors involved remainpoorly understood. LPA has been identified as one of the major

constituents in serum that induces Ca2@ signals and cell proliferationor differentiation in various cell types (23, 26, 27). Here we demonstrate that LPA, or an LPA-like molecule, is responsible for theCa2@-mobiizing effect of serum in SCLC call lines: (a) LPA induced

@2+mobilization to the same level as that stimulated by serum; and

(b) phospholipase B pretreatment of either LPA or serum destroyedthe ability of these agents to stimulate Ca2@ mobilization. An important question that emerged from these findings is whether LPA was

also responsible for the potent colony-stimulating effect of serum inSCLC.

Several lines of evidence demonstrate that LPA is not the majorgrowth factor for SCLC cell lines present in serum: (a) LPA does notstimulate colony growth, even at concentrations 100-fold higher thanthose required to induce half-maximum stimulation of Ca2@ mobilization; (b) The dose-response curves of serum-induced Ca2@ mobilization were shifted more than 50-fold to the left of the dose-responsecurves for colony growth. This is in marked contrast to neuropeptideswhich stimulate both Co2@ mobilization and colony growth withidentical dose-response curves (13); and (c) phospholipase B pretreatment to destroy lysophosphatidates including LPA did not reduce theability of serum to induce colony growth. These findings demonstratethat LPA is not the major growth factor for SCLC cell lines present inserum. Since our results showed that LPA mediates the Ca2@-mobilizing effects of serum it follows that the potent colony-stimulatingactivity of serum can be dissociated from its ability to mobilize Ca2@.

An important implication of these results is that colony growthcould be mediated by distinct signal transduction pathways in SCLCcell lines. Thus, neuropeptides are known to signal through receptorscoupled to Gq (reviewed in Refs. 28, 29) and thereby stimulatePIP2-PLC-mediated hydrolysis of polyphosphoinositides, leading to acascade of molecular events including the mobilization of Ca2@ frominternal stores (30). In contrast, serum would stimulate growth of

SCLC cell lines through a PIP2-PLC-independent pathway. However,

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POLYPEPTIDEGROWTHFACtORS AND SCLC COLONYGROWTH

cells: delineation of alternative pathways. Proc. Natl. Acad. Sci. USA, 87:2162—2166,1990.

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14. Sethi, T., and Rozengurt, E. Gastrin stimulates Ca2@mobilization and clonal growthin small cell lung cancer cells. Cancer Res., 52: 6031—6035, 1992.

15. Sethi, T., Herget, T., Wu, S. V., Walsh, I. H., and Rozengurt, E. CCKA and CCKBreceptors are expressed in small cell lung cancer lines and mediate Ca2@mobilizationand clonal growth. Cancer Res., 53: 5208—5213,1993.

16. Rozengurt. E. Polypeptide and neuropeptide growth factors: signalling pathways androle in cancer. In: M. Peckham (ed), The Oxford Textbook of Oncology. Oxford,United Kingdom: Oxford University Press, 1994.

17. Hibi, H., Takahashi, T., Sekido, Y., Ueda, R., Hide, T., Ariyoshi, Y., Takagi, H., andTakahashi, T. Coexpression of the stem cell factor and the c-kit genes in small-celllung cancer. Oncogene, 6: 2291—2296,1991.

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22. Bums, P. A., Chan, D., Dienhart, D. 0., Tolley, R., Tagawa, M., and iewett, P. B.Neuropeptide signal transduction in lung cancer: clinical implications of bradykininsensitivity and overall heterogeneity. Cancer Rex., 52: 24—31,1992.

23. Moolenaar, W. H. LPA: a novel lipid mediator with diverse biological actions. TrendsCell. Biol., 4: 213—219,1994.

24. Van der Bend, R. L, Bruner, i., ialink, K., Van Corven, E. J., Moolenaar, W. H., andVan Blitterswijk, W. i. Identification of a putative membrane receptor for thebioactive phospholipid, lysophosphatidic acid. EMBO J., ii: 2495—2501,1992.

25. Tsien, R. Y., Pozzan, T., and Rink, T. J. T-cell mitogens cause early changes incytoplasmic free @2+and membrane potential in lymphocytes. Nature (Lond.), 295:68—71,1982.

26. Tigyi, G., and Miledi, R. Lysophosphatidates bound to serum albumin activatemembrane currents in xenopus oocytes and neurite retraction in PC12 phaeochromocytoma cells. J. Biol. Chem., 267: 21360—21367, 1992.

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6147

an alternative interpretation is that the colony-stimulating effect ofserum is not mediated by a single factor but by the interactive effects

of multiple factors. To prove that colony growth of SCLC call linescould be stimulated through a Ca2@-independent pathway, it wasnecessary to identify defined factor(s) that substitute phospholipaseB-treated serum in promoting colony growth without causingconcomitant @2+mobilization in SCLC cells.

In the present study we demonstrate that HGF and SCF inducecolony growth in SCLC cells but failed to promote Ca2@mobilization,even at concentrations that induced maximum proliferation. HGF alsopurified as an activity that induces cell locomotion (i.e., scatterfactor), binds to c-met (31, 32), the specific receptor that mediates thegrowth-promoting and motogenic effects of HGF in endothelial andepithelial cells (33, 34). SCF, the gene product of the steel locus of themouse (35), has been identffied as the ligand of c-kit and acts onhematopoeitic, melanocyte, and germ cell lineages (36, 37). Interestingly, expression of c-met and c-kit has been documented in SCLC bydifferent laboratories (17—21),but the effect of their ligands on cellgrowth of these cells has not been explored in detail. Our resultsdemonstrating that HGF and SCF can promote colony growth raisethe possibility that these factors act in an autocrine manner in SCLC.Indeed, SCF has been shown to be expressed by many SCLC cell lines(17, 19). Although HGF has been detected in a minority of SCLCcells, the expression of this factor is prominent in the lung (38), infibroblasts (34), and in normal bronchial epithelial cells (39), suggesting a paracrine mode of action. Thus, SCLC growth may be stimulated by multiple autocrine and paracrine loops involving bothneuropeptide and polypeptide growth factors that act throughdifferent intracellular signaling pathways.

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1994;54:6143-6147. Cancer Res   Michael J. Seckl, Thomas Seufferlein and Enrique Rozengurt  PathwaySmall Cell Lung Cancer Cells through a Calcium-independent

ofFactor and Stem Cell Growth Factor Stimulate Colony Growth Lysophosphatidic Acid-depleted Serum, Hepatocyte Growth

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