(CANCER RESEARCH 50, 5257-5262, September 1, 1990]

Effect of Guanine and Adenine Nucleotides on Bombesin-stimulated Phospholipase

C Activity in Membranes from Swiss 3T3 and Small Cell Lung CarcinomaCells1

Yoav Sharoni, Jean Viallet, Jane B. Trepel, and Edward A. Sausville2

NCI-Navy Medical Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20814 [Y. S., J. V., J. B. T., E. A. S.]; Faculty of Health Sciences, Ben-Gurion University of the Neger, Beer-Sheva, Israel [Y. S.]; and Department of Medicine, Division of Medical Oncology, Vincent T. Lombardi Cancer Research Center,Georgetown University School of Medicine, Washington, DC 20007 fE. A. S.J

ABSTRACT

In |3H|inositol-labeled membranes prepared from Swiss mouse 3T3and human small cell lung carcinoma cells, (Tyr4|-bombesin stimulatedproduction of water-soluble inositol phosphates. The reaction was stimulated by guanosine 5'-O-[3-thiotriphosphate] and was specifically inhibited by both [Leu13-^-CH2NHLeu14l-bombesin and the antibombesinantibody 2All. (Tyr4|-homhcsin-ind uccd activation of phospholipase C ismost apparent in Ca2+-depleted conditions (

BOMBESIN-STIMULATED PHOSPHOLIPASE C

ml /7iyo-['H]inositol. Human SCLC cells were labeled for 5 days inKI'MI 1640 medium formulated without inositol, supplemented asdescribed for cell culture, with addition of 5 ¿¿Ci/mlmj-o-[]H]inositol.

SCLC were harvested by centrifugation, and 3T3 cells were scrapedinto their media, washed once in 0.15 M NaCl, 0.015 M Na phosphate,pH 7.4, and once in a buffer containing 50 mM Tris-HCl (pH 8.0), 1miMEGTA, 2 mM MgCl2, 2 Mg/ml leupeptin, and 0.5 miviPMSF. Cellswere resuspended at 4°Cin 2 ml/plate homogenization buffer formu

lated as above but with 20 mM Tris-HHCl. After 10 min, the cellsuspension was homogenized with a Teflon and glass homogenizer andcentrifuged at 800 x g for 5 min. The pellet was homogenized again inhalf the original volume and the combined supernatant was centrifugedat 40,000 x g for 10 min. The pellet was resuspended in 40 mM Tris-HCl (pH 6.9), 1 mM EGTA, 10 mM LiCl, 4 //g/ml leupeptin, and 0.5mM PMSF. Membranes were maintained on ice and used directlywithout freezing.

Incubation of the membrane suspension for measurement of phos-pholipase C activity was done essentially as described by SträubandGershengorn (28). Unless otherwise noted in the figure legends, 20 ¿il(20-50 ng of membrane protein) of membranes were added to obtain100 n\ with the following conditions: 40 mM Tris (pH 6.9), 2 mMEGTA, 2 mM EDTA, 10 mM LiCl, 2 ¿ig/mlleupeptin, 0.5 mM PMSF,1 mM ouabain, 2 mM dithiothreitol, 2 mM ATP, and 6 mM MgCl2 (2mM [Mg2+'fr„or no added MgCl2 (0.2 UM [Mg2*]fre«).[Ca2*]frrewasadjusted to 0.1 ¿¿Mwith CaCl2. The [Ca2*]fr„and [Mg2*)rretwere calcu

lated using the computer program described by Jean and Klee (29).All components except the membranes were preincubated at 37°C

for 2 min. After addition of the membranes, the reaction was continuedfor 1 min unless otherwise indicated and was terminated by addition of1 ml of CHC13/CH,OH/HC1 (100/100/1). Phases were separated byaddition of 0.25 ml of 0.01 M EDTA, vigorous mixing, and centrifugation. An aliquot (0.7 ml) of the aqueous phase was added to 5 ml ofHjO, neutralized with 50 ^1 of l M NaOH, and adsorbed to Dowex 1as described (30). IP, was eluted with 0.1 M formic acid/0.2 M ammonium formate, IP2 was eluted with 0.1 M formic acid/0.4 M ammoniumformate, and IP3 was eluted with 0.1 M formic acid/0.75 M ammoniumformate. Six-mi fractions were mixed with 12 ml Aquassure and conversion to dpiii accomplished using a series of quenched standards afterscintillation counting.

RESULTS

Phospholipase C in Membrane Fractions. In a typical membrane preparation incubated without other additions for 1 minat 37°C,the ['HJinositoI label in the IP, fraction did not change,

while the IP2 and IP., fractions increased from 14 to 22% and5 to 6% of the label, respectively. While accumulation of IP2may reflect direct hydrolysis of phosphatidylinositol-4-phos-phate, IP2 may also arise by degradation of IP.,. The latterprocess was clearly evident in the membrane preparation, because addition of ['H]IP, demonstrated a rate of hydrolysis ofIP., to IP2 of 0.1 pmol/min with 1 ¿¿MIP, and no added Mg2*and 40 pmol/min with 10 MMIP.i and 2 mM [Mg2+]frcc.Thus,our results will be presented as dpm in the inositol polyphos-phate (IP2 plus IP.,) fraction after agonist treatment.

Stimulation of Phospholipase C by Bombesin and GTP-yS.Fig. 1 demonstrates that addition of the bombesin agonist [Tyr4]-BN (100 nM) to ['Hjinositol-labeled Swiss 3T3 membranes inthe absence of added Mg2+, followed by incubation for I min,

causes a small increase in inositol poly phosphates, as does 10Õ/MGTPiS. However, GTP>S and [Tyr4]-BN at the above

concentrations together act to increase inositol polyphosphatesto a 2-fold greater extent than the sum of the separate effects.

Stimulation of phospholipase C by [Tyr4]-BN in the presenceof 10 n\\ GTP-yS is concentration dependent, with maximalactivation at about 1000 n\i and half-maximal activation at 10nM (Fig. 1). Activation of phospholipase C by [Tyr4]-BN and

10 100 1000

[Tyr4-BN] nMFig. I. Stimulation of phospholipase C activity by [Tyr4]-BN and GTP-yS:

inhibition by a bombesin antagonist. [3H|Inositol-labeled Swiss 3T3 membranes(32 >S was detected as early as 8 s (not shown), which was theshortest possible time for the assay. The capacity of 100 nM[Tyr4]-BN to increase phospholipase C activity is specificallyinhibited by 10 MMof the antagonist [Leu"--¿-CH2NH-Leu14]-

bombesin. The inhibition by this antagonist is partially reversedby increasing the [Tyr4]-BN to 1000 nM.

As shown in Fig. 2, [Tyr4]-BN also activates phospholipase

C in membranes derived from the SCLC cell line NCI-H345 inthe presence of 10 MMGTP-yS. Fig. 2 also demonstrates the

specificity of the interaction of these membranes for the bombesin agonist. Inclusion of the monoclonal antibody 2AI1 (50Mg/ml) directed against bombesin in the incubation completelyabolished the ligand-stimulated phospholipase C activity, reducing it to the value achieved with 10 MMGTP7S alone. Thisinhibition was evident up to a concentration of 100 nM [Tyr4]-BN but was partially reversed by an increase of the [Tyr4]-BN

concentration to 1000 nM.Effect of GDP/3S and Fluoride. The experiments presented in

Fig. 1 suggest that an element responsive to guanine nucleotideparticipates in bombesin-mediated signal transduction. Fig. 3demonstrates using Swiss 3T3 membranes that the inclusion of1 mM GDP/3S inhibits basal phospholipase C activity and theactivity stimulated by [Tyr4]-BN alone, by GTP-yS alone, and

5258

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BOMBESIN-STIMULATED PHOSPHOLIPASE C

6000 -i • no addition

! ÃœGDPUS ImM

5."3CO

CUrç 600 -

800 -

400 -

200 -

basai Tyr4BN GTPyS GTP|S +

l|iM lOuM Tyr BN

Al-F

Fig. 3. Inhibition of basal, [Tyr4)-BN-stimulated. and GTP-xS-stimulatedphospholipase C activity by GDPfÃ-S.Reactions with ['Hjinositol-labeled Swiss3T3 membranes (28 ng) were conducted in duplicate, as described in the legendto Fig. 1. Incubations under the control conditions (basal) or with the indicatedconcentrations of [Tyr4l-BN and GTP-yS were with 0.2 »IM|Mg!*]fr„.For the[ALF4|~ complex (Al-F), 10 UM AlClj and 10 mM NaF were incubated in areaction with 2 mM [Mg2*)fr„in the absence (darker bars] or presence of I mM

GDP0S (lighter bars).

2110(1,

5.—

100(1.

O basal

• GTPyS+Tyr4-BN

10"'10 10

|Ca'2+]free(M)

10'

Fig. 4. Effect of |Ca2*] on phospholipase C activity. Reactions with [JH|inositol-labeled Swiss 3T3 membranes (22 ng) were conducted in duplicate, asdescribed in the legend to Fig. 1. To achieve [Ca2*]rretat or below 10~' M, 8 mMEGTA was added. Two mM EGTA was used at all other |Ca2*| concentrations.The MgClz concentration was adjusted to give about 2 mM [Mg2*]r„,at all Ca2*concentrations. Incubations were with no additions (O) or with 10 UMGTP-ySplus 1000 nM [Tyr'l-BN (•).

by [Tyr4]-BN plus GTP7S. Inhibition of the basal activity andof the [Tyr4]-BN-stimulated activity by GDP/3S to a similar

level suggests that phospholipase C activation achieved withoutaddition of GTP7S is probably attributable to the presence ofendogenous guanine nucleotides in the membrane fraction.

Phosphatidylinositide hydrolysis can be stimulated in a li-gand-independent fashion by [A1F4]~.This species is postulated

to activate G proteins by mimicking the terminal phosphate ofGTP, without being subject to hydrolysis, resulting in persistentG protein activation (31). It can be seen in Fig. 3 that theactivity in the presence of 10 ^M AlClj and 10 mM NaF wasnot significantly inhibited by GDP/3S.

Effect of Ca2* and Mg2*. Phosphatidylinositide hydrolysiswas specifically activated by guanine nucleotides and [Tyr4]-bombesin only when [Ca2*]freewas below 10~6M (Fig. 4). Higher

calcium concentrations increased the basal activity to suchlevels that the addition of GTP7S and [Tyr4]-BN did not cause

any additional effect. Activation of phospholipase C by GT?7Splus [Tyr4]-BN was evident ad [Ca2+]freebelow 10~9 M, which

was achieved with 8 mM EGTA. This activation was maximalat about 10~7M [Ca2+]free,which is the concentration used in all

other experiments.Since guanine nucleotides may act as a Mg-GTP complex,

the effect of [Mg2*] on the activation of phospholipase C was

examined (Fig. 5). In membranes from Swiss 3T3 cells, basaland GTP7S-stimulated phospholipase C activity was greater inthe presence of 2 mM [Mg2*]frcc,as compared with 0.2 /¿M[Mg2*]frec.However, [Tyr4]-BN-dependent stimulation of phospholipase C was much more evident at 0.2 /tM [Mg2*]frccthan

1

-

3

3000 -i

2000 -

1000 -

I basal

D GTPyS

•

1

GTPyS + Tyr -BN

[Mg: 0.2nM 2mMCell line: Swiss 3T3

2mMNCI-H345

0.2nM 2mMNCI-N417

Fig. 5. Effect of Mg2* on phospholipase C activation in Swiss 3T3 and SCLCcell lines. Reactions with ['Hjinositol-labeled membranes were conducted in

triplicate, as described in the legend to Fig. 1. Results are the average ±SE of 10experiments with Swiss 3T3 cells, 6 experiments with NCI-H345, and 2 withNCI-H417. The results were normalized to the same total radioactivity in membranes per assay to allow calculation of average and SE and to allow comparisonof the three cell lines.

at 2 mM [Mg2+]free.With 2 mM [Mg2+]free,addition of 1 n\t[Tyr4]-BN causes only small additional stimulation of the activity (1.4-fold that evoked by GTP>S alone). By contrast, at 0.2fiM [Mg2+]frce,the addition of [Tyr4]-BN caused a larger stimu

lation (3.5-fold the activation by GTP7S alone). Entirely analogous results, particularly the dependence on low [Mg2*] fordemonstration of optimal [Tyr4]-BN activation, are shown inFig. 5 for the bombesin-responsive SCLC cell line NCI-H345.In contrast, in the bombesin nonresponsive SCLC cell lineNCI-N417 the activation by GT?7S is similar to the other twocell lines, but there was no response to bombesin of the membrane fraction at either 0.2 ¿ÌMor 2 mM [Mg2+]free.

Kinetics of Phospholipase C Activation by GTP7S and |Tyr4]-

BN. Since the experiments of Fig. 5 suggest an importantinfluence of Mg2+ in regulating response to [Tyr4]-BN, furtherexamination of the role of Mg2+ was undertaken. In order to

follow the onset of activation, the incubation temperature wasreduced to 25°C,since at 37°Cthe reaction was at maximal

velocity at the earliest possible measurement (8 s; not shown).At 0.2 fiM [Mg2+]fre(.and with GTP7S alone, a significant lag

phase is observed prior to phospholipase C activation (Fig. 6a).In contrast, at 0.2 ¿IM[Mg2+]fre

BOMBESIN-STIMULATED PHOSPHOLIPASE C

a. |Mg++M).2uM

time (sec)Fig. 6. Time course of activation of phospholipase C by [Tyr']-BN and GTP>S.

Reactions with [JH)inositol-labeled Swiss 3T3 membranes (33 jjg) were conductedin duplicate, as described in the legend to Fig. 1 but at 25°C.The experiment

shown is one of three similar experiments. The IP2 plus IP3 formed in the controlincubation at the indicated time points was subtracted from the results with 10>iMGTP-xS (O) or with GTP>S plus 100 nM [Tyi"]-BN (•).

1500-

1000-

500 -i

1500-

1000 H

500 i

basalTyr4BNGTP-ßGTPi6+Tyr4BN

b.[Mg++]=2mM

•

ATP no ATP APP(NH)PFig. 7. Effect of adenine nucleotidcs on phospholipase C activation. Reactions

with [3H]inositol-labeled Swiss 3T3 membranes (27 ^g) were conducted in dupli

cate, as described in the legend to Fig. 1. The omission of ATP was taken intoaccount when calculating the concentration of [CaI*]r«.and |Mg!*]r,„.The|Mg2*]r,„in a and b is shown. App(NH)p was added at the same concentration

as ATP (2 mm).

the absence of ATP, especially with high [Mg2+]frce,whereas thebasal activity is 2-fold higher in the presence of ATP. Theactivation of phospholipase C by GTP7S and [Tyr4]-BN was

not affected by the omission of ATP. The replacement of ATPby App(NH)p, a nonhydrolyzable analogue of ATP, does notinhibit the GTP^S- and [Tyr4]-BN-stimulated activation of

phospholipase C.

DISCUSSION

Although it is clear that bombesin-like peptides transducesignals using a calcium- and inositol-related signal transductionpathway (7-12), the mechanism of this process has not yet beenelucidated. Initial experiments showed that pertussis toxin inhibited bombesin-mediated DNA synthesis (23) but not phospholipase C activation (11, 24, 25). Inhibition by pertussis toxinof bombesin-stimulated DNA synthesis was partially reversedby concomitant insulin treatment (24). These results could beinterpreted to indicate that a G protein-mediated step occursafter phospholipase C activation. However, Fischer and Schon-brunn (32) have demonstrated a decrease in the affinity ofbombesin congeners for membranes from a variety of cell types

by incubation with guanine nucleotides. Inhibition by guaninenucleotides of agonist binding was not reversed by treatmentwith pertussis or cholera toxin. These latter results, therefore,argue in favor of the existence of a cholera and pertussis toxin-insensitive G protein which would participate in bombesin-induced signal transduction. Further evidence in favor of thispoint of view includes the recent observation (33) in permeabi-lized Swiss 3T3 cells that nonhydrolyzable guanine nucleotideanalogues potentiate the appearance of bombesin-dependentand protein kinase C-mediated M, 80,000 protein phosphoryl-ation. Hasegawa-Sasaki et al. (34) have also shown stimulationby GTP of bombesin and vasopressin but not PDGF-stimulatedinositol phosphate production in WFB rat fibroblast membranes.

The experiments presented here extend these observations tomembrane fractions derived from murine Swiss 3T3 cells andhuman SCLC cells. In addition, the experiments presented heredemonstrate that, in membranes from both types of cell, poten-tiation of phospholipase C by bombesin and GTP-yS occurredwith greatest dependence on added ligand at

BOMBESIN-STIMULATED PHOSPHOLIPASE C

phospholipase C. Bombesin, however, did not induce this phos- REFERENCESphorylation. Margolis et al. (42) have demonstrated that thetyrosine kinase antagonist tyrphostin inhibited epidermalgrowth factor-induced increases in [Ca2+]¡in HER cells but notthe increase in [Ca2+]¡induced by bombesin or bradykinin. In

contrast, Cirillo et al. (43) have presented evidence that bombesin treatment of Swiss 3T3 cells results in tyrosine phos-phorylation of a M, 115,000 membrane-associated protein.Isacke et al. (44), however, did not observe increases in totalcellular tyrosine phosphates after bombesin treatment, andaffinity cross-linking studies (45, 46) have clearly shown thatbombesin cross-linked to a M, ~75,000 heavily glycosylatedspecies. A clear demonstration of kinase activation as an obligatory step in bombesin-mediated signalling has, therefore, notyet been shown. In our experiments, the inclusion of ATP or anon-hydrolyzable analogue of ATP did not influence bombesin-stimulated phospholipase C activation. While this result isconsistent with the observations of Margolis et al. (42), adetermination of whether a kinase participates in bombesinreceptor-mediated signalling must await better structural characterization of the receptor.

The influence of bombesin or its antagonists on the proliferation of SCLC is controversial. Takuwa et al. (47) and Laytonet al. (48) demonstrated no influence of bombesin agonists onDNA synthesis of SCLC grown in liquid mass culture. Thebombesin antagonists [D-Arg',D-Phes,D-Trp7-9,Leu"]-substanceP and [D-Arg',D-Pro2,D-Trp7-9,Leu"]-substance P inhibitedDNA synthesis at high (~10~4M) concentrations (47-49). Thebombesin antagonist [Leu"-i¿-CH2NH,Leu'4]-bombesin inhib

ited SCLC proliferation at 1 /¿Mconcentration [where it wasshown to inhibit bombesin-mediated signal transactions (50)]only in serum-free soft agar cloning and did not inhibit SCLCproliferation in liquid media.4 An inherent difficulty, however,is the stability or accessibility of bombesin-related antagoniststo the bombesin receptor in the liquid culture system, becauseSCLC tend to grow in large clumps of adherent cells. Also, asoutlined by Takuwa et al. (47) and Woll and Rozengurt (51),SCLC can respond biochemically to a variety of neuropeptides.Thus, blockade of several receptors may be necessary to inhibitgrowth-stimulatory influences definitively. Definition of elements of the bombesin-related signal transduction pathwaycould reveal a common pathway of activation used by otherneuropeptides.

The experiments presented here offer independent corrobo-ration of the importance of a guanine nucleotide-mediatedpathway in bombesin-induced signalling in murine fibroblastand lung carcinoma cell lines. [Tyr"]-BN in the presence of the

nonhydrolyzable GTP analogue GTP7S potentiated phospholipase C activity, which was specifically inhibited by a bombesinantagonist and a bombesin-specific monoclonal antibody.GDP0S inhibited this process. The presence or absence of ATPor App(NH)p did not influence the reaction. Further experiments will focus on identifying the G protein active in thetransduction of the bombesin signal. Therapeutic agents whichcould disrupt the coupling of this G protein to intracellularsignals or uncouple it from the hormone receptor could be ofgreat interest in the development of novel approaches to thetherapy of SCLC.

ACKNOWLEDGMENTS

The authors acknowledge the support of Drs. Marc Lippman andJohn Minna during the course of this work and the thoughtful comments of Drs. Jorge Laborda and Peter Worland.

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1990;50:5257-5262. Cancer Res Yoav Sharoni, Jean Viallet, Jane B. Trepel, et al. from Swiss 3T3 and Small Cell Lung Carcinoma CellsBombesin-stimulated Phospholipase C Activity in Membranes Effect of Guanine and Adenine Nucleotides on

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