Int. J . Cancer: 46, 293-298 (1990) 0 1990 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer

CHARACTERIZATION, IN SOME HUMAN BREAST CANCER CELL LINES, OF GASTRIN-RELEASING PEPTIDE-LIKE RECEPTORS WHICH ARE ABSENT IN NORMAL BREAST EPITHELIAL CELLS sylvie GIACCHETTI',Christiane GAUVILLI?,Patricia DE C ~ M O U X Laurent ~ , BERTIN',Phihppe BERTHON',Jean-Piem ABITA', Franck CUTTITTA~ and Fabien CALVO'.~ 'Laboratory of Pharmacology and Insem U.204, Institut de Gknktique Molkculaire, Hopital Saint-Louis, 2 7 rue Juliette Dodu, F-75010 Paris, France; and 2National Cancer Institute-NavyMedical Oncology Branch (NU-Navy MOB), Naval Hospital, Bethesda, MD 20814 USA. The binding of IzSl-Tyr4bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinS, single-site class of receptors in 3 cell lines (K, of 0.75 X 10 and IO-' M, B ,, of 0.75 x lo-" and 9.7 X M/mg protein in MDAMB23I and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analo ues (natural or synthetic) inhibited the binding of 1251-Ty~bombe~in in the following order of potency: gastrin-releasing peptide (GRP, EC,, = 1.7 X M) > BIM 26159 > bombesin, T y r 4 bombesin > BIM 26147 > litorin > neuromedin C. In contrast, lZ5l-Tyr'bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex IV-Tyr' bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

The peptide bombesin (Anastasi et al., 1971) and structurally related peptides like gastrin-releasing peptide (GRP) (McDonald et al., 1979) and neuromedins (Minamino et al., 1983) which are known for their hormonal or neurotransmitter nature, act as potent mitogens for some cell types in culture such as mouse fibroblasts 3T3 (Rozengurt and Sinnet-Smith, 1983), normal bronchial epithelium (Willey et al., 1984) and smallcell lung cancers (SCLC) (Cuttita et al., 1985; Weber et al., 1985). These peptides have been detected in various normal tissues in mammals (Westendorf and Schownbrun, 1985; Polak et a l . , 1976) together with specific cell-surface receptors (Zachary and Rozengurt, 1985; Jensen et al., 1978: Moody et al., 1978; Scemama et al., 1986). They have also been detected in tumoral specimens of SCLC and cell lines derived from SCLC (Moody et al., 1981, 1985; Sausville et al., 1986; Erisman et al., 1982), and shown to function as autocrine growth factors involved in the abnormal growth of these tumors (Cuttita et al., 1985; Carney et al., 1987; Woll and Rozengurt, 1988). High levels of neuropeptides such as vasoactive intestinal peptide (VIP) and bombesin-like immunoreactivity have been detected in human and animal milk (Jahnke and Lazarus, 1984; Werner et al., 1985) and in rat mammary tumors (Gaudino et al., 1988). We previously reported the presence of VIP receptors in human breast cancer (Gespach et al., 1988) and normal epithelial cells (Berthon et al., 1989). We therefore investigated the presence of specific receptors to bombesin and their potential involvement in an autocrine loop, in both normal and tumoral cell cultures of human breast. We report here the presence of single-class, high-affinity receptors to bombesin-

related peptides in 3 out of 8 breast cancer cell lines tested. We found no receptors in 2 cultures of normal breast epithelium. In all specimens studied, neither GRP nor neuromedin B mRNA transcription was detected, and GRP had no effect on proliferation of breast cancer cells. MATERIAL AND METHODS

Human breast cancer cell lines and normal breast epithelial cells Eight well-characterized human breast carcinoma cell lines were studied: MCF 7 (Soule et al., 1973), T47D (Keydar et al., 1979), ZR 75-1 (Engel et al., 1978), MDA-MB 231 (Cailleau et al., 1974), and H466B (Calvo et al., 1986) are derived from metastatic breast carcinomas. HSL 53, RIB (both established in our laboratory) and BT20 (Lasfargues and Ozzello, 1958) are derived from primitive human breast cancers. All cells except H466B were cultured in Dulbecco's modified medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Boehringer, Mannheim, FRG), and 2 m~ glutamine. The H466B cells were cultured in DMEM/F12 (l:l), 10 m~ HEPES, supplemented with hormones, growth factors and 5% fetal bovine serum (Calvo et al., 1984). Human normal mammary epithelial cells in long-term culture were derived from reduction mammoplasty. After enzymatic dissociation, released organoids were cultured for several months in DMEM/F12 (1:l) 10 m~ HEPES containing low calcium concentrations (60 FM) supplemented with 5% calcium-free horse serum, 10 mg/ml insulin, 5 x lop6M cortisol, 100 ng/ml cholera toxin, 20 ng/ml epidermal growth factor, 2 m~ glutamine, 50 IU/ml penicillin and 50 mglrnl streptomycin (Soule and McGrath, 1986). Normal cells were tested for binding experiments after 1 to 2 months in culture and were 100% positive by immunocytochemistry with anti-cytokeratin antibodies. For experiments, exponentially growing cells were removed by treatment with %O NaCl (wlv) and 2.5 m~ EDTA (PH: 7.4) for 3 min at 37°C. Cell viability, determined by Trypan blue exclusion, was

>85%. Cell growth experiments T47D and MDA-MB 231 cells in log-phase growth were harvested with trypsin-EDTA (trypsin 0.5 g/l, EDTA 0.2 g/l). After 2 washes in serum-free medium, cells were plated in DMEMIF12 (1:l) with 10 m~ HEPES supplemented with 10 m~ triiodothyronine, 1 mg/l prostaglandin F2a, 10 nM dexamethasone, 3 mg/l insulin and 25 mg/l transfemn, on collagencoated plastic flasks (Calvo et al., 1984), in the presence or

3To whom reprint requests should be sent. Received: February 15, 1990 and in revised form April 9, 1990.

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GIACCHETTI ET AL.

absence of various concentrations of bombesin; 3 x lo5 cells were seeded at day 0 in 25-cm2 flasks in triplicate and cultures were counted on days 6 and 12. Preparation of plasma membranes Cells of cancer or normal breast origin were suspended at the concentration of 2 X lo7 cells/ml in 10 m~ Tris HCl buffer (PH: 7.5) containing 1 m~ EDTA, 30 m~ NaCl and 5 m~ phenylmethylsulfonyl fluoride. Cells were disrupted with a polytron homogenizer (Kinematica, Lucerne, Switzerland) using 3 bursts of 5 sec each at 4°C and centrifuged at 600 g for 10 min at 4°C. Plasma membrane preparation was obtained from low-speed supernatant recentrifugation at 20,000 g (30 min, 4°C) as described by Gespach et al. (1988). Plasma membranes were stored frozen at - 80°C prior to running binding experiments. Protein content was determined using the method of Lowry et al. (1951); 100 pg protein from plasma membranes were equivalent to 2 X lo6 T47D cells. Before binding experiments, plasma membranes were homogenized in the binding buffer by 10 sec sonication (Sonics, Danbury, CT). (1251-Tyfl)bombesin receptor binding Plasma membranes were incubated in 0.2 ml binding assay medium containing 50 m~ Tris HC1 (PH: 7.4), 5 m~ MgCl,, 0.1% bacitracin, 1% bovine serum albumin and 5 X M (1251-Tyr")bombesin. To this reaction mixture, various concentrations of unlabelled bombesin were added for calculation of a ligand binding curve. Incubation was stopped by addition of 1 ml ice-cold phosphate buffer. Membranes were then washed twice in the same buffer and the radioactivity bound to membranes was determined in a gamma counter (LKB, Wallach, Bromma, Sweden). Specific binding was the difference between the total radioactivity of (Iz5I-Tyr") bombesin bound in the absence (total binding) or presence (non-specific binding) of l o p 7 M unlabelled bombesin. The specific binding data were Scatchard plotted and, in addition, analyzed by the LIGAND program.

H345 small-cell lung carcinoma cell line (a gift from Dr. J. Minna, Bethesda) was used as positive control. Chemicals Materials were obtained from the following sources: synthetic bombesin, (TyF) bombesin, litorin, neuromedin B and C, insulin, somatostatin, bradykinin, (D ArgI-D Pro2-D P and EGS were obtained from Sigma, T ~ p ~ , ~ - L e usubstance ") gastrin-releasing peptide (GRP) from Peninsula (St. Helens, UK) and (12'I-TyP) Bombesin (2000 Ci/mmol) from Novabiochem, Laufelfingen, Switzerland. Bombesin receptor inhibitors (BM 26159 and BM 26147) were gifts from Biomeasures, Hopkinton, MA. RESULTS

1 . Association of (1251-TyJ')bombesin with T47D cell membranes The specific binding of ( 1251-Ty~) bombesin by T47D membranes reached equilibrium after 60 min at 20°C. This occurred after 30 min at 37°C and 110 min at 4°C (not shown). Nonspecific binding was consistently below 5% of total (1251-Tyr") bombesin bound. Specific binding of (Iz5I-TyP)bombesin during 1 hr incubation at 20°C was proportional to membrane protein concentration from 50 to 200 pglml. Therefore, further experiments were performed in cells incubated at 20°C for 60 min, with 100 pg/ml plasma membrane proteins. The binding of (Iz5I-Tyf') bombesin to T47D membranes was inhibited in a similar manner by increasing concentrations of either unlabelled (Tyr") bombesin or bombesin. Subsequently, bombesin was used for competition experiments (Fig. 1). When specific binding data from 10 experiments were Scatchard plotted a linear relationship was obtained, suggesting that T47D cells have a single class of non-interacting binding M sites with a dissociation constant (K,) estimated at 1 X and a concentration of binding sites at 0.97 pmoVmg of protein corresponding to 36,000 binding sites per cell (Fig. 1, inset).

Chemical cross-linking of ('251-Tyfl)bombesin to T47D cells Covalent affinity labelling of bombesin binding sites was performed according to Kris et al. (1987) and Zachary and Rozengurt (1987). Briefly, lo5 T47D cells were seeded in 35-mm well tissue culture dishes (Corning, Coming, NY) and cultured to subconfluence. The cells were washed 3 times with ice-cold cross-linking buffer containing DMEM and 20 m~ HEPES (PH: 7.4), 100 p,g/ml bacitracin, then incubated at 4°C in 0.5 ml of the same buffer containing 0.2% bovine serum albumin and 5 X M (lZ5I-Tyr")bombesin, in the presence or absence of an excess of unlabelled bombesin. After 90 min, the cells were washed 3 times with ice-cold cross-linking buffer and once with phosphate buffer. The cross-linking agent, EGS, (Sigma, St Louis, MO) was dissolved in DMSO immediately before use at a concentration of 30 m~ and then diluted to 1 m~ in phosphate buffer. One ml of the EGS final dilution was added to each well for 20 min at 4°C. The reaction was stopped by addition of 10 I J . ~of 2 M Tris HC1 (PH:8). Cells were rapidly rinsed twice with ice-cold phosphate buffer and then solubilized in 80 p.1 Laemmli (1970) sample buffer. Samples were sonicated and boiled for 3 min, then applied on an 8% SDS-PAGE. The gel was dried and exposed on Kodak XAR-5 film using an intensifying screen, for 5 days at - 80°C.

2 . Binding of ('251-Tyfl)bornbesin to cancer and normal epithelial cells ( 1251-Tytqbombesin binding experiments were performed with 7 other human breast cancer cell lines and cultures derived from normal mammary epithelial cells from 2 different individuals. Table I summarizes the characteristics of (1251-TyF) bombesin receptors in these cells. Three out of the 8 cell lines possess bombesin receptors: T47D, MDA-MB23 1 and HSL53. Specific binding sites could be demonstrated neither in H466B, ZR 75-1, BT20, RIB and MCF7 cancer cells nor in the 2 normal samples.

Northern blot analysis Northern blot analysis was performed on T47D, MCF 7, ZR 75-1, MDA-MB231 and normal cells, using the guanidium isothiocyanate technique (Davis et al., 1986). The cDNA probe (0.9 kb) for human GRP was kindly provided by Dr. J. Battey (Laboratory of Neurosciences, NICNSD, NIH Bethesda, MD) inserted at the Hind11 site of pSp64 plasmid.

4 . Molecular characterizationof (I2?-Tyfl) bombesin binding sites in T47D cells The distribution of (1251-Tyr") bombesin bound to plasma membranes was analyzed by SDS PAGE after cross-linking with EGS (Fig. 2). A major band of (1251-Tyr")bombesin covalently linked to T47D membranes was observed, with an apparent M, of 75,000 (lane A). The labelling of this compo-

3 . Specijicity of binding sites for (1251-Tyfl) bombesin To determine the pharmacological specificity of the (Iz5I-Tyr") bombesin binding sites in T47D, various related and non-related peptides were tested for their ability to inhibit (1251-Tyr") bombesin binding. The relative potency of each peptide (1C5J is shown in Table II. If we assign a value of 100 to bombesin, the relative potencies of the peptides are: GRP (600) > BM 26259 (250) > bombesin = TyF-bombesin (100) > BM 26147 (50) > litorin (3.75) = neuromedin u'~) P %- neuroC = (D Argl- D Pro2-D T ~ p ~ , ~ - L esubstance medin B , insulin, somatostatin, bradikinin.

295

BOMBESIN B I N D I N G I N H U M A N BREAST CELLS

L

0441 CJ d

I

I

0

1;

10"O

I

10-8

-9

10-7

10-6

BBS Concentration (M) FIGURE1 - Competitionfor (1251-TyP)bornbesin binding by increasing concentrations of unlabelled bornbesin. T47D membranes (100 kg/ml) were incubated with (I2TTyf) bombesin ( 5 X lo-" M) in the presence of increasing concentrations of unlabelled bombesin at 20°C for 1 hr. Specific binding was determined at each concentration as described in "Methods". Each point represents the mean SD of 10 experiments performed in duplicate. Inset, Scatchard analysis of the binding data.

*

TABLE I - ('*'I-TYR4) BOMBESIN BINDING CHARACTERISTICS [KD, BINDING CAPACITY AND SITE NUMBER PER CELL) IN DIFFERENT MAMMARY EPITHELIAL CELLS Specific binding

Cell line

+

T47D MDA-MB 231 HSL53 MCF7 ZR75- 1 H466B RIB BT20

+ +-

-

Normal mammary epithelial cells

Binding capacity Wmg)

Site number per cell

36,000 2.276

-

9.7 x 10-13 0.75 x 10-13 NT -

-

-

I x 10-~ 0.75 x 10-9

NT

-

-

-

-

-

-

-

-

NT -

Membranes (100 &ml) of T47D, MDA-MB231, MCW, ZR75-I, H466B and normal mammary epithelial cells (2 different specimens) were incubated with ("'1-Tyr4) bombesin (5 X lo-" M) in the presence of increasing concentrations of unlabelled bombesin at 20°C for 1 hr. The ratio of specifically bound peptide was plotted as a function of specific binding as described in Fig. 1. Analysis was performed according to the method of Scatchard. Results are the mean ? SD of 5 experiments in duplicate. Different membrane concentrations of HSL53, RIB and BT20 were tested for binding after incubation with ('*'I-Tyr4) bombesin (5 X lo-" M) in the presence or absence of l o - ' M bombesin. Only HSL53 exhibited specific binding which was correlated to the membrane concentrations (total bound radioactivity by 100 kg protein; 1,500 cpm, non-specific binding: 120 cpm).

nent was completely inhibited by lo-' (lane B).

M

unlabelled bombesin

5 . GRP transcripts in human breast cancer cell lines and normal breast epithelial cells

GRP transcription was studied in T47D and MDA-MB231 cells which possess bombesin receptors, and ZR 75- 1, MCF7 and 2 normal samples which do not. None of these cells showed mRNA transcripts when hybridized with the cDNA

probe for human GRP. H345 human small-cell lung carcinoma cell line, the positive control, expressed appreciable quantities of human GRP mRNA. 6 . Cell growth experiments T47D and MDA-MB231 human breast cancer cell lines which possess bombesin receptors were cultured in serum-free medium containing hormones and growth factors. Under these serum-free conditions doubling time is 48 hr and 28 hr for

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GIACCHETTI ET AL.

TABLE U - INHIBITION OF (1251-~R4) BOMBESIN BINDING TO T47D PLASMA MEMBRANES BY RELATED PEF’l’IDESAND BOMBESIN ANTAGONISTS Binding inhibition

Peptides

IC50 (M)

GRP BM 26159

1.7 4 1 1

Bombesin TyF bombesin

x 1O-Io x 10-10

x 10-9 x lo-; 2 x 101.5 x 1.5 x lo-*

BM 26147

Litorin Neuromedin C

T47D membranes were incubated with (’251-T$) bombesin (5 X lo-” M) and the designated peptide (lo-“ to 10K7 M) at 20°C for 1 hr. Specifically bound radioactivity was determined as described in “Methods”.Insulin, somatostatin, bradykinin, neuromedin B and (D Argl-D F’ro*-D Trp’.9-Leu11)substance P did not inhibit (Iz5I-Tyfi) bombesin binding.

T47D and MDA-MMB231 respectively, compared to 32 hr and 24 hr in serum-supplemented medium. No significant reproducible effect on cell number at day 6 or 12 could be observed after treatment with various concentrations of bombesin under these experimental conditions. DISCUSSION

Bombesin-like peptides comprise a large family of peptides initially described in amphibians (Anastasi et al., 1971) and later found in mammalian cells (Walsh and Wong, 1978; Moody and Pert, 1979). In amphibians, bombesin-like peptides have been classified into 3 subfamilies: the bombesins, the ranatensins and the phyllolitorins (Erspamer et al., 1985). Each subfamily is characterized by a common amino-acid sequence near its carboxyl terminus. At present, whereas a mammalian homologue of phyllolitorin has yet to be demonstrated, homologues of bombesin and ranatensin have been identified as gastrin-releasing peptide (McDonald et al., 1979) and neuromedin B (Minamino et al., 1983; Krane et al., 1988) respectively. Little is yet known about their respective roles in mammals but the different peptides bind to their specific receptors and to the bombesin-GRP receptors. In this study, identification and characterization of bombesin receptors in human breast cancer cells are reported. Three out of 8 cell lines tested were shown to possess such receptors on their cell surface while receptors were absent in 2 normal human epithelial mammary cell cultures tested. Scatchard analysis showed the presence of a single class of binding sites of high affinity (K, of lop9 M and 0.75 X lop9M in T47D and MDA-MB231 cells, respectively), similar to that observed in human small-cell lung cancer (SCLC) cell lines (Moody et al., 1985). The number of binding sites per cell was variable (2,000 sites in MDA-MB231 and 36,000 in T47D), but similar to that observed in human SCLC (Moody et al., 1985, Woll and Rozengurt, 1988). No correlation between the presence of bombesin receptors and the hormonal or EGF receptor status was observed for the breast cell lines tested. The specificity of the receptor for bombesin binding was studied using competition analysis with structurally related peptides. Gastrin-releasing peptide and a synthetic antagonist of GRP, BM 26159, were 10 times more potent than bombesin and Tyr“ bombesin. Another antagonist of GRP, BM 26149, and litorin were weaker competitors for binding. Similarly, Neuromedin C (GRP,,,,) which contains the carboxyl terminal moiety responsible for maximal binding to the GRP receptor, weakly blocked binding. This may relate to the stability of this ligand which is rapidly degraded by aminopeptidases. In contrast, neither unrelated peptides (somatostatin, bradykinin and insulin) nor neuromedin B and (D Argl-D Pro2-D T r ~ ~ . ~ - L e usubstance ll) P, yet a potent bombesin antagonist in fibroblast 3T3 cells (Woll and Rozengurt, 1988) interfered with (*251-Tfl) bombesin binding. These results suggest that

FIGURE2 - Molecular identification of the J251-TyPbombesin binding sites in T47D cells. Cross-linking was realized as described in “Methods”. Binding of (1251-TyF)bombesin to the T47D cells was carried out at 4°C in the absence of unlabelled bombesin (lane A) and in the presence of lo-’ M bombesin (lane B). The molecular weight of (1251-Ty14)bombesin is 1300 daltons; the apparent molecular weights of the (lZ5I-TyF)bombesin binding proteins are 75,000, according to their electrophoretic mobility.

these receptors are more specific to the bombesin-GRP peptide sub-family. The apparent molecular weight of the complex ligandreceptor as determined by SDS-PAGE after cross-linking of (1251-Tyr“)bombesin to cell membranes from T47D cells with EGS was 75 kDa and appeared as a single band. This complex was sensitive to bombesin, and the cross-linked receptors in T47D cells were similar in molecular weight to those described with the same techniques in human glioma (Kris et al., 1987) and 3T3 cells (Zachary and Rozengurt, 1987). The significance of bombesin-GRP receptors in some human breast cancer cell lines and their absence in others and normal human breast cells remains intriguing. Our attempts to evi-

BOMBESIN BINDING IN HUMAN BREAST CELLS

dence transcription or secretion of GRP, GRP-related peptides and neuromedin B, in all these cell lines, were unsuccessful. Exogenously added bombesin, when tested on the growth of 2 cell lines which have receptors, T47D and MDA-MB23 1, did not modify their proliferation rate in a serum-free medium liquid culture assay. Therefore, a proliferative autocrine or exocrine loop of bombesin is not present in these cells contrary to that shown earlier in normal and malignant bronchial epithelial cells. Three hypotheses, at least, may explain the absence of such a biological effect. First, GRP may have effects other than proliferative ones in this cell system; while this report was being submitted, Pate1 and Schrey (1990) showed that BBS induced phospholipase C-mediated inositol phospholipid hydrolysis and elevation of cytosolic Ca2+ levels in MCM and T47D cells and had no effects on growth of MCW.

297

Therefore, a second hypothesis is that transduction signals could be blocked at other stages (Yarden and Ulrich, 1988). Third, the receptors identified here as GRP receptors could be specific receptors of a GRP-sub-family peptide as yet unidentified. These hypotheses are now under investigation. ACKNOWLEDGEMENTS

This work was supported by the “Association pour la Recherche contre le Cancer” (ARC) and the “FBdBration Nationale des Groupements des Entreprises Franpises dans la Lutte contre le Cancer” (FEGEFLUC). We are grateful to Dr. C. Gespach (Inserm U 55, France) and Dr. F. Thomas (IPSEN Biotech, France) for helpful discussions and Dr.J.-P. Moreau for providing bombesin antagonists.

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