High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells.

Abstract

Gastrin-releasing peptide (GRP) Peptides related to bombesin, including gastrin-releasing peptide (GRP) and the neuromedins, are widely distributed in normal mammalian tissues (10-16) and also are found in high levels in human pulmonary (17)(18)(19)(20) and thyroid (21) tumors. The finding that bombesin is mitogenic (9) and the wide distribution of endogenous mammalian analogues of bombesin strongly suggest that this family of peptides may play a role in the control of both normal and abnormal animal cell growth.An important step in the elucidation of the mechanism of bombesin-stimulated mitogenesis is to determine the presence of specific receptors for this peptide in Swiss 3T3 cells. We have used biologically active, radiolabeled GRP, the mammalian counterpart of bombesin, to establish the presence of receptors for this molecule.In the present paper, we show that the mitogenic stimulation of Swiss 3T3 cells by peptides of the bombesin family is mediated by a single class of high-affinity receptors. This receptor is distinct from those of other mitogens for these cells. Further, a substance P (SP) analogue recently described as a bombesin antagonist (22) selectively blocks both GRP binding and GRP-induced mitogenesis. Since there is evidence that bombesin-like peptides may contribute to the self-stimulatory (autocrine) growth of small-cell carcinoma of the lung (18-20, 23, 24, 47), our findings may suggest novel clinical approaches to the treatment of this important cancer. MATERIALS AND METHODSCell culture procedures (9,25,26) and assays of DNA synthesis by [3H]thymidine incorporation (26) were carried out as described.Binding Assay. For binding at 37°C, confluent and quiescent cultures of Swiss 3T3 cells in 33-mm dishes were washed twice with Dulbecco's modified Eagle's medium (DME medium) and incubated with 1 ml of binding medium, which consisted of 1:1 (vol/vol) DME and Waymouth media supplemented with 1 mg of bovine serum albumin per ml, 50 mM 2-[bis(2-hydroxyethyl)-2-amino]ethanesulfonic acid (Bes) (pH 7.0), and GRP labeled with 125I at tyrosine-15 at the concentrations indicated. After 30 min of incubation (unless otherwise stated), cultures were washed rapidly four times with cold (4°C) wash solution (0.15 M NaCl/5 mM KCl/0.02 M Na2HPO4/1.8 mM KH2PO4, pH 7.2, supplemented with bovine serum albumin at 1 mg/ml). Washed cultures were extracted in 0.5 ml of 0.1 M NaOH containing 2% Na2CO3 and 1% NaDodSO4, and total cell-associated radioactivity was determined in a y counter. Nonspecific binding, defined as the cell-associated radioactivity not displaced in the presence of 360 nM GRP, was proportional to the concentration of 1251-GRP and varied from 23% of total binding at the highest concentrations (3 nM) of 125I-GRP to 5% at low concentrations (0.05 nM). For binding at 4°C, cells were washed once with DME medium at 4°C and incubated at 4°C for 5 min prior to the binding assay. The binding assay at 4°C was performed in 0.14 M NaCl/5 mM KCl/0.01 M Na2-HPO4/1.8 mM KH2PO4/1.8 mM CaCl2/1 mM MgCl2/25 ...

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells

“…returned close to unstimulated control values after 6 min. We have found (results not shown) that the stimulation of 45Ca2+ release by bombesin could be almost completely blocked by a short preincubation with [D-Arg1,D-Pro2,D-Trp7 9,Leu'1]substance P, which has been found to be a competitive antagonist of bombesin action in these (Corps et al, 1985;Zachary & Rozengurt, 1985) and other cells (Jensen et al, 1984). The bombesin-stimulated release of 45Ca2+ was not affected by the addition of EGTA to the medium (Fig.…”

“…Swiss 3T3 cells, which possess specific high-affinity receptors for radiolabelled gastrin-releasing peptide (Zachary & Rozengurt, 1985) and [Tyr4]bombesin (Brown & Laurie, 1986), should prove useful for this purpose. The binding of bombesin to its receptors on Swiss 3T3 cells results in a rapid stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) Heslop et al, 1986), generating inositol 1,4,5-trisphosphate (1,4,5-4P3) and diacylglycerol, an endogenous activator of protein kinase C (see Nishizuka, 1984).…”

Characterization of ligand binding and processing by gastrin-releasing peptide receptors in a small-cell lung cancer cell line

“…Only bombesin-and ranatensin-like peptides have mammalian counterparts, namely gastrin-releasing peptide (GRP) and neuromedin B (NMB) respectively (McDonald et al, 1978;Minamino et al, 1983Minamino et al, , 1985. GRP is a potent mitogen for Swiss 3T3 cells (Rozengurt & Sinnett-Smith, 1983;Zachary & Rozengurt, 1985) and also has been shown to be an autocrine growth factor for small-cell lung cancer (SCLC) . SCLC cells synthesize (Moody et al, 1981) and secrete (Korman et al, 1986) GRP, express high-affinity GRP receptors Gaudino et al, 1988;Layton et al, 1988;Kado-Fong & Malfroy, 1989) and respond mitogenically to GRP stimulation (Carney et al, 1987).…”

Tissue-specific regulation of gastrin-releasing peptide synthesis, storage and secretion by oestrogen and progesterone

“…GRP is also found in the brain (Lynn et al 1996), in pulmonary endocrine cells of the foetal lung (Spindel et al 1987) and is produced by a variety of human tumours (Preston et al 1996). Mitogenic effects of GRPlike peptides have been demonstrated for a number of different cell types, including Swiss 3T3 fibroblast cells (Zachary & Rozengurt 1985), ovine foetal chondrocytes (Hill & McDonald 1992) and human endometrial stromal cells in culture (Endo et al 1991).…”