Gastrin Binding Research Articles

A network map of the gastrin signaling pathway

Gastrin is the primary hormone that induces gastric acid secretion (Edkins 1906). In humans, the gene encoding for gastrin is located on chromosome 17q21 (Lund et al. 1986). This hormone is produced by the G-cells of the antrum of stomach as preprogastrin, which comprises of 101 amino acids and is cleaved between Ala-21 and Ser-22 to yield progastrin (Reeve et al. 1984). Progastrin is then sequentially cleaved by prohormone convertase and carboxypeptidase E to yield glycine-extended gastrins- a 35-amino acid gastrin-34-Gly (G34-Gly) or an 18-amino acid gastrin-17-Gly (G17-Gly) in endocrine cells (Varro et al. 1995; Lacourse et al. 1997). G34-Gly and G17-Gly are amidated at their carboxyl terminal groups by the enzyme peptidyl alpha-amidating mono-oxygenase to form amidated gastrin. Gastrin interacts with the membrane-bound G-protein coupled cholecystokinin receptor group consisting of Cholecystokinin A receptor (CCKAR) and Cholecystokinin B receptor (CCKBR). CCKAR possesses high affinity for cholecystokinin and has a negligible affinity for gastrin (Ferrand and Wang 2006; Dufresne et al. 2006). However, CCKBR has a high affinity for gastrin and their carboxyl amidated analogues. CCKBR is expressed in the brain, smooth muscle cells, and parietal cells (Kopin et al. 1992; Berna et al. 2007). In addition to this, gastrin has also been reported to interact with annexin 2, and thereby exerts proliferation effects in gastrointestinal cancers (Singh et al. 2007; Singh 2007; Sarkar et al. 2012). The release of gastrin is induced by gastrin-releasing peptide, a neurotransmitter which acts on its basolateral receptor in the G-cells. Binding of gastrin to CCKBR present in parietal and enterochromaffin-like (ECL) cells (Schmitz et al. 2001; Kulaksiz et al. 2000)induces gastric acid secretion by parietal cells as well as histamine release by ECL cells (Dockray et al. 2005). The released histamine reaches parietal cells by paracrine diffusion where it binds H2 receptors and induces gastric acid secretion. The secretion of gastric acid inhibits the release of gastrin hormone rendering a negative feedback control, further preventing excess acid secretion. In addition, low pH value in the stomach inhibits gastrin release through stimulating somatostatin secretion by antral D-cells (Bloom et al. 1974). Gastrin is a well-known growth factor for the gastrointestinal tract. Gastrin stimulates proliferation of gastric mucosal cells (Hansen et al. 1976), maturation of parietal cells and enterochromaffin-like cells (Jain and Samuelson 2006), and promotes islet differentiation in the pancreas (Wang et al. 1993). It has also been shown to stimulate proliferation of gastric (Ishizuka et al. 1992) and colon cancer cells (Watson et al. 1989). Gastrin modulates invasion (Wroblewski et al. 2002), apoptosis (Przemeck et al. 2008; Todisco et al. 2001) and migration (Noble et al. 2003) in epithelial cells. Both glycine-extended gastrin and amidated gastrin have been reported to induce angiogenesis (Clarke et al. 2006; Lefranc et al. 2004). Moreover, the hypergastrinemic state has been associated with diverse physiological disorders in humans and other mammals. These include atrophic gastritis (Lehy et al. 2000), pernicious anemia (Orlando et al. 2007), excess acid secretion leading to duodenal ulcer disease in Helicobacter pylori infection (Berna et al. 2006; Jensen 2002; Scarpignato et al. 1996) and gastrinoma (Kloppel and Anlauf 2007). Recently, a number of research groups have explored the feasibility of using gastrin to treat various diseases. Gastrin has been used in combination therapy with epidermal growth factor to increase beta-cell mass which reversed hyperglycemia in diabetic mice (Suarez-Pinzon et al. 2005). Gastrin-stimulated beta cell neogenesis when used in combination with glucagon-like peptide 1 in human pancreatic duct cell transplanted in immunodeficient diabetic mice (Suarez-Pinzon et al. 2008). Gastrins and their receptors have been suggested as potential targets to treat gastrointestinal and pancreatic cancers (Rengifo-Cam and Singh 2004). Gastrin has also been shown to have therapeutic promise as it inhibits the growth of cholangiocarcinoma cells by promoting apoptosis (Kanno et al. 2001). On account of the functional significance of different natural forms of gastrin, we have assembled signaling pathway reactions induced by them upon binding to gastrin receptor(s) in different human cell types. These manually curated signaling events are made available through NetPath (http://www.netpath.org/) (Kandasamy et al. 2010) in different formats for analysis by the scientific community.

Paradoxically Augmented Anti-Tumorigenic Action of Proton Pump Inhibitor and Gastrin in APCMin/+ Intestinal Polyposis Model1

Though long-term administration of proton pump inhibitor (PPI) imposed the risk of gastrointestinal track tumorigenesis by accompanied hypergastrinemia, no overt increases of colon cancer risk were witnessed after a long-term cohort study. Our recent investigation revealed that PPI prevented colitis-associated carcinogenesis through antiinflammatory, anti-oxidative, and anti-mutagenic mechanisms in spite of hypergastrinemia. Therefore, we hypothesized that PPI might either antagonize the trophic action of gastrin on gastrointestinal tumorigenesis or synergize to exert augmented anti-tumorigenic actions.We challenged APCMin/+ mice with gastrin, PPI, PPI and gastrin together for 10 weeks and counted intestinal polyposis accompanied with molecular changes. Gastrin significantly increased intestinal polyposis, but combination of PPI and gastrin markedly attenuated intestinal polyposis compared to gastrinpromoted APCMin/+ mice (P < .001), in which significant β-catenin phosphorylation and inhibition of β-catenin nuclear translocation were observed with PPI alone or combination of PPI and gastrin, whereas gastrin treatment significantly increased β-catenin nuclear translocation. Significant footprints of apoptosis, G0/G1 accumulation, inactivation of p38 and extracellular signal-regulated kinase, decreased expressions of CD31, and inhibition of tumor necrosis factor-α and cyclooxygenase-2 were noted in the combination group. In vitro investigations were similar to in vivo findings as shown that PPI treatment inhibited the binding of gastrin to its receptor, inactivated β-catenin-associated signaling including Tcf/Lef and glycogen synthase kinase β, and paradoxically inhibited β-catenin-associated proliferative activities. Our investigations explain why colon cancer risk has not increased despite long-term use of PPIs and provide a rationale for using PPI to achieve anti-tumorigenesis beyond acid suppression.

Competitive solid-phase immunoassay of gastrin in serum using time-resolved fluorometry

Background. A competitive solid-phase assay for the measurement of gastrin in serum using time-resolved fluorescence was developed as an alternative to conventional radioimmunoassay (RIA) technology. Methods. The assay depends on the competitive binding of unlabelled versus Eu-labelled gastrin to specific gastrin antibodies – bound to anti-rabbit IgG immobilized on polystyrene microtitration strips. The bound Eu3+-label was dissociated from the bound gastrin and converted to a fluorescent β-diketone chelate which was measured by fluorometry with time-resolution. Results. Using a sample volume of 50 μl the lower limit of detection was below10 pmol/L. Dilution of samples showed an excellent linearity. Spiking with gastrin-17 in known concentrations showed a recovery of 103% indicating that there is no bias inherent in the assay. The method correlated fully with the routine in-house radioimmunoassay in the concentration range 10–400 pmol/L (slope = 0.98 with r2 = 0.98). Thus the reference interval for clinical samples does not require modification when changing from one method to the other. Conclusion. We have described a convenient and accurate competitive assay for measurement of gastrin in serum based on solid-phase technology using time-resolved fluorometric detection as a realistic alternative to the established state-of-the-art RIA-technology.

Abstract 4132: Down Regulation of the CCK-B/C Receptor Inhibits Growth of Pancreatic Cancer

Abstract Background: Pancreatic cancer has a dismal prognosis with only 1% surviving 5 years. Reasons for this poor prognosis include the inability to diagnosis pancreatic cancer in early stages and its resistance to standard chemotherapy. The gastrointestinal peptide, gastrin, has been identified as a growth factor in pancreatic cancer. Gastrin stimulates growth of pancreatic cancer through the cholecystokinin (CCK)-B receptor and a splice variant of this receptor (CCK-C), which is expressed only in cancer cells. Hypothesis: It is hypothesized that down regulation of the gastrin binding receptors, CCK-B/C, by small interfering RNAs (siRNAs) will inhibit growth of pancreatic cancer. Methods: A series of human pancreatic cancer cells were screened by Real-Time RT-PCR for the presence and quantity of CCK-B/C receptor mRNA. Receptor binding was identified in pancreatic cancer cells with a CCK-B polyclonal antibody (1:10,000) by immunocytochemistry. PANC-1 cells were transfected with siRNAs that target a region of mRNA common to both the CCK-B and CCK-C receptor with either Lipofectamine 2000 or pegylated cationic nanoliposomes previously shown to be nontoxic after systemic intravenous administration. RNA was extracted and analyzed by TaqMan Real-Time RT-PCR for the receptor expression 3 days after treatment. Control cells were untreated, vehicle only treated (Lipofectamine 2000 or pegylated cationic nanoliposomes), or treated with a scrambled siRNA control. The effects of CCK-B/C knock-down on growth were evaluated by counting cells at various time points by trypan blue dye exclusion. Results: The CCK-B/C receptor was detected in several human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, AsPC-1, BxPC-3, and Capan-1) by Real-Time RT-PCR. The receptor was identified by immunofluorescence in the plasma membrane. CCK-B/C receptor expression was down regulated by CCK-B/C siRNA with both Lipofectamine 2000 and pegylated cationic nanoliposomes. PANC-1 cell growth was significantly decreased in CCK-B/C receptor targeted siRNA treated cells compared to untreated cells, vehicle only, and scrambled control on day 3 (p&amp;lt;0.0001, n=9). Control cells more than doubled in number while CCK-B/C siRNA treated cells increased by only 10%. Conclusion: The CCK-B/C receptor is present in human pancreatic cancer cells and knock-down significantly decreases cell growth. Systemic administration of CCK-B/C siRNAs by pegylated cationic nanoliposomes may be a potential therapeutic modality for pancreatic cancer. Supported by NIH CA117926. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4132.

A Combinatorial Approach for the Design of Complementarity-determining Region-derived Peptidomimetics with in Vitro Anti-tumoral Activity

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH(2)) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (K(d) values in micromolar range) and the parental monoclonal antibodies (K(d) values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.

Designing antibodies for the inhibition of gastrin activity in tumoral cell lines

Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin-2 (CCK-2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK-2 receptor. The gastrin neutralizing activity assay showed that N-terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti-C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody-based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activity.

Valine-286 residue in the third intracellular loop of the cholecystokinin 2 receptor exerts a pivotal role in cholecystokinin 2 receptor mediated intracellular signal transduction in human colon cancer cells

Although expression of the gastrin/cholecystokinin-2 receptor (CCK2R) is widely reported in human colorectal cancer, little is known on its role in mediating mature amidated gastrin (gastrin-17 amide, G-17) induced intracellular signal transduction in colon cancer cells. The purpose of this study was to explore the intracellular events of colorectal cancer cells after gastrin binding to CCK2R. Meanwhile, the influence of a natural point mutation 286V→F in the third intracellular loop of CCK2R on gastrin-envoked intracellular signal transduction was also investigated. Firstly, Colo320 cells were stably transfected with wild type (Colo320 WT) and mutant CCK2R (Colo320 M), respectively. The intracellular signal transduction events in response to gastrin were investigated in both Colo320 WT and Colo320 M cells. In Colo320 WT cells, G-17 induced formation of intracellular cyclic AMP and inositol 1,4,5-trisphosphate, and stimulated intracellular calcium mobilization. G-17 also stimulated tyrosine phosphorylation of ERKl/2, p38, FAK, and paxillin, and up-regulated the mRNA expression of early response gene c-Jun and c-Fos. However, G-17 inhibited proliferation and induced apoptosis in Colo320 WT cells. Mutation 286V→F in the third intracellular loop of CCK2R blocked G-17 induced biological without affecting binding affinity of CCK2R to G-17. Our results suggest that activation of CCK2R by gastrin stimulates heterotrimeric G-protein Gq and G12/13 mediated intracellular signal transduction pathway in colon cancer cells. The valine-287 residue in third intracellular loop of CCK2R plays a pivotal role in CCK2R mediated intracellular signal transduction.

Gastrin stimulates receptor-mediated proliferation of human esophageal adenocarcinoma cells

The prevalence of esophageal adenocarcinoma in the setting of Barrett's metaplasia continues to increase in Western nations at a rate greater than any other cancer. The trophic properties of gastrin have been documented in gastric, pancreatic and colon cancer cell lines, suggesting a potential role for this regulatory peptide in the growth of these malignancies. The aims of these studies were to identify and characterize the presence of functional cholecystokinin type-2 (gastrin) receptors on the membranes of human esophageal adenocarcinoma cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of cholecystokinin type-2 receptor transcripts in human esophageal adenocarcinoma cell lines. Competitive binding assays revealed specific binding of gastrin in SEG-1 cells (IC 50 of 2.4×10 −8 M). This finding was confirmed by laser scanning confocal microscopy through internalization of rhodamine green labeled gastrin heptapeptide in SEG-1 cells. Gastrin caused a dose-dependent increase in proliferation of SEG-1 cells when compared to controls. This effect was abolished by co-incubation with L365,260, a CCK-2-specific receptor antagonist. Gastrin-induced phosphorylation of the p44 and p42 mitogen-activated protein kinases was demonstrated by Western blot analysis. In conclusion, the studied human esophageal adenocarcinoma cell lines possess cholecystokinin type-2 (gastrin) receptors. Receptors bind gastrin, resulting in increased proliferation in SEG-1 cells.

Brain tumors might not be able to escape gastrin

One of the most terrifying characteristics of malignant tumors is the motility of transformed cells, which enables them to spread to adjacent tissue. If they are then able to travel in the bloodstream, they can invade other organs giving rise to secondary tumors. However, in the brain it is enough that primary tumor cells can disseminate quickly to surrounding structures, thus restricting the surgical removal of transformed tissue. Saving healthy, normal tissue is very important because each area of the brain is functionally unique and therefore precious, despite the brain's enormous plasticity.Neoplastic transformation occurs in glial cells, which then exhibit a remarkable capacity to invade normal tissue diffusely. Fortunately, enough cell lines obtained from glioblastoma tumor biopsies retain this ability, allowing in vitro studies of proliferation and migration. Among various factors studied in this context, cholecystokinin (CCK) and its close relative gastrin were candidate growth factors because they control the growth of both normal tissue and some cancers. It was found that various tumors express CCK2-type gastrin receptors. Indeed, in the case of human glioblastoma cells it was found that gastrin acts via CCK2 receptors; however, it was not possible to block the effect of gastrin completely using known gastrin receptor antagonists.Recent results indicating that the most malignant forms of glial brain tumors do not express the CCK2 receptor caught the attention of Pannequin and co-workers [1xc-Terminal heptapeptide of gastrin inhibits astrocytomas motility by interacting with new gastrin binding sites. Pannequin, J. et al. J. Pharmacol. Exp. Ther. 2002; 302: 274–282Crossref | PubMed | Scopus (5)See all References][1]. They hypothesized that gastrin exerted its anti-motility action through a new, as yet unknown, gastrin receptor. When testing several human glioma cell lines, they did not observe any binding of various CCK or gastrin receptor agonists, and the presence of mRNAs encoding either CCK1 or CCK2 receptors were consistently undetected. Screening various modified gastrin analogs showed that only C-terminal heptapeptide G-7 had the capacity to bind to cell membranes. Kinetic studies and pharmacological profiling confirmed the existence of a single specific site, different from the classical CCK1 or CCK2 receptors. This new target is probably a member of the G-protein-coupled receptor class. However, G-7 did not cause accumulation of inositol phosphate or cAMP second messengers, nor did it stimulate the proliferation of the cells. It did, however, have a strong stimulatory action on the expression of c-Fos – the transcription factor that is an important element of a proliferation-controlling mechanism. The most intriguing observation was a dose-dependent, significant decrease of tumor cell migration.The authors propose the existence of a new, specific gastrin-binding receptor and suggest that it might be coupled to the Rho protein. Although this is a very interesting result, it is not rare that new receptors are discovered during studies of atypical responses to novel ligands. The true achievement of the authors is the discovery of a new agent that can decrease the motility of malignant astrogliomas. This might constitute a novel target for the development of new therapeutic approaches against these tumors; today no effective treatment is available.

Molecular Complementarity III. Peptide Complementarity as a Basis for Peptide Receptor Evolution: A Bioinformatic Case Study of Insulin, Glucagon and Gastrin

Dwyer has suggested that peptide receptors evolved from self-aggregating peptides so that peptide receptors should incorporate regions of high homology with the peptide ligand. If one considers self-aggregation to be a particular manifestation of molecular complementarity in general, then it is possible to extend Dwyer's hypothesis to a broader set of peptides: complementary peptides that bind to each other. In the latter case, one would expect to find homologous copies of the complementary peptide in the receptor. Thirteen peptides, 10 of which are not known to self-aggregate (amylin, ACTH, LHRH, angiotensin II, atrial natriuretic peptide, somatostatin, oxytocin, neurotensin, vasopressin, and substance P), and three that are known to self-aggregate (insulin, glucagon, and gastrin), were chosen. In addition to being self-aggregating, insulin and glucagon are also known to bind to each other, making them a mutually complementary pair. All possible combinations of the 13 peptides and the extracellular regions of their receptors were investigated using bioinformatic tools (a total of 325 combinations). Multiple, statistically significant homologies were found for insulin in the insulin receptor; insulin in the glucagon receptor; glucagon in the glucagon receptor; glucagon in the insulin receptor; and gastrin in gastrin binding protein and its receptor. Most of these homologies are in regions or sequences known to contribute to receptor binding of the respective hormone. These results suggest that the Dwyer hypothesis for receptor evolution may be generalizable beyond self-aggregating to complementary peptides. The evolution of receptors may have been driven by small molecule complementarity augmented by modular evolutionary processes that left a “molecular paleontology” that is still evident in the genome today. This “paleontology” may allow identification of peptide receptor sites.

C-terminal heptapeptide of gastrin inhibits astrocytomas motility by interacting with a new gastrin binding site.

It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.

Gastrin interferes with the differentiation of gastric pit cells and parietal cells.

Gastrin is known to have stimulatory effects on gastric mucosa; however, long-term effect of gastrin stimulation is not well known. To investigate the long-term effect of hypergastrinaemia, we established hypergastrinaemic transgenic mice by introducing a mutated human gastrin gene. Homozygously transgene-expressing mice showed serum gastrin levels of more than 600 pg/mL. Neither progastrin nor glycine-extended gastrin titre elevation were observed in hypergastrinaemic transgenic mice. Stomachs from the 30-35-week-old transgenic mice were 30-50% heavier and their mucosa were markedly thicker than those of the controls. The hypertrophic gastric mucosa of hypergastrinaemic transgenic mice consisted of elongated pits with widespread proliferative zones, and comprised depleted glandular regions. In situ hybridization study indicated that expression of H, K-ATPase mRNA in parietal cells of hypergastrinaemic transgenic mice was markedly decreased. By gastrin binding assay in vivo, specific gastrin binding sites were observed in the mid-glandular region of hypergastrinaemic transgenic mice that consisted mainly of prepit cells. These results suggest that long-term stimulation of gastrin increases the expression of CCK-B/gastrin receptors in the less-differentiated pit cells that are the main component of elongated gastric units, and lessens the well-differentiated characteristics of parietal cells.

Gastrin stimulates the growth of gastric pit cell precursors by inducing its own receptors.

Gastrin/CCK-B receptors (CCKB-Rs) are present on parietal and enterochromaffin-like cells in the gastric mucosa but not on pit cells in the proliferative zone. Because serum gastrin levels are well correlated with the growth of the gastric pit, we examined whether pit precursor cells express CCKB-Rs using hypergastrinemic transgenic mice and a mouse pit precursor cell line, GSM06. In situ hybridization indicated that CCKB-R mRNA was limited to the lower one-third of the mucosa in control mice, whereas it was faintly distributed along the mid- to low glandular region in the hypergastrinemic transgenic mouse mucosa. CCKB-R-positive midglandular cells appear to have a pit cell lineage; therefore, GSM06 cells were used for an [(125)I]gastrin binding study. [(125)I]gastrin bound to the membrane fraction of the GSM06 cells when precultured with gastrin. Gastrin dose dependently induced CCKB-R expression in GSM06 cells and stimulated their growth. Thus these findings suggest that gastrin directly stimulates the growth of the pit cell lineage by inducing its own receptor in pit cell precursors.

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells

Gastrin 17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin 17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin 17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met 15]gastrin 17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin 17gly or gastrin 17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin 17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle 15]gastrin 2,17gly to cells or membranes with disuccinimidyl suberate. The IC 50 value for binding of gastrin 17gly to DLD-1 cells was 2.1±0.4 μM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin 17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.

Mutation of lysine residues of the 78-kDa gastrin-binding protein reduces gastrin binding.

The 78-kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. Both the N- and C-terminal halves of the GBP bind gastrin, but the affinity of the N-terminal half for gastrin is 7.2-fold higher than the affinity of the C-terminal half. In order to define the gastrin-binding sites of the GBP in greater detail, we have constructed a truncation mutant lacking residues 221-318 of the N-terminal domain and a series of point mutants in which the lysine residues in the first 220 residues of the N-terminal domain were mutated to arginine residues. The effect of these mutations on both the extent of covalent cross-linking of iodinated gastrin2,17 and on the affinity for gastrin17 was investigated. Deletion of residues 221-318 of the GBP decreased the affinity 5.5-fold and reduced, but did not abolish, the extent of covalent cross-linking. Mutation of the 17 lysines in residues 1-220 of the GBP decreased the affinity for gastrin between 1.7- and 3.5-fold and in some cases reduced, but did not abolish, the extent of covalent cross-linking. We conclude that one or more lysine residues are involved in binding of gastrin to the GBP, but that no single lysine residue is the preferred target for covalent cross-linking of iodinated gastrin2,17 to the GBP.

Gastrin, CCK, signaling, and cancer.

Gastrin, produced by G cells in the gastric antrum, has been identified as the circulating hormone responsible for stimulation of acid secretion from the parietal cell. Gastrin also acts as a potent cell-growth factor that has been implicated in a variety of normal and abnormal biological processes including maintenance of the gastric mucosa, proliferation of enterochromaffin-like cells, and neoplastic transformation. Here, we review the models used to study the effects of gastrin on cell proliferation in vivo and in vitro with respect to mechanisms by which this hormone might influence normal and cancerous cell growth. Specifically, human and animal models of hypergastrinemia and hypogastrinemia have been described in vivo, and several cells that express cholecystokinin (CCK)B/gastrin receptors have been used for analysis of intracellular signaling pathways initiated by biologically active amidated gastrins. The binding of gastrin or CCK to their common cognate receptor triggers the activation of multiple signal transduction pathways that relay the mitogenic signal to the nucleus and promote cell proliferation. A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades, including mitogen-activated protein kinase, is an important early response to these signaling peptides. Gastrin and CCK also induce rapid Rho-dependent actin remodeling and coordinate tyrosine phosphorylation of cellular proteins including the non-receptor tyrosine kinases p125fak and Src and the adaptor proteins p130cas and paxillin. This article reviews recent advances in defining the role of gastrin and CCK in the control of cell proliferation in normal and cancer cells and in dissecting the signal transduction pathways that mediate the proliferative responses induced by these hormonal GI peptides in a variety of normal and cancer cell model systems.

Effect of phosphorylation on glycine-extended gastrin (G-Gly) binding to cells

Effect of phosphorylation on glycine-extended gastrin (G-Gly) binding to cells

Affinity of cholecystokinin receptor antagonists for the gastrin-binding protein

A 78 kDa gastrin-binding protein is a likely target for the anti-proliferative effects of the cholecystokinin (CCK) receptor antagonists d, l-4-benzamido- N, N-dipropylglutaramic acid (proglumide) and N-4-chlorobenzoyl- l-tryptophan (benzotript) on colorectal carcinoma cell lines [Baldwin, G.S., 1994. Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein. Proc. Natl. Acad. Sci. USA 91, 7593–7597.]. Definition of the physiological role of the gastrin-binding protein has been hampered by the very low affinity of benzotript for the gastrin-binding protein. Benzotript analogues were therefore tested for their ability to inhibit the binding of iodinated gastrin to the gastrin-binding protein. The affinity of the most potent analogue (the d-isomer of benzotript, CR 665) was similar to the value reported previously for the l-isomer. In order to isolate more potent binding inhibitors, several selective CCK receptor antagonists were also tested as inhibitors of the binding of gastrin to the gastrin-binding protein. The affinity of the most potent binding inhibitor PD 149164 (benzenebutanoic acid, 4-fluoro-!b/-[[3-(1 H-indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo-[3.3.1.1 3,7]dec-2-yloxy)carbonyl]amino]propyl]amino]-, [ R-( R*, S*)]-) was approximately 10-fold higher than the l-isomer of benzotript. PD 149164 may serve as the lead compound for the future development of more potent and selective gastrin-binding protein inhibitors.

Expression of Gastrin/CCK-B Receptors Does Not Lead to a Mitogenic Response to Gastrin in Two Colon Cell Lines

To clarify the impact of the classic gastrin/CCK-B receptor on the growth of benign and malignant colonic cells, two permanent cell lines expressing this receptor have been established. The conditionally immortalized nonmalignant colonic cell line YAMC and the colonic carcinoma cell line SW 403 were stably transfected with a plasmid encoding the gastrin/CCK-B receptor (GR), or with plasmid alone (V). Expression of the gastrin/CCK-B receptor in the transfected YAMC-GR and SW 403-GR cells was demonstrated by gastrin binding experiments. The YAMC-GR cell line did not respond mitogenically to pentagastrin or gastrin17in vitro and was not tumorigenic. The SW 403-GR cell line was stimulated by gastrin17in vitro, but the growth patterns of SW 403-GR and SW 403-V were the same in nude mice with cells injected either subcutaneously or into the spleen. These results provide further evidence that the gastrin/CCK-B receptor is not responsible for gastrin-stimulated growth in colonic tumors.

Structural requirements for the binding of non-steroidal anti-inflammatory drugs to the 78 kDa gastrin binding protein

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the proliferation of colorectal carcinoma cell lines in vitro and reduce the risk of colorectal carcinoma in vivo. The good correlation observed between the potency of NSAIDs as inhibitors of colorectal carcinoma cell proliferation and as antagonists of a 78 kDa gastrin binding protein (GBP) suggested that blockade of the GBP might contribute to the anti-proliferative effects of NSAIDs [G.S. Baldwin, V.J. Murphy, Z. Yang, T. Hashimoto, J. Pharmacol. Exp. Ther. 286 (1998) 1110–1114]. The most potent NSAID investigated was sulindac sulphide, which had an IC 50 value of 40 μM. In order to investigate the structural requirements for binding to the GBP, 26 analogues of sulindac sulphide and sulindac sulphoxide were tested for their ability to inhibit the binding of iodinated gastrin to the GBP. Six of the analogues inhibited gastrin binding by more than 50% at a concentration of 1 mM. The IC 50 values estimated by computer fitting of titration data were in the range of 280–940 μM. Comparison of the analogue structures suggests that a substituent with a carboxyl group is preferred in the R 2 position. In addition the location of the NSAID binding site within the GBP structure was investigated. NSAIDs bound to both the N- and C-terminal halves of the GBP, and the affinities determined were similar to the values previously reported for the full-length GBP. The results reported herein represent the first step in the rational design of more potent GBP antagonists, some of which may be useful for the treatment of colorectal carcinoma.