Mice overexpressing progastrin are predisposed for developing aberrant colonic crypt foci in response to AOM

Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.     

Sulfation of gastrin: Effect on immunoreactivity

The effect of sulfuric acid esterification of Tyr-12 in gastrin-17 on immunoreactivity was evaluated by the ability of seventeen antisera raised against non-sulfated gastrin-17 to bind sulfated gastrins in extracts of gastrinoma and antral tissue. Using non-sulfated Tyr-12 iodinated gastrin as tracer, and non-sulfated gastrin-17 as standard the antisera showed three different patterns of reactivity: Three antisera (Nos. 2602, 2605 and 4562) bound sulfated gastrins with low (4–23%) potency; four antisera (Nos. 2604, 2720, 4710 and 4713) measured sulfated gastrins with a potency similar to that of non-sulfated gastrins (81–100% crossreactivity); whereas ten antisera (Nos. 2601, 2606, 2609, 2716, 2717, 2718, 4556, 4559, 4560 and 4563) displayed enhanced reactivity with sulfated gastrins (130–373% cross-reactivity). Using Gly-2 iodinated gastrin as tracer, the latter type of antisera reacted almost equally with sulfated and non-sulfated gastrins, suggesting that the apparent increase in binding of sulfated gastrins rather is due to increased displacement of Tyr-12 iodinated gastrin. The results show that derivatization of amino acid residues greatly influences antibody binding.     

Opossum (Didelphis virginiana) "little" and "big" gastrins

1. "Little" gastrins from most mammalian species are 17 amino acid peptides and the precursor "big" gastrins are 34 amino acid peptides. 2. "Little" gastrins of the New World hystricomorphs, guinea-pig and chinchilla, are 16 amino acid peptides due to deletion of a glutamic acid in the region 6-9 from their NH2-terminus and the corresponding "big" gastrins are 33 amino acid peptides. 3. Antral gastrins from the opossum, a New World marsupial, have a glutamic acid deletion in the same region as the hystricomorph gastrins. 4. Opossum "big" gastrin is a 33 amino acid peptide with the following sequence: less than ELGPQDLPYLTADLSKKQGPWLEEEEAYGWMDF#.     

Gastrins, iron and colorectal cancer

This minireview explores the connections between circulating gastrins, iron status and colorectal cancer. The peptide hormone gastrin is a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. Gastrins bind two ferric ions with μM affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity. The ferric ion ligands have been identified as glutamates 7, 8 and 9 in the 18 amino acid peptide glycine-extended gastrin. An interaction between gastrin and transferrin was first demonstrated by covalent crosslinking techniques, and has been recently confirmed by surface plasmon resonance. We have therefore proposed that gastrins act as catalysts in the loading of transferrin with iron. Several recent lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease haemochromatosis, and that transferrin saturation positively correlates with circulating gastrin concentrations, suggest that gastrins may be involved in iron homeostasis. In addition the recognition that ferric ions may play an unexpected role in the biological activity of non-amidated gastrins may assist in the development of new therapies for colorectal carcinoma.     

Antral content, secretion and peripheral metabolism of N-terminal progastrin fragments

OBJECTIVES: In addition to the acid-stimulatory gastrins, progastrin also release N-terminal fragments. In order to examine the cellular content, secretion and peripheral metabolism of these fragments, we developed an immunoassay specific for the N-terminal sequence of human progastrin. RESULTS: The concentration of N-terminal progastrin fragments in human antral tissue was 6.7 nmol/g tissue (n=5), which was only half of that of acid-stimulatory gastrins (12 nmol/g tissue). Gel chromatography of antral extracts showed that the progastrin fragment 1-35 and 1-19 constitute the major part of the N-terminal progastrin fragments. The basal concentration of N-terminal fragments in normal human plasma was almost 30-fold higher than that of the amidated, acid-stimulatory gastrins (286 pmol/l versus 9.8 pmol/l, n=26, P<0.001). In contrast, the concentration of N-terminal fragments in hypergastrinemic plasma was only 2.7-fold higher than the concentration of amidated gastrins (540 pmol vs. 198 pmol/l, P=0.02). During meal stimulation, the plasma concentrations of N-terminal progastrin fragments and amidated gastrins increased in a correlated manner (r=0.97, P=0.005). The half life for progastrin 1-35 in circulation was 30 min, and a pig model revealed the kidneys and the vasculature to the head as the primary sites of degradation. CONCLUSION: The cellular and circulatory concentration profiles of N-terminal progastrin fragments differ markedly from those of the acid-stimulatory gastrins. The high basal plasma concentrations of N-terminal progastrin fragments cannot be explained by differences in elimination.     

Alpha-carboxyamidation of antral progastrin. Relation to other post-translational modifications

Using radioimmunoassays for amidated and glycine-extended gastrin before and after trypsin-carboxypeptidase B cleavage and chromatography, alpha-carboxyamidation of porcine antral progastrin has been related to tyrosine-O-sulfation and proteolytic cleavages. Corresponding to the sequence at the proteolysis and amidation site, -Gly-Arg-Arg-, antrum contained three COOH-terminally extended precursor types. The glycine-extended gastrins were present in the highest concentrations (241 +/- 58 pmol/g). The degree of tyrosine-O-sulfation was identical for amidated and precursor gastrins irrespective of component size, whereas the component size differed for glycine-extended and amidated forms. For instance, gastrin-34-Gly constituted 54% of the glycine-extended gastrins, while gastrin-34 comprised 8% of the amidated gastrins. The results indicate that tyrosine-O-sulfation occurs prior to NH2-terminal cleavages, which again precede carboxyamidation; but a significant correlation between tyrosine-O-sulfation and proteolytic cleavages or alpha-carboxy-amidation of antral gastrin could not be demonstrated. Furthermore, our results suggest that the immediate precursor of the principal hormonal form, gastrin-17, is gastrin-17-Gly rather than gastrin-34 as previously believed.     

COOH-terminal extended endogenous gastrins

Extracts of antral mucosa, gastrinoma tissue and a gastrinoma serum were subjected to Sephadex G-50 high-resolution chromatography monitored by two radioimmunoassays specific for the NH₂- and the COOH-terminal sequence of heptadecapeptide gastrin. Both tissue extracts and serum contained in addition to known NH₂-terminal extended forms also gastrins corresponding to heptadecapeptide gastrin extended at the COOH-terminus. Four COOH-terminal extended gastrins with mean elution constants (K_av) of 0.14, 0.32, 0.44 and 0.49 were encountered. Re-chromatography in urea gradient and tryptic cleavage corroborated their existence. The COOH-terminal extended gastrins are supposed to represent early stages in the modification of the gastrin m-RNA product.     

Sulfation of gastrin in Zollinger-Ellison sera: evidence for association between sulfation and proteolytic processing

Sulfated gastrins were quantitated in sera from 15 patients with the Zollinger-Ellison syndrome (ZES) by specific radioimmunoassays. The total concentration of gastrin varied from 174 to 285000 pmol/1. Sulfated gastrins constituted $\text{44.8±5.5\%}$ (mean ± S.E.M.) of the gastrins in ZES sera compared with $\text{37.7±1.9\%}$ in sera from 100 control subjects ($\text{P>0.1}$). There was no correlation between gastrin concentration and sulfation ($\text{r=0.40}$). Gel and ion-exchange chromatography showed that up to 90% of the gastrins could be in the sulfated form. The highest degree of sulfation was found in sera where the small gastrin components dominated. Thus, the percentage of small gastrins (G-17 and G-14) correlated with the degree of sulfation ($\text{N = 15}$, $\text{r=0.75}$, $\text{P<0.01}$). We suggest therefore that proteolytic processing of the gastrin precursor and sulfation of tyrosyl are associated.     

Gastrins, iron homeostasis and colorectal cancer

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract.     

Gastrin increases its own synthesis in gastrointestinal cancer cells via the CCK2 receptor

The involvement of the gastrointestinal hormone gastrin in the development of gastrointestinal cancer is highly controversial. Here we demonstrate a positive-feedback loop whereby gastrin, acting via the CCK2 receptor, increases its own expression. Such an autocrine loop has not previously been reported for any other gastrointestinal hormone. Gastrin promoter activation was dependent on the MAP kinase pathway and did not involve Sp1 binding sites or epidermal growth factor receptor transactivation. As the treatment of gastrointestinal cancer cells with amidated gastrin led to increased expression of non-amidated gastrins, the positive-feedback loop may contribute to the sustained increase in circulating gastrins observed in colorectal cancer patients.     

Post-poly(Glu) cleavage and degradation modified by O-sulfated tyrosine: a novel post-translational processing mechanism.

Expression of bioactive peptides requires several modifications of the primary translation product. Gastrin, a vertebrate gut hormone, occurs in multiple forms, including a bioactive fragment of the predominant gastrin-17. Gastrin-17 is, however, without known cleavage sites. In order to identify the new site, we therefore isolated, from antral mucosa, fragments of gastrin-34 and -17 monitored by monospecific immunoassays. After three steps of reverse-phase chromatography, the short gastrins were identified as hepta-, hexa- and pentapeptide amides. By far the most abundant of these was tyrosine O-sulfated gastrin-6. The near complete sulfation contrasts with the larger gastrins, of which only half are sulfated. The longest N-terminal fragment of gastrin-34 was a hexadecapeptide without complementarity to the short gastrins. Instead, the predominant N-terminal fragment of gastrin-17 was the decapeptide complementary to gastrin-7. Therefore the novel processing site is the Glu10-Ala11 bond that follows a poly(Glu6-10) sequence. Moreover, gastrin-7 is apparently trimmed, with subsequent accumulation of sulfated gastrin-6. Consequently, O-sulfated tyrosine ensures production of a new hormone which stimulates gastric acid secretion as potently as gastrin-17.     

Identification of gastrin molecular variants in gastrinoma syndrome

The molecular species of gastrin in the circulation and in tumor extracts were studied in two groups of patients: (1) with benign gastrinoma and (2) with gastrinoma with liver metastases. Radioimmunoassays (RIAs) and immunoaffinity chromatography for the amino (NH2)- and amidated COOH-terminus of gastrin-17 (antiserum G17) and the NH2-terminus of gastrin-34 (antiserum G34) were employed. In both benign and metastatic tumors the molecular forms of gastrin in boiling water extracts measured by the gastrin-17 NH2- and COOH-terminal assays were similar. In addition to a molecular component resembling the amidated gastrin-17, there were also significant amounts of larger molecular weight (mol. wt.) forms. The larger mol. wt. forms absorbed by the NH2-terminus of G17 antiserum corresponded to the COOH-terminus-extended forms of gastrin-17. Furthermore, larger mol. wt. gastrins immunopurified by antiserum to the NH2-terminus of gastrin-34 corresponded to gastrin-34 extended molecules. Sera of patients with liver metastases had higher concentrations of the NH2-terminal of gastrin-17 whereas sera of patients with benign gastrinoma contained predominantly gastrins detected by the COOH-terminal assay. These results suggest that: (a) there are differences in the molecular pattern of gastrin in the circulation of patients with benign and metastatic gastrinomas; (b) gastrins which are fully processed with carboxy-terminal amidation predominate in the circulation of patients with benign gastrinoma; and (c) gastrins containing the gastrin-17 and COOH-terminally extended gastrin-17 and gastrin-34 precursor molecules occur in high concentration in the circulation of gastrinoma patients with metastases to the liver.     

Complete sulfation of jejunal gastrin in the human fetus

The degree of tyrosine-O-sulfation and the ratio between large (gastrin-34 and component I) and small (gastrin-17 and -14) molecular forms of gastrin were studied in extracts of human fetal (n = 14) and adult (n = 9) antrum, duodenum, jejunum and pancreas. Boiled water extracts were applied to gel- and ion-exchange chromatography before and after treatment with trypsin and arylsulfatase. The fractions were monitored with sequence-specific radioimmunoassays that distinguish sulfated from non-sulfated gastrins. In antrum and duodenum about half the gastrins were sulfated at all stages of development. In the fetal jejunum gastrin occurred in sulfated form only while in the adult 72% (range, 64–88%) of the jejunal gastrins were sulfated. The larger molecular forms of gastrin predominated in the fetal compared with the adult antrum. In duodenum and jejunum, however, the ratio between small and large forms was the same in fetus and adult. Gastrin was undetectable in both fetal and adult pancreas. The results show that the degree of sulfation of gastrin varies substantially in the different parts of the gut at different stages of development. The differences may have functional significance, since sulfation increases the pancreozyminic and cholecystokinetic potency of gastrin.     

Molecular forms of gastrin in antral mucosa and serum of dogs.

Instillation of liver extract into antral pouches produced an increase in the serum concentrations of both little (G-17) and big (G-34) gastrins. The molar fraction of G-17 plus G-34 represented by G-17 was about 0.9 in antral mucosa and about 0.3 in serum 3 hr after initiating release with liver extract. The predominance of G-34 in serum can be accounted for only in part by its slower rate of removal from the blood so other factors probably also contribute. Although G-17 contributed only about 30% of the total molar concentration of gastrins in serum, it accounted for about 70% of the acid stimulatory activity because on a molar basis it is about five times more bioactive than G-34.     

Gastrin mediates resistance to hypoxia-induced cell death in xenografts of the human colorectal cancer cell line LoVo

Aim: Gastrins act as growth factors for the normal and neoplastic colorectal mucosa. The aim of this study was to determine the role of gastrins in the response of human colorectal cancer (CRC) cells to hypoxia in vitro and in vivo. Methods: Expression of the gastrin gene in the human CRC cell line LoVo was examined under normoxia and hypoxia by quantitative PCR and by radioimmunoassay. Gastrin expression was knocked down with shRNA, and the effect on cell proliferation was measured by cell counting, on cell apoptosis by annexin V staining, and on cell migration by Boyden chamber assay. The effect of gastrin knockdown on tumourigenesis in mouse xenografts was analysed by measurement of tumour volumes and weights, and by immunohistochemistry. Results: Gastrin gene expression in LoVo cells was stimulated by hypoxia via binding of hypoxia-inducible factor-1α to the gastrin promoter. The viability of gastrin knockdown cells exposed to hypoxia (1% O₂) in vitro was diminished because of loss of resistance against hypoxia-induced apoptosis, and the effect was partly reversed by treatment with non-amidated, but not amidated, gastrin. Conditioned medium from control LoVo cells under hypoxia simulated proliferation but not migration, and the effect was blocked by an inhibitor of non-amidated gastrins, but not by an inhibitor of amidated gastrins. In xenografts in mice exposed to hypoxia (10% O₂) for 21 days, tumour necrosis was significantly increased by knocking down gastrin expression. Conclusion: These results provide evidence that non-amidated gastrins are involved in the adaptation of CRCs to hypoxic microenvironments through increasing resistance to apoptosis.     

A novel effect of bismuth ions: selective inhibition of the biological activity of glycine-extended gastrin

Although bismuth salts have been used for over two centuries for the treatment of various gastrointestinal disorders, the mechanism of their therapeutic action remains controversial. Because gastrins bind two trivalent ferric ions with high affinity, and because ferric ions are essential for the biological activity of glycine-extended gastrin 17, we have investigated the hypothesis that trivalent bismuth ions influence the biological activity of gastrins. Binding of bismuth ions to gastrins was measured by fluorescence quenching and NMR spectroscopy. The effects of bismuth ions on gastrin-stimulated biological activities were measured in inositol phosphate, cell proliferation, and cell migration assays. Fluorescence quenching experiments indicated that both glycine-extended and amidated gastrin 17 bound two bismuth ions. The NMR spectral changes observed on addition of bismuth ions revealed that Glu-7 acted as a ligand at the first bismuth ion binding site. In the presence of bismuth ions the ability of glycine-extended gastrin 17 to stimulate inositol phosphate production, cell proliferation, and cell migration was markedly reduced. In contrast, bismuth ions had little effect on the affinity of the CCK-2 receptor for amidated gastrin 17, or on the stimulation of inositol phosphate production by amidated gastrin 17. We conclude that bismuth ions may act, at least in part, by blocking the effects of glycine-extended gastrin 17 on cell proliferation and cell migration in the gastrointestinal tract. This is the first report of a specific inhibitory effect of bismuth ions on the action of a gastrointestinal hormone.     

Processing-independent radioimmunoanalysis: a general analytical principle applied to progastrin and its products

Most peptide hormone assays measure only fully processed bioactive peptides. Such assays are unsuited to detect hormone gene expression by alternative or attenuated prohormone processing (tissue- or cell-specific processing). The gastrin system is expressed in several different tissues and is therefore useful for studies of tissue-specific processing. Consequently we have developed a simple processing-independent radioimmunoanalysis for progastrin. Using antisera against the NH2-terminus of a sequence, devoid of processing sites (preprogastrin76-86) after trypsination of neighboring cleavage sites, the assay quantitates the mRNA product irrespective of degree of processing. Used together with a conventional assay for the mature carboxyamidated gastrins, the processing-independent analysis shows that in different tissues only 1 to 55% of the total translation product is processed to bioactive gastrins. Thus processing-independent analysis greatly improves the detection of gastrin gene expression at the peptide level. The principle of the assay should be applicable to all protein and peptide systems.     

Guinea pig “little” gastrin is a hexadecapeptide

Gastrin heptadecapeptides (gastrins I and II which differ in the presence of sulfatte on the tyrosine of the latter) have been purified and sequenced from several mammalian species including pig, dog, cat, sheep, cow, human and, more recently, rat. This report describes the purification of “little” gastrin from guinea pig (GP) antra. GP antra were defatted with acetone and the acetone cakes were extracted with 0.1M NH₄HCO₃. The extract was concentrated by adsorption to DE53 anion exchange cellulose and the peak eluates were fractionated on a Sephadex G5OF column. The peptides were brought to final purity by 3 successive HPLC steps. The GP sequence compared to other species is shown: Guinea Pig A E E E A Pig <E G P W M E E E E E A Y G W M D F Human L Thus GP “little” gastrins I and II are hexadecapeptides due to a deletion of a glutamic acid in the region 6–9 from the N-terminus.