Biomedical subjects

Andrea Varro

Publications and source records attributed to Andrea Varro.

27 records · Page 2Linked to original sources

PubMed2003

Progastrin stimulates murine colonic epithelial mitosis after DNA damage.

BACKGROUND & AIMS: Transgenic mice that overexpress progastrin are more susceptible than either wild-type mice or mice that overexpress amidated gastrin to chemical carcinogen-induced colonic adenomas. We have investigated whether alterations in the regulation of apoptosis or mitosis after DNA damage contribute to the effects of progastrin on murine colonic epithelium. METHODS: Apoptosis and mitosis were assessed on a cell positional basis in murine intestinal epithelium after gamma-irradiation. Mice analyzed were progastrin overexpressing, gastrin overexpressing, gastrin knockout, and their wild-type counterparts. The expression of cell cycle regulators was analyzed by gene array and Western blotting. RESULTS: Apoptosis was induced to similar levels in the small intestinal and colonic crypts of all mice 4.5 hours after 8 Gy gamma-radiation. Colonic mitosis was inhibited to almost undetectable levels by 8Gy gamma-radiation in wild-type, gastrin-knockout, and gastrin-overexpressing mice. However, significant colonic mitosis persisted in progastrin-overexpressing mice up to 24 hours after 8Gy gamma-radiation. Increased levels of cdk4 and cyclin D1 proteins were found in the colonic epithelium of progastrin-overexpressing mice relative to wild-type animals after gamma-radiation. CONCLUSIONS: After DNA damage by gamma-radiation, mice with elevated progastrin exhibit significantly higher levels of colonic mitosis than wild-type or gastrin-overexpressing mice. Persistently elevated cdk4 and cyclin D1 in progastrin overexpressing mice accounts for the capacity of colon cells to continue with the cell cycle after DNA damage.

Animals↗

PubMed2003

Localization of orexin-1 receptors to vagal afferent neurons in the rat and humans.

BACKGROUND & AIMS: Orexin-A and -B are brain-gut peptides that stimulate food intake via orexin-R1 and -R2 receptors. Cholecystokinin (CCK) inhibits food intake via CCK(A) receptors expressed on vagal afferent neurons. The purpose of the study was to determine whether vagal afferent neurons express OX-R1 and OX-R2 and whether orexin-A inhibits responses to CCK. METHODS: OX-R1 and -R2 expression by rat and human nodose ganglia was examined by reverse-transcriptase polymerase chain reaction (RT-PCR). Receptor localization was determined by immunohistochemistry. Responses of rat jejunal afferent fibers were examined by electrophysiology. RESULTS: Both rat and human nodose ganglia expressed OX-R1 as detected by RT-PCR, and humans also expressed OX-R2. The identity of the products was confirmed by sequencing. Immunohistochemistry indicated expression of OX-R1 in both species in neurons that also expressed CCK(A) and leptin receptors. In human ganglia there was also expression in glial cells that was absent in rats. Orexin-A had no effect on the resting discharge of afferent nerve fibers but inhibited responses to CCK. CONCLUSIONS: OX-R1 and CCK(A) receptors are expressed by human and rat vagal afferent neurons. Orexin inhibits responses to CCK suggesting a role in modulation of gut to brain signaling.

Animals↗

PubMed2003

Gastrin induces proliferation in Barrett's metaplasia through activation of the CCK2 receptor.

BACKGROUND AND AIMS: Factors associated with the development and malignant progression of Barrett's esophagus are poorly understood. Gastrin is a mitogen capable of inducing growth in normal and malignant gastrointestinal mucosa. It is unknown whether gastrin can influence cellular events in the esophagus in Barrett's. METHODS: Reverse-transcription polymerase chain reaction (RT-PCR) and northern analysis for the cholecystokinin (CCK(2)) receptor were performed on normal, inflamed, metaplastic, and malignant esophageal mucosa. Real-time PCR quantified expression of the receptor. [(125)I]-G17-autoradiography localized the CCK(2) receptor in mucosal sections. [(3)H]-thymidine and bromodeoxyuridine (BrdU) incorporation determined proliferation in response to G17 in biopsy specimens incubated ex vivo. Proliferation and signaling studies were performed on OE33(E) cells transfected with the CCK(2) receptor. RESULTS: RT-PCR identified receptor expression in 3 of 9 controls, 5 of 7 patients with esophagitis, 10 of 10 patients with Barrett's metaplasia, and 7 of 12 esophageal adenocarcinomas. Real-time PCR quantified expression in 10 patients with Barrett's showing a level of expression 2 orders of magnitude higher than in 12 control patients. [(125)I]-G17 bound to epithelia within glandular regions of Barrett's mucosa. Ten nmol/L G17 induced a 2-fold (n = 7, P = 0.0257, t test) increase in [(3)H]-thymidine incorporation in mucosal biopsy specimens, abolished by the addition of the CCK(2) receptor antagonist L-740, 093. One nmol/L G17 induced a 1.94- +/- 0.13-fold (n = 6, t test, P = 0.001) increase in [(3)H]-thymidine incorporation in OE33(E)(GR) cells, abolished by L-740, 093. CONCLUSIONS: Gastrin induces proliferation via the CCK(2) receptor in Barrett's mucosa. This may have implications for the management of patients with Barrett's esophagus in whom gastrin is elevated by acid-suppression therapy.

Adult↗

PubMed2003

Stimulation of gastrin-CCKB receptor promotes migration of gastric AGS cells via multiple paracrine pathways.

Responses to G protein-coupled receptor stimulation may be mediated by paracrine factors. We have developed a coculture system to study paracrine regulation of migration of gastric epithelial (AGS) cells after stimulation of gastrin-CCK(B) receptors. In cells expressing this receptor, G-17 stimulated migration by activation of protein kinase C. However, G-17 also stimulated the migration of cells expressing green fluorescent protein, but not the receptor, when they were cocultured with receptor-expressing cells consistent with activation of paracrine signals. The use of various pharmacological inhibitors indicated that gastrin stimulated migration via activation of the EGF receptor (EGR-R), the erbB-2 receptor tyrosine kinase, and the MAP kinase pathway. However, gastrin also released fibroblast growth factor (FGF)-1, and migration was inhibited by the FGF receptor tyrosine kinase inhibitor SU-5402. Flow cytometry indicated that in both cell types, gastrin increased MAP kinase via activation of EGF-R but not FGF-R1 or erbB-2. We conclude that gastrin-CCK(B) receptors stimulate epithelial cell migration partly via paracrine mechanisms; transactivation of EGF-R is only one component of the paracrine pathway.

Cell Movement↗

PubMed2003

Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency.

We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A's negligible PC1 activity, some mature ACTH and glucagon-like peptide 17-36(amide) were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.

Animals↗

PubMed2003

Gastrin: an analytical review.

The gastric hormone gastrin is produced in multiple forms that vary in their biological properties. In this analytical review, the strategies available for the assay of different gastrins in plasma are considered. Except for research purposes, it is seldom necessary or even desirable to employ assays that are specific for an individual molecular form of gastrin. Instead, routine clinical assays of plasma gastrin should ideally react equally with the sulphated and unsulphated forms of the COOH-terminally amidated peptides, the most important of which are peptides of 17 and 34 amino acid residues (i.e. G17 and G34), and should not react with the related hormone cholecystokinin. Methods appropriate for the use of such assays are reviewed. These assays are important in the diagnosis of gastrinoma. Although it is recognized that circulating concentrations of gastrin are elevated in other conditions, including chronic atrophic gastritis, Helicobacter pylori infection and long-term administration of proton pump inhibitors, it is not clear whether gastrin radioimmunoassay is important for the clinical management in these conditions.

Animals↗

PubMed2002

Gastrin-stimulated gastric epithelial cell invasion: the role and mechanism of increased matrix metalloproteinase 9 expression.

The gastric hormone gastrin regulates the organization of the gastric epithelium, but the cellular control mechanisms are yet unknown. Epithelial remodelling typically involves extracellular proteolysis mediated by the matrix metalloproteinases (MMPs). Since a gene-array analysis of the gastric cancer cell line AGS-G(R) suggested that gastrin increased MMP-9 expression, we examined the control of MMP-9 expression by gastrin. Gelatin zymography confirmed gastrin induction of MMP-9 in AGS-G(R) cells, but showed a small inhibition of MMP-2. Immunocytochemical studies showed that MMP-9 was localized to vesicles that appeared to traffic along the processes that were extended in response to gastrin. Gastrin stimulated the invasion of AGS-G(R) cells through artificial basement membrane, which was reduced by an inhibitor of MMP-2/-9. There was also an increase in MMP-9 in the stomach of patients with elevated plasma gastrin and multiple-endocrine neoplasia type 1 (MEN-1) syndrome, suggesting in vivo regulation of MMP-9 expression by gastrin. Finally, we showed that the expression of 1.9 kb of human MMP-9 gene promoter coupled with luciferase (MMP-9-luc) was increased 7.65+/-1.2-fold by gastrin, via a pathway which includes stimulation of protein kinase C, and activation of Raf and the mitogen-activated protein (MAP) kinase pathway. The tumour suppressor menin (which is mutated in MEN-1 syndrome) inhibited the expression of MMP-9-luc by gastrin. These results suggest that gastrin increases MMP-9 expression, which is associated with increased invasion, and this is a putative mechanism regulating remodelling of the gastric epithelium.

Animals↗

PubMed2002

Identification of plasminogen activator inhibitor-2 as a gastrin-regulated gene: Role of Rho GTPase and menin.

BACKGROUND & AIMS: The gastric hormone gastrin regulates acid secretion, gene expression, and the functional development and cellular composition of the gastric mucosa. Using a gene array, we sought to identify major, novel, gastrin-regulated genes. METHODS: A cancer gene array was probed with samples from the gastric cancer cell line AGS, expressing the gastrin-cholecystokinin(B) receptor and stimulated with gastrin. The expression of gastrin-regulated genes was further characterized by Western blots and enzyme-linked immunosorbent assay in tissue and blood of hypergastrinemic patients. Gene expression was studied using promoter-luciferase reporter constructs. RESULTS: Plasminogen activator inhibitor 2 (PAI-2) was identified as a major, previously unknown target of gastrin in the gastric cancer cell line AGS. The relevance was confirmed by showing elevated tissue and plasma PAI-2 in hypergastrinemic patients (pernicious anemia and multiple endocrine neoplasia type 1). PAI-2 promoter-luciferase constructs showed that gastrin stimulated expression via pathways involving Galpha and Gbetagamma subunits, protein kinase C, RhoA, and the transcription factors CREB and AP1. The tumor suppressor menin inhibited transcription. In addition, gastrin stimulated expression in adjacent cells via a paracrine mechanism involving protein kinase C and RhoA but not CREB. CONCLUSIONS: A gene array showed PAI-2 to be a novel gastrin-regulated gene, stimulated in part through CREB and AP-1 and inhibited by the tumor suppressor menin.

Aged↗

PubMed2002

Deletion of functional gastrin gene markedly increases colon carcinogenesis in response to azoxymethane in mice.

BACKGROUND & AIMS: We recently reported that transgenic mice overexpressing progastrin were at a higher risk for developing colon cancers in response to azoxymethane (AOM), whereas mice overexpressing gastrin-17 were at a reduced risk. To examine further the role of gastrins in colon carcinogenesis, we generated gastrin gene knockout mice (GAS-KO). METHODS: The height and proliferative index (PI) of colonic crypts were similar in GAS-KO and wild-type (WT) mice, suggesting that the absence of gastrins in GAS-KO mice did not significantly affect the growth of colonic mucosa. GAS-KO and WT mice were treated with AOM for 3-4 weeks; control mice received saline. RESULTS: Colonic proliferation in response to AOM was significantly increased in GAS-KO vs. WT mice. Aberrant crypt foci (ACFs) were similarly increased significantly by approximately 2-5-fold in GAS-KO vs. WT mice after 2 weeks of AOM treatment. Female GAS-KO mice developed adenomas (Ads) and adenocarcinomas (AdCAs) at earlier times ( approximately 10 months) than the male GAS-KO mice and the male and female WT mice ( approximately 12 months). The total numbers of Ads and AdCAs were significantly higher in GAS-KO than in WT mice. CONCLUSIONS: These results suggest the novel possibility that loss of gastrin expression (and hence amidated gastrins) significantly increases susceptibility to colon carcinogenesis in response to AOM. Previous studies with FVB/N transgenic mice similarly suggested a protective role of amidated gastrins against colon carcinogenesis, which supports the present findings of an increase in colon carcinogenesis in GAS-KO mice lacking normal physiological levels of amidated gastrins.

Animals↗