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Graham S. Baldwin - One of the best experts on this subject based on the ideXlab platform.

  • Progastrin a potential predictive marker of liver metastasis in colorectal cancer
    International Journal of Colorectal Disease, 2017
    Co-Authors: David A Westwood, Oneel Patel, Arthur Shulkes, Christopher Christophi, Graham S. Baldwin
    Abstract:

    Staging of colorectal cancer often fails to discriminate outcomes of patients with morphologically similar tumours that exhibit different clinical behaviours. Data from several studies suggest that the gastrin family of growth factors potentiates colorectal cancer tumourigenesis. The aim of this study was to investigate whether Progastrin expression may predict clinical outcome in colorectal cancer. Patients with colorectal adenocarcinoma of identical depth of invasion who had not received neoadjuvant therapy were included. The patients either had stage IIa disease with greater than 3-year disease-free survival without adjuvant therapy or stage IV disease with liver metastases on staging CT. Progastrin expression in tumour sections was scored with reference to the intensity and area of immunohistochemical staining. Progastrin expression by stage IV tumours was significantly greater than stage IIa tumours with mean Progastrin immunopositivity scores of 2.1 ± 0.2 versus 0.5 ± 0.2, respectively (P < 0.001). This is the first study to show that Progastrin expression may be predictive of aggressive tumour behaviour in patients with colorectal cancer and supports its clinical relevance and potential use as a biomarker.

  • Progastrin: a potential predictive marker of liver metastasis in colorectal cancer.
    International journal of colorectal disease, 2017
    Co-Authors: David A Westwood, Oneel Patel, Arthur Shulkes, Christopher Christophi, Graham S. Baldwin
    Abstract:

    Staging of colorectal cancer often fails to discriminate outcomes of patients with morphologically similar tumours that exhibit different clinical behaviours. Data from several studies suggest that the gastrin family of growth factors potentiates colorectal cancer tumourigenesis. The aim of this study was to investigate whether Progastrin expression may predict clinical outcome in colorectal cancer. Patients with colorectal adenocarcinoma of identical depth of invasion who had not received neoadjuvant therapy were included. The patients either had stage IIa disease with greater than 3-year disease-free survival without adjuvant therapy or stage IV disease with liver metastases on staging CT. Progastrin expression in tumour sections was scored with reference to the intensity and area of immunohistochemical staining. Progastrin expression by stage IV tumours was significantly greater than stage IIa tumours with mean Progastrin immunopositivity scores of 2.1 ± 0.2 versus 0.5 ± 0.2, respectively (P 

  • Ferric ions inhibit proteolytic processing of Progastrin.
    Biochemical and biophysical research communications, 2010
    Co-Authors: Gianni Bramante, Arthur Shulkes, Oneel Patel, Graham S. Baldwin
    Abstract:

    The gastrointestinal hormone gastrin is generated from an 80 amino acid precursor (Progastrin) by cleavage after dibasic residues by prohormone convertase 1. Phosphorylation of Ser(75) has previously been suggested, on the basis of indirect evidence, to inhibit cleavage of Progastrin after Arg(73)Arg(74). Gastrins bind two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. This study directly investigated the effect of iron binding and of serine phosphorylation on the cleavage of synthetic Progastrin-derived peptides. The affinity of synthetic Progastrin(55-80) for ferric ions, and the rate of cleavage by prohormone convertase 1, were not affected by phosphorylation of Ser(75). In contrast, in the presence of ferric ions the rate of cleavage of both Progastrin(55-80) and phosphoSer(75)Progastrin(55-80) by prohormone convertase 1 was significantly reduced. Hence iron binding to Progastrin may regulate processing and secretion in vivo, and regulation may be particularly important in diseases with altered iron homeostasis.

  • The C-terminal flanking peptide (CTFP) of Progastrin inhibits apoptosis via a PI3-kinase-dependent pathway.
    Regulatory peptides, 2010
    Co-Authors: Oneel Patel, Graham S. Baldwin, Gianni Bramante, Kathryn M Marshall, Arthur Shulkes
    Abstract:

    Progastrin is processed to a number of peptides including glycine-extended gastrin, amidated gastrin and the C-terminal flanking peptide (CTFP). Progastrin and gastrin-gly are pro-proliferative and anti-apoptotic in gastric and colorectal cancer cell lines. The CTFP is a major form of Progastrin in the stomach and colon and stimulates proliferation. However the effect of CTFP on apoptosis has not been examined. Using the human gastric carcinoma cell line AGS we show that CTFP attenuates apoptosis through a PI3-kinase pathway by stimulating the phosphorylation of Akt leading to sustained increases in the concentrations of Bcl-xL and phosphorylated Bad protein and by reducing caspase 3 activity. The anti-apoptotic effect represents an important potential mechanism for the growth promoting action of CTFP.

  • Production, secretion, and biological activity of the C-terminal flanking peptide of human Progastrin
    Gastroenterology, 2006
    Co-Authors: Kelly A. Smith, Graham S. Baldwin, Oneel Patel, Shamilah Lachal, Ian G. Jennings, Bruce E. Kemp, John Burgess, Arthur Shulkes
    Abstract:

    Background & Aims: Processing of Progastrin, the 80-amino acid precursor of the hormone gastrin, generates a variety of peptides with distinct distributions and biological activities. However, little is known regarding the expression, secretion, and biological activity of the 6-amino acid C-terminal flanking peptide (CTFP) of Progastrin. The objectives were to determine the concentration of CTFP in normal subjects and patients with gastrointestinal diseases and to investigate the biological activity of CTFP. Methods: CTFP, gastrin-amide (Gamide), glycine-extended gastrin (Ggly), and Progastrin were measured using region-specific radioimmunoassay (RIA) in antral extracts and resected colorectal cancers (CRC) and in plasma from normal subjects (fasting and meal stimulated) and from patients with CRC, multiple endocrine neoplasia type 1 (MEN-1), or pernicious anemia. The effect of CTFP on proliferation, migration, and activation of the mitogen-activated protein kinase (MAPK) pathway in several types of gastrointestinal cell lines was determined. Results: CTFP is by far the predominant Progastrin-derived peptide found in the antrum (4-fold higher than Gamide), resected CRC, and circulation (60-fold higher than Gamide) and is released after meal stimulation. The hypergastrinemic patients (MEN-1, pernicious anemia) had elevated plasma Gamide but unaltered CTFP demonstrating differential secretion of these 2 Progastrin-derived peptides. Finally, CTFP stimulated proliferation and migration and activated MAPK of cells in culture. Conclusions: The high and regulated expression of CTFP in healthy and diseased subjects combined with the evidence for biological activity of CTFP demonstrates that CTFP is not an inactive metabolite of Progastrin processing but is a bioactive peptide with potential roles in the normal and diseased gastrointestinal tract.

Pomila Singh - One of the best experts on this subject based on the ideXlab platform.

  • Clathrin mediates endocytosis of Progastrin and activates MAPKs: role of cell surface annexin A2.
    American journal of physiology. Gastrointestinal and liver physiology, 2012
    Co-Authors: Shubhashish Sarkar, Carla Kantara, Pomila Singh
    Abstract:

    Cell-surface-associated annexin A2 (CS-ANXA2) is a nonconventional “receptor” for Progastrin; expression levels of both are elevated in colon cancers, and downregulation of either reduces tumorigen...

  • Annexin A2 Mediates Up-regulation of NF-κB, β-catenin, and Stem Cell in Response to Progastrin in Mice and HEK-293 Cells
    Gastroenterology, 2010
    Co-Authors: Shubhashish Sarkar, Rafal Swiercz, Carla Kantara, Katherine A. Hajjar, Pomila Singh
    Abstract:

    Background & Aims Prograstrin induces proliferation in colon crypts by activating p65nuclear factor-κB (NF-κB) (p65) and β-catenin. We investigated whether Annexin A2 (AnxA2), a Progastrin receptor, activates NF-κB and β-catenin in vivo. Methods ANXA2-null (ANXA2 −/− ) and wild-type (ANXA2 +/+ ) mice were studied, along with clones of Progastrin-responsive HEK-293 cells that stably expressed full-length Progastrin (HEK-mGAS) or an empty vector (HEK-C). Small interfering RNA was used to down-regulate AnxA2, p65NF-κB, and β-catenin in cells. Results Proliferation and activation of p65 and β-catenin increased significantly in HEK-mGAS compared with HEK-C clones. HEK-mGAS cells had a 2- to 4-fold increase in relative levels of c-Myc, cyclooxygenase (COX)-2, CyclinD1, double cortin CAM kinase-like 1 (DCAMKL+1), and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and β-catenin, as well as levels of DCAMKL+1. Surprisingly, down-regulation of β-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of β-catenin in HEK-mGAS clones. Loss of either p65 or β-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of Progastrin. Lengths of colon crypts and levels of p65, β-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2 +/+ mice compared with either ANXA2 −/− mice given Progastrin or ANXA2 +/+ and ANXA2 −/− mice given saline. Conclusions AnxA2 expression is required for the biologic effects of Progastrin in vivo and in vitro and mediates the stimulatory effect of Progastrin on p65NF-κ, β-catenin, and the putative stem cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and cocarcinogenic effects of Progastrin.

  • Insulin-like growth factors are more effective than Progastrin in reversing proapoptotic effects of curcumin: critical role of p38MAPK
    American journal of physiology. Gastrointestinal and liver physiology, 2010
    Co-Authors: Pomila Singh, Shubhashish Sarkar, William Rengifo-cam, Shahid Umar, Amar Singh, Thomas G. Wood
    Abstract:

    Progastrin and insulin-like growth factors (IGFs) stimulate hyperproliferation of intestinal epithelial cells (IECs) via endocrine/paracrine routes; hyperproliferation is a known risk factor for co...

  • Functional Cross-talk Between β-Catenin and NFκB Signaling Pathways in Colonic Crypts of Mice in Response to Progastrin
    The Journal of biological chemistry, 2009
    Co-Authors: Shahid Umar, Shubhashish Sarkar, Yu Wang, Pomila Singh
    Abstract:

    We recently reported a critical role of NFκB in mediating hyperproliferative and anti-apoptotic effects of Progastrin on proximal colonic crypts of transgenic mice overexpressing Progastrin (Fabp-PG mice). We now report activation of β-catenin in colonic crypts of mice in response to chronic (Fabp-PG mice) and acute (wild type FVB/N mice) Progastrin stimulation. Significant increases were measured in relative levels of cellular and nuclear β-catenin and pβ-cat45 in proximal colonic crypts of Fabp-PG mice compared with that in wild type littermates. Distal colonic crypts were less responsive. Interestingly, β-catenin activation was downstream of IKKα,β/NFκB, because treatment of Fabp-PG mice with the NFκB essential modulator (NEMO) peptide (inhibitor of IKKα,β/NFκB activation) significantly blocked increases in cellular/nuclear levels of total β-catenin/pβ-cat45/and pβ-cat552 in proximal colons. Cellular levels of pβ-cat33,37,41, however, increased in proximal colons in response to NEMO, probably because of a significant increase in pGSK-3βTyr216, facilitating degradation of β-catenin. NEMO peptide significantly blocked increases in cyclin D1 expression, thereby, abrogating hyperplasia of proximal crypts. Goblet cell hyperplasia in colonic crypts of Fabp-PG mice was abrogated by NEMO treatment, suggesting a cross-talk between the NFκB/β-catenin and Notch pathways. Cellular proliferation and crypt lengths increased significantly in proximal but not distal crypts of FVB/N mice injected with 1 nm Progastrin associated with a significant increase in cellular/nuclear levels of total β-catenin and cyclin D1. Thus, intracellular signals, activated in response to acute and chronic stimulation with Progastrin, were similar and specific to proximal colons. Our studies suggest a novel possibility that activation of β-catenin, downstream to the IKKα,β/NFκB pathway, may be integral to the hyperproliferative effects of Progastrin on proximal colonic crypts.

  • Beta-catenin/Tcf-4 inhibition after Progastrin targeting reduces growth and drives differentiation of intestinal tumors.
    Gastroenterology, 2007
    Co-Authors: Julie Pannequin, Nathalie Delaunay, Michael Buchert, Fanny Surrel, Jean-françois Bourgaux, Joanne Ryan, Stéphanie Boireau, Jessica Coelho, André Pélegrin, Pomila Singh
    Abstract:

    BACKGROUND & AIMS: Aberrant activation of the beta-catenin/Tcf-4 transcriptional complex represents an initiating event for colorectal carcinogenesis, shifting the balance from differentiation toward proliferation in colonic crypts. Here, we assessed whether endogenous Progastrin, encoded by a target gene of this complex, was in turn able to regulate beta-catenin/Tcf-4 activity in adenomatous polyposis coli (APC)-mutated cells, and we analyzed the impact of topical Progastrin depletion on intestinal tumor growth in vivo. METHODS: Stable or transient RNA silencing of the GAST gene was induced in human tumor cells and in mice carrying a heterozygous Apc mutation (APCDelta14), which overexpress Progastrin but not amidated or glycine-extended gastrin. RESULTS: Depletion of endogenous Progastrin production strongly decreased intestinal tumor growth in vivo through a marked inhibition of constitutive beta-catenin/Tcf-4 activity in tumor cells. This effect was mediated by the de novo expression of the inhibitor of beta-catenin and Tcf-4 (ICAT), resulting from a down-regulation of integrin-linked kinase in Progastrin-depleted cells. Accordingly, ICAT down-regulation was correlated with Progastrin overexpression and Tcf-4 target gene activation in human colorectal tumors, and ICAT repression was detected in the colon epithelium of tumor-prone, Progastrin-overexpressing mice. In APCDelta14 mice, small interfering RNA-mediated Progastrin depletion not only reduced intestinal tumor size and numbers, but also increased goblet cell lineage differentiation and cell apoptosis in the remaining adenomas. CONCLUSIONS: Thus, depletion of endogenous Progastrin inhibits the tumorigenicity of APC-mutated colorectal cancer cells in vivo by promoting ICAT expression, thereby counteracting Tcf-4 activity. Progastrin targeting strategies should provide an exciting prospect for the differentiation therapy of colorectal cancer.

Frédéric Hollande - One of the best experts on this subject based on the ideXlab platform.

  • The Wnt Target Jagged-1 Mediates the Activation of Notch Signaling by Progastrin in Human Colorectal Cancer Cells
    Cancer Research, 2009
    Co-Authors: Julie Pannequin, Nathalie Delaunay, Jean-françois Bourgaux, Joanne Ryan, Caroline Bonnans, Dominique Joubert, Frédéric Hollande
    Abstract:

    The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that Progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that Progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of beta-catenin/Tcf-4. Accordingly, recombinant Progastrin sequentially activated the transcription of Wnt and Notch target genes in Progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that Progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of Progastrin targeting for the clinical management of CRC.

  • The wnt target jagged-1 mediates the activation of notch signaling by Progastrin in human colorectal cancer cells.
    Cancer research, 2009
    Co-Authors: Julie Pannequin, Nathalie Delaunay, Jean-françois Bourgaux, Joanne Ryan, Caroline Bonnans, Dominique Joubert, Frédéric Hollande
    Abstract:

    The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that Progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that Progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of B-catenin/Tcf-4. Accordingly, recombinant Progastrin sequentially activated the transcription of Wnt and Notch target genes in Progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that Progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of Progastrin targeting for the clinical management of CRC. [Cancer Res 2009;69(15): 6065–73]

  • Adherens junctions and tight junctions are regulated via different pathways by Progastrin in epithelial cells
    Journal of Cell Science, 2003
    Co-Authors: Frédéric Hollande, Debra J Lee, Armelle Choquet, S Roche, Graham S. Baldwin
    Abstract:

    Adhesion between neighbouring epithelial cells is a crucial and tightly controlled process. In the gastrointestinal tract, the integrity of cell-cell contacts is essential for the regulation of electrolyte absorption and for the prevention of tumour metastasis. We recently showed that migration of the gastric epithelial cell line IMGE-5 is stimulated by the nonamidated form of the hormone gastrin 17 . Here, we examine the effect on cell-cell adhesion of the prohormone Progastrin, the concentration of which is increased in the plasma of patients with colorectal carcinoma. Progastrin induced the dissociation of both tight junction (TJ) and adherens junction (AJ) complexes in IMGE-5 cells. In Progastrin-secreting DLD-1 human colorectal carcinoma cells, expression of an antisense gastrin construct restored membrane localisation of zonula occludens-1 (ZO-1), occludin, β-catenin and E-cadherin. This restoration was reversed by treatment with exogenous Progastrin. Endogenous or exogenous Progastrin also increased the paracellular flux of mannitol, and induced cell migration of several gastrointestinal cell lines. In addition, Progastrin enhanced Src tyrosine kinase activity and induced a spatial delocalisation of protein kinase Cα. Using dominant-negative mutants and pharmacological inhibitors, we showed that the stimulation of Src kinase activity was essential for the regulation of TJs. By contrast, the dissociation of AJs involved phosphatidylinositol 3-kinase, partly through the formation of a complex with protein kinase Cα. We conclude that separate pathways mediate the disruption of AJs and TJs by Progastrin. Either pathway may contribute to the co-carcinogenic role of this prohormone in colorectal carcinoma.

  • Adherens junctions and tight junctions are regulated via different pathways by Progastrin in epithelial cells.
    Journal of cell science, 2003
    Co-Authors: Frédéric Hollande, Debra J Lee, Armelle Choquet, S Roche, Graham S. Baldwin
    Abstract:

    Adhesion between neighbouring epithelial cells is a crucial and tightly controlled process. In the gastrointestinal tract, the integrity of cell-cell contacts is essential for the regulation of electrolyte absorption and for the prevention of tumour metastasis. We recently showed that migration of the gastric epithelial cell line IMGE-5 is stimulated by the nonamidated form of the hormone gastrin(17). Here, we examine the effect on cell-cell adhesion of the prohormone Progastrin, the concentration of which is increased in the plasma of patients with colorectal carcinoma. Progastrin induced the dissociation of both tight junction (TJ) and adherens junction (AJ) complexes in IMGE-5 cells. In Progastrin-secreting DLD-1 human colorectal carcinoma cells, expression of an antisense gastrin construct restored membrane localisation of zonula occludens-1 (ZO-1), occludin, beta-catenin and E-cadherin. This restoration was reversed by treatment with exogenous Progastrin. Endogenous or exogenous Progastrin also increased the paracellular flux of mannitol, and induced cell migration of several gastrointestinal cell lines. In addition, Progastrin enhanced Src tyrosine kinase activity and induced a spatial delocalisation of protein kinase C alpha. Using dominant-negative mutants and pharmacological inhibitors, we showed that the stimulation of Src kinase activity was essential for the regulation of TJs. By contrast, the dissociation of AJs involved phosphatidylinositol 3-kinase, partly through the formation of a complex with protein kinase C alpha. We conclude that separate pathways mediate the disruption of AJs and TJs by Progastrin. Either pathway may contribute to the co-carcinogenic role of this prohormone in colorectal carcinoma.

  • biologically active recombinant human Progastrin 6 80 contains a tightly bound calcium ion
    Journal of Biological Chemistry, 2001
    Co-Authors: Graham S. Baldwin, Frédéric Hollande, Adrienne C. Paterson, Greg Neumann, Zhiyu Yang, Yulia Karelina, Rosslyn Strang, Daniel Fourmy, Arthur Shulkes
    Abstract:

    Abstract Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human Progastrin6–80 and to investigate its structure and biological activities in vitro. Human Progastrin6–80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage Progastrin6–80 was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin6–80 at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that Progastrin6–80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of Progastrin6–80suggested that Progastrin6–80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human Progastrin6–80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Arthur Shulkes - One of the best experts on this subject based on the ideXlab platform.

  • Progastrin a potential predictive marker of liver metastasis in colorectal cancer
    International Journal of Colorectal Disease, 2017
    Co-Authors: David A Westwood, Oneel Patel, Arthur Shulkes, Christopher Christophi, Graham S. Baldwin
    Abstract:

    Staging of colorectal cancer often fails to discriminate outcomes of patients with morphologically similar tumours that exhibit different clinical behaviours. Data from several studies suggest that the gastrin family of growth factors potentiates colorectal cancer tumourigenesis. The aim of this study was to investigate whether Progastrin expression may predict clinical outcome in colorectal cancer. Patients with colorectal adenocarcinoma of identical depth of invasion who had not received neoadjuvant therapy were included. The patients either had stage IIa disease with greater than 3-year disease-free survival without adjuvant therapy or stage IV disease with liver metastases on staging CT. Progastrin expression in tumour sections was scored with reference to the intensity and area of immunohistochemical staining. Progastrin expression by stage IV tumours was significantly greater than stage IIa tumours with mean Progastrin immunopositivity scores of 2.1 ± 0.2 versus 0.5 ± 0.2, respectively (P < 0.001). This is the first study to show that Progastrin expression may be predictive of aggressive tumour behaviour in patients with colorectal cancer and supports its clinical relevance and potential use as a biomarker.

  • Progastrin: a potential predictive marker of liver metastasis in colorectal cancer.
    International journal of colorectal disease, 2017
    Co-Authors: David A Westwood, Oneel Patel, Arthur Shulkes, Christopher Christophi, Graham S. Baldwin
    Abstract:

    Staging of colorectal cancer often fails to discriminate outcomes of patients with morphologically similar tumours that exhibit different clinical behaviours. Data from several studies suggest that the gastrin family of growth factors potentiates colorectal cancer tumourigenesis. The aim of this study was to investigate whether Progastrin expression may predict clinical outcome in colorectal cancer. Patients with colorectal adenocarcinoma of identical depth of invasion who had not received neoadjuvant therapy were included. The patients either had stage IIa disease with greater than 3-year disease-free survival without adjuvant therapy or stage IV disease with liver metastases on staging CT. Progastrin expression in tumour sections was scored with reference to the intensity and area of immunohistochemical staining. Progastrin expression by stage IV tumours was significantly greater than stage IIa tumours with mean Progastrin immunopositivity scores of 2.1 ± 0.2 versus 0.5 ± 0.2, respectively (P 

  • Ferric ions inhibit proteolytic processing of Progastrin.
    Biochemical and biophysical research communications, 2010
    Co-Authors: Gianni Bramante, Arthur Shulkes, Oneel Patel, Graham S. Baldwin
    Abstract:

    The gastrointestinal hormone gastrin is generated from an 80 amino acid precursor (Progastrin) by cleavage after dibasic residues by prohormone convertase 1. Phosphorylation of Ser(75) has previously been suggested, on the basis of indirect evidence, to inhibit cleavage of Progastrin after Arg(73)Arg(74). Gastrins bind two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. This study directly investigated the effect of iron binding and of serine phosphorylation on the cleavage of synthetic Progastrin-derived peptides. The affinity of synthetic Progastrin(55-80) for ferric ions, and the rate of cleavage by prohormone convertase 1, were not affected by phosphorylation of Ser(75). In contrast, in the presence of ferric ions the rate of cleavage of both Progastrin(55-80) and phosphoSer(75)Progastrin(55-80) by prohormone convertase 1 was significantly reduced. Hence iron binding to Progastrin may regulate processing and secretion in vivo, and regulation may be particularly important in diseases with altered iron homeostasis.

  • The C-terminal flanking peptide (CTFP) of Progastrin inhibits apoptosis via a PI3-kinase-dependent pathway.
    Regulatory peptides, 2010
    Co-Authors: Oneel Patel, Graham S. Baldwin, Gianni Bramante, Kathryn M Marshall, Arthur Shulkes
    Abstract:

    Progastrin is processed to a number of peptides including glycine-extended gastrin, amidated gastrin and the C-terminal flanking peptide (CTFP). Progastrin and gastrin-gly are pro-proliferative and anti-apoptotic in gastric and colorectal cancer cell lines. The CTFP is a major form of Progastrin in the stomach and colon and stimulates proliferation. However the effect of CTFP on apoptosis has not been examined. Using the human gastric carcinoma cell line AGS we show that CTFP attenuates apoptosis through a PI3-kinase pathway by stimulating the phosphorylation of Akt leading to sustained increases in the concentrations of Bcl-xL and phosphorylated Bad protein and by reducing caspase 3 activity. The anti-apoptotic effect represents an important potential mechanism for the growth promoting action of CTFP.

  • inactivating cholecystokinin 2 receptor inhibits Progastrin dependent colonic crypt fission proliferation and colorectal cancer in mice
    Journal of Clinical Investigation, 2009
    Co-Authors: Guangchun Jin, Andrea Varro, Xiangdong Yang, Vigneshwaran Ramanathan, Michael Quante, Gwang Ho Baik, Sophie S W Wang, Shanisha A Gordon, D M Pritchard, Arthur Shulkes
    Abstract:

    Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of Progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human Progastrin. Murine deletion of Cck2r abrogated Progastrin-dependent increases in colonic proliferation, mucosal thickness, and β-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of Progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase–like-1 (DCAMKL1), stem cells expressing leucine rich repeat–containing G protein–coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human Progastrin–overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that Progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

Graham J. Dockray - One of the best experts on this subject based on the ideXlab platform.

  • COOH-terminal 26-amino acid residues of Progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo.
    American journal of physiology. Gastrointestinal and liver physiology, 2004
    Co-Authors: Penelope D. Ottewell, Graham J. Dockray, Andrea Varro, Timothy C. Wang, Alastair J.m. Watson, Rod Dimaline, Christopher M. Kirton, David M. Pritchard
    Abstract:

    Transgenic mice (hGAS) that overexpress human Progastrin are more susceptible than wild-type mice (FVB/N) to the induction of colonic aberrant crypt foci (ACF) and adenomas by the chemical carcinog...

  • Progastrin stimulates murine colonic epithelial mitosis after DNA damage
    Gastroenterology, 2003
    Co-Authors: Penelope D. Ottewell, Graham J. Dockray, Andrea Varro, Timothy C. Wang, Alastair J.m. Watson, D. Mark Pritchard
    Abstract:

    Abstract 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 γ-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 γ-radiation. Colonic mitosis was inhibited to almost undetectable levels by 8Gy γ-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 γ-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 γ-radiation. Conclusions: After DNA damage by γ-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.

  • regulation by gastric acid of the processing of Progastrin derived peptides in rat antral mucosa
    The Journal of Physiology, 1997
    Co-Authors: J A Macro, Andrea Varro, G W Bate, C Vaillant, Nabil G Seidah, R Dimaline, Graham J. Dockray
    Abstract:

    1. Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with Progastrin processing in rat antral mucosa. 2. Progastrin processing was studied using a pulse-chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on-line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone-processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3. Cleavage of [3H]- and [35S]-labelled Progastrins at Arg-94-95 or Arg-57-58, and amidation at Phe-92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty-four amino acid amidated gastrin) at Lys-74-75 to give G17 (the seventeen amino acid amidated gastrin), and of G34-Gly to G17-Gly (G34 and G17 with COOH-terminal glycine), was increased 3-fold after treatment with omeprazole for either 1 or 5 days. 4. Approximately 20% of newly synthesized amidated and Gly-extended gastrins were secreted within 240 min of the labelling period in omeprazole-treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5. The amidating enzyme, peptidyglycine alpha-amidating mono-oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6. The main consequence of increased cleavage at Lys-74-75 is the production of G17 and G17-Gly at the expense of G34 and G34-Gly, respectively. The latter have longer plasma half-lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone-processing enzymes cannot account for the rapidity of the changes in cleavage of Progastrin at Lys residues after omeprazole.

  • Processing and proliferative effects of human Progastrin in transgenic mice.
    The Journal of clinical investigation, 1996
    Co-Authors: Timothy C. Wang, Andrea Varro, Theodore J. Koh, R J Cahill, Charles A. Dangler, James G. Fox, Graham J. Dockray
    Abstract:

    Incompletely processed gastrins have been postulated to play a role in growth of the gastrointestinal tract, but few studies have examined the effects of Progastrin on mucosal proliferation in vivo. Human gastrin gene expression and Progastrin processing were therefore studied in transgenic mice containing a human gastrin (hGAS) minigene, and compared to processing in mice bearing an insulin gastrin (INS-GAS) transgene that overexpresses amidated gastrin. Progastrin processing was studied using region-specific antisera and radioimmunoassays, biosynthetic labeling, immunoprecipitation, and HPLC. Proliferative effects due to overexpression of processed and unprocessed gastrin in INS-GAS and hGAS mice, respectively, were determined using routine histology and BrdU incorporation. The pancreatic islets of INS-GAS mice were able to produce carboxyamidated G-17, resulting in a twofold elevation of serum amidated gastrin, marked thickening of the oxyntic mucosa, and an increased BrdU labeling index (LI) of the gastric body. In contrast, livers of adult hGAS mice expressed abundant human gastrin mRNA and human Progastrin but were unable to process this peptide to the mature amidated form, resulting in markedly elevated serum Progastrin levels and normal amidated gastrin levels. Nevertheless, there was a marked increase in the BrdU labeling index of the colon in hGAS mice (LI 7.46+/-1.90%), as well as in INS-GAS mice (LI 6.16+/-1.17%), compared to age-matched, wild type control mice (LI 4.01+/-0.98%, P < 0.05). These studies suggest that incompletely processed gastrin precursors may contribute to colonic mucosal proliferation in vivo.

  • Pathways of processing of the gastrin precursor in rat antral mucosa.
    Journal of Clinical Investigation, 1995
    Co-Authors: Andrea Varro, Svetlana Voronina, Graham J. Dockray
    Abstract:

    Abstract The precursor of the acid-stimulating hormone gastrin gives rise to multiple peptides differing markedly in biological activity, but the relevant biosynthetic pathways are poorly understood. We have used antibodies to amidated gastrins, gastrins with COOH-terminal glycine (Gly) gastrins with COOH-terminal hydroxyglycine (GlyOH) and to the COOH terminus of Progastrin, to immunoprecipitate peptides labeled with [35S]sulfate or [3H]tyrosine during incubation of rat antral mucosa in vitro. Labeled Progastrin was detectable after 30 min of continuous incubation with isotopic precursors, G34 and G34-Gly after 60 min, and G17 and G17-Gly after 120 min. Pulse chase experiments indicated that Progastrin is converted to G34-Gly which then follows one of two pathways: (a) hydroxylation of COOH-terminal Gly and conversion to G34 followed by cleavage yielding G17, or (b) cleavage to G17-Gly. The kinetics of G17-Gly and G17 labeling were similar, suggesting that G17-Gly is a product in its own right, and not simply an intermediate in G17 synthesis. Since the two peptides are reported to have distinct biological activities, they appear to be alternative mature products of Progastrin processing.