[Show abstract][Hide abstract] ABSTRACT: Background & aims:
Many colon cancers produce the hormone progastrin, which signals via autocrine and paracrine pathways to promote tumor growth. Transgenic mice that produce high circulating levels of progastrin (hGAS) have increased proliferation of colonic epithelial cells and are more susceptible to colon carcinogenesis than control mice. We investigated whether progastrin affects signaling between colonic epithelial and myofibroblast compartments to regulate tissue homeostasis and cancer susceptibility.
Colonic myofibroblast numbers were assessed in hGAS and C57BL/6 mice by immunohistochemistry. Human CCD18Co myofibroblasts were incubated with recombinant human progastrin (rhPG)(1-80) for 18 hours, and proliferation was assessed in the presence of pharmacologic inhibitors. The proliferation of human HT29 colonic epithelial cells was assessed after addition of conditioned media from CCD18Co cells incubated with progastrin. The effects of the insulin-like growth factor (IGF)-I receptor antagonist AG1024 were investigated in cultured HT29 cells and on the colonic epithelium of hGAS mice compared with mice that did not express transgenic progastrin (controls).
The colonic mucosa of hGAS mice contained greater numbers of myofibroblasts that expressed α-smooth muscle actin and vimentin than controls. Incubation of CCD18Co myofibroblasts with 0.1 nmol/L rhPG(1-80) increased their proliferation, which required activation of protein kinase C and phosphatidylinositol-3 kinase. CCD18Co cells secreted IGF-II in response to rhPG(1-80), and conditioned media from CCD18Co cells that had been incubated with rhPG(1-80) increased the proliferation of HT29 cells. The colonic epithelial phenotype of hGAS mice (crypt hyperplasia, increased proliferation, and altered proportions of goblet and enteroendocrine cells) was inhibited by AG1024.
Progastrin stimulates colonic myofibroblasts to release IGF-II, which increases proliferation of colonic epithelial cells. Progastrin might therefore alter colonic epithelial cells via indirect mechanisms to promote neoplasia.
[Show abstract][Hide abstract] ABSTRACT: CD24 is expressed in the putative stem cells within several tissues and is overexpressed in gastric and colonic adenocarcinomas. Perturbed CD24 expression may therefore alter the response of gastrointestinal epithelia to damage-inducing stimuli that induce cancer. We have investigated the effects of CD24 deletion on gastric responses to Helicobacter felis infection and γ-irradiation using CD24-null mice. Gastric CD24 expression was determined by immunohistochemistry in C57BL/6 mice. Female CD24-null and C57BL/6 mice were infected with H. felis for 6 wk, and inflammation, proliferation, apoptosis, and parietal cell numbers were assessed in gastric tissue sections. Apoptosis and proliferation were analyzed on a cell-positional basis in stomach, small intestine, and colon of CD24-null and C57BL/6 mice following γ-irradiation. Apoptosis was also assessed in HT29 cells following CD24 siRNA transfection. Of CD24-positive cells in the gastric corpus, 98% were H(+)-K(+)-ATPase-expressing parietal cells. CD24-null mice showed more prominent gastric H. felis colonization than C57BL/6 mice but displayed a marked reduction in corpus inflammation, reduced Ki67 labeling, and less gastric atrophy 6 wk following infection. Corpus apoptosis was elevated in CD24-null mice, but this did not increase further with H. felis infection as observed in C57BL/6 mice. More apoptotic cells were found following γ-irradiation in the stomach, small intestine, and colon of CD24-null mice and following CD24 knockdown in vitro. In conclusion, CD24 is expressed in gastric parietal cells, where it modulates gastric responses to H. felis and γ-radiation. CD24 also regulates susceptibility to apoptosis in the distal murine gastrointestinal tract.
Preview · Article · Aug 2012 · AJP Gastrointestinal and Liver Physiology
[Show abstract][Hide abstract] ABSTRACT: Amidated gastrin-releasing peptide (GRP) is the prototypical autocrine growth factor. Nonamidated peptides derived from the C terminus of pro-GRP are also biologically active in colorectal cancer (CRC) cell lines in vitro, via a receptor distinct from the GRP receptor. The aims of this study were to measure the amounts of pro-GRP-derived peptides in human CRC cell lines and tumors, characterize the immunoreactive peptide, and investigate its effect on proliferation in vitro and in vivo. Pro-GRP-derived peptides were quantitated by region-specific ELISA in extracts of five human CRC cell lines and 20 tumors. The immunoreactive material was purified by HPLC and its mass and sequence established by mass spectrometry. The concentration of GRPamide was determined by RIA. Proliferation of DLD-1 cells and murine gastrointestinal mucosa was measured by [(3)H]-thymidine incorporation and mitotic index, respectively. In CRC cell extracts, ELISA for pro-GRP-derived peptides detected 3-152 fmol/10(6) cells. The immunoreactive peptide was purified and identified as pro-GRP42-98. Resected stage III tumors contained significantly less pro-GRP immunoreactivity than stage II tumors, and no amidated GRP was detected in cell lines or tumors. Stable transfection of DLD-1 cells with pro-GRP short hairpin RNA, or treatment with a monoclonal anti-pro-GRP antibody, significantly reduced proliferation. Pro-GRP42-98, pro-GRP47-68, and pro-GRP80-97 significantly stimulated mitosis in colonic, but not small intestinal, mucosa of 10-wk-old mice. We conclude that nonamidated peptides derived from the C terminus of pro-GRP are expressed in significant quantities in CRC cell lines and tumors and stimulate the proliferation of CRC cells and of normal colonic mucosa. Such peptides are attractive targets for novel CRC therapies.
[Show abstract][Hide abstract] ABSTRACT: Introduction Transgenic hGAS mice, which have high circulating progastrin concentrations show increased colonic epithelial proliferation and increased susceptibility to colorectal carcinogenesis. As signalling between colonic epithelial cells and their underlying myofibroblasts is known to regulate homeostasis and the colonic stem cell niche, we hypothesise that progastrin may regulate colonic carcinogenesis by exerting effects upon both myofibroblasts and epithelial cells. We have therefore assessed the numbers of pericryptal myofibroblasts in the colonic mucosa of hGAS mice and have investigated the effects of treating a human colonic myofibroblast cell line with exogenous recombinant progastrin.
Methods hGAS mice were backcrossed onto a C57BL/6 genetic background and epithelial mitotic index and crypt length were assessed in the distal colon on a cell positional basis using H&E sections. The numbers of total and proliferating myofibroblasts were assessed by dual immunohistochemistry with anti-α-smooth muscle actin or vimentin antibodies (to stain myofibroblasts); and anti-Ki67 antibody (to stain proliferating cells). CCD18co human colonic myofibroblasts were treated with 0-10 nM progastrin, gastrin-17 (G-17) or glycine-extended gastrin (G-Gly) for 18 h and proliferation was assessed using a luminescence-based assay. Signalling pathways were investigated by pre-treating cells with inhibitors for the CCK-2 receptor (YM022), Protein kinase C (RO-32-0432), MAP kinase (PD98059) or PI3 kinase (LY294002).
Results Consistent with previous observations in hGAS mice on the FVB/N genetic background the percentage of colonic epithelial mitotic cells was increased by 90% in hGAS mice (p<0.001), which also showed a 22% increase in colonic crypt length (p<0.01). The percentage of colonic mucosal cells which were myofibroblasts was increased in hGAS mice by α-smooth muscle actin and vimentin staining (26% and 25%, respectively, p<0.001); but no Ki67 staining was observed in myofibroblasts. 0.1 nM PG maximally increased proliferation of CCD18co cells by 86% (p<0.001) and this was inhibited by RO-032-0432 (p<0.001). G-17 and G-Gly did not affect CCD18co cell proliferation.
Conclusion Progastrin overexpressing hGAS mice show increased numbers of colonic pericryptal myofibroblasts as well as increased colonic epithelial proliferation. Progastrin also stimulates the proliferation of colonic myofibroblasts in vitro via Protein kinase C pathway signalling. Progastrin therefore appears to increase colonic susceptibility to carcinogenesis by affecting both stromal and epithelial compartments.