Progastrin_1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

Abstract

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG(1-80)) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK(1) and CCK(2) receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK(1)-R and CCK(2)-R on IEC cells. High-affinity (K(d) = 0.5-1.0 nM) binding sites for (125)I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG >or= COOH-terminally extended G17 >or= G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Intestinal expression of mutant and wild‐type progastrin significantly increases colon carcinogenesis in response to azoxymethane in transgenic mice

“…15 Colonic mucosae were homogenized in 0.5 mL lysis buffer (Promega, Madison, WI) and centrifuged at 13,000g for 30 minutes (Beckman GS-15R centrifuge; Beckman Instruments, Fullerton, CA). The supernatant then was removed with a Pasteur pipette, and the protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL).…”

“…Colonic mucosal protein samples (20 g) from transgenic mice and their wild-type littermates were processed for WIB using anti-PG antibodies (L516) at a 1:10,000 dilution, as is described elsewhere. 15 The full-length (9-kilodalton [kD]) PG peptide was present in significant quantities in colonic mucosal samples from heterozygous transgenic (Tg/ϩ) and homozygous transgenic (Tg/Tg) mice but was absent in colonic mucosal samples from wild-type (ϩ/ϩ) mice (Fig. 2C).…”

“…Therefore, the relative concentrations of mutant hPG and wild-type hPG in plasma samples were determined by WIB analysis, as is described elsewhere. 42 In brief, 0.1-2.5 ng of recombinant hPG (rhPG) 15 was dot-blotted on polyvinylidene fluoride membranes and then processed for WIB analysis. A standard curve was generated using the densitometric readings for the recombinant peptide.…”

“…8,9 In recent years, it has been discovered that nonamidated gastrins (e.g., G-Gly) exert potent growth factor effects in vitro on rat intestinal cells, 10 3T3 fibroblasts, 10 pancreatic carcinoma cells, 11 and colon carcinoma cells. 10,12 More recently, the growth factor effects of the full-length precursor peptide, PG, were demonstrated in vitro [13][14][15] and in vivo 16 -20 on small and large intestinal mucosal cells and on gastrointestinal carcinoma cell lines. Azoxymethane (AOM)-induced colonic tumors in rats, as well as a significant percentage of adenomas (Ads) and adenocarcinomas (AdCas) in humans, express the gastrin gene.…”

Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells

“…PG, GG and G17 exert proliferative effects on normal and cancerous gastrointestinal (GI) cells in vitro and in vivo (Wang et al, 1996;Hollande et al, 1997;Koh et al, 1999;Baldwin et al, 2001;Brown et al, 2003;Singh et al, 2003;Ottewell et al, 2005). PG and GG exert cocarcinogenic effects on colon carcinogenesis in response to azoxymethane (Singh et al, 2000a, b;Aly et al, 2001;Cobb et al, 2004).…”