Novel Gastrin Receptors Mediate Mitogenic Effects of Gastrin and Processing Intermediates of Gastrin on Swiss 3T3 Fibroblasts

Abstract

We have reported previously mitogenic effects of gastrin on several immortalized and neoplastic cell lines, including Swiss 3T3 fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for a closely related peptide, cholecystokinin, were recently cloned. These studies were undertaken to investigate if CCK-A- and CCK-B receptors were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3 cells. Receptor antagonists that inhibit the biological effects and binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260 (L60)) receptors were ineffective toward inhibiting the binding and proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18 and L60 demonstrated no binding to the cells, indicating that CCK-A and CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8, gastrin, L18, and L60, on the other hand, demonstrated specific binding to a pancreatic cancer cell line (AR42J cells) (used as a positive control). In cross-linking studies the molecular mass of the major band of gastrin receptors (GR) on Swiss 3T3 cells was determined to be approximately 45 kDa. The mitogenic potency of 0.1-1.0 nM gastrin-like peptides on Swiss 3T3 cells was in the order of G1-17 > or = G1-17-Gly > G5-17 > or = G5-17-Gly > G2-17 > CCK-8-Gly > or = G1-17-Lys > or = CCK-8. The relative binding affinity of the peptides (based on the dose-dependent inhibition of binding of 125I-G1-17 to Swiss 3T3 cells) was similar to the relative mitogenic potency of the peptides as given above. Furthermore, G1-17-Gly was equally effective as G1-17 in displacing the binding of 125I-G1-17 to the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it became evident that the novel gastrin preferring GR, expressed by Swiss 3T3 cells, binds and mediates the mitogenic effects of not only the mature (amidated) forms of gastrin-like peptides but also binds and mediates the mitogenic effects of glycine-extended forms of gastrin-like peptides. Possible mRNA expression of CCK-A and CCK-B receptor subtypes by gastrin-responsive rodent intestinal and fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the methods of Northern blot analysis and reverse transcriptase-polymerase chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum served as positive controls.(ABSTRACT TRUNCATED AT 400 WORDS)

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

“…The AS clones were downregulated for the expression of ANX II by >70%. The total number of specific high-affinity binding sites for PG (measured by a single point assay, as described previously (Singh et al, 1995), were significantly reduced by B65-78% in AS-ANX II clones compared to that in C cells and S clones (data now shown). (b) The representative S 2 and AS 2 clones were stimulated to grow in the presence of optimal concentrations of rhPG (0.1, 1.0 nM).…”

“…For example high-affinity (K d ¼ B1.0 nM) binding sites for PG are present on IEC and Swiss 3T3 cells, that demonstrate a relative binding affinity (RBA) for gastrins in the order of PG>G17>CCK8 (Singh et al, 1995. The binding affinity of PG for colon cancer cells was determined by Scatchard analysis, and the RBA of gastrin-like peptides for the PG binding sites determined.…”

“…The cell lines were obtained and grown as monolayer cultures in the appropriate growth medium, and regularly monitored for the absence of mycoplasma as described previously (Singh et al, 1995(Singh et al, , 1996. Stock cultures of cells were subcultured at appropriate intervals to maintain the cells at subconfluent densities.…”

“…High-affinity binding sites (receptors?) were identified that were distinct from CCK 1 R, CCK 2 R and CCKC-R (Chicone et al, 1989;Narayan et al, 1992;Seva et al, 1994;Singh et al, 1995Singh et al, , 2003Baldwin et al, 2001). These novel binding sites remain unidentified to-date.…”

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

“…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.…”