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.

We and others have reported the presence of novel progastrin (PG)/gastrin receptors on normal and cancerous intestinal cells. We had earlier reported the presence of 33-36 kDa gastrin-binding proteins on cellular membranes of colon cancer cells. The goal of the current study was to identify the protein(s) in the 33-36 kDa band, and analyse its functional significance. A carbodiimide crosslinker was used for crosslinking radio-labeled gastrins to membrane proteins from gastrin/PG responsive cell lines. Native membrane proteins, crosslinked to the ligand, were solubulized and enriched by >1000-fold, and analysed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. The peptide masses were researched against the NCBInr database using the ProFound search engine. Annexin II (ANX II) was identified, and confirmed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. As HCT-116 cells express autocrine PG, the in situ association of PG with ANX II was demonstrated in pulldown assays. Direct binding of PG with ANX II was confirmed in an in vitro binding assay. In order to confirm a functional importance of these observations, sense and anti-sense (AS) ANX II RNA-expressing clones of intestinal epithelial (IEC-18) and human colon cancer (HCT-116) cell lines were generated. AS clones demonstrated a significant loss in the growth response to exogenous (IEC-18) and autocrine (HCT-116) PG. We have thus discovered that membrane-associated ANX II binds PG/gastrins, and partially mediates growth factor effects of the peptides.

Progastrin1-80 stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites.

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.

pp60c-Src Kinase mediates growth effects of the full-length precursor progastrin1-80 peptide on rat intestinal epithelial cells, in vitro.

Growth factor effects of precursor forms of gastrins have become evident in recent years. However, intracellular pathways that mediate growth effects of the precursor molecules are not known. In previous studies, we reported an increase in Tyr phosphorylation of pp60(c-Src) in intestinal epithelial cells (IEC) in response to the fully processed form of gastrin [gastrin(1-17) (G17)]. We have now examined whether c-Src kinase is similarly phosphorylated and activated in response to the full-length precursor molecule, progastrin (PG)(1-80), (recombinant human PG) in IEC cells. We found a significant increase in pp60(c-Src) kinase activity in response to both G17 and PG (0.1-1.0 nM), suggesting that growth effects of both the precursor and fully processed gastrin molecules may be mediated via similar pathways. On the other hand, pp62(c-Yes) was not phosphorylated or activated in response to either G17 or PG. To examine whether c-Src kinase mediates proliferative effects of PG, IEC cells were microinjected with anti-Src-IgG and (3)H-thymidine ((3)H-Tdr) uptake of the cells measured. Control cells received nonimmune IgG. The (3)H-Tdr uptake of cells stimulated with 1.0 nM PG was significantly reduced in cells microinjected with anti-c-Src-IgG; control IgG had no effect. In cells stimulated with 1.0% fetal calf serum, microinjection with c-Src-IgG had no effect on (3)H-Tdr uptake. The specificity of the effect was further confirmed by blocking the inhibitory effect of anti-c-Src-IgG with antigenic Src peptide. These results suggest that activation of c-Src kinase likely represents a critical step in mediating proliferative effects of both the precursor and fully processed forms of gastrins on IEC.

Role of autocrine and endocrine gastrin-like peptides in colonic carcinogenesis.

Colon carcinogenesis is a multistep process that involves deletions, mutations, and changes in expression of genes that regulate growth, differentiation, and apoptosis. Hyperproliferation can initiate dysplastic growth, resulting in accumulation of genetic defects and progression of colon cancer. Although genetic instability, because of inheritance of specific genetic defects, plays a dominant role in familial cancers, in the majority of sporadic cancers hyperproliferation is likely to play a permissive role in initiation and progression of the disease. Thus factors that regulate growth, differentiation, and apoptosis are likely to play an important role in colon carcinogenesis. Autocrine gastrins, insulin-like growth factor-II, transforming growth factor-alpha, and endocrine gastrins have been implicated in the tumorigenic potential of colon cancer cells. In this article we focus on the role of endocrine and autocrine gastrins in colon cancer and review recent advances that suggest a role of processing intermediates of gastrin in colon carcinogenesis.

Gastrin gene expression is required for the proliferation and tumorigenicity of human colon cancer cells.

The majority of human colon cancers express the gastrin gene, and a significant percentage bind gastrin-like peptides. However, it is not known if gastrin gene products are physiologically relevant to the growth and proliferation of human colon cancers. To investigate the functional role of gastrin gene expression, we examined the effect of gastrin antisense (AS) RNA expression on the growth and tumorigenicity of colon cancer cells. The full-length human gastrin cDNA was cloned in the AS direction in a retroviral vector under the transcriptional control of human cytomegalovirus promoter. Three representative human colon cancer cell lines that expressed negligible (Colo-205A) to significant (Colo-320 and HCT-116) levels of gastrin mRNA were transfected with either AS or control vectors and subjected to various growth studies in vitro and in vivo. The proliferative and tumorigenic potential of the AS clones from the gastrin-expressing cell lines was significantly suppressed compared to that of the control clones, whereas the growth of Colo-205A-AS cells (the negative control) was similar to that of the Colo-205A-C-cells, indicating the relative specificity of the antitumorigenic effects of AS gastrin RNA expression. We believe that this is the first evidence that supports a possible critical role of gastrin gene expression in the tumorigenicity of human colon cancers that express the gastrin gene. Because > 60-80% of human colon cancers express the gastrin gene, it can be expected that the growth of a significant percentage of these cancers may be critically dependent on the expression of gastrin gene products. Therapeutic measures, such as the AS strategy used in the present study, may therefore prove to be useful in treating human colon cancers in the future.

Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts. Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors.

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)

Simulation of two-electron homogeneous electrocatalysis for steady-state voltammetry at hemispherical microelectrodes.

Expanded space grid digital simulation of second-order, two-electron homogeneous electrocatalysis was extended to slow scan voltammetry at hemispherical microelectrodes. Predictions of the simulations are examined for reversible and quasireversible heterogeneous charge transfer of catalyst for a range of homogeneous catalytic rate constants (k1) and electrode radii. Working curves of catalytic efficiency vs long k1 were generated assuming reacting species with equal diffusion coefficients. As electrode radii in the less than 10-microns range decrease, progressively larger homogeneous catalytic rates are needed to yield analytically significant amplification of limiting currents. Simulations using hemispherical radii of (2/pi)rd can be used to predict catalytic efficiencies for microdisk electrodes with radii rd. Simulated working curves were used to estimate a log k1 of 3.88 +/- 0.55 (M-1 s-1) for electron transfer from the anion radical of 9,10-diphenylanthracene to 4,4'-dibromobiphenyl from steady-state catalytic efficiencies obtained at carbon microdisk electrodes. This value was in good agreement with 3.90 +/- 0.16 M-1 s-1 found previously by cyclic voltammetry.

Somatostatin inhibits VIP-stimulated amylase release from perifused guinea pig pancreatic acini.

We have examined the direct effect of somatostatin (SRIF) on basal and stimulated amylase release from guinea pig pancreatic acini using the in vitro method of continuous perifusion. The optimal conditions of flow rate, chamber size, acinar cell volume per chamber, and period of secretagogue infusion were defined for the perifusion system. The kinetic profile of amylase release in response to cholecystokinin-octapeptide (CCK-8), vasoactive intestinal peptide (VIP), and SRIF was studied. Under optimal conditions, the acini were found to remain equally responsive to an ED50 dose of CCK-8 (0.5-0.8 nM) for 12 h of perifusion. The duration of amylase response to any given dose of CCK-8, given for the optimal period of 5 min, was 80-100 min. The total amylase released minus the basal release divided by 90 min (delta response) in response to the maximum effective (Maxeff) dose of CCK-8 (100 nM) was 14,667 +/- 1,433 U/l (amounting to a 10-fold increase compared with basal values). When compared with the amount of total delta amylase released in response to the Maxeff dose of CCK, the total amylase released in response to the Maxeff doses of SRIF (1 microM) and VIP (10 nM) was 10-21% and 51-59%, respectively. SRIF (100 nM) significantly decreased VIP- (0.1-1.0 nM) stimulated amylase release by 45-70% in the perifusion method of study but had no significant effect on the CCK-stimulated amylase release. This suggests that the perifusion method can be used for investigating the mechanism of SRIF-mediated inhibition of VIP effects on amylase release in an in vitro system.