Human Progastrin Research Articles

The G-protein coupled receptor 56, expressed in colonic stem and cancer cells, binds progastrin to promote proliferation and carcinogenesis.

Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56−/− mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Progastrin Stimulates Colonic Cell Proliferation via CCK2R- and β-Arrestin–Dependent Suppression of BMP2

Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)-partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of β-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by β-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44(+), bromodeoxyuridine+, and NUMB(+) cells, indicating an increase in symmetric divisions of putative cancer stem cells. Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and β-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.

Progastrin-Induced Secretion of Insulin-Like Growth Factor 2 From Colonic Myofibroblasts Stimulates Colonic Epithelial Proliferation in Mice

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.

P53 Gene Mutation Increases Progastrin Dependent Colonic Proliferation and Colon Cancer Formation in Mice

ABSTRACTTransgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.

Inactivating cholecystokinin-2 receptor inhibits progastrin-dependent colonic crypt fission, proliferation, and colorectal cancer in mice

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 beta-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.

Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95

Recombinant human progastrin(6-80) binds two ferric ions with an apparent dissociation constant of 2.2 +/- 0.1 microM [Baldwin (2004) Protein J 23:65-70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin(57-95) were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin(57-95) did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin(57-95) was at least 40-fold weaker than the binding of ferric ions to progastrin(6-80).

Production, Secretion, and Biological Activity of the C-Terminal Flanking Peptide of Human Progastrin

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. 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. 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. 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.

Antral content, secretion and peripheral metabolism of N-terminal progastrin fragments

Objectives In addition to the acid-stimulatory gastrins, progastrin also release N-terminal fragments. In order to examine the cellular content, secretion and peripheral metabolism of these fragments, we developed an immunoassay specific for the N-terminal sequence of human progastrin. Results The concentration of N-terminal progastrin fragments in human antral tissue was 6.7 nmol/g tissue ( n = 5), which was only half of that of acid-stimulatory gastrins (12 nmol/g tissue). Gel chromatography of antral extracts showed that the progastrin fragment 1–35 and 1–19 constitute the major part of the N-terminal progastrin fragments. The basal concentration of N-terminal fragments in normal human plasma was almost 30-fold higher than that of the amidated, acid-stimulatory gastrins (286 pmol/l versus 9.8 pmol/l, n = 26, P < 0.001). In contrast, the concentration of N-terminal fragments in hypergastrinemic plasma was only 2.7-fold higher than the concentration of amidated gastrins (540 pmol vs. 198 pmol/l, P = 0.02). During meal stimulation, the plasma concentrations of N-terminal progastrin fragments and amidated gastrins increased in a correlated manner ( r = 0.97, P = 0.005). The half life for progastrin 1–35 in circulation was 30 min, and a pig model revealed the kidneys and the vasculature to the head as the primary sites of degradation. Conclusion The cellular and circulatory concentration profiles of N-terminal progastrin fragments differ markedly from those of the acid-stimulatory gastrins. The high basal plasma concentrations of N-terminal progastrin fragments cannot be explained by differences in elimination.

COOH-terminal 26-amino acid residues of progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo.

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 carcinogen azoxymethane. We have previously shown significantly increased levels of colonic mitosis in hGAS compared with FVB/N mice after gamma-radiation. To investigate whether the effects of progastrin observed in hGAS colon require the presence of other forms of circulating gastrin, we have crossed hGAS (hg(+/+)) with gastrin knockout (G(-/-)) mice to generate mice that express progastrin and no murine gastrin (G(-/-)hg(+/+)). After azoxymethane, G(-/-)hg(+/+) mice developed significantly more ACF than control G(-/-)hg(-/-) mice (which do not express any forms of gastrin). G(-/-)hg(+/+) mice also exhibited significantly increased colonic mitosis both before and after exposure to 8 Gray Gy gamma-radiation or 50 mg/kg azoxymethane compared with G(-/-)hg(-/-). Treatment of G(-/-)hg(-/-) mice with synthetic progastrin (residues 21-101 of human preprogastrin) or G17 extended at its COOH terminus corresponding to the COOH-terminal 26-amino-acid residues of human preprogastrin (residues 76-101, G17-CFP) resulted in continued colonic epithelial mitosis after gamma-radiation, whereas glycine-extended gastrin-17 and the COOH-terminal tryptic fragment of progastrin [human preprogastrin-(96-101)] had no effect. Immunoneutralization with an antibody against G17-CFP before gamma-radiation significantly decreased colonic mitosis in G(-/-)hg(+/+) mice to levels similar to G(-/-)hg(-/-). We conclude that progastrin does not require the presence of other forms of gastrin to exert proliferative effects on colonic epithelia and that the portion of the peptide responsible for these effects is contained within amino acid residues 76-101 of human preprogastrin.

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.

Metabolism of recombinant progastrin in sheep.

Precursor forms of peptide hormones may be biologically active with effects distinct from the mature end product. Nonamidated progastrin-derived peptides stimulate growth of colonic epithelium and are elevated in the circulation of patients with colorectal carcinomas, whereas the amidated end product is the major regulator of gastric acidity. Using region-specific radioimmunoassays, we here compared the in vitro and in vivo metabolism of recombinant human progastrin-(6-80) and two other nonamidated gastrins, gastrin-17-Gly and Tyr(70)-progastrin-(71-80). Although progastrin-(6-80) was very stable in vitro, both progastrin-(6-80) and gastrin-17-Gly were degraded in vivo. The in vivo data were best fitted by a double-exponential decay curve, and the half-lives for progastrin-(6-80) (t1/2alpha = 5.1 +/- 1.1, t(1/2)beta = 42 +/- 11 min) were significantly (P < 0.05) longer than for gastrin-17-Gly (t(1/2)alpha = 2.2 +/- 0.6, t(1/2)beta = 13 +/- 1 min). Tyr(70)-progastrin-(71-80) was degraded more rapidly. Comparison with amidated gastrins suggests that peptide length, rather than sequence, is the critical determinant of clearance. Progastrin has the clearance characteristics to be considered a circulating hormone.

Biologically Active Recombinant Human Progastrin6–80Contains a Tightly Bound Calcium Ion

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 progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) 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. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) 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 progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) 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.

Overexpression of sense or antisense human gastrin mRNA does not affect proliferation of normal rat kidney fibroblasts

Progastrin-derived peptides have been reported to stimulate mitogenesis in Swiss 3T3 fibroblasts [P. Singh, A. Owlia, R. Espeijo, B. Dai, 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. J. Biol. Chem. 270 (1995) 8429-8438]. The aim of the present study was to determine the generality of these findings, by investigating the effect of endogenous and exogenous progastrin-derived peptides on the proliferation of the normal rat kidney fibroblast cell line NRK. Levels of endogenous progastrin-derived peptides were modified by stable transfection of NRK cells with tetracycline-repressible plasmids containing sequences encoding human gastrin in either the sense or antisense orientation. Expression of sense and antisense gastrin mRNA was demonstrated by reverse transcriptase PCR and by radioimmunoassay, and cell proliferation rates were determined by the colorimetric MTT assay. Sense clones produced full length human progastrin, but significant quantities of glycine-extended or amidated gastrin17 were not detected. Concentrations of endogenous rat progastrin in antisense clones were significantly lower than concentrations in clones transfected with vector only. However no difference in proliferation rate was observed between sense, antisense and vector-transfected clones. No stimulation of proliferation was observed in synchronised untransfected NRK cells after supplementation of media with gastrin17 or gastrin17gly in the concentration range 0.3 to 100 nM. Our results do not provide evidence in support of the hypothesis that endogenous or exogenous progastrin-derived peptides act as growth factors in NRK fibroblasts.

New Consensus Features for Tyrosine O-Sulfation Determined by Mutational Analysis

Tyrosine sulfation is an ubiquitous modification of proteins synthesized along the secretory pathway. It enhances protein-protein interactions and may be necessary for the bioactivity of secreted proteins and peptides. To predict tyrosine sulfation, a consensus has been proposed based on sequence comparisons of known substrates and on in vitro studies using synthetic peptides. This consensus predicts the presence of acidic residues on the amino-terminal side of the target tyrosine as the key feature. Using site-directed mutagenesis, we have examined the role of residues neighboring the sulfation site of an intact protein, human progastrin, in vivo. The results show that the charge of the residue in the amino-terminal position (-1) of the tyrosine is critical and can be neutral or acidic, whereas a basic residue abolishes sulfation. In addition, the degree of sulfation is influenced by the residues in positions -2 and -3. Hence, surprisingly a basic residue in position -2 enhances sulfation. Our data suggest a considerably broader range of substrates for the tyrosylprotein sulfotransferase than hitherto assumed and that the tyrosylprotein sulfotransferase is cell-specifically expressed.

Processing and proliferative effects of human progastrin in transgenic mice.

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.

Determination of sorting related structures in human progastrin

Determination of sorting related structures in human progastrin

An efficient cellular system for mutational analysis of prohormone processing.

A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the corresponding glycine-extended forms. In contrast, the media contained more incompletely processed gastrin forms. This suggests that gastrin was directed to the regulated secretory pathway but that some progastrin products were constitutively secreted. Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insulin cells. Mutational analysis of tyrosine sulfation of gastrin demonstrated that substitution of the uncharged residue carboxy-terminal to the tyrosine with an acidic residue does not increase sulfation in contrast to previous results, where the amino-terminal residue was replaced with an acidic residue. The mutant peptides displayed sulfation-dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones.

Identification of gastrin component I as gastrin-71. The largest possible bioactive progastrin product.

Gastrin component I is the largest hormonally active form of gastrin. In order to determine its structure, we isolated progastrin-derived peptides from normal human antral tissue. A radioimmunoassay specific for sequence 20-25 of human progastrin was developed to monitor the purifications. After four or five steps of reverse-phase chromatography, the peptides were pure and could be identified by a combination of microsequence, amino acid and mass spectral analysis as well as by a library of sequence-specific immunoassays. In addition to intact progastrin 1-80, fragments 1-71, 1-35, 6-35, 20-35, and 20-36 of progastrin were identified. Only the 71-amino-acid peptide contained at its C-terminus the alpha-amidated bioactive site (Trp-Met-Asp-Phe-NH2). This unoheptacontapeptide amide (gastrin-71) corresponds to component I and is the largest possible bioactive product of progastrin. Its structure shows that progastrin is used in its entirety for biosynthesis of active peptides. The occurrence of fragments 6-35, 20-35, and 20-36 demonstrate that antral progastrin is partially cleaved at two monobasic sites (Arg5 and Arg19) in addition to processing at the three C-terminal dibasic sites. The results show that both the N- and C-terminal parts of antral progastrin undergo extensive processing. The results also suggest that progastrin may follow two different processing pathways of which the less trafficked releases gastrin-71.

Phosphorylation of human progastrin by Golgi membrane kinase

Phosphorylation of human progastrin by Golgi membrane kinase

Incomplete processing of progastrin expressed by human colon cancer cells: role of noncarboxyamidated gastrins.

Gastrin is mitogenic for several colon cancers. To assess a possible autocrine role of gastrin in colon cancers, we examined human colon cancer cell lines for expression of gastrin mRNA and various forms of gastrin. Gastrin mRNA was not detected in the majority of colon cancer cell lines by Northern hybridization but was detected in all human colon cancer lines by the sensitive method of reverse transcriptase-polymerase chain reaction (PCR). Gastrin mRNA was quantitated by the competitive PCR method. The majority of cell lines expressed very low levels of gastrin mRNA (< 1-5 copies/cell); only one cell line expressed > 20 copies/cell. The mature carboxyamidated form of gastrin was not detected in any of the cell lines by radioimmunoassay or immunocytochemistry. Results suggested that either gastrin mRNA expressed by colon cancer cells was altered (mutated) or posttranslational processing of progastrin was incomplete. Gastrin cDNA from all the colon cancer cell lines had an identical sequence to the published sequence of human gastrin cDNA. Specific antibodies against precursor forms of gastrin were used, and significant concentrations of nonamidated (glycine-extended) and prepro forms of gastrin were measured in tumor extracts of representative colon cancer cell lines. The presence of precursor forms of gastrin suggested a lack of one or more of the processing enzymes and/or cofactors. Significant concentrations of the processing enzyme (peptidylglycine alpha-amidating monooxygenase) were detected in colon cancer cells by immunocytochemistry. Therefore, lack of other cofactors or enzymes may be contributing to incomplete processing of precursor forms of gastrin, which merits further investigation. Since low levels of gastrin mRNA were expressed by the majority of human colon cancer cell lines and progastrin was incompletely processed, it seems unlikely that gastrin can function as a viable autocrine growth factor for colon cancer cells. High concentrations of glycine-extended gastrin-17 (GG) (> 10(-6) M) were mitogenic for a gastrin-responsive human colon cancer (DLD-1) cell line in vitro. It remains to be seen if GG or other precursor forms of gastrin are similarly mitogenic in vivo, which may then lend credibility to a possible autocrine role of gastrinlike peptides in colon cancers.