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

GASTROENTEROLOGY 2006;131:1463–1474

BASIC–ALIMENTARY TRACT Production, Secretion, and Biological Activity of the C-Terminal Flanking Peptide of Human Progastrin KELLY A. SMITH,* ONEEL PATEL,* SHAMILAH LACHAL,* IAN JENNINGS,‡ BRUCE KEMP,‡ JOHN BURGESS,§ GRAHAM S. BALDWIN,* and ARTHUR SHULKES*

See editorial on page 1638. Background & Aims: Processing of progastrin, the 80amino 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 Cterminal 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. Methods: 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. Results: 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. Conclusions: 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.

G

astrin amide (Gamide) is a gastrointestinal hormone produced in antral and duodenal G cells.1,2 Gamide was initially identified as a major acid secretagogue of the stomach and has also been characterized as a growth factor for the gastric mucosa.2 Gamide is produced from the precursor preprogastrin (101 amino acids in humans), by a series of posttranslational modifications.3 As preprogastrin is translated, the

peptide enters the endoplasmic reticulum (ER) where the 80amino acid peptide, progastrin, is generated by cleavage of the signal peptide from the N-terminus.4 The molecule then enters the Golgi network where it may undergo sulfation and phosphorylation.5 The modified peptide is packaged into secretory vesicles where the molecule is cleaved after dibasic residues to yield peptides with C-terminal glycine (G34gly and G17gly)6 and the 6-amino acid C-terminal flanking peptide (CTFP) (progastrin75– 80). The final processing step involves amidation to yield G34amide or G17amide (Figure 1). Thus, there are 3 distinct regions of the progastrin molecule flanked by dibasic cleavage sites: the N-terminal flanking fragment (progastrin1–35), the central region containing G34gly, and the C-terminal flanking region (progastrin75– 80) (Figure 1). The extent of progastrin processing is dependent on a number of factors, including the site of synthesis, the residence time within secretory vesicles, and the abundance of processing enzymes.5,7 Following the discovery that the progastrin-derived peptides, gastrin glycine (Ggly) and progastrin, are present in normal antrum, gastrinomas, and colorectal cancers (CRC)8 –12 and are biologically active,13,14 these progastrin-derived peptides received substantial attention. The accumulating evidence from in vitro and in vivo studies supports an involvement of these peptides in the accelerated development of gastric and colonic cancers and the maintenance of gastric acid secretion (reviewed by Dockray et al,5 Baldwin and Shulkes,15 Koh and Chen,16 and Smith and Watson17). However little attention has been paid to the CTFP, although early studies performed before the knowledge that nonamidated peptides were biologically active demonstrated that the CTFP was present in the antrum of a number of species, including human, in amounts at least equivalent to Gamide.9,18 –20 In the sheep, we showed that the concentration of antral CTFP was 3-fold higher than Gamide and, furthermore, that CTFP was by far the major circulating form (308 pmol/L vs 35 pmol/L for CTFP and Gamide, respectively).21 However, there have been no similar studies in human, and, in Abbreviations used in this paper: ACN, acetonitrile; CTFP, C-terminal flanking peptide; ER, endoplasmic reticulum; Gamide, gastrin amide; Ggly, gastrin glycine; CRC, colorectal cancer; MAPK, mitogen-activated protein kinase; MEN-1, multiple endocrine neoplasia type 1; RIA, radioimmunoassay. © 2006 by the AGA Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.08.040

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*Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia; ‡St. Vincent’s Institute for Medical Research, Melbourne, Victoria, Australia; and §Department of Diabetes and Endocrinology, Royal Hobart Hospital, Tasmania, Australia

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Hobart, and Royal Melbourne Hospital Human Ethics Committees.

Meal Study Blood samples were collected from 7 healthy adult males following an overnight fast (t ⫽ 0 minutes). After the initial sample collection, individuals were provided with a protein and carbohydrate-rich breakfast (1675 kJ) and blood samples taken at 30, 60, 120, 180, and 240 minutes.

Tissue Extracts Figure 1.

Structure of human progastrin and progastrin-derived peptides. Preprogastrin (101 amino acids) is converted to progastrin (80 amino acids) by removal of the signal peptide. The sequential actions of prohormone convertases converts progastrin to glycine-extended forms and the C-terminal flanking peptide (CTFP, shown by hatched box) by cleavage at paired basic residues. The C-terminus of Ggly is then amidated by peptidyl ␣-amidating monooxygenase. For simplicity, only the 17 amino acid forms of Ggly and Gamide are depicted.

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particular, the concentration of CTFP has not been compared with the other major recognized forms of gastrin (progastrin, Ggly, Gamide) in the normal circulation or antrum or in patients with hypergastrinemia or CRC. The biological activity of CTFP has also not being examined in a systematic fashion as has Ggly.22–25 Here, we report the results of such investigations using a newly developed radioimmunoassay (RIA) for human CTFP and region-specific assays for progastrin, Ggly, and Gamide.

Materials and Methods Peptides and Antagonists The peptides Gastrin-17 gly (Ggly), CTFP, tyr-CTFP (or YCTFP), and tyr-PG71– 80 were custom synthesized by Auspep Pty Ltd (Parkville, Australia) and received at a purity of 89%, 95%, 97%, and 62%, respectively. Gamide was synthesized by Research Plus (Bayonne, NJ) at a purity of 86%. Recombinant progastrin6 – 80 was prepared in our laboratory as reported previously.26 G17 extended at the C terminus (progastrin55– 80 (G17-CTFP) was synthesized at the School of Biological Sciences, University of Liverpool, United Kingdom. The CCK2 receptor antagonist RP 73870A was obtained from RhonePoulenc Rorer (Vitry-Sur-Seine, France).

Patient Samples Antral biopsy specimens were obtained from subjects with nonulcer dyspepsia who were not on any medication that might affect their gastrin status.27 Control plasma was obtained from fasting subjects presenting for colonoscopy who were found negative for CRC and polyps and had no family history of either of these.28 Plasma from 21 CRC patients was collected before tumor resection. The tumors from 10 of these patients were also collected. Plasma was also collected from 18 patients who were about to undergo nongastrointestinal surgery. Plasma from 26 patients with pernicious anemia was retrieved from our gastrin diagnostic service. Forty-two samples from patients with multiple endocrine neoplasia type 1 were obtained from the Endocrinology Laboratory, Royal Hobart Hospital (Hobart, Australia). Human samples were utilized with patient consent under the guidelines of Austin Health, Royal

Frozen tissue was boiled in 5 volumes of water (vol/wt) for 5 minutes. Cells were disrupted by sonification (Branson Ultrasonics, Danbury, CT) for 10 seconds then boiled for an additional 5 minutes. Cell lysates were centrifuged at 3000 rpm at 4°C for 15 minutes, and the supernatant was collected and stored at ⫺20°C.

Plasma Extraction Both untreated and ethanol extracted plasma were assayed for CTFP. For ethanol extraction, 2 volumes of 100% ethanol was added to plasma (vol/vol), vortexed, and centrifuged at 15,000 rpm for 15 minutes at 4°C, and 1-mL aliquots of the supernatant were transferred into tubes and dried down under air streams. Tubes were stored at ⫺20°C.

Radioimmunoassays Radiolabeling CTFP. YCTFP was iodinated by the chloramine T method and then purified by high-performance liquid chromatography (HPLC) with an acetonitrile (ACN) gradient. HPLC purification separated monoiodo- or diiodoYCTFP at approximately 13% and 17% ACN, respectively. The monoiodinated peptide was utilized in binding studies during antibody evaluation and subsequent RIA. CTFP Antibody Development. YCTFP was conjugated to keyhole limpet hemocyanin (Calbiochem, San Diego, CA) with glutaraldehyde at a ratio of 600:1:6000, peptide:hemocyanin:glutaraldehyde. Rabbits were immunized with 1:1 emulsions of conjugate to Freund’s Adjuvant (Complete for the initial and Incomplete for all subsequent injections) at 6-week intervals for 56 weeks. Seven days following immunization, blood was collected by ear vein bleed. One rabbit (rabbit C14) generated an antibody with sufficient affinity for CTFP, as determined by binding with labelled YCTFP. Binding studies with dilutions of antisera C14 indicated that a final concentration of 1:1500 gave a Bound/Free of radiolabelled CTFP of 1. Construction of a standard curve with unlabeled CTFP gave an accurate detection range of between 35 and 1600 fmol/tube and a 50% inhibitory dose (ID50) of 125 ⫾ 4 fmol/tube. Competition with progastrin6 – 80 demonstrated that C14 also detected intact progastrin with equal affinity to CTFP. No immunoreactivity was detected with standard curves of Gamide and Ggly up to concentrations of 100 nmol/mL. C14 cross-reactivity with Gamide and Ggly is therefore ⬍ 0.0001%. Extracts of some tumors and antra were also measured with an antiserum (LW60), which is directed against progastrin66 – 80 and has a 50% immunochemical potency for CTFP.10 Gamide, Ggly, and Progastrin RIA. Gamide, Ggly, and progastrin were measured by RIA using antisera 1296, 7270, and 1137, respectively, as previously reported.12 The Gamide antiserum does not detect Ggly, and the Ggly antisera

does not detect Gamide. Antisera 1137 was raised against human progastrin71– 80 and has a high affinity for intact progastrin and no affinity for C-terminal fragments with less than 8 residues.12

Sizing Chromatography Synthetic peptide standard, antral tissue extracts, plasma, and ethanol-extracted plasma were separated by size on a calibrated Sephadex G-50 superfine column (10 ⫻ 1200 mm; Pharmacia Biotech, Uppsala, Sweden). Samples were eluted with 0.02 mol/L veronal/0.05% BSA/0.005% sodium azide, pH 8.7, at 4°C at a flow rate of 5.5 mL/h in 1-mL fractions. Blue dextran (Pharmacia Biotech) and Na-125I (ICN, Sydney, Australia) were used to determine void and total volumes, respectively.

Reverse-Phase HPLC Progastrin-derived peptides were purified from antral extracts on a C18 ␮Bondapak column (8 mm ⫻ 100 mm, Waters Associates, Melbourne, Australia) connected to a dual pump series 1100 HPLC system (Hewlett Packard, Waldbron, Germany) at a flow rate of 1 mL/min with a gradient from 0% to 70% ACN/0.05% trifluroacetic acid over 45 minutes and washout period of 10 minutes at 70% ACN/0.05% TFA. One-milliliter fractions were collected.

Normal-Phase HPLC Normal phase chromatography was conducted using a TSK gel amide-80 column (2.0 ⫻ 250 mm, Tosoh Corporation, Nanyo, Japan) at a flow rate of 100 ␮L/min with a gradient from 100% to 40% ACN/2.6 mmol/L ammonium acetate, pH 5.5, over 80 minutes.29

Mass Spectrometry Mass spectrometric analysis was conducted using the Matrix Assisted Laser Desorption Ionization Time-of-flight (Maldi-TOF) method on a Qstar mass spectrometer (Applied Biosystems, Foster City, CA).

Immunohistochemistry Sections from the same CRC used for peptide extraction were twice deparaffinized with Histolene (Fronine, NSW, Australia) and rehydrated with alcohol. The sections were permeabilized with 0.1% trypsin in 0.1% CaCl2, pH 7.6, for 1 minute. Endogenous peroxidase activity was quenched using the DAKO EnVision⫹ System-HRP (DAB) kit (DakoCytomation, Carpenteria, CA). Sections were incubated for 5 minutes with the blocking solution at room temperature. Following washes with phosphate-buffered saline (PBS), the sections were incubated with CTFP antiserum (C14, 1:500) at room temperature for 1 hour. The sections were visualized using the DAKO EnVision⫹ System-HRP (DAB) kit according to the manufacturer’s instructions. The sections were also counterstained with hematoxylin (Sigma Aldrich, St. Louis, MO). Following washing, the sections were dehyrated in alcohol and cleared in histolene. Normal rabbit serum was used as negative control.

Growth and Maintenance of Cell Lines The gastric (LIM 1839) and colonic (SW1222, HCT-15) cell lines were a gift from the Ludwig Institute (Parkville, Australia). Cells were maintained in RPMI growth medium containing 10% FBS at 37°C in 5% CO2. IMGE-5 cells, a nontrans-

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formed mouse gastric epithelial cell line,30 were grown in DMEM containing 1 U/mL interferon-␥ and 10% FBS at 33°C in 5% CO2 (permissive conditions). For proliferation and migration assays, upon serum starvation, IMGE-5 cells were shifted to 39°C without interferon-␥ (nonpermissive conditions) and kept under these conditions for the remainder of the experiments.

Proliferation Assays Ninety-six-well plates were inoculated with 2500 cells per well in media containing 10% FBS. Cells were incubated overnight, and serum was starved the following day. After 24 hours, peptides were diluted to the required concentration in media containing 0.5% FBS and 5 ␮Ci/mL tritiated thymidine, and cells were incubated in 100 ␮L for 24 hours for LIM1839 and HCT-15 cells and 48 hours for SW1222 cells. After incubation, cells were washed twice with PBS, trypsinized until detachment, and then harvested with 2 washes with water on glass filters with a cell harvester (Nunc, Roskilde, Denmark). Filters were dissolved in scintillant and ␤-emissions counted.

Migration Assay Wound healing assays were performed as described previously.30 Cells were seeded into 12-well plates and grown to 80%–90% confluence. At 24 hours, a linear wound was made across the cell monolayer using a 20-␮L pipette tip. Cells were washed twice with PBS and peptides (diluted in serum-free DMEM) applied. Wounds were photographed using a Nikon Coolpix camera (model 995) with a microscope coupler at 0, 17, and 24 hours posttreatment. Wound regions were marked and photographed at the same region each time. Six measurements of each wound size were taken per image.

Western Blot Detection of Mitogen-Activated Protein Kinase HCT-15 cells were seeded at 80%–90% confluence into Petri dishes, and serum starved for 24 hours. One hour prior to treatment, sodium vanadate (1 mmol/L) was added. Cells were incubated with 10 nmol/L CTFP for each time point, followed by washes with Tris-buffered saline (TBS) at 4°C then lysed with lysis buffer. Cell lysates were collected, vortexed, and centrifuged at 10,000 rpm for 10 minutes at 4°C. Supernatants were removed, and protein content was quantitated by Bradford assay. Thirty micrograms of cell lysate protein and loading dye were denatured at 95°C for 5 minutes and separated by electrophoresis on stacked 10% and 12% SDS-polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Membranes were blocked in 5% skim milk/0.1% Tween 20/TBS then washed and incubated overnight with primary antibody (either rabbit antitotal mitogen-activated protein kinase [MAPK] or rabbit antiphosphorylated MAPK; Cell Signaling, Danvers, MA), diluted 1:1000 in 5% BSA/0.1% Tween 20/TBS. Membranes were incubated with rabbit anti-rabbit IgG conjugated to alkaline phosphatase (1:1000, Sigma-Aldrich, Castle Hill, Australia). Membranes were washed with 0.1% Tween 20/TBS prior to and after incubation with secondary antibody. MAPK p42/44 was visualized by incubation with BCIP/NBT solution (SigmaAldrich, Castle Hill, Australia) and the intensity of each band quantified by densitometry. The proportion of MAPK phos-

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Table 1. Peptide Concentrations in Human Antral Tissue and Plasma

Peptides CTFP Gamide Ggly Progastrin Ratio CTFP/Gamide CTFP/Ggly CTFP/progastrin

Antrum (nmol/g)

Plasma (pmol/L)

[Plasma] [Antrum]

8.69 ⫾ 2.01 1.97 ⫾ 0.44 0.021 ⫾ 0.005 0.43 ⫾ 0.14

800 ⫾ 18 13 ⫾ 1 25 ⫾ 1 1.2 ⫾ 0.4

0.09 0.007 1.23 0.003

4.4 420 20

62 32 666

NOTE. Values are expressed as mean ⫾ standard error of mean, where n ⫽ 42 for plasma and n ⫽ 6 for antral extracts.

not retained on the column, as was also observed for a synthetic CTFP standard (Figure 2). For 2 of the 6 individuals, a minor amount of immunoreactive material eluted during the 70% ACN wash at the end of the gradient. No intact progastrin was detected in the late eluting peak by RIA. The immunoreactive material from the synthetic standard and the early eluting immunoreactive material of the antral extract had similar absorption profiles with major peaks detected at approximately 60 minutes when subjected to normal phase chromatography (Figure 3). Mass spectrometry of these peaks revealed that both the standard and the antral extract contained species with 2 distinct molecular masses: 664.1 daltons, approximately the theoretic mass of CTFP (663.6), and 686.1 daltons, believed to be a sodium adduct of the peptide. Mass spectrometry of the late eluting immunoreactive material from the reverse-phase HPLC purification showed no specific peaks (data not shown).

phorylated was determined by comparing the amount of phosphorylated and total MAPK at each time point.

Gamide and CTFP were not detected in human fundic extracts. Measurement of progastrin-derived peptides in human antral extracts (n ⫽ 6) demonstrated that CTFP is the most abundant of the peptides assayed, at 8.69 ⫾ 2.01 nmol/g (Table 1). This concentration was more than 4 times greater than the next highest peptide concentration (Gamide, 1.97 ⫾ 0.44 nmol/ g). Using an antiserum that detects intact progastrin but not CTFP (No. 1137),12 progastrin (0.43 ⫾ 0.14 nmol/g) made little contribution to the immunoreactive material, and only a small amount of Ggly was detected (0.021 ⫾ 0.005 nmol/g). Despite vastly different concentrations, linear regression demonstrated that the concentrations of antral progastrin and Ggly were both correlated with antral CTFP concentrations (r2 value ⫽ 0.89; P ⬍ .01 and r2 value ⫽ 0.92; P ⬍ .01, respectively). Conversely, although a trend was observed between Gamide and CTFP concentration (r2 value ⫽ 0.39), the relationship was not significant. There was no correlation between the concentrations of Gamide and Ggly or of Gamide and progastrin. Remeasurement of 4 antral extracts with antiserum LW60, which has a 50% immunochemical potency for CTFP, gave values of a similar order (4.5 ⫾ 2.5 nmol/g) to those measured with the CTFP-specific antiserum. The CTFP concentration measured with LW60 was more than double the Gamide concentration. Separation of antral extracts by reverse-phase HPLC demonstrated that the majority of CTFP immunoreactive material was

80

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Results CTFP Is the Most Abundant ProgastrinDerived Peptide in Human Antral Tissue

100

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Results are expressed as mean ⫾ SEM. Statistical significance was determined by a t test when the number of groups was 2, or by 1-way analysis of variance, followed by a Bonferroni t test, when the number of groups was greater than 2, using the program SigmaStat version 2.03 (Jandel Scientific, San Rafael, CA). Standard curves were fitted using a 4-parameter logistic curve fit with the program SigmaPlot version 8 (Jandel Scientific). Linear regression curves were also fitted using SigmaPlot, and the statistical measures were obtained from linear regression analysis with SigmaStat.

250

CTFP immunoreactivity (pmol/fraction)

Statistical Analysis

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Figure 2.

Purification of progastrin-derived peptides from human antral tissue extracts by reverse phase HPLC. (A) Two nanomoles synthetic CTFP standard. (B and C) Human antral tissue extract from 2 different individuals. CTFP immunoreactivity (measured by RIA with antibody C14) is depicted by shaded bars, intact progastrin immunoreactivity (antibody 1137) is depicted by the dotted line, and the ACN gradient is depicted by the dashed line. A single unretained major peak of CTFP immunoreactivity was found in the column flow through in A and C. In B, a late eluting peak was observed in addition to the unretained peak.

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100

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0.20 80

0.15

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Absorbance 214 nm

0.05

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extracted plasma are presented. As was observed for antral tissue, RIA of plasma from normal individuals showed CTFP to be the most abundant of the progastrin-derived peptides assayed (Table 1). The circulating concentration of CTFP was 800 ⫾ 18 pmol/L, with progastrin (1.2 ⫾ 0.4 pmol/L) making little contribution to the material detected. Gamide and Ggly concentrations were 13 ⫾ 1 pmol/L and 25 ⫾ 1 pmol/L, respectively. CTFP is thus more than 30 times more abundant than Ggly, the next most abundant progastrin-derived peptide in plasma. Linear regression of the concentration of CTFP vs the concentration of Gamide, Ggly, or progastrin in plasma demonstrated no correlation between any of the peptides (data not shown). Interestingly, the plasma to antral ratios of CTFP, Gamide, Ggly, and progastrin concentration were quite different, suggesting differential secretion and/or clearance of each of these peptides. Gamide and progastrin had the lowest plasma to antrum ratio with values of 0.007 and 0.003, respectively. The plasma to antrum ratio for CTFP was 0.09, whereas Ggly, although being the immediate precursor of Gamide, had a much higher ratio of 1.23 (Table 1).

CTFP and Gamide Are Coreleased but Differentially Regulated After Meal Stimulation

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Figure 3.

Purification of CTFP by normal phase chromatography. (A) Synthetic CTFP standard and (B) unretained peak from the reverse phase HPLC from antral extract. The solid line depicts absorbance at 214 nm, and the dashed line depicts the ACN gradient.

Plasma was assayed for CTFP and Gamide after an overnight fast and 30, 60, 120, 180, and 240 minutes after eating a normal breakfast (t ⫽ 0 minutes) (Figure 6). The concentration of Gamide increased with a peak at 30 minutes after commencement of the meal, returned to basal levels at 60 minutes, and then decreased to below basal levels from 120 to 240 minutes. The concentration of CTFP also increased 30 minutes following commencement of the meal, but the eleva-

CTFP Is the Most Abundant ProgastrinDerived Peptide in Human Plasma Coefficient of binding (B/F)

Our initial results using unextracted plasma demonstrated very high concentrations of CTFP (4390 ⫾ 190 pmol/ L). To check for specificity, plasma competition curves, ethanol extraction to remove large proteins, and sizing chromatography were conducted. Dosage curves generated from unextracted and ethanol-extracted plasma samples from a control subject demonstrated that dilutions of both forms of plasma displaced bound radiolabelled CTFP in parallel with the CTFP standard (Figure 4). Recovery of synthetic CTFP added to plasma and ethanol extracted was 64%, a value comparable with those previously reported for progastrin and somatostatin RIA.31,32 The basal CTFP concentration in ethanol-extracted plasma, uncorrected for losses during extraction, was 800 ⫾ 18 pmol/L. Sizing chromatography of untreated or ethanol-extracted plasma suggested that CTFP in plasma comprised an immunoreactive peak eluting in the void volume plus a broad peak eluting late in the profile, with the early part of the latter peak coeluting with synthetic CTFP (Figure 5). The observation that the peak in the void volume was absent from ethanol-extracted plasma indicated that this immunoreactivity was probably the result of either assay interference by albumin or other plasma proteins or of noncovalent binding of CTFP to tissue proteins.33,34 The nature of the CTFP immunoreactivity in the void volume awaits further characterization, and, in the present work, only data from ethanol-

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

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Figure 4.

Plasma displaced label from the anti-CTFP antibody in parallel with a CTFP standard. A CTFP standard curve (solid circles and solid line) was compared with dose response curves of plasma (open diamonds and dashed line) and extracted plasma (shaded triangles and dotted line) from a control subject. The standard curve is expressed as fmol/tube vs B/F, and the dose response curves are expressed as volume of plasma (␮L) vs B/F. Dot points indicate individual data points, and lines represent curves of best fit for each data set generated by the program Sigmaplot as described in the Materials and Methods section.

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centration ⬍50 pmol/L; mean, 30 ⫾ 3 pmol/L) and hypergastrinemic patients (patients with Gamide concentration ⬎50 pmol/L; mean, 146 ⫾ 19 pmol/L) (Figure 7A). As for CTFP, plasma Ggly or progastrin was similar in the normo- and hyper-gastrinemic groups. Because only mild hypergastrinemia was observed in MEN-1 patients, the concentrations of CTFP were also investigated in pernicious anemia patients with more extreme hypergastrinemia (mean Gamide, 471 ⫾ 44 pmol/L; Ggly, 214 ⫾ 25 pmol/L). As was observed in the hypergastrinemic MEN-1 patients, no difference in CTFP concentration was found in the plasma of pernicious anemia patients compared with controls (Figure 7B). CTFP is found in high concentration in colorectal tumors but is not increased in the plasma of CRC patients. The concentration of CTFP was the highest of the 4 peptides assayed in resected CRC with a mean concentration of 19 ⫾ 2 pmol/g. In contrast, the mean concentrations of Gamide, Ggly, and progastrin in tumor extracts were low at 0.07 ⫾ 0.01, 0.03 ⫾ 0.02, and 0.2 ⫾ 0.1 pmol/g, respectively. Remeasurement of 4 CRC extracts with antiserum LW60, which has a 50% immunochemical potency for CTFP, gave values of a similar order (9.5 ⫾ 1.2 pmol/g) to that measured with the CTFP-specific antiserum. BASIC– ALIMENTARY TRACT

840

A

820 800

Figure 5.

Purification of CTFP by sizing chromatography. (A) Elution profile of 2 pmoles CTFP, 1 pmole progastrin, and 1 pmole Gamide standards detected by RIA with antibodies C14 (CTFP, progastrin) and 1296 (Gamide). (B) Human antral tissue extract. (C) One milliliter human plasma from a control subject. (D) One milliliter ethanol extracted plasma from a control subject. The elution profile of CTFP immunoreactivity is represented by solid circles and solid line and Gamide immunoreactivity by open circles and dotted line. CTFP immunoreactivity is detected in the early eluting material in both the standard and the plasma profiles but is not present in the extracted plasma. Plasma CTFP immunoreactivity elutes in a broad peak starting at the elution position of the synthetic standard.

tion was sustained and reached a maximum at 120 minutes before returning to basal levels at 180 minutes.

CTFP Concentration in Patients With Various Gastrointestinal Diseases CTFP concentration is unchanged in the plasma from hypergastrinemic multiple endocrine neoplasia type 1 patients or pernicious anemia patients. The concentrations of CTFP, Gamide, Ggly, and progastrin were measured in the plasma from patients with multiple endocrine neoplasia type 1 (MEN1). No difference was observed in the concentration of CTFP between normogastrinemic patients (patients with Gamide con-

Peptide concentration (pmol/L)

780 760 740 720

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Figure 6. The concentration of CTFP and Gamide in the plasma of individuals following meal stimulation after an overnight fast. (A) The concentration of CTFP (solid circles and solid line) and (B) Gamide (open circles and dotted line) in pmol/l. Expressed as mean ⫾ SEM, where n ⫽ 4 for Gamide and 7 for CTFP. The maximum concentration of CTFP is observed at 120 minutes after feeding, whereas Gamide concentrations reach a maximum earlier at 30 minutes after feeding.

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CTFP Stimulates Cellular Proliferation in Cultured Cells

Peptide concentration (pmol/L)

800

A

700 600 500 200

*

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Gamide

Ggly

PG

CTFP

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To investigate whether CTFP is biologically active, 2 gastric (MKN-28 and LIM1839) and 2 colonic (SW1222 and HCT-15) tumor cell lines were tested for proliferation in response to increasing concentrations of CTFP. Although no effect of CTFP was observed on MKN-28 cells (data not shown), the proliferation of LIM1839 cells was increased in a dosedependent manner, with a maximum stimulation of proliferation of 161% ⫾ 6% at 10 nmol/L CTFP (Figure 9A). Similar results were obtained with SW1222 and HCT-15 cells, with a maximum stimulation of proliferation of 113% ⫾ 3% and 129% ⫾ 7% at 1 nmol/L and 1 ␮mol/L CTFP, respectively (Figure 9B and 9C). CTFP was more effective than 10 nmol/L Ggly in LIM1839 and HCT-15 cells and had a similar proliferative effect in SW1222 cells (Figure 9). The 10 nmol/L dose of Ggly was chosen because it resulted in a maximal response in a variety of cell lines.24,26,30 Significant stimulation of proliferation (145% ⫾ 10%) of the nontransformed gastric cell line IMGE-5 was also observed in response to 10 nmol/L CTFP, and the magnitude of the effect was similar to the effect of Ggly (Figure 10A).

*

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Peptide concentration (pmol/L)

1469

400

* 200

0

Gamide

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(A) The concentration (mean ⫾ SEM) of Gamide, Ggly, progastrin, and CTFP in the plasma of normogastrinemic (solid bars, n ⫽ 14) and hypergastrinemic (shaded bars, n ⫽ 28) MEN-1 patients. Although Gamide was significantly higher in the hypergastrinemic compared with normogastrinemic patients (*P ⬍ .001), no difference was observed in the plasma concentration of CTFP and Ggly between groups. (B) The concentration (mean ⫾ SEM) of Gamide, Ggly, and CTFP in the plasma of control subjects (solid bars, n ⫽ 42) and pernicious anemia patients (shaded bars, n ⫽ 26). Peptide concentrations (in pmol/L) were determined by RIA and are expressed as mean ⫾ SEM. Gamide and Ggly were significantly higher in pernicious anemia patients compared with controls (*P ⬍ .001); however, no difference was observed in the concentration of CTFP.

Figure 7.

Immunohistochemistry of a resected CRC using the CTFP antiserum C14 demonstrated high levels of staining (Figure 8A), which disappeared when the primary antibody was omitted (Figure 8B). The concentrations of CTFP, Gamide, Ggly, and progastrin were also determined in plasma from CRC patients before tumor resection and compared with the corresponding concentrations in plasma from patients admitted for an unrelated surgical procedure. No difference was observed in the concentrations of any of the 4 peptides measured. The concentrations of peptides in plasma (pmol/L) in control and in CRC patients were 730 ⫾ 23 and 700 ⫾ 28 for CTFP, 35 ⫾ 11 and 40 ⫾ 10 for Gamide, 24 ⫾ 3 and 27 ⫾ 3 for Ggly, and 2 ⫾ 0.7 and 3 ⫾ 1.5 for progastrin, respectively.

Figure 8.

(A) CTFP immunostaining in a representative section of a human CRC (original magnification, ⫻200). (B) Control section in which nonimmune rabbit serum was used instead of the primary CTFP antibody.

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Figure 9. CTFP stimulates proliferation in gastric and colorectal carcinoma cell lines. Proliferation of (A) LIM1839, (B) SW1222, and (C) HCT-15 cells was measured by tritiated thymidine incorporation after 24, 48, and 24 hours incubation, respectively, with increasing concentrations of CTFP (100 pmol/L to 1 ␮mol/L; solid bars) and a single concentration of Ggly (10 nmol/L; hatched shaded bar) and compared with control (solid bar). Proliferation was measured in counts (cpm) and is expressed as a percentage of control. Percentages are depicted as mean ⫾ SEM, where n ⫽ 3 experiments (6 replicates per experiment). Statistical significance, compared with control, was determined by a 1-way analysis of variance, followed by a Bonferroni t test, where ⫹P ⬍ .01 and *P ⬍ .001.

CTFP Stimulates Migration of the Gastric Cell Line IMGE-5 To determine whether CTFP affected cell migration, a wound-healing assay was used with the IMGE-5 cells. After 24 hours of treatment with 10 nmol/L CTFP, wound sizes were significantly reduced compared with control (16% ⫾ 1% of the original size compared with a control of 49% ⫾ 5% (*P ⬍ .001;

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Figure 10. CTFP stimulates proliferation and migration of IMGE-5 cells. (A) Proliferation was measured by tritiated thymidine incorporation after 24 hours incubation with 10 nmol/L Ggly or CTFP and is expressed as a percentage of control. Percentages are depicted as mean ⫾ SEM, where n ⫽ 5 experiments (4 replicates per experiment). (B) Images of wounded IMGE-5 monolayers in a migration assay at 0, 17, and 24 hours of treatment with 10 nmol/L Ggly or CTFP. (C) Graphical representation of wound healing assay on IMGE-5 cells. Cellular migration was determined by the extent of wound closure. Wound size at 17 and 24 hours is expressed as percentage of original wound size at t ⫽ 0, and the value at each time point is compared with the corresponding control at the same time point. Percentages are depicted as mean ⫾ SEM, where n ⫽ 3 experiments (6 replicates per experiment). Statistical significance, compared with control, was determined by a 1-way analysis of variance, followed by a Bonferroni t test, where #P ⬍ .05, and *P ⬍ .001. ⫹P ⬍ .05 compared with 24-hour treatment with 10 nmol/L Ggly.

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antrum and in most gastrinomas and CRC.7,9,12,19,35 In the present study, we have shown that the C-terminal hexapeptide of progastrin, progastrin75– 80, (structure confirmed by HPLC and mass spectroscopy), is by far the major stored form of progastrin in antrum and in resected CRC. Similarly, the CTFP circulates at 60-fold higher concentration compared with Gamide. Circulating CTFP is released by a meal in a similar fashion to Gamide but is maintained at higher levels for a longer period. Furthermore, CTFP is not elevated in the plasma of patients with CRC or with hypergastrinemia resulting from gastrin-secreting tumors or pernicious anemia. Finally, CTFP is biologically active as it stimulates the proliferation of gastric and CRC cell lines and the proliferation and migration of a nontransformed gastric cell line and activates MAPK in a CRC cell line. We found that the CTFP concentration in the antrum was approximately 3.5-fold higher than the sum of the concentrations of Gamide, Ggly, and unprocessed progastrin (Table 1). Analysis of antral extracts by sizing and reverse-phase and normal-phase chromatography and by mass spectrometry confirmed that CTFP, progastrin75– 80, is the component responsible for the immunoreactivity detected. We found no evidence

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Figure 11.

CTFP stimulates MAPK phosphorylation in HCT-15 cells. (A) Western detection of 42- and 44-kilodalton forms of MAPK (p42 and p44, respectively) probed for phospho-MAPK (top panel) and total MAPK (bottom panel). Cells were treated with 10 nmol/L CTFP for 0, 3, 5, 10, 15, and 30 minutes. (B) Graphical representation of data from 3 independent experiments, mean ⫾ SEM. The percentage of MAPK phosphorylation was determined as the ratio of optical density of the phosphorylated MAPK band compared with the total MAPK band. MAPK phosphorylation was significantly stimulated in HCT-15 cells after 3 and 5 minutes of treatment with 10 nmol/L CTFP (#P ⬍ .05).

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Figure 10B and 10C). Ten nanomolars Ggly was used as a positive control and, interestingly, CTFP exerted a significantly greater effect on cell migration than the same concentration of Ggly (⫹P ⬍ .05; Figure 10C).

CTFP Stimulates Phosphorylation of MAPK CTFP stimulated activation of the MAPK signal transduction pathway in the colon tumor cell line HCT-15 (Figure 11). The MAPK phosphorylation increased to 50% and 131% of the value observed in untreated cells after 3 and 5 minutes incubation with CTFP, respectively. Phosphorylation of MAPK returned to basal levels after a 10-minute incubation. The CTFP-stimulated increase in MAPK phosphorylation was not blocked by a CCK-2 receptor antagonist (Figure 12). Progastrin6 – 80 and G17-CTFP also stimulated MAPK phosphorylation (Figure 12).

Discussion Since the biological activity of Ggly and progastrin was first described,13,14 there has been intense interest in the physiologic and pathophysiologic roles of these peptides. However, it is not generally appreciated that they constitute relatively small proportions of the final products of the gastrin gene in

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Comparison of effects of progastrin6 – 80, G17-CTFP, and CTFP on MAPK phosphorylation in HCT-15 cells. (A) Western detection of 42- and 44-kilodalton forms of MAPK (p42 and p44, respectively) probed for phospho-MAPK (top panel) and total MAPK (bottom panel). Cells were treated with the indicated peptides with and without the CCK-2 antagonist for 3 minutes. (B) Graphical representation of data from 3 independent experiments, mean ⫾ SEM. The percentage of MAPK phosphorylation was determined as the ratio of intensity of the phosphorylated MAPK band compared with the total MAPK band. MAPK phosphorylation in HCT-15 cells was significantly stimulated by all peptides. The CCK-2 antagonist had no effect alone and did not alter the stimulatory effect of CTFP (#P ⬍ .05).

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that Ser75 was phosphorylated.9 In the only previous measurement of antral CTFP in human, Ggly or unprocessed progastrin was not assayed, but the CTFP concentration was approximately 1.5-fold higher than antral Gamide concentrations.19 We previously reported using an RIA specific for sheep CTFP that the antral CTFP concentration in sheep was 2.5-fold higher than the sum of the concentrations of Gamide, Ggly, and unprocessed progastrin.21 In pig, rat, and dog, the disparities were less marked, but CTFP was always present at a higher concentration than Gamide.18,20,36 Thus, in all studies reported to date, with extracts from a number of species and with a variety of RIAs, the CTFP is the predominant stored species in the normal antrum. We extended our studies to measure CTFP in resected CRC because previous reports have suggested that there is altered processing in gastrin-producing cells of tumors.7,10 –12 Consistent with these observations, the concentration of CTFP was 270-fold higher than Gamide and comprised 98% of total gastrin immunoreactivity of resected CRC. Immunohistochemistry confirmed the very high expression of the CTFP in the resected CRC. Using an antiserum (LW60) generated against progastrin66 – 80 with a 50% immunochemical potency for CTFP, Nemeth et al10 also reported that a CTFP-like peptide was the major gastrin species detected in CRC, although there was only an 8 to 1 ratio between CTFP and Gamide. Using this antiserum (LW60) we obtained a similar result to Nemeth et al.10 Because the sequences of Gamide and Ggly overlap and Gamide, Ggly, and CTFP are derived from the same precursor, the sum of Gamide and its immediate precursor, Ggly, should be approximately equal to the CTFP concentration. There are a number of possible explanations for the excess CTFP immunoreactivity detected in antrum and CRC. In gastrinomas, the ratio of CTFP to Gamide ranges from 0.4 to 18 with some of the excess being attributed to the CTFP extended at the amino terminus by G34 and G17.19 However, these extended forms comprise less than 5% in normal antrum10,19 and indeed were not detected in the present study. The presence of nonimmunoreactive metabolites offers a possible explanation for the apparent excess of CTFP because variable amounts of N-terminal fragments of G17, such as G14, or of G17 extended by Gly-Arg or Gly-Arg-Arg at the C-terminus have been reported in the antrum and in gastrinomas.8,35,37,38 Alternative explanations that require testing include the presence of nonimmunoreactive breakdown products of Gamide or Ggly, heterogeneity of the secretory granules, differences in processing within secretory granules, and preferential sorting of CTFP into secretory granules.5,39,40 Interestingly, Goetze et al recently reported that, in human, the antral concentration of N-terminal progastrins (assayed with an antiserum directed against progastrin1–10) was only ⬃60% of Gamide and ascribed this difference in part to endoproteolytic cleavage after Arg5 of progastrin.41 The present study is the first to report the circulating concentrations of CTFP in man. Consistent with the findings in the antrum, plasma CTFP was by far the predominant circulating form of progastrin-derived peptides. Because cross-reactivity with the high concentrations of proteins and globulins present in plasma is a potential problem with plasma RIA,34 a number of control experiments and chromatographic characterizations were performed. First, the plasma was ethanol extracted to remove large interfering proteins. Recovery of synthetic CTFP added to plasma and then ethanol extracted was 64%, a value

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comparable with previously reported RIAs.31,32 Ethanolextracted plasma also inhibited binding of label to antibody C14 in parallel with the synthetic standard with no depression in maximum binding. Sizing chromatography of ethanolextracted plasma revealed a broad peak, elution of which began in the position of synthetic CTFP and of immunoreactive CTFP from antral extracts. Furthermore, ethanol extraction removed the immunoreactive peak that eluted at the void volume. The plasma concentration of CTFP in normal subjects was 800 ⫾ 18 pmol/L, 60-fold higher than Gamide. The proportion of stored CTFP peptide being secreted was also high, with a plasma to antral concentration ratio of 0.09 compared with 0.007 for Gamide (Table 1). The lower ratio for Gamide is probably a reflection of the facts that the half-life for Gamide disappearance is longer than for CTFP21 and that most of the secreted Gamide comes from newly synthesized gastrin, with stores of gastrin being of lesser importance.42 The relationship between synthesis, storage, and secretion for CTFP and Ggly, which both have higher plasma to antrum ratios than Gamide, has not been explored. Gamide and Ggly circulate at similar concentrations, but Gamide is found in vast excess in the antrum (Table 1). The high concentration of circulating CTFP is also observed in sheep using a sheep-specific CTFP RIA with a basal concentration of 308 pmol/L, 10-fold higher than plasma Gamide.21 The previous report of a high concentration of N-terminal progastrin fragments in human plasma (286 pmol/L, 30-fold higher than Gamide)41 supports our finding that multiple gastrin peptides derived from a single progastrin precursor can circulate at quite different concentrations. Meal simulation studies suggest that there is cosecretion of Gamide and CTFP. However, as Gamide concentrations diminish, coinciding with the predicted increase in acid secretion,43 elevated CTFP concentrations are sustained for at least 4 times longer than Gamide. A similar effect was reported for N-terminal progastrin fragments in which concentrations remained elevated following meal stimulation, despite a decrease in Gamide concentration.41 No difference in CTFP concentrations is observed between normo- and hypergastrinemic MEN-1 patients with gastrinsecreting tumors. The CTFP to Gamide ratio is therefore decreased in these patients. Because these MEN-1 patients had only mild hypergastrinemia, pernicious anemia patients with more extreme hypergastrinemia (derived from antral sources) were also investigated. No difference in CTFP concentration between control subjects and pernicious anemia patients was observed either. This is an interesting finding, considering that the 2 forms of hypergastrinemia originate from distinct sources.44 Similarly, Goetze et al41 recently reported that the concentration of N-terminal progastrin in hypergastrinemic patients was not greater than in control patients. Whether this shift in ratios is indicative of a change to constitutive secretion or of differential regulation remains to be determined. In any case, it is apparent that CTFP concentrations do not necessarily reflect Gamide concentrations. The observation that patients with CRC did not have elevated circulating concentrations of CTFP, despite the high concentrations of CTFP in the resected CRC, demonstrates that there is not an obligatory relationship between content and secretion. It is now clear that progastrin and nonamidated intermediates such as Ggly are biologically active (reviewed by Dockray et al,5 Baldwin and Shulkes,15 Koh and Chen,16 and Smith and

Watson17). These peptides stimulate the proliferation of various cell lines and activate several signal transduction pathways, such as the MAPK pathway. Studies with transgenic animals overexpressing these peptides or after peptide infusion have shown that nonamidated gastrins can stimulate colonic proliferation, accelerate the development of colon cancer, and potentiate gastric acid secretion.16 However, as shown in the present study, the amount of Ggly and progastrin in antrum, in resected CRC, and in the circulation is very low compared with CTFP. In contrast to Ggly and progastrin, there have been few studies examining the biological activity of CTFP. C-terminal extended gastrin but not CTFP induced histamine release in rats with 1% the potency of Gamide,45 and short-term (5 minutes) intravenous infusions of CTFP into rats did not stimulate gastric acid secretion.46 A recent study using a single peptide injection demonstrated that continued colonic epithelial mitosis after ␥ radiation requires G17 extended through the CTFP (G17-CTFP) but that CTFP by itself was not mitogenic.47 The limitations of these studies are that they are short-term and investigate very specific peptide functions. The present study provides in vitro evidence for the biological activity of CTFP using 3 different measures. First, CTFP stimulated proliferation in 2 gastric and 2 colonic cell lines. Second, CTFP stimulated migration of the IMGE-5 gastric cell line in a wound-healing assay. It is noteworthy that CTFP had similar or greater effect than Ggly for both proliferation and migration, although formal dose response curves are required to substantiate this observation. The third measure of biological activity was activation of MAPK. The MAPK pathway is triggered by a variety of receptors to transduce signals for the initiation of growth and differentiation.48 The utilization of the MAPK pathway by both Gamide and Ggly is well recognized.30,49 Treatment of HCT-15 CRC cells with CTFP produced a rapid increase in MAPK phosphorylation that was not dependent on the CCK-2 receptor. Other nonamidated gastrin peptides, progastrin6 – 80 and G17-CTFP, also stimulated MAPK phosphorylation. Importantly, CTFP also stimulated proliferation in these cells. A similar transient activation of MAPK was previously shown to be sufficient for the proliferative effect of Gamide and Ggly in the murine gastric cell line IMGE-5.30 Dissection of the signal transduction pathways will require assessment in a variety of cell lines because the transduction pathways triggered by amidated and nonamidated gastrins appear to be cell-type specific.30,50 There are a number of implications from these studies. First, there are major differences in the amounts of progastrinderived peptides stored in the antrum, and these differences are not necessarily reflected in the circulating concentrations. Further studies on the regulation of antral progastrin synthesis, processing, and secretion are required to explain these differentials. Second, with the demonstration that CTFP is biologically active in CRC cell lines and is the major gastrin component in resected CRC, studies on the role of CTFP in the development of CRC assume greater importance. Infusion of CTFP or administration of CTFP antibody to murine models of CRC should be informative, but it will be critical to determine the nature of the receptor involved because the present work suggests that it is not the CCK2 receptor. A final implication is that many of the studies using full-length progastrin or Ggly in transgenic animals, animal models of colon cancer, and cell

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lines should also be performed with the C-terminal flanking peptide because this is the major expressed form. To conclude, the high and regulated expression and secretion of CTFP in healthy subjects and in patients with a variety of diseases, combined with the evidence for biological activity of CTFP, demonstrates that CTFP is not an inactive metabolite of progastrin processing but a bioactive peptide with potential roles in the normal and diseased gastrointestinal tract. References 1. Gregory RA, Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;46:103– 114. 2. Dockray G, Dimaline R, Varro A. Gastrin: old hormone, new functions. Pflugers Arch 2005;449:344 –355. 3. Rehfeld JF, Bundgaard JR, Goetze JP, Friis-Hansen L, Hilsted L, Johnsen AH. Naming progastrin-derived peptides. Regul Pept 2004;120:177–183. 4. Varro A, Dockray GJ. Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A. Biochem J 1993;295:813– 819. 5. Dockray GJ, Varro A, Dimaline R, Wang T. The gastrins: their production and biological activities. Annu Rev Physiol 2001;63: 119 –139. 6. Varro A, Voronina S, Dockray GJ. Pathways of processing of the gastrin precursor in rat antral mucosa. J Clin Invest 1995;95: 1642–1649. 7. Rehfeld JF, van Solinge WW. The tumor biology of gastrin and cholecystokinin. Adv Cancer Res 1994;63:295–347. 8. Del Valle J, Sugano K, Yamada T. Progastrin and its glycineextended posttranslational processing intermediates in human gastrointestinal tissues. Gastroenterology 1987;92:1908 – 1912. 9. Varro A, Desmond H, Pauwels S, Gregory H, Young J, Dockray GJ. The human gastrin precursor. Characterization of phosphorylated forms and fragments. Biochem J 1988;256:951–957. 10. Nemeth J, Taylor B, Pauwels S, Varro A, Dockray GJ. Identification of progastrin derived peptides in colorectal carcinoma extracts. Gut 1993;34:90 –95. 11. Van Solinge WW, Nielsen FC, Friis-Hansen L, Falkmer UG, Rehfeld JF. Expression but incomplete maturation of progastrin in colorectal carcinomas. Gastroenterology 1993;104:1099 –1107. 12. Ciccotosto GD, McLeish A, Hardy KJ, Shulkes A. Expression, processing, and secretion of gastrin in patients with colorectal carcinoma. Gastroenterology 1995;109:1142–1153. 13. Seva C, Dickinson CJ, Yamada T. Growth-promoting effects of glycine-extended progastrin. Science 1994;265:410 – 412. 14. Wang TC, Koh TJ, Varro A, Cahill RJ, Dangler CA, Fox JG, Dockray GJ. Processing and proliferative effects of human progastrin in transgenic mice. J Clin Invest 1996;98:1918 –1929. 15. Baldwin GS, Shulkes A. Gastrin, gastrin receptors, and colorectal carcinoma. Gut 1998;42:581–584. 16. Koh TJ, Chen D. Gastrin as a growth factor in the gastrointestinal tract. Regul Pept 2000;93:37– 44. 17. Smith AM, Watson SA. Review article: gastrin and colorectal cancer. Aliment Pharmacol Ther 2000;14:1231–1247. 18. Desmond H, Dockray GJ, Spurdens M. Identification by specific radioimmunoassay of two novel peptides derived from the Cterminus of porcine preprogastrin. Regul Pept 1985;11:133– 142. 19. Pauwels S, Desmond H, Dimaline R, Dockray GJ. Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay. J Clin Invest 1986;77:376 –381. 20. Varro A, Nemeth J, Bridson J, Lee C, Moore S, Dockray GJ. Processing of the gastrin precursor. Modulation of phosphory-

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lated, sulfated, and amidated products. J Biol Chem 1990;265: 21476 –21481. Paterson AC, Lockhart SM, Baker J, Neumann G, Baldwin GS, Shulkes A. Identity and regulation of stored and secreted progastrin-derived peptides in sheep. Endocrinology 2004;145:5129 – 5140. Aly A, Shulkes A, Baldwin GS. Short term infusion of glycineextended gastrin(17) stimulates both proliferation and formation of aberrant crypt foci in rat colonic mucosa. Int J Cancer 2001; 94:307–313. Chen D, Zhao CM, Dockray GJ, Varro A, Van Hoek A, Sinclair NF, Wang TC, Koh TJ. Glycine-extended gastrin synergizes with gastrin 17 to stimulate acid secretion in gastrin-deficient mice. Gastroenterology 2000;119:756 –765. Hollande F, Imdahl A, Mantamadiotis T, Ciccotosto GD, Shulkes A, Baldwin GS. Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line. Gastroenterology 1997;113:1576 –1588. Koh TJ, Dockray GJ, Varro A, Cahill RJ, Dangler CA, Fox JG, Wang TC. Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation. J Clin Invest 1999;103:1119 –1126. Baldwin GS, Hollande F, Yang Z, Karelina Y, Paterson A, Strang R, Fourmy D, Neumann G, Shulkes A. Biologically active recombinant human progastrin(6-80) contains a tightly bound calcium ion. J Biol Chem 2001;276:7791–7796. Zavros Y, Paterson A, Lambert J, Shulkes A. Expression of progastrin-derived peptides and somatostatin in fundus and antrum of nonulcer dyspepsia subjects with and without Helicobacter pylori infection. Dig Dis Sci 2000;45:2058 –2064. Paterson AC, Leeding KS, Bach LA, Baldwin GS, Macrae FA, Shulkes A. More about: prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J Natl Cancer Inst 2000;92:1947– 1950. Schlichtherle-Cerny H, Affolter M, Cerny C. Hydrophilic interaction liquid chromatography coupled to electrospray mass spectrometry of small polar compounds in food analysis. Anal Chem 2003; 75:2349 –2354. Hollande F, Choquet A, Blanc EM, Lee DJ, Bali JP, Baldwin GS. Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells. J Biol Chem 2001;276:40402– 40410. Reasbeck PG, Burns SM, Shulkes A. Calcitonin gene-related peptide: enteric and cardiovascular effects in the dog. Gastroenterology 1988;95:966 –971. Paterson AC, Baldwin GS, Shulkes A. Metabolism of recombinant progastrin in sheep. Am J Physiol Endocrinol Metab 2002;283: E449 –E456. Rehfeld JF, Schwartz TW, Stadil F. Immunochemical studies on macromolecular gastrins: evidence that “big big gastrins” are artifacts in blood and mucosa, but truly present in some large gastrinomas. Gastroenterology 1977;73:469 – 477. Rehfeld JF. How to measure cholecystokinin in tissue, plasma and cerebrospinal fluid. Regul Pept 1998;78:31–39. Jensen S, Borch K, Hilsted L, Rehfeld JF. Progastrin processing during antral G-cell hypersecretion in humans. Gastroenterology 1989;96:1063–1070. Varro A, Nemeth J, Bridson J, Lonovics J, Dockray GJ. Modulation of posttranslational processing of gastrin precursor in dogs. Am J Physiol 1990;258:G904 –G909.

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37. Power DM, Dimaline R, Balaspiri L, Dockray GJ. A novel gastrinprocessing pathway in mammalian antrum. Biochim Biophys Acta 1988;954:141–147. 38. Kothary PC, Mahoney WC, Vinik AI. Identification of gastrin molecular variants in gastrinoma syndrome. Regul Pept 1987;17: 71– 84. 39. Rahier J, Pauwels S, Dockray GJ. Biosynthesis of gastrin. Localization of the precursor and peptide products using electron microscopic-immunogold methods. Gastroenterology 1987;92: 1146 –1152. 40. Bundgaard JR, Birkedal H, Rehfeld JF. Progastrin is directed to the regulated secretory pathway by synergistically acting basic and acidic motifs. J Biol Chem 2004;279:5488 –5493. 41. Goetze JP, Hansen CP, Rehfeld JF. Antral content, secretion and peripheral metabolism of N-terminal progastrin fragments. Regul Pept 2005;133:47–53. 42. Dockray GJ, Hamer C, Evans D, Varro A, Dimaline R. The secretory kinetics of the G cell in omeprazole-treated rats. Gastroenterology 1991;100:1187–1194. 43. Richardson CT, Walsh JH, Hicks MI, Fordtran JS. Studies on the mechanisms of food-stimulated gastric acid secretion in normal human subjects. J Clin Invest 1976;58:623– 631. 44. Lehy T, Roucayrol AM, Mignon M. Histomorphological characteristics of gastric mucosa in patients with Zollinger-Ellison syndrome or autoimmune gastric atrophy: role of gastrin and atrophying gastritis. Microsc Res Tech 2000;48:327–338. 45. Sandvik AK, Dockray GJ. Biological activity of carboxy-terminal gastrin analogs. Eur J Pharmacol 1999;364:199 –203. 46. Desmond H, Varro A, Young J, Gregory H, Nemeth J, Dockray GJ. The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum. Regul Pept 1989;25:223–233. 47. Ottewell PD, Varro A, Dockray GJ, Kirton CM, Watson AJ, Wang TC, Dimaline R, Pritchard DM. COOH-terminal 26-amino acid residues of progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo. Am J Physiol Gastrointest Liver Physiol 2005;288:G541–G549. 48. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A 1993;90:5889 –5892. 49. Yassin RR. Signaling pathways mediating gastrin’s growth-promoting effects. Peptides 1999;20:885– 898. 50. Aly A, Shulkes A, Baldwin GS. Gastrins, cholecystokinins and gastrointestinal cancer. Biochim Biophys Acta 2004;1704:1–10. Received December 21, 2005. Accepted July 12, 2006. Address requests for reprints to: Arthur Shulkes, DSc, University of Melbourne Department of Surgery, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia. e-mail: [email protected]; fax: (613) 9458 1650. Supported in part by grants from the National Health and Medical Research Council of Australia and the Austin Hospital Medical Research Foundation. The authors thank Professor Joe Proietto, Austin Health, and Dr Venkat Parameswaran, Royal Hobart Hospital, for provision of the plasma from the meal study and patients with multiple endocrine neoplasia, respectively; Dr Adrienne Paterson and Mildred Yim for some of the gastrin RIAs; CURE/Digestive Diseases Research Center Antibody/RIA Core, Los Angeles, for antibody 1296 (for Gamide RIA); Professor Jens Rehfeld, Rigshospitalet, Denmark, for antibody 7270 (for Ggly RIA); and Professors Dockray and Varro for the kind donation of antibody LW60 (CTFP assay) and G17-CTFP peptide. K.A.S.’s current address is Hubrecht Laboratory, Netherlands Institute of Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.