1 Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555-1043; 2 Elim Biopharmaceuticals, South San Francisco, California 94080; 3 Department of Physiology, University of Liverpool, Liverpool L69 3BX, United Kingdom; and 4 National Cancer Institute, Fredericksburg, Maryland 21702
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ABSTRACT |
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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 (rhPG1-80) 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 CCK1 and CCK2 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 CCK1-R and CCK2-R on IEC
cells. High-affinity (Kd = 0.5-1.0 nM)
binding sites for 125I-rhPG were discovered on IEC cells
that demonstrated relative binding affinity for gastrin-like peptides
in the order PG COOH-terminally extended G17
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.
progastrin-preferring receptors; confocal microscopy; in vitro growth effects; Gly-gastrin
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INTRODUCTION |
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UNDER PHYSIOLOGICAL CONDITIONS, the completely processed amidated forms of gastrin (G17, G34) are present as the major circulating forms of gastrin and play an important role in acid secretion from parietal cells (41). COOH-terminal amidation of gastrin-like peptides (G17, G34, CCK-8) is required for measuring maximum acid response (20, 41). Early on, it was discovered that gastrins are trophic for gastrointestinal mucosal cells and play an important role in the renewal of colonic mucosa (10, 11, 13, 16). In vitro studies revealed direct proliferative effects of G17 on normal and neoplastic gastrointestinal epithelial cells (6, 16, 36, 46). In vivo growth-promoting effects of G17 on colon cancer cells was also established in rodent models (25, 34). In the late 1980s, we learned that COOH-terminal amidation was not crucial for measuring biological effects (24). Glycine-extended gastrin (G-Gly) was demonstrated to be mitogenic for rat intestinal epithelial cells (IEC) (29), Swiss 3T3 fibroblasts (29), pancreatic cancer cells (22), colon cancer cells (29, 35), and gastric epithelial cells (7) to promote invasiveness of human colon cancer cells (12). More recently, we learned that the full-length precursor molecule, progastrin (PG), may also be biologically active. Transgenic mice overexpressing PG (hGAS) demonstrated hyperproliferation of colonic crypt cells (44), and PG-expressing mice were at a higher risk for developing preneoplastic and neoplastic lesions in the colonic mucosa in response to azoxymethane (32, 33).
On the basis of the above results, we speculated that nonamidated precursor gastrins (PG) may play an aggressive mitogenic role in vivo, resulting in hyperproliferation of colonic crypt cells, thus making them more susceptible to carcinogenic effects of azoxymethane (discussed in Refs. 23, 32, and 33). Factors contributing to proliferative and cocarcinogenic effects of PG in vivo may be due to indirect or direct effects of the precursor peptides on the large intestinal epithelial cells. To investigate possible direct effects of PG on IEC cells, we generated the full-length precursor recombinant human PG (rhPG1-80) molecule and examined direct growth effects of rhPG on IEC cells in vitro. We report for the first time significant growth effects of rhPG on IEC cells in vitro. Surprisingly, rhPG was more effective as a growth factor than the incompletely processed G-Gly peptide and the fully processed G17 peptide; CCK-8 was significantly less effective, suggesting the possibility that IEC cells lack the expression of CCK1 and CCK2 receptors (-R). Using RT-PCR and confocal microscopy with fluorescently-labeled gastrin-like peptides (G17, G-Gly, CCK-8), we confirmed the notion that IEC cells lack the expression of CCK1-R and CCK2-R. Biologically active 125I-rhPG was generated to examine if specific binding sites for the precursor peptide were present on IEC cells. High-affinity binding sites for rhPG were discovered on IEC cells, sites that are apparently specific for PG and gastrin-like peptides. Our studies thus suggest, for the first time, the novel possibility that the high-affinity PG binding sites may directly mediate the growth factor effects of the prohormone PG on IEC cells.
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MATERIALS AND METHODS |
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Generation of the full-length recombinant human
PG1-80 in an Escherichia coli expression system.
The DNA encoding the mature peptide (80 amino acids) of human PG
(corresponding to amino acid residues 22-101 of the
Swiss-PROT:P01350) was synthesized by PCR-mediated assembly of
oligonucleotides as described by Jayaraman and Puccini
(9). The codons were optimized for efficient tRNA usage in
E. coli. The synthesized PG DNA was cloned in-frame into
pET32 (Novagen, Madison, WI) downstream of the thioredoxin gene and
His* tag sequence under the control of a phage T7 RNA polymerase
promoter. The cDNA for the seven-amino-acid recognition site of
recombinant TEV protease (rTEV) was incorporated just before
the PG gene to create a unique cleavage site within the thioredoxin-PG
fusion protein (FP) product. The structure of the construct was
confirmed by DNA sequencing. E. coli strain BL21(DE3)
(Novagen) containing the engineered isopropyl
-D-thiogalactopyranoside (IPTG)-inducible T7 RNA
polymerase gene was transformed with the plasmid thus constructed. The
expression of thioredoxin-PG FP was confirmed by inducing logarithmic
5-ml culture of recombinant BL21(DE3) for 4 h with 1 mM IPTG
(Sigma-Aldrich, St. Louis, MO) and separation of the expressed protein
by 15% SDS-PAGE. The presence of a protein band corresponding to the
molecular mass of thioredoxin-PG (~20 kDa) in samples stimulated by
IPTG indicated the expression of the FP by the recombinant E. coli clones.
Purification of recombinant PG. The FP produced was mostly in a soluble form. The FP was separated, under native conditions and using metal affinity chromatography, from IPTG-stimulated cultures of recombinant BL21(DE3) growing in Luria-Bertani medium (Fisher Scientific, Houston, TX) plus ampicillin (100 µg/ml) by using standard procedures. The FP, bound to the column, was eluted with three column volumes of 1× elution buffer (20 mM Tris, pH 8.0, 400 mM NaCl, 500 mM imidazole). The elute was desalted and subjected to proteolytic cleavage by using recombinant TEV protease (10 U/ml; GIBCO Life Technologies, Carlsbad, CA) at room temperature for 1 h. The reaction mixture, at the end of proteolytic cleavage, was adjusted to 1× washing buffer and once again passed through a nickel-agarose column to remove the TEV protease, uncleaved FP, and all other contaminants bound to the nickel-agarose resin. The flow through and wash containing the released PG was once again desalted through Sephadex G25 by using 20 mM Tris · HCl buffer (pH 7.5) or 100 mM ammonium bicarbonate (for subsequent lyophilization). The desalted protein solution in 100 mM ammonium bicarbonate was lyophilized and resuspended in 10% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) and separated by reverse phase (RP)-HPLC, using the column Vydac 214TP510, C4, 300-Å (10 mm ID × 250 mm length). Gradient of 20-40% ACN in 1% TFA was applied over 60 min. The peak containing PG was lyophilized and characterized by amino acid analysis and mass spectrometry as described below.
Amino acid analysis and mass spectrometry.
Amino acid analysis of the HPLC-purified peptides (nonlabeled and
125I-labeled) was carried out in the University of Texas
Medical Branch (UTMB) Protein Chemistry Laboratory on an Applied
Biosystems (Foster City, CA) model 420H PTC-amino acid analyzer system
as previously described (14, 15). The
NH2-terminal amino acids of purified rhPG were found to be
identical to those of the full-length human PG. HPLC-purified peptides
were further analyzed by mass spectrometry on an Applied
Biosystems Voyager-DE STR matrix-assisted laser-desorption
time-of-flight (MALDI-TOF) instrument in the UTMB Protein Chemistry
Laboratory. The mass spectrophotometric analysis as seen in Fig.
1A confirmed rhPG. rhPG was
further confirmed by Coomassie blue staining of purified rhPG samples,
separated by SDS-PAGE (Fig. 1B). The purified rhPG samples
were additionally subjected to Western immunoblot analysis using
specific anti-PG antibodies as described in the legend of Fig.
1. The standard yield was ~1-4 mg/l of culture. For
large-scale preparations, we used the services of the Protein
Expression Core Facility at UTMB. For all growth and binding assays,
the purified and lyophilized rhPG was dissolved in either serum-free
medium (for in vitro growth assays) or in HBSS (GIBCO, Grand Island,
NY; for binding assays) at the indicated concentrations.
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Cell culture. Rat intestinal cell lines IEC-6 and IEC-18 (American Type Culture Collection, Manassas, VA), which are pluripotent and can be induced to differentiate into either large or small IEC (19), were grown as monolayer cultures in DMEM (GIBCO) supplemented with glutamine (2 nM) and 10% heat-inactivated FCS (Hyclone, Logan, UT) in an atmosphere of 95% air-5% CO2 at 37°C. In a few experiments, rat pancreatic acinar cells (AR42J) were also used and grown in DMEM with low glucose, as described previously (29). The cell lines were regularly monitored for the absence of mycoplasma by using a mycoplasma detection kit (Boehringer-Mannheim, Indianapolis, IN). Stock cultures of cells were subcultured at appropriate intervals to maintain the cells at subconfluent densities. For cell counting and subculturing, the cells were dispersed with a solution of 0.05% trypsin and 0.02% EDTA.
In vitro growth assays. Cell numbers were measured directly by counting the total number of cells with a Coulter electronic particle counter (model Z1), as described previously (28, 29). Briefly, an optimal number of cells (4 × 104 cells) were plated in 35-mm dishes with 2 ml of normal growth medium containing 10% FCS. After 24 h, the medium was changed to serum-free medium and cells were cultured for an additional 24 h. Cells were then treated with various peptides with or without receptor antagonists for 48 h in serum-free medium. At the end of the treatment, cells were disbursed with trypsin-EDTA solution as given above and cells were counted by using either a Coulter electronic counter or with the help of a hemacytometer under light microscopy.
Radiolabeling of rhPG. rhPG was radiolabeled with Na125I (Amersham, Chicago, IL) by using IodoGen, by methods similar to those described previously for G17 (31), with modifications. IodoGen (4 µg/100 µl CH2Cl2) in total volume of 100 µl was aliquoted to the bottom of 12 × 75-mm glass tubes and dried under N2 gas, followed by the addition of 25 µl of 0.25 M phosphate buffer (pH 7.4) and 20 µl of rhPG (2 nmol). Reaction was started by the addition of 10 µl Na125I (1 mCi). The reaction was carried out at room temperature for 4 min and was stopped by the addition of 50 µl of 0.1% TFA. The reaction mixture was loaded on a SepPak column that had been washed with 2 ml of 100% ACN and 10 ml PBS through a 2-ml syringe that was tightly attached to the SepPak column. Reaction mixture absorbed by the SepPak column was washed with ~1 l PBS. Fifty-milliliter aliquots (toward the end of the wash) were counted until very low counts remained in the eluate, as a measure of loss of free 125I. The SepPak column was finally washed with 0.5 ml of 50% TFA 4 times, and counts were measured in each fraction in a gamma counter (model 5500; Beckman, Fullerton, CA). The fractions containing the maximum counts (usually fractions 2 and 3) were then dried in a Savant Speed Vac for 1 h and dissolved in HBSS containing 0.1% BSA and 25 mM HEPES (0.5 ml). The 125I-rhPG solution was then further purified by HPLC as described below.
HPLC purification of biologically active, intact 125I- rhPG. HPLC of labeled and nonlabeled rhPG was carried out on a Vydac analytical C18 RP column (4.6 × 25 cm columns; The Separations Group, Hesperia, CA), connected to a Beckman analytical gradient workstation. The solvents used were 0.1% TFA in water (solvent A) and 0.1% TFA in ACN (solvent B). Peptides were eluted from the column with increasing linear gradients of solvent B at a flow rate of 1 ml/min. The column eluate was monitored at 215 nm, and 1-min fractions were collected and pooled on the basis of absorbance and were subsequently dried in a Savant Speed Vac before amino acid analysis and mass spectrometry. In separations involving radiolabeled peptides, 10-µl aliquots of the 1-min fractions were counted in the gamma counter to identify the peaks corresponding to the iodinated peptides.
The HPLC profile of nonlabeled and 125I-labeled rhPG thus obtained from a representative experiment of more than 20 experiments is shown in Fig. 1, C and D. The retention time of peak 3 was almost identical to that of unlabeled, intact rhPG (Fig. 1, C and D). Peak 3 was also associated with the highest counts. On the basis of total counts associated with peptide in the peak 3 fraction (as determined from spectrophotometric readings of HPLC eluates), specific activity of 125I-rhPG was calculated to be ~3,000 dpm/fmol. To confirm that the 125I-rhPG in peak 3 represented intact molecules, in duplicate experiments rhPG was labeled with Na127I rather than Na125I by using a similar protocol as described above for 125I-rhPG. Peak 3 samples containing 127I-rhPG were subjected to NH2-terminal amino acid analysis and Western blot analysis with specific anti-PG antibodies. NH2-terminal amino acid analysis confirmed that iodinated rhPG was intact at the NH2-terminal end, whereas Western blot analysis confirmed the presence of an intact COOH-terminal end (Fig. 1, E and F). The specificity of the anti-PG antibodies used for detecting and confirming PG-like peptides [COOH-terminally extended G17 (G17-CT) and rhPG] that contain COOH-terminally extended 8 amino acids beyond G17 (Fig. 2) by Western blot analysis is presented in Fig. 1, E and F and described in detail in the legend. G17-CT and G-Gly used in these studies were synthesized by Dr. J. Smith (University of Liverpool). G17 was obtained from Bachem (Torrance, CA). The biological activity of 125I-rhPG in peak 3 was further confirmed by a single-point binding assay as described below. More than 70-90% of 125I-rhPG was determined to be specifically bound to IEC cells (wherein >70-90% of 125I-rhPG bound to the cells was completely displaced by 1,000× excess of nonlabeled rhPG). The presence of full-length, biologically active 125I-rhPG in peak 3 (Fig. 1D) was thus confirmed by NH2-terminal amino acid sequencing, Western blot analysis, and binding assays.
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Relative binding affinity, specificity, and binding affinity of gastrin-like peptides for 125I-rhPG binding sites. For measuring specific binding of the full-length precursor molecule PG, we used fraction 3 (Fig. 1D) of 125I-rhPG. Cells were subcultured in 160-mm flasks and grown to subconfluence. All binding assays were performed 36 or 48 h after cell seeding in culture medium containing 10% FCS and 2% glutamine. Before the start of the binding assays, the cells in culture were washed with HBSS containing 0.1% BSA and 25 mM HEPES (Sigma) and scraped with a rubber policeman into conical tissue culture polystyrene tubes. Cells were centrifuged at 500 g for 5 min and resuspended in HBSS at a concentration of 1-2 × 106 cells/ml. Aliquots (~1.0 ml) of suspended cells in polystyrene tubes were used in all binding assays. All binding assays were conducted essentially as described previously for 125I-G17 (29). Either one of two types of binding assays was performed. For purposes of determining binding affinity, a multipoint (7-12 points) saturation analysis was performed by using increasing concentrations (0.03-1.0 nM) of 125I-rhPG with (specific binding) or without (total binding) 1,000-fold excess of radio-inert homologous ligand. The binding data were analyzed by a Scatchard plot (21). For all other purposes, a single-point saturation analysis was performed by using 1.0 nM of radioactive ligand with or without a 1,000-fold excess of radio-inert ligand in triplicate, as described previously (29). The optimal time, temperature, and pH for measuring the highest number of specific binding sites for 125I-rhPG on IEC-6 and -18 cells was determined as described previously by using single-point assays at different time points, temperatures, and pH (17, 28). The highest number of specific binding sites for 125I-rhPG on IEC-6 cells was measured at 37°C after 0.5-1.0 h of incubation (data not shown). A pH of 6.5 was determined to be optimal (data not shown). Cells in suspension were incubated with 0.5-1.0 nM 125I-rhPG in the presence or absence of increasing concentrations (0.1 nM-10.0 µM) of either the homologous or the heterologous peptide/antagonist. The antagonists L-365260 (L-60) and L-360718 (L-18) (Merck Sharp and Dohme) were added at a final dilution of 0.01% Me2SO in the binding assay tubes, as described previously (29). L-60 is a specific antagonist of CCK2-R, whereas L-18 is a specific antagonist of CCK1-R (29). All control and binding assay tubes received the same concentration of Me2SO. Nonspecific binding was determined in all assays in the presence of 1,000× excess of the nonlabeled peptide. Binding assays were performed at 37°C for 30-60 min at pH 6.5. At the end of the incubation, the cells were pelleted and washed twice with 1 ml of fresh ice-cold HBSS plus 0.1% BSA. Cell pellets were counted for 125I in a gamma counter with ~70% efficiency for 125I. The binding affinity and the total binding capacity of 125I-rhPG for binding IEC cells was determined from a Scatchard plot of specific binding data, obtained by conducting a multiple-saturation-point assay as described above (29). The relative binding affinities of the gastrin-like peptides for binding the specific binding sites for rhPG on IEC cells were determined from a log-dose inhibition of specific binding of 125I-rhPG by various peptides and receptor antagonists, as described previously (29).
Preparation of total cellular RNA from IEC cells, cDNA synthesis, and PCR amplification. Total cellular RNA was isolated as described previously (18) from IEC-6, IEC-18, and AR42J cells. cDNA synthesis from RNA was conducted as described previously (35). For PCR amplification of the cDNA, 10 pmol each of upstream and downstream primers and 1 unit of AmpliTaq DNA polymerase (Perkin-Elmer) were added to the reaction mixture. The PCR reaction was conducted essentially as described previously (29, 35). The upstream and downstream primers were designed on the basis of published cDNA sequences of rat CCK1 (previously CCK-A) and CCK2 (previously CCK-B) receptor genes, to cover different parts of the coding sequences, along the length of the entire cDNA as described in the legend of Fig. 4.
Preparation of fluorescently labeled G17, G-Gly, and CCK
peptides.
The unlabeled peptides were synthesized on a 433A Applied Biosystems
peptide synthesizer equipped with a conductivity monitoring unit using
Fmoc amino acid derivatives. The purity of the peptides was assessed by
RP-HPLC on a C18 column, and the structures were confirmed by MALDI-TOF
mass spectrometry as described previously (38). The
labeling of the peptides with rhodamine green (RG) was
conducted as described previously (4, 39, 40). Briefly, peptides were dissolved in a mixture of ACN and an aqueous solution of
sodium bicarbonate (0.2 M, pH 9.0). A solution of
N-hydroxysuccinimidyl ester of RG (Molecular Probes, Eugene,
OR) in dimethylformamide was added under nitrogen in the dark. The
reaction mixture was stirred at room temperature overnight. A fresh
aqueous solution of hydroxylamine was added to quench the formation of
side-chain-labeling products. After a 2-h reaction, the mixture was
dissolved in 35% aqueous ACN and products were separated by RP-HPLC
using a preparative Waters -Bondapak RP-18 column (19 × 150 mm; eluent: ACN/water/0.05% TFA, 35-70% gradient of ACN, 30 min). Yields were 50-75%. The structures of the products were
confirmed by MALDI-TOF and ion-spray mass spectrometry.
Binding of fluorescently labeled peptides to the IEC cells. Cells were incubated for 1 h at 37°C with the optimal concentrations of the fluorescently labeled peptide (1 nM) in the presence or absence of 100-1,000× excess nonlabeled peptides in neutral red-free DMEM containing 0.1% BSA in a CO2 incubator. Cells were rinsed three times with medium and observed under a microscope using identical parameters for all compounds as described below. In separate experiments, the biological efficacies of fluorescently labeled peptides were examined in growth assays as described above. The bioefficacies of fluorescently labeled peptides were additionally compared with those of the nonlabeled peptides in binding assays as follows. Briefly, radiolabeled 125I-G17 was prepared as described previously (31). IEC cells in suspension were incubated with 1.0 nM 125I-G17 in the presence or absence of increasing concentrations (1.0-1,000 nM) of either the fluorescently labeled peptide or the nonlabeled peptide. Binding assays were performed essentially as described above for measuring the relative binding affinity of peptides for 125I-rhPG binding sites. On the basis of the results of the binding assays, the relative binding affinity of fluorescently labeled peptide was compared with that of the nonlabeled peptides for displacing the binding of 125I-G17 to IEC cells, as described above. In a few experiments, the specificity of the binding of RG-labeled gastrin2-17 (G2-17) and G-Gly peptides to IEC cells was examined as follows. IEC cells in suspension were incubated with either RG-labeled G2-17 or RG-labeled G-Gly in the presence or absence of 100 or 1,000-fold excess of gastrin-like peptides (G17, G-Gly, CCK-8) at 37°C for 30 min as described above. The cells were then washed three times with ice-cold HBSS buffer containing 0.1% BSA. The washed cell pellets were resuspended in 0.5 ml of washing buffer, and the relative levels of fluorescent peptide in each sample were analyzed with a microplate fluorescence reader (Flx 800; Bio-Tek Instruments, Winooski, VT) using the optimal excitation and emission wavelengths for RG derivatives as described below.
Laser scanning confocal microscopy. Cells were grown in Nunc cover glass chamber slides in medium without phenol red. After incubation with fluorescent derivatives (as described above), the cells were observed on a Zeiss inverted LSM 410 laser scanning confocal microscope. Fluorescence of RG derivatives was excited using a 588-nm argon/krypton laser, and emitted fluorescence was detected through a 515- to 540-nm band pass filter. All observations were done using a pinhole of 40, a ×63 oil immersion lens, and an electronic zoom of 3.2 to yield a magnification of ×2,016 of the stored images. Images were stored on an optical disk and analyzed with Zeiss LSM software.
Other analytical procedures. Protein was measured by using the BCA protein assay kit (Pierce).
Statistical analysis. Differences between means were compared with the use of the unpaired Student's t-test, and differences were considered statistically significant at the level of P < 0.05.
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RESULTS |
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In vitro growth effects of rhPG on IEC cells.
Growth effects of increasing concentrations of rhPG (0.1-1,000 nM)
were examined on IEC-6 and -18 cells as described in MATERIALS AND METHODS. Data from 3-6 separate experiments are
presented in Fig. 3. As can be seen from
Fig. 3, rhPG was maximally effective at low concentrations
(0.1-1.0 nM) on both of these cell lines. In previous studies
(28), we had similarly reported significant growth effects
of G17 on IEC-6 cells at concentrations of 0.1-1.0 nM, with 1.0 nM
G17 being the most effective. To examine the relative potency of
different gastrin-like peptides (shown in Fig. 2), we used the optimal
dose of 1.0 nM of each peptide in the presence or absence of
CCK1-R or CCK2-R antagonists, and the results
are presented in Table 1. In all
experiments, the relative growth effects of CCK-8 (a closely related
peptide that shares four amino acids at the COOH-terminal end with G17)
were significantly lower than that of either G17 or rhPG (Table 1). The
growth effects of all peptides, including CCK-8, were slightly to
significantly increased in the presence of CCK2-R
antagonist L-60 (Table 1), indicating that the growth effects of
gastrin-like peptides on IEC cells were mediated via receptor subtypes
other than CCK2-R. L-60 had no growth effect on its own on
IEC cells (data not shown). CCK1-R antagonist had no effect
on the growth-promoting effects of rhPG on IEC cells (Table 1) and had
no effect on the growth of IEC cells in the presence or absence of any
of the gastrin-like peptides (data not shown). These results provided
evidence that the growth-promoting effects of gastrin-like peptides,
including CCK-8, were independent of CCK1-R and
CCK2-R subtypes. The relative growth potency of
gastrin-like peptides on IEC cells was in the order of PG G-CT
G-Gly > G17 > *CCK-8 (* significant
difference; P < 0.05; Table 1). In previous studies,
we had observed that growth effects of G34 were similar to those of G17
(6, 29), suggesting that the extension of G17 at the
NH2-terminal end by 17 amino acids was not effective in
increasing the growth potency of the gastrin-like peptides. However,
extension of G17 at the COOH-terminal end and further extension at the
NH2-terminal end (as in PG) resulted in significantly
increasing the growth potency of the peptide (Table 1).
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Absence of CCK1-R and CCK2-R expression at
the RNA level in IEC cells.
Since pharmacological inhibitors of CCK1-R and
CCK2-R did not inhibit growth effects of either rhPG (Table
1) or other gastrin-like peptides (data not shown), the results
suggested the presence of binding sites/proteins other than
CCK1-R and CCK2-R on IEC cells that may be
mediating growth-promoting effects of PG and other gastrin-like
peptides on these cells. To confirm that the growth effects of
gastrin-like peptides on IEC cells were not mediated by
CCK1-R and CCK2-R subtypes, we first examined
the presence or absence of CCK1-R and CCK2-R in
IEC-6 and IEC-18 cells by RT-PCR, as described in MATERIALS AND
METHODS (and as detailed in the legend of Fig.
4). AR42J cells were used as a positive control for these studies, because they are known to express high concentrations of both CCK1-R and CCK2-R
(29). Representative data from AR42J and IEC-6 cells is
presented in Fig. 4. AR42J cells were positive for the presence of both
CCK1-R and CCK2-R (Fig. 4A), whereas IEC-6
cells were completely devoid of either CCK1-R or
CCK2-R (Fig. 4B). IEC-18 cells were similarly
devoid of either CCK1-R or CCK2-R (data not
shown).
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Binding of fluorescently labeled gastrins to IEC cells by confocal
microscopy.
To further visualize a possible difference in the pharmacokinetics of
binding of gastrin-like peptides to cell lines that are either positive
(AR42J) or negative (IEC) for CCK receptor subtypes, we examined the
binding of fluorescently labeled G17 and G-Gly by confocal microscopy,
as described in MATERIALS AND METHODS. Fluorescently
labeled rhPG was not used in these studies because it was technically
difficult to prepare fluorescently labeled rhPG. NIH/3T3 cells were
used as a negative control that, unlike Swiss 3T3 cells, does not
express CCK1-R and CCK2-R subtypes and does not
respond to gastrins in growth assays, suggesting an absence of binding
sites for gastrin-like peptides (data not shown). Fluorescently labeled
CCK-8 was used for identifying the presence of CCK receptor subtypes
(CCK1-R and/or CCK2-R), since CCK-8
preferentially binds CCK receptor subtypes with over 100 times higher
affinity than it binds all other gastrin receptor subtypes described to
date (reviewed in Ref. 23). Fluorescently labeled G-Gly
was used for identifying the presence of receptors, other than
CCK1-R and CCK2-R, since G-Gly binds CCK-R
subtypes with negligible affinity but binds the putative novel
receptors with high affinity on several cell types (22,
29). Fluorescently labeled G2-17 molecule was also used to
identify specific binding sites for gastrin-like peptides, irrespective
of amidation and nonamidation, since G17 (unlike CCK-8 and G-Gly) binds
not only CCK receptor subtypes with very high affinity but also binds
the novel gastrin receptors with significant affinity (23,
29). Confocal data with the fluorescently labeled peptides is
presented in Fig. 5. A high degree of
G2-17 binding was detected in both IEC and AR42J cells; NIH/3T3
cells that served as the negative control lacked any binding for
G2-17 (Fig. 5A). G-Gly binding, at almost equal levels
to that of G2-17, was detected in IEC cells, but low levels of
G-Gly binding were detected in AR42J cells and no binding was detected
in NIH/3T3 cells (Fig. 5A). Relatively high levels of
G2-17 and G-Gly were bound to IEC cells, but low-to-negligible levels of fluorescently labeled CCK-8 were bound to IEC cells (Fig.
5B). A very high level of labeling with fluorescently
labeled CCK-8 was reported in AR42J cells in previous studies (4,
38-40), confirming that the absence of binding of
fluorescently labeled CCK-8 to IEC cells in the current studies did not
reflect loss of biological activity of RG-labeled CCK-8 molecule. The
biological activity of the RG-labeled peptides was confirmed in growth
assays and binding assays. RG-labeled G2-17 and RG-labeled G-Gly
(1.0 nM) were as effective as the corresponding nonlabeled peptide in
stimulating the growth of IEC-6 cells (data not shown). Importantly, the relative binding affinity of RG-labeled peptides for displacing the
binding of 125I-G17 to IEC-6 cells was similar that of the
corresponding nonlabeled peptide (data not shown). The specificity of
the binding of RG-labeled gastrin molecule to IEC cells was further
confirmed by examining the binding of RG-labeled G2-17
and RG-labeled G-Gly in the presence or absence of increasing
concentrations of nonlabeled gastrin-like peptides to IEC cells, which
was either visualized by confocal microscopy or quantitatively assessed
by using the microplate fluorescence reader as described in
MATERIALS AND METHODS. Representative confocal data for
binding of RG-labeled G2-17 in the presence or absence of 100- to
1,000-fold excess G17 is shown in Fig. 5C. G17 at increasing
concentrations completely displaced binding of fluorescently labeled
G2-17 to IEC cells (Fig. 5C). The percent displacement
of the binding of fluorescently labeled peptides to IEC cells by
increasing concentrations of gastrin-like peptides was also
quantitatively assessed, as described in MATERIALS AND METHODS, and the data are presented in Table
2. At 100- to 1,000-fold excess
concentrations, G17 and G-Gly increasingly displaced the binding of the
corresponding RG-labeled peptide to IEC cells; CCK-8 was once again
relatively ineffective (Table 2).
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Specific binding of 125I-rhPG and relative binding
affinity of rhPG binding sites for gastrin-like peptides on IEC cells.
The presence of specific binding sites for 125I-rhPG on IEC
cell lines was measured by single-point assays as described in
MATERIALS AND METHODS. IEC-6 and IEC-18 cells were positive
for specific binding to 125I-rhPG (Table
3). Displacement of binding of the
radiolabeled peptide in the presence of increasing concentrations of
the nonlabeled gastrin-like peptides (presented as %inhibition of
binding) is shown in Fig. 6. rhPG was
most effective in displacing the binding of 125I-rhPG to
both of the cell lines (Fig. 6). G-Gly and G17-CT (Fig. 2) demonstrated
binding affinities from almost equal to only 10-20% reduced for
PG binding sites on IEC cells (Table 4).
The amidated gastrin, G17, on the other hand, demonstrated almost
30-50% reduced affinity for PG binding sites (Fig. 6 and Table
4). The binding affinity of a closely related peptide, CCK-8, was
significantly reduced to <1% for the PG binding sites on IEC cells
(Fig. 6; Table 4). The relative binding affinity of gastrin-like
peptides for displacing 125I-rhPG binding to IEC cells was
in the order of PG G17-CT
G-Gly > G17 > *CCK-8
(* significantly different; P = 0.05). Thus the data
presented in Fig. 6 and Table 4 indicated the novel possibility that
the full-length precursor molecule has the highest binding affinity for
GP-R subtypes on IEC cells.
|
|
|
Binding affinity (Kd) of 125I-rhPG binding
sites for PG on IEC cells.
Binding affinity in terms of Kd of PG for the
125I-rhPG binding sites was measured by using a multipoint
saturation assay, as described in MATERIALS AND METHODS.
Kd was determined from a Scatchard plot of the
specific binding data as described previously (29). Only
one class of high-affinity binding sites was detected for 125I-rhPG on IEC cells, with binding affinities of
~0.5-1.0 nM (Fig. 7). The binding
affinity values thus measured correlated with the concentrations at
which PG was biologically active, in vitro, on the growth of these
cells (Table 1), indicating that the growth effects measured on these
cells were probably mediated via the high-affinity GP-R binding sites.
|
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DISCUSSION |
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Significant growth-inducing effects of G17 and G-Gly have been previously reported on rat IEC-6 (3, 28, 29, 42, 43, 48). More recently, we and others reported a significant increase in the proliferation and carcinogenesis of large IEC in transgenic mice overexpressing the full-length PG molecule (32, 33, 44), suggesting the novel possibility that the precursor PG peptide may also function as a growth factor for IEC. To examine this novel possibility, we successfully generated the full-length 80-amino acid PG molecule and used the immortalized IEC cell lines as in the in vitro models. We report for the first time direct growth effects of rhPG1-80 on IEC lines in vitro. Baldwin et al. (1) recently generated a slighted truncated rhPG6-80 molecule, which was reported to exert significant growth effects on a temperature-dependent transformed gastric epithelial cell line (YMAC cells). In our studies, we preferentially used nontransformed IEC as the in vitro models rather than transformed intestinal cell lines (such as colon cancer cell lines) since a majority of the transformed cell lines express autocrine growth factors (such as gastrins and IGFs in the case of colon cancer cells; see Ref. 23) and are generally nonresponsive to growth effects of exogenous peptides/growth factors (5, 23, 47).
In previous studies, we and others have, however, reported a significant loss in the proliferative and/or tumorigenic potential of transformed (30) and nontransformed (8) colonic cell lines and in pancreatic cancer cell lines (37) on downregulation of the expression of autocrine gastrins (mainly PG; reviewed in Ref. 23). More recently, we confirmed a potent growth-promoting role of autocrine PG by using specific anti-PG antibodies that completely inhibited the growth of gastrin-dependent human colon cancer cells in vitro to basal levels (unpublished observations). In addition, in preliminary studies (26, 27) we have reported significant growth effects of rhPG on several other gastrin-responsive cells, including AR42J cells and mouse colon cancer cells, and similarly observed that rhPG was more effective than G17 as a growth factor on these cell lines. It thus appears likely that the full-length precursor peptide, PG, can potentially function as a potent growth factor for several cell types in vitro and in vivo.
The method used by us for generating the full-length PG molecule was different from the method used by Baldwin et al. (1). An important difference was that we synthesized the full-length PG cDNA by optimizing the codons to remove possible secondary structure of RNA for efficient usage by E. coli expression system. In addition, we used thioredoxin, rather than glutathione S-transferase (1) as the fusion partner to increase stability and solubility and reduce any toxic effects of the intact peptide. All of these steps, we believe, resulted in the generation of high concentrations of the full-length molecule in a soluble form rather than the generation of a truncated molecule in an insoluble form.
Surprisingly, our results suggested that the full-length PG molecule was more potent as a growth factor for IEC cells than the incompletely processed (G-Gly) and the completely processed (G17) peptides. The biological potency of PG and gastrin-like peptides is known to be changed significantly on the basis of amidation at the COOH-terminal end and the amino acid extension at the COOH-, and perhaps NH2-, terminal ends (20, 23, 41). A case in point is the fact that loss of amidation and the extension of one amino acid at the COOH-terminal end (in the incompletely processed G-Gly peptide) results in reducing the acid-secretory activity of the peptide compared with that of the amidated G17 peptide. However, a further extension of amino acids at the COOH-terminal end (as in the precursor PG-like peptides) results in complete loss of acid secretory activity of the peptide. We were therefore surprised to find that the precursor PG peptide was more effective than the incompletely processed peptides as a growth factor for IEC cells in vitro. More importantly, CCK-8 was the least effective as a growth factor for IEC cells, suggesting that the growth effects of rhPG and all other gastrin-like peptides were in all probability mediated via binding sites other than CCK1-R and CCK2-R.
We used the method of RT-PCR and confirmed for the first time that IEC
cells do not express detectable levels of CCK1-R and CCK2-R transcripts. In spite of an absence of
CCK1-R and CCK2-R in IEC cells, we measured
significant and specific binding of fluorescently labeled gastrin-like
peptide to IEC cells, providing further evidence that gastrin-like
peptides may be mediating growth effects on IEC cells via binding
proteins other than CCK1-R and CCK2-R. We
therefore generated biologically active 125I-rhPG for
examining possible presence of specific binding sites for PG-like
peptides and determined if the incompletely processed and completely
processed gastrin peptides recognize the same binding sites as
125I-rhPG. In the current studies, we report for the first
time the presence of high-affinity PG binding sites, which surprisingly demonstrated the highest affinity for the full-length PG molecule itself and demonstrated reduced affinity for all other gastrin-like peptides in the order of PG G17-CT
G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In preliminary studies, we have similarly measured high-affinity
PG binding sites (Kd ~ 1.0 nM) on several
other target cells, including AR42J cells and mouse and human colon
cancer cells (26, 27). The relative binding affinity of
G17 and CCK-8 for binding the specific 125I-rhPG binding
sites on AR42J cells and on the colon cancer cell lines was also
significantly lower than that of the full-length PG peptide (26,
27).
Importantly, the relative growth potency of gastrin-like peptides mimicked the relative binding affinity of these peptides for 125I-rhPG binding sites on IEC cells in the current studies, strongly suggesting that the high-affinity binding sites may be mediating the growth effects of gastrin-like peptides. The binding studies further suggest that COOH-terminal extension of the G17 molecule, as in PG-like molecules (rhPG, G17-CT), perhaps stabilizes the binding reaction, resulting in a higher binding affinity and higher growth potency of the molecules. Since in previous studies we did not measure a significant difference in the growth potency of G34 and G17 (26, 29), it suggested that the NH2-terminal extension of the G17 peptide by 17 amino acids did not significantly impact the growth potency of these peptides. Thus the extension of G17 at the COOH-terminal end, and perhaps a further extension at the NH2-terminal end (as in PG-like molecules), appears to impart a higher binding affinity and growth potency to the gastrin-like molecules.
The fact that G17 demonstrated a significant binding affinity for rhPG binding sites suggests that the structural attributes present within the G17 molecule may be required for binding the rhPG binding sites (GP-R?). Additional evidence for the latter possibility was obtained from our preliminary cross-linking studies. We had previously reported cross-linking of G17 to several proteins with molecular mass of ~30-35, 45-50, and 72-80 kDa in crude membrane preparations from colon cancer cells (2) and from normal colonic mucosal membranes (17). These studies suggested that proteins in bands 30-35 and 45-50 kDa had a relatively high binding affinity for gastrin-like peptides with poor-to-negligible affinity for CCK-8 (17). More recently, in preliminary studies we examined the relative binding affinity of rhPG for displacing the binding of 125I-G17 to the 30-35 and 45-50 kDa proteins in colon cancer and IEC cells in cross-linking studies. rhPG was significantly more effective than G17 in binding the proteins in bands 30-35 and 45-50 kDa (unpublished observations). In a few preliminary studies, we have also used 125I-rhPG in cross-linking studies and confirmed that rhPG is similarly cross-linked to proteins in bands 30-35, 45-50, and 72-80 kDa; the binding to 72- to 80-kDa proteins, however, was largely nonspecific (unpublished observations).
In summary, in the current studies we have for the first time demonstrated that the precursor PG peptide exerts direct growth effects in vitro on intestinal epithelial cells at physiological "concentrations" and is relatively more potent as a growth factor than the completely processed form of gastrin, G17. High-affinity binding sites that were specific for PG and gastrin-like peptides were measured on IEC cells, which demonstrated negligible affinity for CCK-8. An important finding was that the relative growth potencies of PG and gastrin-like peptides mimic the relative binding affinity of these peptides for the rhPG binding sites, suggesting that these novel binding sites in all probability mediate the growth effects of PG and gastrin-like peptides on IEC cells.
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ACKNOWLEDGEMENTS |
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The secretarial help of P. Gazzoli is acknowledged.
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FOOTNOTES |
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Some of the studies presented in this manuscript have previously been presented in their preliminary form as an abstract (27).
This work was supported by National Cancer Institute Grants CA-60087 and CA-72992 to P. Singh.
Address for reprint requests and other correspondence: P. Singh, Dept. of Anatomy and Neurosciences, The Univ. of Texas Medical Branch, Galveston, TX 77555-1043 (E-mail: posingh{at}utmb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 16, 2002;10.1152/ajpgi.00351.2002
Received 20 August 2002; accepted in final form 7 October 2007.
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