1Section of Gastroenterology, Boston University School of Medicine, Boston Medical Center, Boston 02118; 2Gastrointestinal Division, University of Massachusetts Medical School, Worcester, Massachusetts 01605; and 3Department of Oncology and the Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia 20057
Submitted 6 December 2002 ; accepted in final form 17 February 2003
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ABSTRACT |
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gastrin-17; AGS-B cells; proliferation
The polypeptide hormone gastrin is still considered the most potent substance known to stimulate gastric acid secretion (41). However, another biological property attributed to gastrin is its trophic effect on GI mucosa (14, 30). Numerous studies have demonstrated that gastrin stimulates not only growth of normal GI epithelial cells but also malignant cell lines of colorectal, gastric, and pancreatic etiology (2, 7, 18, 19, 21, 31, 39). A recent large epidemiological study by Thorburn et al. (34) found that prolonged hypergastrinemia comprises a risk factor for the development of colorectal cancer. Moreover, studies by Wang et al. (38) using transgenic mice overexpressing amidated gastrin (G-17) demonstrated a synergistic effect between this important peptide and H. pylori infection in the progression of gastric adenocarcinoma. These studies all suggest a potential role for gastrin in the pathophysiology of these malignancies of the GI tract, whereby elevated levels of circulating gastrin could provide a stimulus for the growth of these tumors.
Despite abundant evidence that gastrin may play an integral role in promoting tumor growth in the stomach, as well as malignancies in the GI tract, the precise mechanisms by which gastrin mediates its trophic properties have not been elucidated. In addition, previous studies aimed at determining a potential role for gastrin in gastric carcinogenesis have yielded conflicting results. These studies involved either the measurement of serum gastrin concentrations in patients with gastric cancer or conversely a determination of the number of individuals with hypergastrinemia who have cancer. Unfortunately, these studies have ignored the fact that gastrin is not mutagenic but rather is mitogenic. This important hormone probably does not cause malignancies to arise, but it does stimulate the growth of preexisting gastric tumors. Size appears to comprise a factor in determining the biological behavior of these tumors, and any factor that enhances their growth could thereby incite their malignant degeneration.
One potential mechanism for the development of gastric and other GI
malignancies involves the multifunctional cytoplasmic protein -catenin,
which under normal physiological circumstances plays a major role in cell-cell
adhesion (20). In addition to
its role in cell-cell adhesion,
-catenin is also a pivotal component of
the Wnt/Wingless (Wg) signaling pathway, which plays a key role in an array of
developmental processes (6,
40). Postnatally, mutations of
the adenomatous polyposis coli gene, which occur not only in
80% of
sporadic colorectal carcinomas but also in other GI malignancies, result in a
truncated protein incapable of forming a complex with
-catenin. As a
result, phosphorylation of
-catenin by the inhibitory complex does not
occur, causing
-catenin to accumulate and the Wg pathway to be activated
(20). In addition, the
increased cytoplasmic
-catenin leads to its association with members of
the T cell factor/lymphocyte enhancer binding factor (TCF/LEF) family of
transcription factors (20).
This
-catenin-TCF/LEF complex is translocated to the nucleus, where it
stimulates the transcription of a variety of downstream target genes including
c-myc and cyclin D1
(6,
11,
20,
29,
33). In particular, cyclin D1,
an important cell cycle regulator, has been reported to be upregulated in
human colorectal tumors with altered
-catenin expression
(37). Abnormalities in cyclin
D1 expression constitute one of many possible avenues that could lead to the
development of uncontrolled cell proliferation. It has been proposed that the
regulation of cyclin D1 may be critical to the normal progression of cells
through G1-S transition by involving cyclin D1-dependent regulatory
proteins (27,
42). In addition, in a study
utilizing a mouse model in which hyperproliferation/hyperplasia was induced,
protein levels of both
-catenin and cyclin D1 measured in tissue
extracts were signifi-cantly enhanced
(26). Moreover, a clinical
analysis demonstrated that nearly 50% of tissue samples examined from 70
colorectal cancer patients expressed increased levels of
-catenin and
cyclin D1 (35). These studies
suggest that one of the potential mechanisms of growth may include the
dysregulation of these proteins.
In the present study, we explored the potential roles of -catenin and
cyclin D1 in mediating the trophic effects of gastrin in gastric
adenocarcinoma. We observed that G-17 stimulated the proliferation of AGS-B
cells and incited a concomitant increase in cyclin D1 and
-catenin mRNA
levels. Western blot analysis demonstrated significant changes in cyclin D1,
but not
-catenin, levels in response to the incubation of AGS-B cells
with G-17. Furthermore, G-17 enhanced activity of the full-length cyclin D1
promoter. These observations indicate that gastrin appears to exert its
mitogenic effects on gastric adenocarcinoma, at least in part, through changes
in cyclin D1 expression.
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MATERIALS AND METHODS |
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Immunocytochemistry. Equal amounts of cells were cultured overnight and were serum starved for 24 h, after which they were further incubated in the presence of 10 nM G-17 for 4 h. After a 4-h incubation, the cells were fixed in paraformaldehyde, washed, and stained for F-actin with rhodamine phalloidin (Molecular Probes, Eugene, OR) for 20 min at 37°C. After being thoroughly washed, the cells were mounted on the slide with Gelvatol and observed under a Nikon epifluorescence microscope.
Proliferation assay. Equal amounts of cells (1 x 105 cells/well) were plated before proliferation assay in a 12-well plate. Following a 24-h serum starvation, cells were pulsed with 1 µCi/well (final concentration) [3H]thymidine for 4 h in the absence or presence of G-17 (1 or 20 nM) and harvested. In addition, to examine specificity, 1 µM L-365260 was added to samples treated with 20 nM G-17. At the end of the incubation period, cells were trypsinized and equal amounts of cell lysates were individually dispensed into scintillation vials. After the addition of scintillation fluid, synthesis of [3H]thymidine-incorporated DNA was measured in triplicate by using an automatic Beckman liquid scintillation counter and was normalized against the corresponding cell numbers.
Northern blot analysis. Cells were serum starved for 24 h and
treated with increasing concentrations of G-17 (10100 nM) in the
absence or presence of 1 µM L-365260. After incubating them for different
periods of time, RNA was extracted by using a modification of the
phenol-chloroform method of Chomczynski and Sacchi
(5,
10,
15,
16,
24). Briefly, cells were
scraped in lysis buffer containing 4 M guanidinium isothiocyanate, 0.5%
sarcosyl, 25 mM sodium citrate (pH 7.0), and 100 mM 2-mercaptoethanol and were
extracted by phenol and chloroform. The RNA extracted in the aqueous phase was
then precipitated with an equal volume of isopropanol, after which samples
were washed with 70% ethanol, dried, and resuspended in diethyl pyrocarbonate
water. Equal amounts of total RNA (20 µg) were fractionated by
electrophoresis in a 1.8% agarose gel in the presence of formaldehyde,
transferred to nylon membranes, and hybridized with radiolabeled cyclin D1,
-catenin, and GAPDH probes (Ambion, Austin, TX).
Western blot analysis. To extract cellular proteins from cultured
cells, cells were washed in 1x PBS, directly lysed in the plate at
4°C, and recovered with a cell scraper. Following lysis in
radioimmunoprecipitation assay protein extraction buffer (20 mM Tris, pH 7.5,
140 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 1% Triton X-100, and 10% glycerol)
supplemented with protease inhibitors, cell debris was pelleted at 4°C and
the supernatant was collected for protein quantification. Bicinchoninic acid
protein assay was used to estimate protein concentration according to the
manufacturer's instructions. Equal amounts of protein were diluted with
5x sample loading buffer, boiled, and loaded onto polyacrylamide gels.
Following electrophoresis, gels were transferred onto PVDF membranes and
incubated with primary antibodies to cyclin D1 (Pharmingen, Chicago, IL) and
-catenin (Transduction Laboratories, Lexington, KY). After incubation
with the primary antibodies, membranes were washed thoroughly in TBS-Tween
buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20), and appropriate
secondary antibodies conjugated with horseradish peroxidase were used to
detect the primary antibodies. Immunoreactive bands were visualized by
chemiluminescence in signaling solution (Pierce, Rockford, IL).
Cyclin D1-luciferase assay. For cyclin D1 promoter analysis, AGS-B cells were first transfected in the presence of Lipofectamine (GIBCO BRL) with the full-length cyclin D1 promoter-luciferase (29) and renilla-luciferase constructs for 5 h, followed by an overnight recovery of the cells in serum-containing medium. Cells were then split into six-well plates, serum starved, and treated in triplicate either with vehicle or with different concentrations of G-17 for 4 and 24 h before being harvested for luciferase assays. Equal volumes of lysate (10 µl) were used to assess cyclin D1-dependent firefly luciferase activity by using the luminometer and were normalized to renilla activity. Respective substrates used in these studies were purchased from Promega (Madison, WI). Each transfection was repeated at least three times, and the samples were analyzed in duplicate.
Statistical analysis. Using SAS 8.0, we performed one-way ANOVA to compare various culture conditions, followed by Tukey's procedure for paired comparisons. Statistical significance was assigned if P < 0.05.
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RESULTS |
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G-17 increases proliferation in AGS-B cells. To examine whether gastrin augments cell proliferation, AGS-B cells were grown in the presence of either vehicle alone, with 1 and 20 nM G-17, or with 20 nM G-17 and the gastrin-specific receptor antagonist L-365260. A fourfold increase in [3H]thymidine uptake was evident after 2 days in cells treated with G-17, an effect that was attenuated significantly by incubation in the presence of L-365260 (Fig. 2).
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G-17 induces a concentration-dependent induction of cyclin D1 and
-catenin mRNA in AGS-B cells. To determine the molecular
mechanisms governing the trophic effects of gastrin in gastric adenocarcinoma,
AGS-B cells were used to determine whether cyclin D1, a protein important for
G1-S transition, was involved in the process. In addition,
-catenin, an important coactivator of Wnt signaling pathway (which has
been implicated in increased transcription of cyclin D1), was also analyzed.
Total RNA samples isolated from the AGS-B cells treated with increasing
concentrations of G-17 (10, 20, and 100 nM) in the presence or absence of 1
µM L-365260 were analyzed by Northern blot analysis. In response to the
incubation of AGS-B cells in the presence of G-17, both
-catenin and
cyclin D1 transcripts were increased, effects that were inhibited by L-365260
(Fig. 3), suggesting that
enhanced proliferation induced by gastrin may involve these two trophic
factors.
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G-17 causes time-dependent induction of cyclin D1 and
-catenin mRNA in AGS-B cells. AGS-B cells were incubated with
or without 10 nM G-17, and total RNA was extracted at different time intervals
(1, 4.5, and 24 h). Northern blot analyses were performed by using
radiolabeled cyclin D1 and
-catenin cDNA probes; GAPDH was used as a
loading control (Fig.
4A). When normalized to GAPDH
(Fig. 4B), a
significant increase in the levels of cyclin D1 and
-catenin mRNA was
detected in RNA extracted from cells that were incubated in the presence of
G-17 at the time points examined.
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G-17-mediated induction of cyclin D1 protein levels in the gastric
carcinoma cell line AGS-B. In addition to mRNA induction, to determine
whether gastrin also augments protein levels of these components, Western blot
analysis was performed on protein extracts isolated from AGS-B cells incubated
for 4.5 h in the absence or presence of 10 nM G-17. G-17 induced a significant
induction of cyclin D1, whereas it did not increase -catenin protein
levels at 4.5 h (Fig. 5).
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G-17 significantly increases cyclin D1 promoter activity. To determine whether gastrin directly affects cyclin D1 transcription, cyclin D1 promoter activity was analyzed. AGS-B cells were transfected with full-length (1745) cyclin D1 promoter-luciferase (1745CD1Luc) and renilla-luciferase constructs and were analyzed for the effects of gastrin on promoter activity. Following normalization of cyclin D1-dependent firefly luciferase to renilla luciferase, a fourfold induction of promoter activity was observed in G-17-treated samples, an effect that was abolished with L-365260 (Fig. 6). In a separate study, we examined the effects of G-17 on LEF-1-dependent transcriptional activity in AGS-B cells. In contrast to its effects on cyclin D1 promoter activity, G-17 did not enhance LEF-1 activity (data not shown).
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DISCUSSION |
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In the present study, the incubation of AGS-B cells in the presence of G-17
not only caused morphological changes but also altered the growth
characteristics of the cells. Recent studies by Kirton et al.
(17) have demonstrated that
gastrin stimulates the movement of parietal cells along the gastric gland
axis. Moreover, using AGS cells, Pagliocca et al.
(23) reported that stimulation
of the CCK-2 (gastrin) receptor promoted branching morphogenesis. These
studies, as well as our observations, all suggest that -catenin, a dual
modulator of cell-cell adhesion and oncogenic events, or its downstream
targets, may be involved in mediating these processes. Interestingly, in the
present study, the upregulation of
-catenin transcripts by G-17 did not
extend to the protein level, as measured by Western blot analysis. We also
determined that G-17 did not enhance LEF-1-dependent transcriptional activity
in these cells. The reasons for this divergence are not clear, but several
possibilities exist. One possibility is that the processing of
-catenin
mRNA to protein might be disrupted in this cell line. Another possible
explanation is that the components associated with ubiquitin-related
degradation mechanism, which has been reported to rapidly degrade
-catenin protein (1), or
any inhibitor of
-catenin stability may be enhanced as a result of
stable overexpression of the CCK-2 (gastrin) receptor. Under such conditions,
-catenin would have been susceptible to degradation at the time point
examined in the present studies. Future studies in which the proteasome
machinery is inhibited will be necessary to determine whether
-catenin
is more susceptible to degradation in these cells. It is also possible that a
barely discernible alteration in the total
-catenin protein pool
resulted in undetectable differences due to saturated basal levels. Finally,
the induction of cyclin D1, one of the target genes of
-catenin-dependent transcription, by gastrin might be mediated directly
by Sp1 and/or cAMP-responsive element sites in the cyclin D1 promoter,
independent of any effect on
-catenin expression. Hocker et al.
(12) recently reported that in
AGS-B cells gastrin-dependent transcriptional response of chromogranin A in
enterochromaffin-like cells is mediated by Sp1 and cAMP-responsive element
binding sites in its promoter.
Gastrin-induced upregulation of cyclin D1, an important component involved in G1-S transition, suggests that this peptide may enhance the proliferation of AGS-B cells through constant turnover of cell cycle machinery. Chen et al. (4) recently reported that the inhibition of cyclin D1 through stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells. In one particular study (22), the degree of overexpression of cyclin D1 mRNA correlated with invasive stages of gastric cancer. Furthermore, following H. pylori infection, one of the major risk factors for developing gastric cancer, the hyperproliferative response observed in the gastric mucosa was associated with increased expression of cyclin D1 (28). These studies all indicate that the upregulation of cyclin D1 appears to be associated with an increase in the malignant potential in gastric adenocarcinoma.
In conclusion, the results of the present studies have demonstrated that
gastrin stimulated the proliferation of gastric adenocarcinoma cells and
incited a concomitant increase in -catenin mRNA levels without any
detected increase at the protein level. Western blot analysis of AGS-B cell
protein extracts did demonstrate significant changes in cyclin D1 transcripts
and protein levels in response to the incubation of AGS-B with G-17. These
observations indicate that gastrin appears to exert its mitogenic effects on
gastric adenocarcinoma, at least in part, via changes in cyclin D1 expression.
Further studies will be required to elucidate the precise intracellular and
molecular pathways that mediate the trophic properties of gastrin in gastric
adenocarcinoma.
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ACKNOWLEDGMENTS |
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Present address for B. Rana: Division of Molecular Cardiology, The Texas A&M University System Health Science Center, College of Medicine, Temple, TX 76504.
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FOOTNOTES |
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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.
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REFERENCES |
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