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Carcinogenesis |
Academic Unit of Cancer Studies, Department of Surgery, University of Nottingham, Nottingham NG7 2UH, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Elevated serum gastrin levels can be a feature of a number of clinical conditions, including ZES3 (4, 5, 6, 7) and PA (8) , but possibly more importantly as a result of infection with Helicobacter pylori (9 , 10) and after the administration of proton pump inhibitors (11 , 12) .
In humans, increased proliferation of colonic crypts has been confirmed in patients with PA and ZES (13) . However, the majority of epidemiological studies performed to date in patients with sustained hypergastrinemia have failed to show an enhanced risk of colorectal cancer. In patients with PA, although conflicting studies have been reported, the majority show that colorectal cancer incidence is not increased (14 , 15) . This has also been shown in patients with ZES (16) . This failure may be attributed to lack of control of confounding factors, such as H. pylori in the control groups. A recent study, in which H. pylori infection was controlled for, showed that hypergastrinemia is associated with a 3.3-fold increase in the relative risk of developing colorectal cancer (17) . It is conceivable, therefore, that hypergastrinemia may promote progression through the adenoma-carcinoma sequence. This hypothesis is strengthened by the preliminary findings of Smith and Watson (18) that human adenomas of all sizes and grades express an isoform of the cholecystokinin B/gastrin receptor.
Thus, the aim of the present study was to evaluate the effect of serum hypergastrinemia induced by omeprazole, a proton pump inhibitor that increases serum gastrin levels 2–4-fold (11 , 12 , 19) , on colonic neoplastic progression in the multiple intestinal neoplasia APCMin-/+ mouse model of APC.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Entry of Mice into Therapeutic Studies.
After results from the PCR analysis were obtained,
APCMin-/+-positive mice were randomized into
each experimental group on an ongoing basis because of the limited
number of mice positive for the Min mutation that are
generated from established breeding colonies. Mice were randomized into
groups on a litter basis with unequal numbers being a result of the
distribution of Min positive and negative mice.
Experiment 1: Effect of Omeprazole-induced Hypergastrinemia on
Adenoma Number and Size and Animal Survival.
An initial study was performed to determine the effect of
hypergastrinemia on tumor burden and survival of
APCMin-/+ mice.
APCMin-/+ mice 4 weeks of age were recruited to
the following groups: group 1, oral vehicle for omeprazole [PBS (pH
7.2)]; group 2, omeprazole (75 mg/kg in a single oral dose;
n = 10).
Tumor size was measured by image analysis by use of Leica Qwinn image processing and analysis system run on a Leica Q5001W PC.
Experiment 2: Effect of Omeprazole-induced Hypergastrinemia on
Survival with or without Immunization with the Gastrin Immunogen,
Gastrimmune.
APCMin-/+ mice 4 weeks of age were recruited to
the following groups: group 1, oral vehicle for omeprazole + control immunogen (n = 20); group 2,
omeprazole (75 mg/kg in a single oral dose) + control
immunogen (n = 20); group 3, omeprazole + Gastrimmune (n = 16).
Gastrimmune is a gastrin immunogen composed of the NH2-terminal nine amino acids of gastrin-17 linked via a peptide spacer to diphtheria toxoid. Treatment with Gastrimmune generates antibodies that neutralize the amidated and glycine-extended forms of gastrin-17 (Aphton Corporation, CA; Ref. 21 ). A rat-specific form of the immunogen was formulated at 2 mg/ml and injected s.c. into mice in 100-µl volumes at week 4, and at three-week intervals thereafter. The rat sequence used within the immunogen differs only by one amino acid from the mouse sequence, and antibodies raised by rat Gastrimmune previously have been shown to neutralize mouse gastrin (data not shown). Control mice receiving immunogen constituents only were given the formulation without the active peptide.
Experiment 3: Effect of Preimmunization with Gastrimmune
Immunogen on the Survival Effect Observed with Omeprazole Treatment.
APCMin-/+ mice 4 weeks of age were recruited to
the following groups: group 1, vehicle + control immunogen
(n = 22 mice); group 2, omeprazole (75
mg/kg) + control immunogen (n = 30
mice); group 3, omeprazole + Gastrimmune
(n = 30 mice); group 4, vehicle + Gastrimmune (n = 18 mice).
Gastrimmune was injected 2 weeks prior to omeprazole. (The latter was administered from week 6 when antibodies against Gastrimmune were known to be detectable.)
Termination of APCMin-/+ Mouse Experiments.
At the end-stage of their disease, APCMin-/+
mice bleed from their multiple polyps, giving rise to anemia, which was
initially confirmed by hematocrit measurements [median
hematocrit, 0.136 (range, 0.058–0.153) compared with 0.385 (range,
0.381–0.391] in wild-type C57/Bl6 mice. Twelve days after the onset
of anemia, mice were terminally anesthetized by a 300-µl injection
containing 0.315 ng/ml Hypnorm and 5 ng/ml Hypnovel (Jannsen-Wickham,
Bucks, United Kingdom, and Roche-Lewis, East Sussex, United
Kingdom) by an experienced, independent observer who was blinded
to the treatment groups. Anemia previously has been validated as a
consistent end point for APCMin-/+ mouse
termination (20)
. After termination, the GI tracts of the
APCMin-/+ mice were removed, and the numbers of
visible tumor nodules in both the small and large bowel were counted
with the aid of a dissecting microscope. The mucosa was fixed in formal
calcium (40% formaldehyde, 10% CaCl2 in
distilled water) prior to embedding in paraffin wax for histological
analysis.
The United Kingdom Coordinating Committee for Cancer Research Guidelines were adhered to throughout all animal experimentation.
Measurement of the Proliferation Index of the GI Mucosa.
One h before termination, mice received i.p. injections of 10 mg/kg
BrdUrd (Sigma). After paraffin-embedding, 4-µm sections were
stained with H&E to confirm correct orientation of the crypts and with
an antibody directed against BrdUrd (mouse monoclonal, used at 1:500
dilution; Dakopatts).
Two experienced observers, blinded to the treatment groups, counted the number of positive nuclei in the crypts from both the small and large intestine. Ten whole crypts from different areas of the colonic mucosa and the small intestinal mucosa of each mouse were identified where possible. The number of total positive nuclei within the epithelial cells was counted, together with the total number of cells per crypt. This was repeated with sections from 4–11 animals. The total number of cells per crypt in the large or small intestine were consistent both within and between experimental animals.
For scoring of the polyps, each section from each mouse was cut at the same depth (2 mm), and each polyp was analyzed by choosing a representative low-power field and by using image analysis to assess the area of BrdUrd staining as a percentage of the total tumor tissue. For assessment of the proliferation of adenoma and carcinoma samples that were taken from the large and small intestines, image analysis was performed by the use of the Leica Qwin image processing and analysis system. BrdUrd staining was specifically measured and expressed as a function of total tumor tissue area. Intraobserver variation, in which different fields of the same section were assessed, was 5.6%, and interobserver variation was 7.5%.
Histological Assessment.
The GI tracts from end-stage APCMin-/+ mice
treated with either vehicle or omeprazole, as detailed in "Experiment
2," were fixed in formal calcium, processed into paraffin, and
stained with H&E. A consultant pathologist assessed the sections in a
blinded manner detailing architectural detail and degree of dysplasia
as well as necrotic and vascular appearance.
RIA to Determine Serum Gastrin Levels.
Prior to termination of the APCMin-/+ mice, the
animals were fasted overnight. Blood was collected by cardiac puncture,
and serum was prepared by centrifugation after clotting at 4°C. The
serum concentrations of total amidated gastrin were measured using the
L2 antiserum and also antiserum directed against progastrin and
glycine-extended gastrin, as described previously (22)
, or
levels were measured by the use of an ELISA-based kit (Invitrogen,
Newcastle, United Kingdom).
To measure "antibody-bound" serum gastrin concentrations in mice immunized with Gastrimmune, serum was collected as described above. The antibody:gastrin complexes were immunoprecipitated using newborn calf serum (Sigma) and 25% polyethylene glycol (8000; Sigma), with a final newborn calf serum-polyethylene glycol-test serum ratio of 1:4:5. The mixture was vortexed, and precipitates were pelleted by centrifugation at 460 x g for 30 min at 4°C. The precipitate was then resuspended in 200 µl of 0.02 M veronal buffer (pH 8.4; 4.12 g/liter sodium barbitone, 0.5 g/liter sodium azide, 1.65 g/liter serum BSA; all reagents from Sigma). The tubes containing the recovered antibody:antigen complexes were then tightly capped and placed in a boiling water bath for 5 min. Recovered gastrin was then measured from a standard gastrin-17 curve prepared with charcoal-stripped pooled mouse serum.
Reverse Transcription-PCR to Confirm CCKBR Expression.
Mouse-specific primers were designed to assess classical CCKBR. The
primers MCK1 (5'-CCCTCCTCAACAGCAGTAGC-3') and MCK2
(5'-GGGTGATTCGAATGGTCAAC-3'), specific for exons 1 and 2, respectively,
were used to amplify cDNA prepared from tumor and nonmalignant mucosa
from both large and small intestine. The 50-µl PCR reactions were set
up using 3 µl of sample cDNA in 1x PCR Buffer (PE Applied
Biosystems), 40 µM deoxynucleotide triphosphates, 20
µM each primer, and 1 unit of AmpliTaq Gold (PE Applied
Biosystems). The PCR cycle consisted of a 95°C activation stage for
10 min followed by 40 cycles of 94°C for 30 s, 60°C for
60 s, and 72°C for 30 s. After a final extension stage of
72°C for 10 min, the PCR products were separated on a 2% (w/v)
agarose gel and stained with ethidium bromide. The mouse colon cancer
cell line MC26 was used as a positive control. A negative control was
also included that was not reverse transcribed and confirmed the
absence of genomic DNA.
Real-Time PCR to Quantify CCKBR Expression.
RNA was extracted from tissue and reverse transcribed from random
hexamer primers (Pharmacia) using Supercript reverse transcriptase
(Life Technologies, United Kingdom). Real-time PCR was performed
using the 5700 Sequence Detection System (PE Applied Biosystems,
Warrington, United Kingdom). Each PCR was performed according to
manufacturer’s instructions using 1 µl of sample cDNA in a 25-µl
reaction volume. The reaction buffer was prepared from the SYBR Green
PCR Core Kit (PE Applied Biosystems) and consisted of 1x SYBR Green
PCR Buffer (PE Applied Biosystems), 3 mM
MgCl2, 0.2 mM dATP, 0.2
mM dCTP, 0.2 mM dGTP, 0.4 mM dUTP,
0.25 units of AmpErase UNG (PE Applied Biosystems), and 0.625 units of
AmpliTaq Gold (PE Applied Biosystems). The murine CCKBR
primers MCK1 and MCK2 and the GAPDH primers (GAPU,
5'-GGTGAAGGTCGGAGTCAACGGA-3'; GAPL, 5'-GAGGGATCTCGCTCCTGGAAGA-3')
were included in parallel reactions at a final concentration of 100
nM each. The fluorescence of the SYBR Green dye
bound to the CCKBR and GAPDH PCR products was
measured after each cycle by the 5700 System, and the cycle number was
recorded when the accumulated signal crossed an arbitrary threshold (Ct
value). The relative gene expression for each sample was determined
using the formula 2Ct(GAPDH) - Ct(CCKBR), and reflected CCKBR gene
expression normalized to GAPDH levels.
Measurement of Serum Omeprazole Levels.
Chilled methanol (0.4 ml) was added to 0.1 ml of serum. After mixing,
samples were centrifuged at 2000 x g for 10
min at 4°C. The supernatant was then analyzed by high-performance
liquid chromatography together with standard samples prepared by
adding known amounts of omeprazole to control serum. The
high-performance liquid chromatography chromatographic conditions were
as follows: Prodigy OGS (250 x 4.5 mm) column; mobile
phase, 42% acetonitrile in 7.5 mM diammonium
hydrogen phosphate (Sigma); flow rate, 1.0 ml/min; UV detection at 304
nm; injection volume, 0.05 ml; analysis time, 30 min; detection time,
6.3 min. The linearity of the standard curve was confirmed over a
sample concentration of 1–20 µg/ml (r = 0.99).
Measurement of Antibodies Raised by Gastrimmune.
The measurement of antibodies raised against Gastrimmune was performed
according to a method described previously (21)
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Statistical Assessment of Results.
Survival analysis was performed by a log-rank test using a multiple
group comparison, and the in vitro results were compared
using a one-way ANOVA, a Student’s t test, or the
Mann-Whitney test with the Minitab statistical package.
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RESULTS |
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No glycine-extended gastrin or progastrin was detected by RIA in either C57/Bl6 or Balb/C mouse strains after omeprazole treatment.
Effect of Omeprazole-induced Hypergastrinemia on Tumor Burden and
Survival of APCMin-/+ Mice.
The results are shown in Table 1
. There was no increase in either small or large intestinal tumor number
at termination (Table 1)
. However, when mean total tumor size
(cumulative size of large and small tumors within either the small or
large intestine) was assessed, there was a significant increase in
large (2.3-fold; P < 0.05) but not small
intestinal tumor load. Survival was significantly reduced in the
omeprazole group (mean survival of 9.6 weeks compared with 13.9 weeks
in the vehicle control-treated group; P < 0.0001). Tumor burden/time also revealed significant differences
between both the small and large intestine (1.46- and 2.6-fold
increases, respectively, in the omeprazole-treated groups;
P < 0.025 for both).
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Omeprazole, together with administration of the control immunogen, was
shown to significantly reduce the survival of
APCMin-/+ mice compared with the vehicle
control (P = 0.00001, log-rank test) with a
median 50% survival of 10 weeks compared with 13 weeks (Fig. 1)
. When Gastrimmune immunization was combined with omeprazole treatment,
there was a increase in the median 50% survival to 14 weeks, which was
significantly different from omeprazole alone (P = 0.0002, log-rank test) but not significantly different from the
vehicle control (P = 0.2602, log-rank test).
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shows the results of this study. Omeprazole significantly reduced
survival (P = 0.0038, log-rank test), with a
median 50% survival of 11 weeks, despite a treatment delay. The
vehicle group had a median 50% survival of 13 weeks, as did the group
receiving the Gastrimmune-omeprazole combination, and there was no
significant difference between these two groups (P = 0.1103, log-rank test). However, the survival of the group
treated with the Gastrimmune-omeprazole combination was
significantly increased from the group treated with omeprazole alone
(P = 0.0002, log-rank test). Gastrimmune
alone was also shown to significantly increase survival when compared
with the vehicle control (P = 0.0017) with a
50% median survival of 19 weeks.
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Antibody-bound gastrin levels were measured after processing of sera as detailed in "Materials and Methods." Animals receiving Gastrimmune + vehicle had a mean bound gastrin level of 18.35 pmol/ml (SD, 2.32 pmol/ml), which approximates the fasting serum gastrin levels measured previously. In the Gastrimmune + omeprazole-treated mice, the bound serum gastrin levels had increased to 55.6 pmol/ml (SD, 9.65 pmol/ml), a 3-fold significant increase over the levels shown in the Gastrimmune + vehicle-treated mice (P < 0.0001).
To determine adenoma proliferation at earlier stages of disease, a third study was initiated in which APCMin-/+ mice were treated with the same treatment regimes as study 2 but sacrificed at week 12. The number of tumors macroscopically visible in the small and large bowel was counted and the LI measured.
Measurement of small intestinal tumors numbers revealed no significant difference between any of the groups. With large intestinal tumor number, there was a 2.5-fold increase, from 0.88 in the vehicle control-treated group to 2.22 in the omeprazole-treated group (P = 0.030). This appeared to be partially reduced when omeprazole was combined with Gastrimmune to give a mean number of 1.65, which was not significantly different from the vehicle control (P = 0.075). The group treated with Gastrimmune alone had a mean large intestinal tumor number of 0.76, a 14% reduction, which again was not significantly different from the vehicle control.
The LI as measured by BrdUrd uptake from the same study is shown in
Table 1
. Omeprazole treatment significantly increased the LI of normal
large intestine (53%; P < 0.020) and large
intestinal tumors (29%; P < 0.025) compared
with the vehicle control. Histological assessment of the large
intestinal crypts after omeprazole treatment revealed a distortion in
their shape attributable to the increased proliferation together with
an increase of the proliferative zone toward the top of the crypt (Fig. 3A)
. This was not observed in the large intestinal crypts from
vehicle-treated APCMin-/+ mice (Fig. 3A)
. There was also an obvious increase in BrdUrd
incorporation in large intestinal adenomas/carcinomas. Fig. 3B
shows a polypoid carcinoma from a control mouse and an
omeprazole-treated mouse at the end-stage of disease progression. The
LI of the small intestinal normal mucosa (from lower and upper regions)
was not significantly affected by omeprazole treatment, whereas that of
small intestinal tumors was increased (35% increase;
P < 0.05).
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Gastrimmune and omeprazole in combination significantly inhibited the
LI increase induced by omeprazole treatment in all tissues responding
to omeprazole (significance levels for normal and malignant large
intestinal mucosa and small intestinal tumors are shown in Table 2
).
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). In most groups in which Gastrimmune significantly inhibited the mucosal LI, the difference was nonsignificant when compared with the respective Gastrimmune-omeprazole combination. However, in large normal intestinal mucosa, there was a significant 30% reduction in the LI after Gastrimmune-omeprazole treatment compared with the LI in the mucosa of the group treated with Gastrimmune alone (P < 0.010).
Histological Evaluation of Intestinal Mucosa.
Polyps in both vehicle control- and omeprazole-treated groups showed
moderate dysplasia without significant pseudostratification of
epithelial cells. Small polyps involved few glands and appeared as
sessile tubular adenomas, with very little villous formation. As
adenomas enlarged, they became polypoid with surface ulceration and
vascular stroma.
Subtle changes in dysplasia between adenomas was difficult to quantify in an objective manner. However, no gross changes in dysplasia were evident between the two groups.
CCKBR mRNA Expression in the APCMin-/+ Mouse
Intestinal Mucosa.
CCKBR was expressed at the gene level on polyps from both the small and
large intestine. Fig. 4
shows a gel in which a specific 99-bp band is shown for the positive
control cell line, MC26, together with polyps and normal mucosa from
the large and small intestines. CCKBR mRNA levels were measured in both
large and small intestinal polyps with and without treatment with
omeprazole. There was a significant 6-fold increase in CCKBR mRNA
levels in hypergastrinemic mice (P < 0.001;
Fig. 5
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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What is not known is how hypergastrinemia affects premalignant change in the colon. This question was addressed in the present study by use of the APCMin-/+ mouse model of familial adenomatous polyposis. A state of hypergastrinemia was induced by the proton pump inhibitor, omeprazole, such that serum gastrin concentrations in the mice were elevated to the same level as patients on a maintenance dose of omeprazole (23 , 24) . The variations in serum gastrin concentrations were large and may have included a postprandial element, despite fasting, because mice are known to be coprophagic. The high omeprazole dose may have been necessary because, unlike rats, which are acutely sensitive to omeprazole in terms of serum gastrin elevation, mice are more refractory, which may relate to enterochromaffin-like cell density and differences in omeprazole metabolism (25) .
Omeprazole-induced hypergastrinemia significantly decreased the survival of APCMin-/+ mice. Examination of the normal and malignant mucosa from both the small and large bowels of these mice revealed an enhanced proliferation in the tumors from both areas. Thus, omeprazole appeared to increase proliferation of the tumors arising within the GI mucosa, which caused the terminal stage of the disease to be reached at an earlier time point. This was confirmed in the first experiment when tumor burden/time was significantly enhanced in both small and large intestinal adenomas from omeprazole-treated mice. In mice terminated at 12 weeks of age, omeprazole had increased the macroscopic tumor number 2.5-fold. This is possibly a result of enhanced proliferation, resulting in earlier visualization of the tumors rather than an effect on tumor incidence. Histological evaluation of the adenomas revealed that smaller lesions were sessile tubular adenomas, becoming polypoid with increasing size. All showed moderate dysplasia, and after treatment with omeprazole, there was no gross change in the dysplasia exhibited by the lesions. Thus, in the time frame of these experiments, an increase in proliferation rather than malignant potential was observed.
In the present study, large but not small bowel mucosal crypts were shown to respond proliferatively in omeprazole-treated mice, which may reflect the differential sensitivity that the GI tract has to gastrin peptides (26) . In the study by Thorburn et al. (17) , the association between gastrin and malignancy was more closely associated in rectal cancer, which lends support to the finding that gastrin has a greater proliferative role in the distal GI tract. In the present study, the large intestinal crypts from omeprazole-treated APCMin-/+ mice had a distorted structure and an increase in the proliferative zone from the lower third to the whole length of the crypt. This has been shown in patients with an increased risk of colon cancer (27 , 28) .
The effect on survival was confirmed as gastrin-mediated by use of the gastrin immunogen, Gastrimmune, which produces antibodies that neutralize both amidated and glycine-extended gastrin-17 with high affinity (21) . Antibodies raised by Gastrimmune do not bind cholecystokinin or other gastrin species such as G34 and pentagastrin. Antigastrin antibodies reversed the reduced survival observed in omeprazole-treated APCMin-/+ mice. Antibodies raised by Gastrimmune were shown to be effective in binding serum gastrin. Antibody-bound gastrin levels were increased in mice treated with omeprazole compared with the vehicle-treated mice. This resulted in a significant reduction in free Gastrimmune-induced antibodies in the omeprazole-treated group. When Gastrimmune was administered at the same time as omeprazole, the decreased survival appeared to be partially reversed at the late stages of the study. This is likely a result of the timing of production of Gastrimmune-induced antibodies, which began to be detectable from week 2 after the initial immunization. Thus, mice had a period of elevated serum gastrin in the initial stages, which could account for only a partial reversal in survival. Omeprazole administration was therefore delayed for a 2-week period to allow anti-Gastrimmune antibodies to be generated, which resulted in the attainment of complete reversal of survival in the Gastrimmune-omeprazole-treated group compared with the mice treated with vehicle.
This study does not delineate the gastrin species that may be inducing the proliferative action on the GI tract because Gastrimmune inhibits only amidated and glycine-extended G17. Omeprazole induced amidated but not glycine-extended gastrin species in the mouse strain used in this study. CCKBR gene and protein expression of the APCMin-/+ tumors has been reported previously (29 , 30) . After omeprazole treatment, classical CCKBR gene expression was increased by intestinal polyps. The role of CCKBR in mediating the effects of hypergastrinemia in APCMin-/+ mice is being tested at present by the use of specific CCKBR antagonists.
Gastrimmune alone had a significant effect in enhancing survival of the APCMin-/+ mice. This indicates that the tumors may have responded in an endocrine manner to basal gastrin levels. Antibody-bound gastrin was indeed confirmed to be present in normogastrinemic APCMin-/+ mice. It previously has been shown that the gastrin gene is activated in normal mucosa and tumors in the APCMin-/+ mouse model and that gastrin immunoreactivity can be detected (31) . If the gastrin is increasing proliferation in an autocrine manner, this may partially explain the increased LI in the normal GI mucosa of APCMin-/+ mice compared with Min-negative mice (31) . Gastrin gene expression and progastrin and glycine-extended gastrin previously have been shown to be expressed in polyps arising within both small and large intestinal mucosa of mice with a mutated APC gene (29 , 32) . Because antibodies raised by Gastrimmune neutralize glycine-extended G17, the antibodies may therefore partially inhibit autocrine pathways established by the tumors. This is relevant because a number of recent publications have shown that in mice in which there is overexpression of precursor gastrin molecules, there is a predisposition to aberrant colonic crypt foci and adenomas and carcinomas in response to a chemical carcinogen (31 , 33) .
In conclusion, gastrin may enhance progression to malignancy when premalignant changes have occurred in the large and small intestines. The findings of these studies highlight an urgent need to investigate the possibility that gastrin may increase progression of existing premalignant colorectal lesions in a clinical setting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Aphton
Corporation, Woodland, CA. 
2 To whom requests for reprints should be
addressed, at Cancer Studies Unit, D Floor, West Block, QMC University
Hospital, Nottingham NG7 2UH, United Kingdom. Phone: 44-115-9709248;
Fax: 44-115-9709902; E-mail: sue.watson{at}nottingham.ac.uk
3 The abbreviations used are: ZES, Zollinger
Ellison syndrome; PA, pernicious anemia; APC, adenomatous polyposis
coli; Min, multiple intestinal neoplasia; GI, gastrointestinal; BrdUrd,
bromodeoxyuridine; CCKBR, cholecystokinin B receptor; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; LI, labeling index.
Received 3/27/00. Accepted 11/ 6/00.
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), omeprazole + immunogen constituents (n = 16);
group 3 (
), omeprazole + Gastrimmune
(n = 20). Log-rank analysis showed that
the survival of omeprazole-treated mice was significantly reduced
compared with the vehicle control (P = 0.00001) and the omeprazole + Gastrimmune-treated group
(P = 0.0002).
), vehicle + Gastrimmune
(n = 18). Omeprazole significantly
reduced survival (P = 0.0038, log-rank
test). Survival of the group treated with Gastrimmune + omeprazole was significantly increased from the group treated with
Omeprazole alone (P = 0.0002), and
treatment with Gastrimmune alone also had significantly increased
survival compared with the control (P = 0.0017).
X174 HaeIII digest);
Lanes 5–8, large intestine polyps; Lanes
10 and 11, small intestine polyps; Lane
12, normal large intestine mucosa; Lane 13,
normal small intestine mucosa. Reverse transcription-PCR was performed
on cDNA prepared from extracted RNA using primers specific for mouse
CCKBR. The product generated was 99 bp.