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6 mice and p53 hemizygous transgenic mice
GASTROENTEROLOGY 1996;110:155–166
Hypertrophic Gastropathy in Helicobacter felis–Infected WildType C57BL/6 Mice and p53 Hemizygous Transgenic Mice JAMES G. FOX,* XIANTANG LI,* RACHEL J. CAHILL,* KARL ANDRUTIS,* ANIL K. RUSTGI,‡ ROBERT ODZE,§ and TIMOTHY C. WANG‡ *Division of Comparative Medicine, Massachusetts Institute of Technology, Boston; ‡Gastrointestinal Unit and Department of Medicine, Massachusetts General Hospital, Boston; and §Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts
Background & Aims: Helicobacter pylori infection causes gastritis and peptic ulcers and is linked epidemiologically to gastric cancer. To analyze host genetic factors and the influence of Helicobacter on cell proliferation, we used an inbred and p53 hemizygous mouse model of Helicobacter felis–induced gastritis. Methods: H. felis was inoculated by gastric intubation into SPF C57BL/6 wild-type and p53 hemizygous mice that were followed up for 1 year and compared with uninfected controls of the same genotype using histology, proliferating cell nuclear antigen (PCNA) staining, and 5-bromo-2ⴕ-deoxyuridine (BrdU) analysis. Results: Infected animals developed sustained anti–H. felis serum immunoglobulin G antibody responses. Six months after infection, both wild-type and p53 hemizygous mice showed active chronic inflammation and marked mucosal hyperplasia compared with uninfected controls. One year after infection with H. felis, the wildtype and p53 hemizygous mice showed severe adenomatous and cystic hyperplasia of the surface foveolar epithelium. BrdU uptake and PCNA staining were markedly increased in both sets of infected mice compared with controls. Infected p53 hemizygous mice had a higher proliferative index than the infected wild-type mice. Conclusions: H. felis can induce a hypertrophic gastropathy in the C57BL/6 genotype; loss of one p53 allele, although insufficient to initiate carcinogenesis at 1 year, enhances the proliferative index, which may lead to an increased risk of cancer induction.
A
clear relationship has been established between Helicobacter pylori infection and a variety of pathological disorders of the stomach, such as chronic gastritis, peptic ulcer disease, gastric mucosa–associated lymphoid tissue lymphoma, and gastric cancer.1,2 Although peptic ulcer disease remains an important health problem in developed countries, gastric cancer, which is the second leading cause of cancer death worldwide, is actually a more important global health problem, particularly in developing countries.3 H. pylori infection has been associated with pathological conditions that predispose to gastric cancer, such as atrophic gastritis and intestinal meta-
plasia.4 Other possible premalignant conditions, such as hypertrophic gastropathy or Me´ne´trier’s disease, are rare, and their relationship to H. pylori is unclear.5,6 Although several studies have shown an epidemiological association between H. pylori and gastric cancer, a definite causative link has not been firmly established and the underlying mechanisms have not been elucidated. One hypothesis proposes that H. pylori induces a chronic inflammatory response that leads to an increased rate of mucosal proliferation7,8 and subsequently to an increased risk of malignant transformation. In fact, recent studies have shown increased proliferating cell nuclear antigen (PCNA) staining, 5-bromo-2ⴕ-deoxyuridine (BrdU) incorporation, and proliferative indices in patients with H. pylori infection.9 – 11 An increased proliferative rate may increase the risk of malignant transformation by allowing more opportunity for mutagenic events to occur. To investigate this possibility, a well-characterized animal model of Helicobacter infection would be useful. While a number of animal models have been introduced, Helicobacter felis–associated gastritis in mice simulates many of the pathological features noted in human H. pylori infection.12 H. felis is a spiral organism originally isolated from the feline gastric tissue. After experimental oral inoculation, H. felis efficiently colonizes the gastric mucosa in mice, rats, and dogs.13 Recent studies in our laboratory indicated that H. felis chronically infected VAF Swiss–Webster mice, leading to an active chronic gastritis characterized by marked polymorphonuclear and mononuclear cell infiltration.14 However, after 1 year, H. felis–infected outbred Swiss–Webster mice did not develop any preneoplastic or neoplastic changes. The response to H. pylori infection varies greatly among individuals, and it is clear that the majority of infected patients do not develop gastric cancer. Although Abbreviations used in this paper: BrdU, 5-bromo-2ⴕ-deoxyuridine; PCNA, proliferating cell nuclear antigen. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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Figure 1. Stomach of a p53 hemizygous C57BL/6 wild-type mouse 6 months after H. felis inoculation. Nodular to plaque-like proliferation of the mucosa in the gastric body markedly increases the thickness of gastric pits and glands with inflammatory cell infiltrates (I) present in the deep mucosa and submucosa but rarely in the muscular layers (M). Mild glandular dilation and intraglandular accumulation of mucus (solid arrows) and/or cells (open arrow) are present in the deep mucosa (bar Å 156 mm).
the clinical outcome of Helicobacter infection may be influenced to some extent by differences in bacterial strains, the susceptibility to cancer development is believed to be determined largely by host genetic factors. Gastric cancer, like many other types of cancer, probably results from an interaction between environmental factors and a genetic predisposition. A number of alterations in tumorsuppressor genes have been described in human gastric cancer specimens, including loss of heterozygosity at the APC, DCC, and p53 loci.15,16 In addition, p53 gene mutations have been reported not only in gastric cancer17 but also in precancerous epithelium, suggesting that the mutation of the p53 gene is an early event in gastric tumorigenesis.18 Activation of oncogenes in gastric cancer has also been reported. However, it is difficult to determine from an analysis of human tumor specimens the true order of tumor progression, i.e., whether there is a causal relationship between each genetic alteration and progression of tumor growth. Thus, to determine the role of genetic factors in the response to gastric Helicobacter infection, we inoculated H. felis into a different strain of mice, the C57BL/6 strain, which is widely used in transgenic and gene knockout studies. In addition, we infected a commercially available strain of hemizygotic knockout mice, the TSG-p53 mice (GenPharm International, Mountain View, CA), which are deficient in one p53 allele and have been used previously in studies of cancer susceptibility.19,20 Taken together, our studies strongly support a role for a genetic basis in determining the response to chronic Helicobacter infection of the stomach.
Materials and Methods Animals Sixteen (9 female and 7 male) 4-week-old hemizygous TSG-p53 mice, deficient in one of the p53 genes, were obtained from GenPharm International. The GenPharm TSG-p53 mice were generated in 129-derived embryonic stem cells (A31) and have been back-crossed onto a C57BL/6 background. Therefore, nine 4-week-old female C57BL/6 mice were also obtained from GenPharm as controls. Hemizygous TSG-p53 mice have a low spontaneous level of tumorigenesis (about 2%) with a much greater survival rate compared with homozygous TSGp53.19,20 The mice were all maintained in an American Association for Accreditation of Laboratory Animal Care–approved facility under barrier conditions as VAF mice for the duration of the 12-month experiment. Animals were housed in microisolater, solid-bottomed polycarbonate cages and fed a commercially prepared pelleted diet and given water ad libitum. The protocol described below was approved by the Animal Care Committee of the Massachusetts Institute of Technology as well as the SRAC Committee of the Massachusetts General Hospital.
Bacteria H. felis (ATCC 49179) was used for oral inoculation as previously described.14 The organism was grown for 48 hours at 37⬚C under microaerobic conditions on 5% lysed horse blood agar supplement with antibiotics. The bacteria were harvested and inoculated (at a titer of 1010 organisms/ mL) into brain-heart infusion broth with 30% glycerol added. The bacterial suspension was frozen at 070⬚C. Before use, aliquots were thawed, analyzed for motility, and cultured for evidence of aerobic or anaerobic bacterial contamination.
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Experimental Infection Of the 16 TSG-p53 mice, 12 were inoculated with H. felis and 4 served as controls. Of the 9 wild-type C57BL/6 mice, 7 were inoculated with H. felis and 2 were controls. Brain-heart infusion broth containing Ç1010 colony-forming units of H. felis per milliliter was used as inoculum. The inocula (0.5 mL) was delivered by mouth into each test mouse three times at 2-day intervals by using a sterile oral catheter. At 6 and 12 months postinfection, infected p53 and wild-type mice and uninfected control mice were killed with CO2 and necropsied.
Histological Evaluation The tissue examined consisted of a section of gastric mucosa taken from the greater curvature of the stomach beginning at the gastroesophageal junction and ending at the gastroduodenal junction. Stomach tissues were fixed in neutral buffered 10% formalin, processed by standard methods, embedded in paraffin, sectioned at 5 mm, and stained with H&E and Warthin–Starry. The glandular mucosae of the body, antrum, and pylorus were examined histologically for a variety of inflammatory and epithelial changes and for the presence of H. felis. Sections of glandular stomach were also examined for diastase-resistant, periodic acid–Schiff positive staining.
Figure 2. Stomach of a p53 hemizygous C57BL/6 mouse 6 months after H. felis inoculation. A marked increase in the lengths of gastric pits with multifocal epithelial erosions are noted (arrows) (bar Å 24 mm).
H. FELIS MOUSE MODEL 157
Quantitation of Parietal Cells in the Gastric Mucosa Gastric parietal cells were semiquantitated with a micrometer (OCM 10 1 10 SG; Olympus, Tokyo, Japan) on the H&E-stained sections from mice analyzed 12 months after the study commenced. Briefly, the gastric mucosa was scanned along the middle zone of the body, where parietal cells densely populate in the normal mucosa. A total of 4–6 nonoverlapping high-power fields (4001) were randomly selected along the midzone of the body for each animal. Parietal cells and nonparietal cells were counted with the guidance of the micrometer (1/16 mm2). The data were expressed as percentages of the parietal cells out of the total epithelial cells in a given field. In the infected animals, the data were further tabulated by the foci of mucous hypertrophy or epithelial hyperplasia of the body mucosa. The data between the control and infected animals were compared by a two-tailed t test.
Immunohistochemistry Gastric mucosa was examined by immunohistochemistry for BrdU and PCNA.
Figure 3. Stomach of a p53 hemizygous C57BL/6 mouse 6 months after H. felis inoculation. The mucous epithelial cells are distended by mucus in their apical cytoplasm with pale mucoid material in the glandular lumina (m). The hyperplastic epithelial cells are round to oval with prominent nuclei and basophilic cytoplasm (H). The parietal cells (arrows ) are markedly decreased. Aggregations of predominantly lymphocytes and macrophages are evident in the deep mucosa (bar Å 27 mm).
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Figure 4. Stomach of a wild-type C57BL/6 mouse 12 months after H. felis inoculation. Adenomatous hyperplasia and cystic dilation of gastric mucosa with lymphocyte aggregates (open arrows) in the deep mucosa and edematous submucosa. The glands penetrate the muscularis mucuosae (M) and invade into the submucosa multifocally (solid arrows). Transition (T) between the hyperplastic mucosa and the antral mucosa seems to be abrupt (bar Å 350 mm).
BrdU immunocytochemistry. In vivo incorporation of BrdU. Animals received a single intraperitoneal injection of
BrdU (50 mg/kg) from a freshly made stock solution (5 mg/ mL) dissolved in phosphate-buffered saline. The mice were killed 1 hour later. At necropsy, a longitudinal section of stomach was taken from the greater curvature extending from the esophageal junction to the gastroduodenal junction. Samples were immediately placed in a cassette, fixed in Carnoy’s fixative overnight, and embedded in paraffin wax. Immunohistochemical detection of BrdU. Immunohistochemical detection of the BrdU incorporation was performed on 4mm sections and visualized using the following avidin-biotin monoclonal antibody immunohistochemical technique. After deparaffinization in xylene through to alcohol, endogenous peroxidase activity in the tissue section was blocked by immersing the slides in 1% hydrogen peroxide in methanol. The slides were then washed in tap water. The BrdU monoclonal antibody only identifies single-stranded DNA. Denaturation of the tissue DNA was achieved by incubation in 1 mol/L hydrochloric acid at 60⬚C for 8 minutes. The slides were washed in tap water and Tris-buffered saline and were incubated with 5% normal rabbit serum to block nonspecific binding of the primary antibody. The tissue was then incubated with the monoclonal antibody to BrdU (Dakopatts, Glostrup, Denmark) diluted to 1:40 in Tris buffer. Control slides, included in each assay, were incubated with mouse serum. The slides were again washed in Tris-buffered saline and incubated with biotinylated anti-mouse immunoglobulin G (1:200 in Tris buffer). After further washings in Tris-buffered saline, slides were incubated with peroxidase-conjugated strepavidin (1:400 in Tris buffer) and washed in Tris-buffered saline, and the labeled cells were visualized by the diaminobenzidine reaction and lightly coun-
Figure 5. Stomach of a wild-type C57BL/6 mouse 12 months after H. felis inoculation. Many epithelial cells are distended by mucus in their apical cytoplasm with nuclei located at the base. The parietal cells (arrows) are markedly decreased. Some of the glandular lumina contained mucoid material (M) and cellular debris (D) (bar Å 27 mm).
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terstained with hematoxylin. The nucleus of cells at the S phase of the cell cycle during the in vivo incorporation phase were stained with brown. Immunohistological analysis. A mean of four entire tissue sections were examined for each animal. Only gastric pits longitudinally sectioned within the upper one third of gastric mucosa and visible in their entire length (ú60 cells) were analyzed. A mean number of 10 well-orientated gastric pits was examined for each specimen. Labeling index was measured by counting the number of BrdU-positive cells and expressing the result as a percentage ratio of the total number of cells in each gastric pit. The relative positions of positive cells in each gastric pit were noted. For cell kinetics evaluation, each gastric pit was divided into five compartments of equal size. The compartments were referred to by the ordinary numbers 1 (luminal zone) to 5 (base of the gastric pit) and the labeling index calculated for each compartment separately. Statistical analysis. The total labeling index and the labeling index per compartment was compared between the
Figure 6. Stomach of a wild-type C57BL/6 mouse 12 months after H. felis inoculation. The glands (G) penetrate the muscularis mucosae (M), invade into the submucosa, and are surrounded by predominantly lymphocytes. A few clusters of the glands reside within the muscularis mucosae (arrows) (bar Å 54 mm).
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study groups. Significance was analyzed using the two-tailed Student’s t test for unpaired data. P values of õ0.05 were considered statistically significant. PCNA immunocytochemistry. Five-micrometer-thick tissue sections were cut, mounted, and dry baked at 60⬚C for 1 hour. The sections were deparaffinized in xylene for 5 minutes, followed by submersion in 100% ethanol and 95% ethanol, respectively. The slides were then placed in preheated (199⬚F) 10 mmol/L citrate buffer for 30 minutes. After a wash in phosphate-buffered saline, the tissue sections were incubated for 15 minutes with horse immunoglobulin G serum provided in the avidin-biotin peroxidase complex kit (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). The sections were then incubated with the PCNA antibody (antihuman, cross-reacts with mouse, immunoglobulin G2a subclass; Pharmingen, San Diego, CA) at a 1:400 dilution (diluted with 2% horse serum in phosphate-buffered saline). After rinsing in phosphate-buffered saline, the slides were incubated with a secondary biotinylated antibody, provided in the ABC kit, for 30–35 minutes. After another rinse in phosphate-buffered saline, the ABC reagent was applied for 40 minutes. Following another rinse in phosphate-buffered saline, the slides were de-
Figure 7. Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Diastase-resistant periodic acid–Schiff stain revealed abundant mucin in the apical cytoplasm of the epithelial cells and within the glandular lumina (bar Å 107 mm).
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ples were obtained and analyzed from the 25 mice included in this study at baseline and 5 and 10 weeks postinoculation. At 26 and 52 weeks after dosing, sera were collected and analyzed from mice being killed.
Results Experimental Design At 6 months postinoculation, 2 wild-type C57BL/ 6 and 4 p53 H. felis-infected mice were killed along with 1 wild-type and 2 p53 uninfected controls; at 12 months, 1 wild-type and 1 p53 controls and 5 H. felis-infected p53 and 3 H. felis-infected C57BL/6 wild-type mice were killed and necropsied. Also during the study, 1 infected C57BL/6 mouse was killed at 6 weeks after dosing, and 3 female p53 mice were killed at 3, 34, and 44 weeks after dosing because of weight loss. One control p53 female mouse was found dead at 50 weeks after dosing. Histopathology
Figure 8. Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Warthin–Starry stain revealed numerous H. felis in the glandular lumina (arrows) (bar Å 10.8 mm).
veloped in 3,3ⴕ-diaminobenzidine tetrahydrochloride (10 mg 3,3ⴕ-diaminobenzidine tetrahydrochloride in 10 mL 0.5 mol/ L Tris buffer, 3 drops 3% H202). Methyl green (2%) was used for 30 minutes to counterstain the tissue sections. Dehydration was performed quickly in 95% ethanol, twice in 100% ethanol, and twice in xylene. Immunostained slides were evaluated for the degree of PCNA staining by noting the location and relative size of the proliferative zone in the gastric antral and corpus epithelium. The proliferative zone corresponds to the segment of epithelium between the level of the highest and lowest PCNA-labeled cell.
By 6 months after infection, mild to moderate nodular to plaque-like thickening of the mucosa was observed in the gastric body with variable sparing of the antrum in both wild-type and transgenic mice in comparison with uninfected controls. The affected mucosa was about 2–3 times the normal thickness in H. felis–infected mice. Microscopically, the nodules and/or plaques were composed of hypertrophic and hyperplastic mucosal epithelium with a variable degree of cystic glandular dilation and inflammatory cell infiltration (Figure 1). There was an increase in lengths of both gastric pits and glands with multifocal erosions on the pit surface. The pit cells
Enzyme-Linked Immunosorbent Assay for H. felis Serum Immunoglobulin G Antibody The enzyme-linked immunosorbent assay for H. felis was performed as previously described.14 Wells of microtiter plates (Dynatech Laboratories, Chantilly, VA) were incubated with 100 mg of H. felis protein per milliliter in carbonate buffer. After being washed, serial twofold dilutions of sera from H. felis–infected or control mice were incubated for 60 minutes at 37⬚C and then given the appropriate dilution of alkaline phosphatase–conjugated goat antimouse immunoglobulin G (Sigma Chemical Co., St. Louis, MO). Serum sam-
Figure 9. Quantitation of parietal cells in the gastric body mucosa. Gastric parietal cells in H. felis –infected mice with either mucous hypertrophy or epithelial hyperplasia were significantly (P õ 0.01) lower than those in control animals. Hypertrophic or hyperplasia mucosa had similar numbers of parietal cells (P ú 0.05) in the infected mice.
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H. FELIS MOUSE MODEL 161
Figure 10. (A ) Stomach of a p53 hemizygous C57BL/6 mouse control (bar Å 54 mm). (B ) Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Immunohistochemical stain detecting BrdU incorporation. Brown cells in A and B are positive for BrdU incorporation (bar Å 54 mm).
were oval to columnar and mostly contained eosinophilic droplets in their atypical cytoplasm (Figure 2). The hyperplastic cells, often located between the pit and glandular regions, were round to oval and smaller in size with prominent nuclei and basophilic cytoplasm (Figure 3). The glandular/mucous cells were columnar and were distended often by mucus in their apical cytoplasm (Figure 3). Some of the dilated glandular lumina contained intraluminal eosinophilic to basophilic mucoid material, neutrophils, mononuclear cells, and/or cellular debris. The squamous epithelial cells of the forestomachs were also hypertrophic and hyperplastic with eosinophilic droplets in the cytoplasm. Mild to marked infiltration of predominantly lymphocytes, as well as some macrophages and neutrophils, were observed in the deep mucosa and submucosa with occasional formation of lymphoid aggregates and interruption of muscularis mucosae (Figure 1). By 12 months after infection, prominent polypoid nodules and cerebriform folds or rugae were observed in the gastric body mucosa with variable extension into the antrum in both wild-type and transgenic mice. The
nodules or folds were distributed predominantly along the greater curvature with focal rigidity. Transition between the nodules or folds and normal-appearing mucosa seemed to be abrupt in most cases. Microscopically, the nodules or folds were composed of hyperplasia proliferation and cystic dilation of gastric mucosa (Figure 4). The affected mucosa was about 3–5 times of the normal thickness, and both gastric pits and glands were markedly increased in length. The dilated glandular cysts contained intraluminal pale mucoid material mixed with variable amounts of neutrophils, mononuclear cells, and/ or debris (Figure 5). The base of the glands broke through the muscularis mucosae and invaded into the submucosa multifocally in some mice (n Å 2) (Figure 6). There was marked infiltration of predominantly lymphocytes and some macrophages as well as neutrophils in the lamina propria and submucosa with frequent formation of lymphoid follicles (Figure 4). The squamous epithelial cells were still hypertrophic and hyperplastic, but intracytoplasmic droplets were not present in squamous or pit cells.
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Figure 11. Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Immunohistochemical detection of PCNA antigen (bar Å 54 mm).
Diastase-resistant periodic acid–Schiff stain revealed abundant mucin in the apical cytoplasm of many glandular cells and in many glandular lumina (Figure 7). Warthin–Starry stain revealed variable numbers of H. felis organisms in the gastric pits and glandular lumina in mice at both 6 and 12 months after infection (Figure 8). No pathological changes were observed in the uninfected control mice at both 6 and 12 months of the study. Gastric parietal cells were markedly decreased in the mucosa of infected animals. Percentages of the parietal cells in the mucosa of the infected animals were approximately the same in wild-type and transgenic mice, but both infected groups were significantly (P õ 0.01) lower in the body than those of the control mice (Figure 9). Hypertrophic and hyperplastic foci of the body mucosa were similarly affected (P ú 0.05) in the H. felis–infected animals. To determine the location and characteristics of the progenitor zone of the stomach, BrdU was administered 1 hour before the mice were killed. The assay measured
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the average number of BrdU-labeled S-phase cells per total number of cells in the gastric pit. In assessment of epithelial cell kinetics of the gastric pit, the zone of maximum proliferation is in the base of the gastric pit, which represents the neck region of the gastric glands. In uninfected mice, BrdU labeling was confined to a limited zone, consistent with the labeling of a discrete progenitor zone (Figure 10A). In infected mice (C57BL/ 6 and p53), the progenitor zone was markedly expanded with labeling in the upper zones (Figure 10B). PCNA staining confirmed this finding of a marked expansion of the proliferative zone in both directions, including at the surface epithelium, in the H. felis–infected mice (Figure 11). Overall, there was marked increase in the proliferative zone, as detected by PCNA staining, in infected p53 and wild-type mice at 6 and 12 months compared with uninfected p53 and wild-type mice. BrdU labeling in 12-month-old mice was quantified (Table 1). These data confirmed the qualitative impression of an increased proliferation rate in infected mice compared with uninfected mice (Figure 12). In addition, they showed that the proliferation index was higher in infected hemizygous p53 mice compared with infected wild-type C57BL/6 control mice, although this did not reach significance (P Å 0.058). At 12 months, there was an extension of the proliferative zone towards the luminal surface of the gastric pits in animals infected with H. felis. This was more marked in the body mucosa (Table 1). Serology Immunoglobulin G antibodies to H. felis were detected in all mice infected with H. felis but not in any control mice or in any of the 25 mice assayed before the study commenced (Figure 13). Interestingly, female mice seemed to have a greater systemic immune response to H. felis than males. The H. felis antibody response began to increase at 5 weeks after dosing, plateaued at 10 weeks after dosing, and remained elevated at 6 and 12 months after H. felis experimental infection.
Discussion Several epidemiological studies strongly implicate H. pylori infection as a carcinogen in the stomach.21,22 Furthermore, a new murine pathogen named Helicobacter hepaticus has been shown to cause a persistent chronic hepatitis and in the A/JCr mouse has been linked to the development of hepatic tumors. In addition to eliciting an immunoglobulin G immune response, H. hepaticus causes an increase in hepatic cell proliferation as measured by PCNA and in this manner has been implicated in the pathogenesis of H. hepaticus–induced hepatic can-
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H. FELIS MOUSE MODEL 163
Table 1. Comparison of the BrdU Labeling Indices of All Experimental Mice at 12 Months Wild-type control
Wild-type H. felis infected
p53 control
Entire
Body
Pyloric region
Entire
Body
Pyloric region
1540
760
780
1836
835
1001
73.3 (8.8)a 4.32 (1.8)
76 (9.7) 4.92 (1.94)
70.9 (8.1) 3.73 (1.5)
90.0 (12.0) 4.15 (1.5)
83.5 (5.7) 3.23 (1.10)
100.1 (11.5) 5.30 (1.3)
LI% per compartment LI% 1 (apex)
0
0
0
0
0
0
LI% 2
0
0
0
0
LI% 3
2.44 (3.2) 6.49 (5.7) 12.86 (8.2)
3.86 (3.3) 6.87 (7.2) 14.1 (8.6)
1.26 (2.6) 5.87 (4.1) 11.75 (8.0)
0.73 (1.9) 3.26 (5.6) 5.69 (7.1) 11.6 (8.0)
1.87 (2.6) 6.53 (7.16) 4.64 (7.2) 14.2 (8.3)
Total cells counted per mouse Cells/gastric pit LI% total
LI% 4 LI% 5
0.64 (2.0) 6.53 (7.2) 8.99 (6.6)
p53 H. felis infected
Entire
Body
Pyloric region
Entire
Body
Pyloric region
2190 (153) 109.5 (6.2) 5.60 (0.83)
1149 (172) 114.9 (17) 5.86 (0.95)
1040 (28) 104.1 (2.8) 5.34 (0.5)
2141 (366) 104.6 (9.7) 8.40 (1.92)
1017 (130) 103.6 (10.8) 8.93 (1.87)
1128 (325) 104.9 (13.1) 7.98 (2.87)
0.06 (0.1) 1.03 (0.7) 4.45 (1.2) 10.65 (3.2) 10.85 (2.07)
0.11 (0.2) 1.35 (1.3) 4.25 (1.8) 11.3 (6.4) 11.6 (3.1)
0
0.05 (0.1) 1.91 (1.0) 7.25 (1.3) 15.15 (3.7) 16.49 (5.9)
0.11 (0.2) 1.48 (0.5) 8.16 (2.6) 17.52 (7.8) 16.39 (7.0)
0
0.71 (0.3) 4.64 (2.2) 9.76 (0.73) 10.14 (1.4)
2.35 (2.3) 7.00 (2.7) 13.69 (6.0) 16.73 (6.6)
NOTE. All results expressed as mean { SD. a A mean of 20 gastric pits were analyzed per mouse, which were subdivided into body and pyloric region. LI%, labeling index.
cer.23 – 25 Previous studies in humans also have suggested that H. pylori infection may contribute to gastric cancer susceptibility by stimulation of mucosal proliferation.7–11 Our study shows that chronic H. felis infection in C57BL/ 6 mice leads to an increase in the proliferation index as measured by both PCNA staining and BrdU incorporation. The proliferative zone of the stomach is normally located at the junction of the base of the pits and the upper region of the gastric glands, the so-called stem cell zone. Under normal conditions, newly formed cells migrate upward from this zone to replenish the surface and foveolar mucous cells or downward to replenish the gastric glandular cells. However, expansion of the proliferative zone has been observed in a number of conditions associated with an increased risk of gastric cancer, such as chronic gastritis,26 pernicious anemia,27 and in the postantrectomy stomach.28 In addition, a similar broadening of the proliferative zone has been found in the rat stomach after exposure to the gastric carcinogen Nmethyl-Nⴕ-nitro-N-nitrosoguanidine. N-methyl-Nⴕ-nitro-N-nitrosoguanidine has been shown to stimulate cell proliferation in the rat stomach and causes a downward expansion of the proliferative zone in both the antrum and fundus.29 In a similar fashion, chronic H. felis infection broadens the proliferative zone in the stomach of the C57BL/6 mouse. However, the mechanism for this proliferative effect is unclear. One possible explanation is that an ongoing cycle of inflammation-mediated epi-
thelial destruction followed by activation of growth factors and subsequent regeneration may lead to increased cell turnover. Nevertheless, regardless of the precise mechanisms involved, an increased proportion of gastric cells undergoing cell division could result in increased mitotic error, an increased mutation rate, and an increased risk of cancer formation. Because loss of heterozygosity at the p53 locus has been found frequently in human gastric cancer specimens and is thought to play a crucial role in cancer development, we infected both hemizygous p53 knockout mice and wild-type C57BL/6 with H. felis. Our results suggest that mice that lack one copy of the p53 gene show a significantly increased proliferative index compared with wild-type controls. Reduction in the gene dosage and consequently in the amount of p53 produced by the normal cell does seem to confer a significant growth advantage in the gastric epithelium in this mouse model. However, despite an increased rate of proliferation, we did not detect any evidence of cancer in the H. felis– infected p53 mouse stomach after a 1-year observation period. The development of frank neoplasia in this mouse model may require either additional mutations, genetic events, or a longer time period. Therefore, loss of p53 may still offer a selective advantage in gastric cancer development. We did not choose to infect homozygous p53 knockout mice in this study because of their known diminished survival (3–6 months).19,20
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Figure 12. Mean labeling index for individual control and experimentally infected mice using the BrdU immunohistochemical technique at 12 months.
Our data are consistent with previous studies of cancer susceptibility in TSG-p53 mice. In particular, a mouse skin model of multistage carcinogenesis has been well characterized, with stages of initiation (with dimethylbenzanthracene) and promotion and progression (with 12-O-tetradecanoyl-phorbol-13-acetate). Both dimethylbenzanthracene and 12-O-tetradecanoyl-phorbol-13-acetate are required for the development of skin cancer because either alone has been shown to be insufficient. In this mouse model of skin cancer, the initial development of tumors by dimethylbenzanthracene and 12-O-tetradecanoyl-phorbol-13-acetate was not increased in p53 hemizygous lines.30 Furthermore, p53 homozygous (null) mice treated with 12-O-tetradecanoyl-phorbol-13-acetate alone did not develop skin tumors either. However, both the hemizygous and the homozygous p53 knockout mice did show faster tumor progression after treatment with dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate with an increase in the rate of benign to malignant conversion. Thus, in this model, the absence of p53 does not augment the frequency of initiation or rate of promotion but greatly enhances the progression of malignancy. Perhaps the most surprising finding in this study was the effect of H. felis infection on the wild-type C57BL/ 6 mouse. Previous studies in which H. felis was inoculated in Swiss–Webster mice revealed active, chronic inflammation with minimal hyperplasia, but the histological changes did not approach the severity of gastric disease shown by the presence of diffuse cystic hyperplasia
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in the C57BL/6 mice after H. felis infection in this study.12,14 However, previous investigators have pointed out that the gastric phenotype (in response to growth factors, for example) is strongly influenced by the genetic background of the mouse. For example, overexpression of transforming growth factor a in inbred FVB/N mice leads to much more severe cystic hyperplasia compared with that observed in the outbred strain CD1.31 Thus, our study as well as preliminary data presented by others suggesting that particular inbred strains of mice have differing severities of H. felis–induced gastritis points to the importance of the genetic makeup in determining the response to chronic infection with H. felis.32 The histological changes observed in infected C57BL/ 6 mice (severe adenomatous and cystic hyperplasia of the surface foveolar epithelium) resemble in some respects those described in patients with hypertrophic gastropathy (Me´ne´trier’s disease). The number of parietal cells in the body mucosa were also significantly decreased in H. felis–infected mice compared with the control uninfected animals. This finding is also a consistent observation noted in Me´ne´trier’s disease. However, given the variation in mucosal thickness between the infected and uninfected mice, these data should be interpreted with caution. Other features typically present in Me´ne´trier’s disease, such as hypoalbuminemia and decreased acid secretion,5 were unfortunately not assessed before the mice were killed in this study; thus, further studies will be required to determine whether the H. felis–infected C57BL/6 mouse is a proper model for this disease. There has been some controversy in the literature regarding a possible association of Me´ne´trier’s disease with H. pylori
Figure 13. Serum immunoglobulin G antibody response in H. felis – infected mice.
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infection.6 One study by Wolfsen et al. found a low frequency of H. pylori (30%–39%) by routine histology and also suggested that Me´ne´trierⴕs disease could be classified into two diseases: hypertrophic lymphocytic gastritis and massive foveolar hyperplasia and minimal inflammation.6 However, a recent study that evaluated 138 patients with hypertrophic gastropathy indicated a high percentage (90%) of cases associated with H. pylori gastritis.33 In that study, there seemed to be marked H. pylori colonization and a higher degree of gastritis in the corpus of the stomach compared with the antrum,33 which is in agreement with our findings in H. felis–infected C57BL/ 6 mice as well as in our earlier studies in Swiss–Webster mice.14 Further evidence for a role of H. pylori in Me´ne´trier’s disease stems from patients who showed marked improvement after treatment with antibiotics.34 – 36 In one case, a 6-week course of amoxicillin and omeprazole led to eradication of H. pylori and normalization of the histological abnormalities.37 Thus, it would be interesting to determine if antibiotic treatment of H. felis–infected mice, as previously described,38 can reverse the hypertrophic gastropathy in our C57BL/6 murine model. Recent data suggest that mice can also be infected with cytotoxin-positive and negative H. pylori strains.39 However, the gastritis induced by H. pylori in mice described to date has been minimal, being limited to focal inflammation and gastric mucosal erosions. The ability of H. pylori to persistently infect mice has not been established; thus, the H. pylori mouse model needs to be studied further to ascertain whether the model will mimic H. pylori disease in humans. Therefore, the H. felis mouse model remains the most viable animal model to study H. pylori pathogenesis. This model may prove useful for studying the role of Helicobacter in inducing epithelial proliferation and the genetic factors that predispose to gastric cancer.
References 1. Blaser MJ. Hypotheses on the pathogenesis and natural history of Helicobacter pylori –induced inflammation. Gastroenterology 1992;102:720–727. 2. Blaser MJ, Parsonnet J. Parasitism by the ‘‘slow’’ bacterium Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J Clin Invest 1994;94:4–8. 3. Correa P. The epidemiology of gastric cancer. World J Surg 1991;15:228–234. 4. Fox JG, Correa P, Taylor NS, Zavala D, Fontham E, Janney F, Rodriguez E, Hunter F, and Diavolitsis S. Campylobacter pyloriassociated gastritis and immune response in a population at increased risk of gastric cancer. Am J Gastroenterol 1989;84:775–781. 5. Scharschmidt BF. The natural history of hypertrophic gastropathy (Menetrier’s disease): report of a case with 16 year follow-up and a review of the pertinent literature. Am J Med 1977;63:644– 652. 6. Wolfsen HC, Carpenter HA, Talley NJ. Menetrier’s disease: a
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form of hypertrophic gastropathy or gastritis? Gastroenterology 1993;104:1310–1319. Correa P, Fox JG. Gastric cancer and Helicobacter pylori. In: Pajares JM, Pena AS, Malfertheiner P, eds. Helicobacter pylori and gastroduodenal pathology. New York: Springer-Verlag, 1993: 239–243. Correa P, Fox J, Fontham E, Ruiz B, Lin Y, Zavala D, Taylor N, Mackinley D, de Lima E, Portilla H, Zarama G. Helicobacter pylori and gastric carcinoma. Cancer 1990;66:2569–2574. Cahill RJ, Sant S, Beattie S, Hamilton H, OⴕMorain C. Helicobacter pylori and increased epithelial cell proliferation: a risk factor for cancer. Eur J Gastroenterol Hepatol 1994;6:1123– 1127. Lynch DAF, Mapstone NP, Clarke AMT, Sobala GM, Jackson P, Morrison L, Dixon MF, Quirke P, Axon ATR. Cell proliferation in Helicobacter pylori associated gastritis and the effect of eradication therapy. Gut 1995;36:346–350. Brenes F, Ruiz B, Correa P, Hunter F, Rhamakrishnan T, Fontham E, Shi T-Y. Helicobacter pylori causes hyperproliferation of the gastric epithelium: pre- and post-eradication indices of proliferating cell nuclear antigen. Am J Gastroenterol 1993;88:1870– 1875. Lee A, Fox JG, Otto G, Murphy J. A small animal model of human Helicobacter pylori active chronic gastritis. Gastroenterology 1990;99:1315–1323. Lee A, Hazell SL, O’Rourke J, Kouprach S. Isolation of a spiralshaped bacterium from the cat stomach. Infect Immun 1988;56:2843–2850. Fox JG, Blanco M, Murphy JC, Taylor NS, Lee A, Kabok Z, Pappo J. Local and systemic immune responses in murine Helicobacter felis active chronic gastritis. Infect Immun 1993;61:2309– 2315. Rhyu M-G, Park W-S, Jung Y-J, Choi S-W, Meltzer SJ. Allelic deletions of MCC/APC and p53 are frequent late events in human gastric carcinogenesis. Gastroenterology 1994;106:1584– 1588. Uchino S, Tsuda H, Noguchi M, Yokota J, Terada M, Saito T, Kobayashi M, Sugimura T, Hirohashi S. Frequent loss of heterozygosity at the DCC locus in gastric cancer. Cancer Res 1992;52:3099–3102. Uchina S, Noguchi M, Ochiai A, Saito T, Kobayashi M, Hirohashi S. p53 mutation in gastric cancer: a genetic model for carcinogenesis is common to gastric an colorectal cancer. Int J Cancer 1993;54:759–764. Shiao Y-H, Rugge M, Correa P, Lehmann HP, Scheer WD. p53 alteration in gastric precancerous lesions. Am J Pathol 1994;144:511–517. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, Butel JS, Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 1992;356:215–221. Harvey M, McArthur MJ, Montgomery CA Jr, Butel JS, Bradley A, Donehower LA. Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nat Genet 1993;5:225–229. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991;325:1127–1131. Nomura A, Stemmermann GN, Chyou P-H, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med 1991;325:1132–1136. Ward JM, Fox JG, Anver MA, Haines DC, George CV, Collins MJ Jr, Gorelick PL, Nagashima K, Gonda MA, Gilden RV, Tully JG, Russell RJ, Benveniste RE, Paster BJ, Dewhirst FE, Donovan JC, Anderson LM, Rice JM. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection
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with a novel Helicobacter species. J Natl Cancer Inst 1994; 86:1222–1227. Fox JG, Zhao Z, Hurley R. Helicobacter hepaticus IgG antibody titer and alanine transminase (ALT) liver enzyme: markers of hepatitis in A/JCr mice. Lab Animal Sci 1994;44:398–399. Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ Jr, Gorelick PL, Ward JM. Helicobacter hepaticus sp nov, a microaerophillic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol 1994;32:1238– 1245. Lipkin M, Correa P, Mikol YB, Higgins PJ, Cuello C, Zarama G, Fontham E, Zavala D. Proliferative and antigenic modifications in epithelial cells in chronic atrophic gastritis. J Natl Cancer Inst 1985;75:613–619. MacDonald WC, Trier JS, Everett NB. Cell proliferation and migration in the stomach, duodenum, and rectum of man: radioautographic studies. Gastroenterology 1964;46:405–417. Assad RT, Eastwood GL. Epithelial proliferation in human fundic mucosa after antrectomy and vagotomy. Gastroenterology 1980;79:807–811. Quimby GF, Eastwood GL. Effect of N-methyl-N-nitrosoguanidine on gastroduodenal epithelial proliferation in Wister/Lewis rats. J Natl Cancer Inst 1981;66:331–337. Kemp CJ, Donehower LA, Bradley A, Balmain A. Reduction of p53 gene dosage does not increase initiation or promotion but enhances malignant progression of chemically induced skin tumors. Cell 1993;74:813–822. Takagi H, Jhappan C, Sharp R, Merlino G. Hypertrophic gastropathy resembling Menetrier’s disease in transgenic mice overexpressing transforming growth factor a in the stomach. J Clin Invest 1992;90:1161–1167. Sakagami T, Shimoyama T, OⴕRourke J, Lee A. Back to the host: severity of inflammation induced by Helicobacter felis in different strains of mice (abstr 241). Am J Gastroenterol 1994;89:1345. Ormand JE, Talley NJ, Shorter RG, Conley CR, Carpenter HA, Rich A, Wilson WR, Phillips SF. Prevalence of Helicobacter pylori in
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specific forms of gastritis: further evidence supporting a pathogenic role for H. pylori in chronic nonspecific gastritis. Dig Dis Sci 1991;36:142–145. Stolte M, Batz C, Eidt S. Giant fold gastritis—a special form of Helicobacter pylori associated gastritis. Z Gastroenterol 1993;31:289–293. Bayerdorffer E, Ritter MM, Hatz R, Brooks W, Ruckdeschel G, Stolte M. Healing of protein losing hypertrophic gastropathy by eradication of Helicobacter pylori: is Helicobacter pylori a pathogenic factor in Menetrier’s disease? Gut 1994;35:701–704. Groisman GM, George J, Berman D, Harpaz N. Resolution of protein-losing hypertrophic lymphocytic gastritis with therapeutic eradication of Helicobacter pylori. Am J Gastroenterol 1994;89:1548–1551. Bayerdorffer E, Ritter MM, Hatz R, Brooks W, Stolte M. Menetrier’s disease and Helicobacter pylori. N Engl J Med 1993;329:60. Dick-Hegedus E, Lee A. Use of a mouse model to examine antiHelicobacter pylori agents. Scand J Gastroenterol 1991;26: 909–915. Marchetti M, Arico B, Burroni D, Figura N, Rappuoli R, Ghiara P. Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 1995;267:1655–1658.
Received July 24, 1995. Accepted September 8, 1995. Address requests for reprints to: James G. Fox, D.V.M., Division of Comparative Medicine, Massachusetts Institute of Technology, Building 45, 37 Vassar Street, Cambridge, Massachusetts 02139. Fax: (617) 258-5708. Supported by the R. Robert and Sally D. Funderburg Research Scholar Award in Gastric Cancer Biology (to T.C.W.), National Institutes of Health grant RO1 CA67463 (to T.C.W., J.G.F., and A.K.R.), and grants RR07036 and RR01046 from the National Center for Research Resources (to J.G.F., X.L., R.J.C., and K.A.). Dr. Rustgi was supported by the Miles and Shirley Fiterman AGA Award (Walter B. Cannon).
Hypertrophic Gastropathy in Helicobacter felis–Infected WildType C57BL/6 Mice and p53 Hemizygous Transgenic Mice JAMES G. FOX,* XIANTANG LI,* RACHEL J. CAHILL,* KARL ANDRUTIS,* ANIL K. RUSTGI,‡ ROBERT ODZE,§ and TIMOTHY C. WANG‡ *Division of Comparative Medicine, Massachusetts Institute of Technology, Boston; ‡Gastrointestinal Unit and Department of Medicine, Massachusetts General Hospital, Boston; and §Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts
Background & Aims: Helicobacter pylori infection causes gastritis and peptic ulcers and is linked epidemiologically to gastric cancer. To analyze host genetic factors and the influence of Helicobacter on cell proliferation, we used an inbred and p53 hemizygous mouse model of Helicobacter felis–induced gastritis. Methods: H. felis was inoculated by gastric intubation into SPF C57BL/6 wild-type and p53 hemizygous mice that were followed up for 1 year and compared with uninfected controls of the same genotype using histology, proliferating cell nuclear antigen (PCNA) staining, and 5-bromo-2ⴕ-deoxyuridine (BrdU) analysis. Results: Infected animals developed sustained anti–H. felis serum immunoglobulin G antibody responses. Six months after infection, both wild-type and p53 hemizygous mice showed active chronic inflammation and marked mucosal hyperplasia compared with uninfected controls. One year after infection with H. felis, the wildtype and p53 hemizygous mice showed severe adenomatous and cystic hyperplasia of the surface foveolar epithelium. BrdU uptake and PCNA staining were markedly increased in both sets of infected mice compared with controls. Infected p53 hemizygous mice had a higher proliferative index than the infected wild-type mice. Conclusions: H. felis can induce a hypertrophic gastropathy in the C57BL/6 genotype; loss of one p53 allele, although insufficient to initiate carcinogenesis at 1 year, enhances the proliferative index, which may lead to an increased risk of cancer induction.
A
clear relationship has been established between Helicobacter pylori infection and a variety of pathological disorders of the stomach, such as chronic gastritis, peptic ulcer disease, gastric mucosa–associated lymphoid tissue lymphoma, and gastric cancer.1,2 Although peptic ulcer disease remains an important health problem in developed countries, gastric cancer, which is the second leading cause of cancer death worldwide, is actually a more important global health problem, particularly in developing countries.3 H. pylori infection has been associated with pathological conditions that predispose to gastric cancer, such as atrophic gastritis and intestinal meta-
plasia.4 Other possible premalignant conditions, such as hypertrophic gastropathy or Me´ne´trier’s disease, are rare, and their relationship to H. pylori is unclear.5,6 Although several studies have shown an epidemiological association between H. pylori and gastric cancer, a definite causative link has not been firmly established and the underlying mechanisms have not been elucidated. One hypothesis proposes that H. pylori induces a chronic inflammatory response that leads to an increased rate of mucosal proliferation7,8 and subsequently to an increased risk of malignant transformation. In fact, recent studies have shown increased proliferating cell nuclear antigen (PCNA) staining, 5-bromo-2ⴕ-deoxyuridine (BrdU) incorporation, and proliferative indices in patients with H. pylori infection.9 – 11 An increased proliferative rate may increase the risk of malignant transformation by allowing more opportunity for mutagenic events to occur. To investigate this possibility, a well-characterized animal model of Helicobacter infection would be useful. While a number of animal models have been introduced, Helicobacter felis–associated gastritis in mice simulates many of the pathological features noted in human H. pylori infection.12 H. felis is a spiral organism originally isolated from the feline gastric tissue. After experimental oral inoculation, H. felis efficiently colonizes the gastric mucosa in mice, rats, and dogs.13 Recent studies in our laboratory indicated that H. felis chronically infected VAF Swiss–Webster mice, leading to an active chronic gastritis characterized by marked polymorphonuclear and mononuclear cell infiltration.14 However, after 1 year, H. felis–infected outbred Swiss–Webster mice did not develop any preneoplastic or neoplastic changes. The response to H. pylori infection varies greatly among individuals, and it is clear that the majority of infected patients do not develop gastric cancer. Although Abbreviations used in this paper: BrdU, 5-bromo-2ⴕ-deoxyuridine; PCNA, proliferating cell nuclear antigen. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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Figure 1. Stomach of a p53 hemizygous C57BL/6 wild-type mouse 6 months after H. felis inoculation. Nodular to plaque-like proliferation of the mucosa in the gastric body markedly increases the thickness of gastric pits and glands with inflammatory cell infiltrates (I) present in the deep mucosa and submucosa but rarely in the muscular layers (M). Mild glandular dilation and intraglandular accumulation of mucus (solid arrows) and/or cells (open arrow) are present in the deep mucosa (bar Å 156 mm).
the clinical outcome of Helicobacter infection may be influenced to some extent by differences in bacterial strains, the susceptibility to cancer development is believed to be determined largely by host genetic factors. Gastric cancer, like many other types of cancer, probably results from an interaction between environmental factors and a genetic predisposition. A number of alterations in tumorsuppressor genes have been described in human gastric cancer specimens, including loss of heterozygosity at the APC, DCC, and p53 loci.15,16 In addition, p53 gene mutations have been reported not only in gastric cancer17 but also in precancerous epithelium, suggesting that the mutation of the p53 gene is an early event in gastric tumorigenesis.18 Activation of oncogenes in gastric cancer has also been reported. However, it is difficult to determine from an analysis of human tumor specimens the true order of tumor progression, i.e., whether there is a causal relationship between each genetic alteration and progression of tumor growth. Thus, to determine the role of genetic factors in the response to gastric Helicobacter infection, we inoculated H. felis into a different strain of mice, the C57BL/6 strain, which is widely used in transgenic and gene knockout studies. In addition, we infected a commercially available strain of hemizygotic knockout mice, the TSG-p53 mice (GenPharm International, Mountain View, CA), which are deficient in one p53 allele and have been used previously in studies of cancer susceptibility.19,20 Taken together, our studies strongly support a role for a genetic basis in determining the response to chronic Helicobacter infection of the stomach.
Materials and Methods Animals Sixteen (9 female and 7 male) 4-week-old hemizygous TSG-p53 mice, deficient in one of the p53 genes, were obtained from GenPharm International. The GenPharm TSG-p53 mice were generated in 129-derived embryonic stem cells (A31) and have been back-crossed onto a C57BL/6 background. Therefore, nine 4-week-old female C57BL/6 mice were also obtained from GenPharm as controls. Hemizygous TSG-p53 mice have a low spontaneous level of tumorigenesis (about 2%) with a much greater survival rate compared with homozygous TSGp53.19,20 The mice were all maintained in an American Association for Accreditation of Laboratory Animal Care–approved facility under barrier conditions as VAF mice for the duration of the 12-month experiment. Animals were housed in microisolater, solid-bottomed polycarbonate cages and fed a commercially prepared pelleted diet and given water ad libitum. The protocol described below was approved by the Animal Care Committee of the Massachusetts Institute of Technology as well as the SRAC Committee of the Massachusetts General Hospital.
Bacteria H. felis (ATCC 49179) was used for oral inoculation as previously described.14 The organism was grown for 48 hours at 37⬚C under microaerobic conditions on 5% lysed horse blood agar supplement with antibiotics. The bacteria were harvested and inoculated (at a titer of 1010 organisms/ mL) into brain-heart infusion broth with 30% glycerol added. The bacterial suspension was frozen at 070⬚C. Before use, aliquots were thawed, analyzed for motility, and cultured for evidence of aerobic or anaerobic bacterial contamination.
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Experimental Infection Of the 16 TSG-p53 mice, 12 were inoculated with H. felis and 4 served as controls. Of the 9 wild-type C57BL/6 mice, 7 were inoculated with H. felis and 2 were controls. Brain-heart infusion broth containing Ç1010 colony-forming units of H. felis per milliliter was used as inoculum. The inocula (0.5 mL) was delivered by mouth into each test mouse three times at 2-day intervals by using a sterile oral catheter. At 6 and 12 months postinfection, infected p53 and wild-type mice and uninfected control mice were killed with CO2 and necropsied.
Histological Evaluation The tissue examined consisted of a section of gastric mucosa taken from the greater curvature of the stomach beginning at the gastroesophageal junction and ending at the gastroduodenal junction. Stomach tissues were fixed in neutral buffered 10% formalin, processed by standard methods, embedded in paraffin, sectioned at 5 mm, and stained with H&E and Warthin–Starry. The glandular mucosae of the body, antrum, and pylorus were examined histologically for a variety of inflammatory and epithelial changes and for the presence of H. felis. Sections of glandular stomach were also examined for diastase-resistant, periodic acid–Schiff positive staining.
Figure 2. Stomach of a p53 hemizygous C57BL/6 mouse 6 months after H. felis inoculation. A marked increase in the lengths of gastric pits with multifocal epithelial erosions are noted (arrows) (bar Å 24 mm).
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Quantitation of Parietal Cells in the Gastric Mucosa Gastric parietal cells were semiquantitated with a micrometer (OCM 10 1 10 SG; Olympus, Tokyo, Japan) on the H&E-stained sections from mice analyzed 12 months after the study commenced. Briefly, the gastric mucosa was scanned along the middle zone of the body, where parietal cells densely populate in the normal mucosa. A total of 4–6 nonoverlapping high-power fields (4001) were randomly selected along the midzone of the body for each animal. Parietal cells and nonparietal cells were counted with the guidance of the micrometer (1/16 mm2). The data were expressed as percentages of the parietal cells out of the total epithelial cells in a given field. In the infected animals, the data were further tabulated by the foci of mucous hypertrophy or epithelial hyperplasia of the body mucosa. The data between the control and infected animals were compared by a two-tailed t test.
Immunohistochemistry Gastric mucosa was examined by immunohistochemistry for BrdU and PCNA.
Figure 3. Stomach of a p53 hemizygous C57BL/6 mouse 6 months after H. felis inoculation. The mucous epithelial cells are distended by mucus in their apical cytoplasm with pale mucoid material in the glandular lumina (m). The hyperplastic epithelial cells are round to oval with prominent nuclei and basophilic cytoplasm (H). The parietal cells (arrows ) are markedly decreased. Aggregations of predominantly lymphocytes and macrophages are evident in the deep mucosa (bar Å 27 mm).
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Figure 4. Stomach of a wild-type C57BL/6 mouse 12 months after H. felis inoculation. Adenomatous hyperplasia and cystic dilation of gastric mucosa with lymphocyte aggregates (open arrows) in the deep mucosa and edematous submucosa. The glands penetrate the muscularis mucuosae (M) and invade into the submucosa multifocally (solid arrows). Transition (T) between the hyperplastic mucosa and the antral mucosa seems to be abrupt (bar Å 350 mm).
BrdU immunocytochemistry. In vivo incorporation of BrdU. Animals received a single intraperitoneal injection of
BrdU (50 mg/kg) from a freshly made stock solution (5 mg/ mL) dissolved in phosphate-buffered saline. The mice were killed 1 hour later. At necropsy, a longitudinal section of stomach was taken from the greater curvature extending from the esophageal junction to the gastroduodenal junction. Samples were immediately placed in a cassette, fixed in Carnoy’s fixative overnight, and embedded in paraffin wax. Immunohistochemical detection of BrdU. Immunohistochemical detection of the BrdU incorporation was performed on 4mm sections and visualized using the following avidin-biotin monoclonal antibody immunohistochemical technique. After deparaffinization in xylene through to alcohol, endogenous peroxidase activity in the tissue section was blocked by immersing the slides in 1% hydrogen peroxide in methanol. The slides were then washed in tap water. The BrdU monoclonal antibody only identifies single-stranded DNA. Denaturation of the tissue DNA was achieved by incubation in 1 mol/L hydrochloric acid at 60⬚C for 8 minutes. The slides were washed in tap water and Tris-buffered saline and were incubated with 5% normal rabbit serum to block nonspecific binding of the primary antibody. The tissue was then incubated with the monoclonal antibody to BrdU (Dakopatts, Glostrup, Denmark) diluted to 1:40 in Tris buffer. Control slides, included in each assay, were incubated with mouse serum. The slides were again washed in Tris-buffered saline and incubated with biotinylated anti-mouse immunoglobulin G (1:200 in Tris buffer). After further washings in Tris-buffered saline, slides were incubated with peroxidase-conjugated strepavidin (1:400 in Tris buffer) and washed in Tris-buffered saline, and the labeled cells were visualized by the diaminobenzidine reaction and lightly coun-
Figure 5. Stomach of a wild-type C57BL/6 mouse 12 months after H. felis inoculation. Many epithelial cells are distended by mucus in their apical cytoplasm with nuclei located at the base. The parietal cells (arrows) are markedly decreased. Some of the glandular lumina contained mucoid material (M) and cellular debris (D) (bar Å 27 mm).
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terstained with hematoxylin. The nucleus of cells at the S phase of the cell cycle during the in vivo incorporation phase were stained with brown. Immunohistological analysis. A mean of four entire tissue sections were examined for each animal. Only gastric pits longitudinally sectioned within the upper one third of gastric mucosa and visible in their entire length (ú60 cells) were analyzed. A mean number of 10 well-orientated gastric pits was examined for each specimen. Labeling index was measured by counting the number of BrdU-positive cells and expressing the result as a percentage ratio of the total number of cells in each gastric pit. The relative positions of positive cells in each gastric pit were noted. For cell kinetics evaluation, each gastric pit was divided into five compartments of equal size. The compartments were referred to by the ordinary numbers 1 (luminal zone) to 5 (base of the gastric pit) and the labeling index calculated for each compartment separately. Statistical analysis. The total labeling index and the labeling index per compartment was compared between the
Figure 6. Stomach of a wild-type C57BL/6 mouse 12 months after H. felis inoculation. The glands (G) penetrate the muscularis mucosae (M), invade into the submucosa, and are surrounded by predominantly lymphocytes. A few clusters of the glands reside within the muscularis mucosae (arrows) (bar Å 54 mm).
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study groups. Significance was analyzed using the two-tailed Student’s t test for unpaired data. P values of õ0.05 were considered statistically significant. PCNA immunocytochemistry. Five-micrometer-thick tissue sections were cut, mounted, and dry baked at 60⬚C for 1 hour. The sections were deparaffinized in xylene for 5 minutes, followed by submersion in 100% ethanol and 95% ethanol, respectively. The slides were then placed in preheated (199⬚F) 10 mmol/L citrate buffer for 30 minutes. After a wash in phosphate-buffered saline, the tissue sections were incubated for 15 minutes with horse immunoglobulin G serum provided in the avidin-biotin peroxidase complex kit (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). The sections were then incubated with the PCNA antibody (antihuman, cross-reacts with mouse, immunoglobulin G2a subclass; Pharmingen, San Diego, CA) at a 1:400 dilution (diluted with 2% horse serum in phosphate-buffered saline). After rinsing in phosphate-buffered saline, the slides were incubated with a secondary biotinylated antibody, provided in the ABC kit, for 30–35 minutes. After another rinse in phosphate-buffered saline, the ABC reagent was applied for 40 minutes. Following another rinse in phosphate-buffered saline, the slides were de-
Figure 7. Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Diastase-resistant periodic acid–Schiff stain revealed abundant mucin in the apical cytoplasm of the epithelial cells and within the glandular lumina (bar Å 107 mm).
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ples were obtained and analyzed from the 25 mice included in this study at baseline and 5 and 10 weeks postinoculation. At 26 and 52 weeks after dosing, sera were collected and analyzed from mice being killed.
Results Experimental Design At 6 months postinoculation, 2 wild-type C57BL/ 6 and 4 p53 H. felis-infected mice were killed along with 1 wild-type and 2 p53 uninfected controls; at 12 months, 1 wild-type and 1 p53 controls and 5 H. felis-infected p53 and 3 H. felis-infected C57BL/6 wild-type mice were killed and necropsied. Also during the study, 1 infected C57BL/6 mouse was killed at 6 weeks after dosing, and 3 female p53 mice were killed at 3, 34, and 44 weeks after dosing because of weight loss. One control p53 female mouse was found dead at 50 weeks after dosing. Histopathology
Figure 8. Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Warthin–Starry stain revealed numerous H. felis in the glandular lumina (arrows) (bar Å 10.8 mm).
veloped in 3,3ⴕ-diaminobenzidine tetrahydrochloride (10 mg 3,3ⴕ-diaminobenzidine tetrahydrochloride in 10 mL 0.5 mol/ L Tris buffer, 3 drops 3% H202). Methyl green (2%) was used for 30 minutes to counterstain the tissue sections. Dehydration was performed quickly in 95% ethanol, twice in 100% ethanol, and twice in xylene. Immunostained slides were evaluated for the degree of PCNA staining by noting the location and relative size of the proliferative zone in the gastric antral and corpus epithelium. The proliferative zone corresponds to the segment of epithelium between the level of the highest and lowest PCNA-labeled cell.
By 6 months after infection, mild to moderate nodular to plaque-like thickening of the mucosa was observed in the gastric body with variable sparing of the antrum in both wild-type and transgenic mice in comparison with uninfected controls. The affected mucosa was about 2–3 times the normal thickness in H. felis–infected mice. Microscopically, the nodules and/or plaques were composed of hypertrophic and hyperplastic mucosal epithelium with a variable degree of cystic glandular dilation and inflammatory cell infiltration (Figure 1). There was an increase in lengths of both gastric pits and glands with multifocal erosions on the pit surface. The pit cells
Enzyme-Linked Immunosorbent Assay for H. felis Serum Immunoglobulin G Antibody The enzyme-linked immunosorbent assay for H. felis was performed as previously described.14 Wells of microtiter plates (Dynatech Laboratories, Chantilly, VA) were incubated with 100 mg of H. felis protein per milliliter in carbonate buffer. After being washed, serial twofold dilutions of sera from H. felis–infected or control mice were incubated for 60 minutes at 37⬚C and then given the appropriate dilution of alkaline phosphatase–conjugated goat antimouse immunoglobulin G (Sigma Chemical Co., St. Louis, MO). Serum sam-
Figure 9. Quantitation of parietal cells in the gastric body mucosa. Gastric parietal cells in H. felis –infected mice with either mucous hypertrophy or epithelial hyperplasia were significantly (P õ 0.01) lower than those in control animals. Hypertrophic or hyperplasia mucosa had similar numbers of parietal cells (P ú 0.05) in the infected mice.
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H. FELIS MOUSE MODEL 161
Figure 10. (A ) Stomach of a p53 hemizygous C57BL/6 mouse control (bar Å 54 mm). (B ) Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Immunohistochemical stain detecting BrdU incorporation. Brown cells in A and B are positive for BrdU incorporation (bar Å 54 mm).
were oval to columnar and mostly contained eosinophilic droplets in their atypical cytoplasm (Figure 2). The hyperplastic cells, often located between the pit and glandular regions, were round to oval and smaller in size with prominent nuclei and basophilic cytoplasm (Figure 3). The glandular/mucous cells were columnar and were distended often by mucus in their apical cytoplasm (Figure 3). Some of the dilated glandular lumina contained intraluminal eosinophilic to basophilic mucoid material, neutrophils, mononuclear cells, and/or cellular debris. The squamous epithelial cells of the forestomachs were also hypertrophic and hyperplastic with eosinophilic droplets in the cytoplasm. Mild to marked infiltration of predominantly lymphocytes, as well as some macrophages and neutrophils, were observed in the deep mucosa and submucosa with occasional formation of lymphoid aggregates and interruption of muscularis mucosae (Figure 1). By 12 months after infection, prominent polypoid nodules and cerebriform folds or rugae were observed in the gastric body mucosa with variable extension into the antrum in both wild-type and transgenic mice. The
nodules or folds were distributed predominantly along the greater curvature with focal rigidity. Transition between the nodules or folds and normal-appearing mucosa seemed to be abrupt in most cases. Microscopically, the nodules or folds were composed of hyperplasia proliferation and cystic dilation of gastric mucosa (Figure 4). The affected mucosa was about 3–5 times of the normal thickness, and both gastric pits and glands were markedly increased in length. The dilated glandular cysts contained intraluminal pale mucoid material mixed with variable amounts of neutrophils, mononuclear cells, and/ or debris (Figure 5). The base of the glands broke through the muscularis mucosae and invaded into the submucosa multifocally in some mice (n Å 2) (Figure 6). There was marked infiltration of predominantly lymphocytes and some macrophages as well as neutrophils in the lamina propria and submucosa with frequent formation of lymphoid follicles (Figure 4). The squamous epithelial cells were still hypertrophic and hyperplastic, but intracytoplasmic droplets were not present in squamous or pit cells.
162 FOX ET AL.
Figure 11. Stomach of a p53 hemizygous C57BL/6 mouse 12 months after H. felis inoculation. Immunohistochemical detection of PCNA antigen (bar Å 54 mm).
Diastase-resistant periodic acid–Schiff stain revealed abundant mucin in the apical cytoplasm of many glandular cells and in many glandular lumina (Figure 7). Warthin–Starry stain revealed variable numbers of H. felis organisms in the gastric pits and glandular lumina in mice at both 6 and 12 months after infection (Figure 8). No pathological changes were observed in the uninfected control mice at both 6 and 12 months of the study. Gastric parietal cells were markedly decreased in the mucosa of infected animals. Percentages of the parietal cells in the mucosa of the infected animals were approximately the same in wild-type and transgenic mice, but both infected groups were significantly (P õ 0.01) lower in the body than those of the control mice (Figure 9). Hypertrophic and hyperplastic foci of the body mucosa were similarly affected (P ú 0.05) in the H. felis–infected animals. To determine the location and characteristics of the progenitor zone of the stomach, BrdU was administered 1 hour before the mice were killed. The assay measured
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the average number of BrdU-labeled S-phase cells per total number of cells in the gastric pit. In assessment of epithelial cell kinetics of the gastric pit, the zone of maximum proliferation is in the base of the gastric pit, which represents the neck region of the gastric glands. In uninfected mice, BrdU labeling was confined to a limited zone, consistent with the labeling of a discrete progenitor zone (Figure 10A). In infected mice (C57BL/ 6 and p53), the progenitor zone was markedly expanded with labeling in the upper zones (Figure 10B). PCNA staining confirmed this finding of a marked expansion of the proliferative zone in both directions, including at the surface epithelium, in the H. felis–infected mice (Figure 11). Overall, there was marked increase in the proliferative zone, as detected by PCNA staining, in infected p53 and wild-type mice at 6 and 12 months compared with uninfected p53 and wild-type mice. BrdU labeling in 12-month-old mice was quantified (Table 1). These data confirmed the qualitative impression of an increased proliferation rate in infected mice compared with uninfected mice (Figure 12). In addition, they showed that the proliferation index was higher in infected hemizygous p53 mice compared with infected wild-type C57BL/6 control mice, although this did not reach significance (P Å 0.058). At 12 months, there was an extension of the proliferative zone towards the luminal surface of the gastric pits in animals infected with H. felis. This was more marked in the body mucosa (Table 1). Serology Immunoglobulin G antibodies to H. felis were detected in all mice infected with H. felis but not in any control mice or in any of the 25 mice assayed before the study commenced (Figure 13). Interestingly, female mice seemed to have a greater systemic immune response to H. felis than males. The H. felis antibody response began to increase at 5 weeks after dosing, plateaued at 10 weeks after dosing, and remained elevated at 6 and 12 months after H. felis experimental infection.
Discussion Several epidemiological studies strongly implicate H. pylori infection as a carcinogen in the stomach.21,22 Furthermore, a new murine pathogen named Helicobacter hepaticus has been shown to cause a persistent chronic hepatitis and in the A/JCr mouse has been linked to the development of hepatic tumors. In addition to eliciting an immunoglobulin G immune response, H. hepaticus causes an increase in hepatic cell proliferation as measured by PCNA and in this manner has been implicated in the pathogenesis of H. hepaticus–induced hepatic can-
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H. FELIS MOUSE MODEL 163
Table 1. Comparison of the BrdU Labeling Indices of All Experimental Mice at 12 Months Wild-type control
Wild-type H. felis infected
p53 control
Entire
Body
Pyloric region
Entire
Body
Pyloric region
1540
760
780
1836
835
1001
73.3 (8.8)a 4.32 (1.8)
76 (9.7) 4.92 (1.94)
70.9 (8.1) 3.73 (1.5)
90.0 (12.0) 4.15 (1.5)
83.5 (5.7) 3.23 (1.10)
100.1 (11.5) 5.30 (1.3)
LI% per compartment LI% 1 (apex)
0
0
0
0
0
0
LI% 2
0
0
0
0
LI% 3
2.44 (3.2) 6.49 (5.7) 12.86 (8.2)
3.86 (3.3) 6.87 (7.2) 14.1 (8.6)
1.26 (2.6) 5.87 (4.1) 11.75 (8.0)
0.73 (1.9) 3.26 (5.6) 5.69 (7.1) 11.6 (8.0)
1.87 (2.6) 6.53 (7.16) 4.64 (7.2) 14.2 (8.3)
Total cells counted per mouse Cells/gastric pit LI% total
LI% 4 LI% 5
0.64 (2.0) 6.53 (7.2) 8.99 (6.6)
p53 H. felis infected
Entire
Body
Pyloric region
Entire
Body
Pyloric region
2190 (153) 109.5 (6.2) 5.60 (0.83)
1149 (172) 114.9 (17) 5.86 (0.95)
1040 (28) 104.1 (2.8) 5.34 (0.5)
2141 (366) 104.6 (9.7) 8.40 (1.92)
1017 (130) 103.6 (10.8) 8.93 (1.87)
1128 (325) 104.9 (13.1) 7.98 (2.87)
0.06 (0.1) 1.03 (0.7) 4.45 (1.2) 10.65 (3.2) 10.85 (2.07)
0.11 (0.2) 1.35 (1.3) 4.25 (1.8) 11.3 (6.4) 11.6 (3.1)
0
0.05 (0.1) 1.91 (1.0) 7.25 (1.3) 15.15 (3.7) 16.49 (5.9)
0.11 (0.2) 1.48 (0.5) 8.16 (2.6) 17.52 (7.8) 16.39 (7.0)
0
0.71 (0.3) 4.64 (2.2) 9.76 (0.73) 10.14 (1.4)
2.35 (2.3) 7.00 (2.7) 13.69 (6.0) 16.73 (6.6)
NOTE. All results expressed as mean { SD. a A mean of 20 gastric pits were analyzed per mouse, which were subdivided into body and pyloric region. LI%, labeling index.
cer.23 – 25 Previous studies in humans also have suggested that H. pylori infection may contribute to gastric cancer susceptibility by stimulation of mucosal proliferation.7–11 Our study shows that chronic H. felis infection in C57BL/ 6 mice leads to an increase in the proliferation index as measured by both PCNA staining and BrdU incorporation. The proliferative zone of the stomach is normally located at the junction of the base of the pits and the upper region of the gastric glands, the so-called stem cell zone. Under normal conditions, newly formed cells migrate upward from this zone to replenish the surface and foveolar mucous cells or downward to replenish the gastric glandular cells. However, expansion of the proliferative zone has been observed in a number of conditions associated with an increased risk of gastric cancer, such as chronic gastritis,26 pernicious anemia,27 and in the postantrectomy stomach.28 In addition, a similar broadening of the proliferative zone has been found in the rat stomach after exposure to the gastric carcinogen Nmethyl-Nⴕ-nitro-N-nitrosoguanidine. N-methyl-Nⴕ-nitro-N-nitrosoguanidine has been shown to stimulate cell proliferation in the rat stomach and causes a downward expansion of the proliferative zone in both the antrum and fundus.29 In a similar fashion, chronic H. felis infection broadens the proliferative zone in the stomach of the C57BL/6 mouse. However, the mechanism for this proliferative effect is unclear. One possible explanation is that an ongoing cycle of inflammation-mediated epi-
thelial destruction followed by activation of growth factors and subsequent regeneration may lead to increased cell turnover. Nevertheless, regardless of the precise mechanisms involved, an increased proportion of gastric cells undergoing cell division could result in increased mitotic error, an increased mutation rate, and an increased risk of cancer formation. Because loss of heterozygosity at the p53 locus has been found frequently in human gastric cancer specimens and is thought to play a crucial role in cancer development, we infected both hemizygous p53 knockout mice and wild-type C57BL/6 with H. felis. Our results suggest that mice that lack one copy of the p53 gene show a significantly increased proliferative index compared with wild-type controls. Reduction in the gene dosage and consequently in the amount of p53 produced by the normal cell does seem to confer a significant growth advantage in the gastric epithelium in this mouse model. However, despite an increased rate of proliferation, we did not detect any evidence of cancer in the H. felis– infected p53 mouse stomach after a 1-year observation period. The development of frank neoplasia in this mouse model may require either additional mutations, genetic events, or a longer time period. Therefore, loss of p53 may still offer a selective advantage in gastric cancer development. We did not choose to infect homozygous p53 knockout mice in this study because of their known diminished survival (3–6 months).19,20
164 FOX ET AL.
Figure 12. Mean labeling index for individual control and experimentally infected mice using the BrdU immunohistochemical technique at 12 months.
Our data are consistent with previous studies of cancer susceptibility in TSG-p53 mice. In particular, a mouse skin model of multistage carcinogenesis has been well characterized, with stages of initiation (with dimethylbenzanthracene) and promotion and progression (with 12-O-tetradecanoyl-phorbol-13-acetate). Both dimethylbenzanthracene and 12-O-tetradecanoyl-phorbol-13-acetate are required for the development of skin cancer because either alone has been shown to be insufficient. In this mouse model of skin cancer, the initial development of tumors by dimethylbenzanthracene and 12-O-tetradecanoyl-phorbol-13-acetate was not increased in p53 hemizygous lines.30 Furthermore, p53 homozygous (null) mice treated with 12-O-tetradecanoyl-phorbol-13-acetate alone did not develop skin tumors either. However, both the hemizygous and the homozygous p53 knockout mice did show faster tumor progression after treatment with dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate with an increase in the rate of benign to malignant conversion. Thus, in this model, the absence of p53 does not augment the frequency of initiation or rate of promotion but greatly enhances the progression of malignancy. Perhaps the most surprising finding in this study was the effect of H. felis infection on the wild-type C57BL/ 6 mouse. Previous studies in which H. felis was inoculated in Swiss–Webster mice revealed active, chronic inflammation with minimal hyperplasia, but the histological changes did not approach the severity of gastric disease shown by the presence of diffuse cystic hyperplasia
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in the C57BL/6 mice after H. felis infection in this study.12,14 However, previous investigators have pointed out that the gastric phenotype (in response to growth factors, for example) is strongly influenced by the genetic background of the mouse. For example, overexpression of transforming growth factor a in inbred FVB/N mice leads to much more severe cystic hyperplasia compared with that observed in the outbred strain CD1.31 Thus, our study as well as preliminary data presented by others suggesting that particular inbred strains of mice have differing severities of H. felis–induced gastritis points to the importance of the genetic makeup in determining the response to chronic infection with H. felis.32 The histological changes observed in infected C57BL/ 6 mice (severe adenomatous and cystic hyperplasia of the surface foveolar epithelium) resemble in some respects those described in patients with hypertrophic gastropathy (Me´ne´trier’s disease). The number of parietal cells in the body mucosa were also significantly decreased in H. felis–infected mice compared with the control uninfected animals. This finding is also a consistent observation noted in Me´ne´trier’s disease. However, given the variation in mucosal thickness between the infected and uninfected mice, these data should be interpreted with caution. Other features typically present in Me´ne´trier’s disease, such as hypoalbuminemia and decreased acid secretion,5 were unfortunately not assessed before the mice were killed in this study; thus, further studies will be required to determine whether the H. felis–infected C57BL/6 mouse is a proper model for this disease. There has been some controversy in the literature regarding a possible association of Me´ne´trier’s disease with H. pylori
Figure 13. Serum immunoglobulin G antibody response in H. felis – infected mice.
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infection.6 One study by Wolfsen et al. found a low frequency of H. pylori (30%–39%) by routine histology and also suggested that Me´ne´trierⴕs disease could be classified into two diseases: hypertrophic lymphocytic gastritis and massive foveolar hyperplasia and minimal inflammation.6 However, a recent study that evaluated 138 patients with hypertrophic gastropathy indicated a high percentage (90%) of cases associated with H. pylori gastritis.33 In that study, there seemed to be marked H. pylori colonization and a higher degree of gastritis in the corpus of the stomach compared with the antrum,33 which is in agreement with our findings in H. felis–infected C57BL/ 6 mice as well as in our earlier studies in Swiss–Webster mice.14 Further evidence for a role of H. pylori in Me´ne´trier’s disease stems from patients who showed marked improvement after treatment with antibiotics.34 – 36 In one case, a 6-week course of amoxicillin and omeprazole led to eradication of H. pylori and normalization of the histological abnormalities.37 Thus, it would be interesting to determine if antibiotic treatment of H. felis–infected mice, as previously described,38 can reverse the hypertrophic gastropathy in our C57BL/6 murine model. Recent data suggest that mice can also be infected with cytotoxin-positive and negative H. pylori strains.39 However, the gastritis induced by H. pylori in mice described to date has been minimal, being limited to focal inflammation and gastric mucosal erosions. The ability of H. pylori to persistently infect mice has not been established; thus, the H. pylori mouse model needs to be studied further to ascertain whether the model will mimic H. pylori disease in humans. Therefore, the H. felis mouse model remains the most viable animal model to study H. pylori pathogenesis. This model may prove useful for studying the role of Helicobacter in inducing epithelial proliferation and the genetic factors that predispose to gastric cancer.
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Received July 24, 1995. Accepted September 8, 1995. Address requests for reprints to: James G. Fox, D.V.M., Division of Comparative Medicine, Massachusetts Institute of Technology, Building 45, 37 Vassar Street, Cambridge, Massachusetts 02139. Fax: (617) 258-5708. Supported by the R. Robert and Sally D. Funderburg Research Scholar Award in Gastric Cancer Biology (to T.C.W.), National Institutes of Health grant RO1 CA67463 (to T.C.W., J.G.F., and A.K.R.), and grants RR07036 and RR01046 from the National Center for Research Resources (to J.G.F., X.L., R.J.C., and K.A.). Dr. Rustgi was supported by the Miles and Shirley Fiterman AGA Award (Walter B. Cannon).