ANTICANCER RESEARCH 25: 903-912 (2005)

Gastric Hamartomatous Tumours in a Transgenic Mouse Model Expressing an Activated Dioxin/Ah Receptor

PATRIK ANDERSSON1,2, CARLOS RUBIO3, LORENZ POELLINGER2 and ANNIKA HANBERG1

1Institute of Environmental Medicine, Departments of 2Cell and Molecular Biology and 3Pathology, Karolinska Institutet, Stockholm, Sweden

Abstract. Background: The dioxin/ Aryl hydrocarbon (Ah) expressing viral oncogenes such as the SV40 large T receptor is a ligand-activated transcription factor known to antigen (7), adenovirus 12 E1A/E1B (8) and the papilloma mediate the toxic effects of the environmental pollutants dioxins. virus 16 early region (9). Materials and Methods: Transgenic mice expressing a More recently, we have reported that tumours of the constitutively active Ah receptor were found to develop gastric glandular develop in transgenic mice that express a tumours. Results: Segments of the normal of the constitutively active dioxin/aryl hydrocarbon receptor corpus region (lined by foveolar, parietal and cardio-pyloral cells) (CA-AhR) (10). This model differs, however, from the developed cystic tumours. Tumours in the and aforementioned experimentally-induced models of gastric muscularis propria consisted of large cysts surrounded by tumours in as much as the mice were not exposed to chemical organized lymphocytic, connective, squamous and adipose tissues carcinogens or viral oncogenes. Instead, the CA-AhR and vessels. Those tumours were obviously hamartomatous in expressed in these mice is a genetically activated form of a nature. Despite the fact that the tumours had an inward growth transcription factor (11), known to mediate the toxic effects and penetrated all stomach layers, no metastases developed. of the environmental pollutants dioxins and polychlorinated Conclusion: The model here described may open new vistas into biphenyls (PCBs) (12). Increased mortality in CA-AhR mice the investigation of the role of environmental pollutants in the was observed, beginning at 6 months of age (10), and the development of gastric hamartomas. results indicated that both the severity of the tumours at a certain age and increased mortality were influenced by the In 1966, Sugimura and Schoental independently found that sex as well as the CA-AhR founder line (10). the administration of N-methyl-N-nitroso-N-nitroguanidine The aim of the present study was to describe, (MNNG) induced tumours in the glandular stomach of rats characterize and illustrate the histological characteristics (1-3). Since then, many animal models have been designed of these gastric tumours in detail. We therefore focused on to investigate the evolution of tumours in the gastric a detailed histopathological characterization of the mucosa by the aid of N-methylnitroseurea (MNU) (4) in tumours, the chronological order of tumour development, mice or 1,2-dimethylhydrazine (DMH) following gastric sex differences and on the comparison between the two irradiation in rats (5). Low doses of MNNG administered different founder lines. to mongolian gerbils (Meriones unguiculatus) infested with Helicobacter pylori caused tumours in the glandular stomach Materials and Methods (6). Stomach tumours were also detected in transgenic mice Animals. Generation of the CA-AhR (11) mice has previously been reported (10). Briefly, CA-AhR mice bred to homozygosity and wild-type control animals were of the same mixed genetic Correspondence to: Annika Hanberg, Institute of Environmental background. The animals were kept in ventilated filter top cages, Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, exposed to a 12-hour light/dark cycle, and received water and Sweden. Tel: +46(0)8-5248 7526, Fax: +46(0)8-34 38 49, e-mail: standard rodent feed (RM3, Special Diet Services, Essex, UK) ad [email protected] libitum. The animals were sacrificed by CO2 asphyxiation followed by cervical dislocation. All experimental procedures were approved Key Words: Gastric tumour, hamartoma, dioxin, Ah receptor. by the local ethical committee on animal experiments.

0250-7005/2005 $2.00+.40 903 ANTICANCER RESEARCH 25: 903-912 (2005)

Figure 1. Macroscopically visible cystic gastric tumours and early changes in the CA-AhR stomachs. (A-D) Photographs of mouse stomachs showing the localization of the forestomach (fs) and the glandular stomach (gs). In C and D arrows show the limiting ridge (lr). (A and B) External view of stomachs from 52-week-old male CA-AhR (Y8 founder line) and wild-type (wt) mice. Arrow shows dark spot. (C) Inside of a stomach from a 52-week-old wild-type male. (D) Inside of a stomach from a 27-week-old CA-AhR male from the A3 founder line. Asterisk indicates bleeding in the mucosa. (E-G) Sections showing gastric zymogenic mucosa from 10-week- old males, wild-type in E and serial sections from CA-AhR mouse of the Y8 founder line in F and G, stained with hematoxylin-eosin (HE; E and F) and the proliferation marker PCNA (G). The foveolar regions are shown in brackets. Tumour penetrating the submucosa and muscularis propria is shown by arrows in F and G. mp = muscularis mucosa; sm = submucosa. Bars = 0.2 mm. (H-J) Stomach sections from 10-week-old CA-AhR (Y8 founder line; H and I) or wild-type (J) male mice stained with HE. Arrows in H show dilated glands basally located in the mucosa Bar = 0.1 mm. In I are glands of cardio-pyloric type seen to expand underneath (arrow) the squamous of the limiting ridge (lr) Bar = 0.35 mm. (J) Limiting ridge of a wild-type animal shown as reference. Bar = 0.3 mm.

Figure 2. Lesions in the mucosa and characteristics of the CA- AhR gastric tumours. (A-E) Stomach section from a 27-week- old male CA-AhR mouse of the A3 founder line stained with HE. The location of the magnified areas in B-E are marked by squares and corresponding letters in A. In A, arrows show multifocal penetration into the submucosa and muscularis propria, asterisks show multiple cystic dilatations in the mucosa. Glands and connective tissue are seen in the limiting ridge (lr). A region showing hamartomatous characteristics is marked by a square (ham). mm = muscularis mucosa, mp = muscularis propria. Bar = 0.75 mm. (B) Occluded pits with accumulation of connective tissue and lymphatic cells. Bar = 0.1 mm. (C) Part of penetrating tumour showing differentiated foveolar cells (fc) and parietal cells (pc). Bar = 0.1 mm. (D) Part of penetrating tumour showing foveolar cells and glands of cardio- pyloral type (cpg). Bar = 0.1 mm. (E) Enlarged tumoural cyst lined with one layer of epithelial cells. Bar = 0.1 mm. (F) Section from a 37-week-old CA-AhR male (Y8 founder line) showing a region with characteristics of a hamartoma, such as well-differentiated cells in a tumour cyst (tc), fatty tissue (ft), lymphatic tissue (lt), connective tissue (ct) and vessels (v). HE; bar = 0.1 mm. (G) Tumour cyst found within the muscularis propria from a 37-week-old CA-AhR mouse (Y8 founder line) showing both foveolar cells and metaplastic squamous epithelium (sq.e). HE; bar = 0.1 mm. (H and I) Adherence to liver (liv) and pancreas (panc) in a 52-week-old CA-AhR male (Y8 founder line). Tumour cells (tc) and connective tissue (ct) are also shown. HE; bars = 0.2 mm. (J) The tumour cysts in G showing positive Alcian Blue pH 2.5 staining (arrows). Bar = 0.1 mm. (K) Occluded pit from the 27-week-old CA-AhR male shown in A-E stained against vimentin showing accumulation of connective tissue (asterisk) and blood-derived cells (arrow). Bar = 0.05 mm. (L) Tumours in the muscularis propria from a 52-week-old CA-AhR male (Y8 founder line) showing glands of cardio-pyloral type by Concanavalin A staining. Bar = 0.3 mm. (M) The tumour cysts in G and J showing positive PAS staining (arrows). Bar = 0.1 mm.

904 Andersson et al: Gastric Tumours in the CA-AhR Transgenic Mouse Model

containing mucous glands. The squamous and the glandular zymogenic portions were separated by a fold, named the limiting ridge (13).

Tissue samples were collected at several ages from both CA-AhR and wild-type mice. Male mice demonstrated a higher susceptibility for developing gastric tumours. Therefore, the study of the time factor was focused on males. The animals were sacrificed at 3, 5, 9, 10, 12, 13, 16, 27, 37 and 52 weeks for males and 3, 9, 10, 16, 27, 38 and 52 weeks for females. The stomachs were removed and rinsed in cold saline solution. An incision was made along the major curvature and the stomach contents were removed by rinsing in cold saline solution. The stomachs were then weighed, gently flattened and pinned on small paraffin blocks and fixed in buffered formalin.

Histochemistry and immunohistochemistry. The fixed tissue samples were dehydrated, embedded in paraffin and cut into 4-Ìm thick sections. Staining with hematoxylin-eosin (HE), Alcian blue pH 2.5, PAS (periodic-acid-schiff) and immunohistochemistry against vimentin was performed according to routine diagnostic procedures at the Karolinska University Hospital, Stockholm, Sweden. Concanavalin A staining has previously been described (14). Detection of parietal cells was performed as follows: sections were de-paraffined, rehydrated and heat-treated for 15 min in Tris- EDTA (pH 9) in a microwave oven. Endogenous peroxidase was blocked in 3% H2O2 in methanol for 15 min and unspecific binding was blocked by incubation for 30 min in PBS-T (=PBS + 0.1 % Triton X-100). Incubation with horseradish peroxidase-conjugated avidin complex (ABC-kit; DakoCytomation, Glostrup, Denmark) and washing was done according to the manufacturer’s protocol. Diaminobenzidine was used as the chromogen substrate. Staining against PCNA (proliferating cell nuclear antigen; Santa Cruz Biotech, Santa Cruz, California, USA) was done as described for parietal cells with the addition of an incubation-step with the primary antibody (Santa Cruz Biotech, Santa Cruz, California, USA) in PBS-T for 30 min and horseradish peroxidase-conjugated Envision+ (DakoCytomation, Glostrup, Denmark) was used as secondary reagent. Figure 3. Age, sex, founder strains and lesion severity. (A-D) Bar graphs showing the percent of those animals analyzed for that group and age that Section analysis and quantification of the parietal/chief cell area. The shows cystic dilatations in the mucosa (A and C) or tumours penetrating identity of hematoxylin-eosin-stained sections was coded. For into the muscularis propria (B and D). Male and female mice of the Y8 quantification of the parietal/chief cell region, high-resolution founder line (A and B) or males of the Y8 or A3 founder lines (C and D) pictures of hematoxylin-eosin-stained sections were taken at the are compared. The number of animals in each group is 2-10. (E and F) same magnification (40x, Leitz Diaplan microscope) of the mucosal Bar graphs showing the average relative weights (stomach/body weight, region approximately 3 mm most proximal to the limiting ridge, BW) of emptied and rinsed stomachs for male (E) or female (F) mice. using a digital camera (Nikon Coolpix 995). The images were Error bars represent standard deviation. Student’s t-test was used for statistical analysis. #, ##, ### (p<0.05, 0.01 and 0.001, respectively) analyzed with the Scion Image program (Version beta 4.0.2) by denotes statistical significant difference compared to wild-type mice and manually circumscribing the total mucosa area and the $, $$ (p<0.05 and 0.01, respectively) denotes statistical significant parietal/chief cell region. The Scion Image program is the Windows difference compared to mice from the Y8 founder line. version of the image analysis program NIH Image, and can be found at http://www.scioncorp.com.

Isolation of RNA and RNA blotting. The glandular part of mouse stomachs was removed and snap-frozen in liquid nitrogen. Total RNA was prepared by tissue homogenization in a guanidinium Tissue sample collection. Following the recommendations by Lee thiocyanate buffer followed by CsCl2-gradient centrifugation (15). et al. (13), the stomach in the mice was divided anatomically into RNA blot analysis was performed by standard methods. The filter three portions: a cephalic portion (the forestomach), covered by was hybridized with specific 32P-labelled cDNA fragments in squamous epithelium, a corpus portion containing zymogenic formamide-containing buffer (15) or Ultrahyb solution (Ambion, glands including parietal and chief cells, and an antral portion, Huntingdon, Cambridgeshire, UK), and exposed to autoradiographic

905 ANTICANCER RESEARCH 25: 903-912 (2005)

Figure 4. Parietal/chief cell region in CA-AhR mice. (A-D) Serial sections from a 70-day-old wild-type male mouse showing staining with HE (A, C), or the surrogate marker for parietal cells described in the text (B, D). (A, B) Sagittal plane section of stomach glandular mucosa showing the region (brackets), muscularis propria (mp), vessels (v) and fatty tissue (ft). Bars = 0.2 mm. (C, D) Transverse plane section of gastric glands showing parietal cells (arrows). Bars = 0.05 mm. (E, F) Stomach sections from 27-week-old (E) wild-type and (F) CA-AhR (A3 founder line) male mice stained with the surrogate marker for parietal cells (marked with brackets). lr = limiting ridge, mp = muscularis propria. Bars = 0.5 mm.

film. The filter was stripped and reprobed with labelled cDNA content along the minor curvature of the stomach. With + + fragments for the H /K -ATPase ‚-subunit. Band intensity was time, these cysts became more numerous and the color of quantified by Phosphoimager analysis (Fuji Film Medical the content of the cysts varied from white and brown to Systems, Stockholm, Sweden), where differences in loading input were corrected for by normalization against the band for the black (Figure 1A). In older animals, including those found house-keeping gene glyceraldehyde-3-phosphate dehydrogenase dead in their cages, dark spots on the peritoneal side of the (GAPDH). stomach were often observed (Figure 1A). Cysts or dark spots were not seen in stomachs from wild-type mice Results (Figure 1B). The limiting ridge is a thin, delicate border between the forestomach and the glandular portion on the Macroscopical observations. The first macroscopically visible of the stomach (Figure 1C). This structure was alterations in the CA-AhR mice were small cysts with clear substantially enlarged in the CA-AhR animals (Figure 1D).

906 Andersson et al: Gastric Tumours in the CA-AhR Transgenic Mouse Model

Figure 5. Decreased expression of H+/K+-ATPase and parietal/chief cell region in CA-AhR mice. (A) RNA blot analysis (30 Ìg total-RNA) showing expression of gastric H+/K+-ATPase ‚-subunit and GAPDH as loading control in male mice (16 and 37 weeks old) from wild-type (wt) and CA-AhR mice from two independent founder lines (Y8 and A3). (B, C) Quantification of the parietal/chief cell area on HE-stained stomach sections from wild- type (wt) and CA-AhR mice from two independent founder lines (Y8 and A3). The graphs show the parietal/chief cell area in percent of the total area in (B) male and (C) female mice. Average values of 3-10 animals in each group are shown and error bars represent standard deviation. Student’s t-test was used for statistical analysis (* p<0.05, **p<0.01, *** p<0.001 compared to wild-type).

In addition, bleeding from the was region mucosa were early alterations in the CA-AhR occasionally seen in the CA-AhR mice (Figure 1D). stomachs. The peculiar lateral expansion of seemingly well- differentiated cells into the limiting ridge was never seen in Microscopical observations wild-type mice. Lesions within the mucosa. Compared to wild-type zymogenic mucosa (Figure 1E), relatively young CA-AhR Tumoural glands invading the submucosa, the muscularis animals (10 weeks) showed irregular glands characterized propria and . Glandular cysts were seen to by hyperplasia of the foveolar epithelium and a reduction of penetrate the muscularis mucosa and the submucosa in CA- the zymogenic glands (Figure 1F). Occasional dilatations of AhR mice at 10 weeks (Figure 1F). This penetration was, at basally-located glands were also found at this age (Figure later stages, multifocal along the corpus region and was 1H). At an early phase of tumour development, the cardia- associated with foveolar hyperplasia and dilated glands in pyloric glands and glandular epithelium resembling foveolar the overlying mucosa (Figure 1F, 2A). The cells in cells showed a lateral expansion underneath the squamous penetrating glands were actively proliferating, as epithelium of the limiting ridge (Figure 1I), which resulted demonstrated by staining for a proliferation marker PCNA in a substantially increased thickness when compared to that (Figure 1G). of wild-type stomachs (Figure 1J). Starting at an age of 16 Tumours continued to penetrate through all muscle layers weeks, cysts were also found throughout the zymogenic of the muscularis propria, thus reaching the subserosal region mucosa (Figure 2A). In contrast to the regular openings of (Figs. 1F, 2A). The penetration through the stomach wall the pits into the lumen of wild-type mice, many pits of CA- was not caused by herniation, as penetrating glands were AhR mice stomachs were occluded (Figure 2B). No lesions surrounded by connective tissue and not by displaced were observed in the antrum. Thus, dilatations of glands muscularis mucosa (10). Tumours found in the muscularis and subsequent formation of cysts exclusively in the corpus propria displayed foveolar, parietal and cardio-pyloral

907 ANTICANCER RESEARCH 25: 903-912 (2005) differentiation characteristics (Figure 2C, D), i.e. indicative occasional goblet cells were seen in the mucosa of both of cells that normally are strictly confined to the mucosa of male and female animals. No tumours penetrating the the gastric corpus and antrum regions, respectively. Goblet stomach wall were observed. cells, normally found in the mucosa of the , were frequently found in some older CA-AhR animals. Once Age, sex, founder strains and lesion severity. Cysts were in the subserosal space the tumoural cysts became larger and initially found in the mucosa of 9-week-old CA-AhR males. thin-walled, covered by only one layer of epithelial cells and At 13 weeks of age, all male mice, and at 27 weeks of age surrounded by a few layers of stromal cells (Figure 2E). all females, had developed cysts in the mucosa. Tumours Other tumoural cysts were surrounded by large amounts of penetrating the muscularis propria and reaching the connective tissue, vessels as well as well-organized lymphatic subserosal region were initially found in one of four foci and fatty tissue (Figure 2F). Squamous metaplasia was 10-week-old CA-AhR males, but were common (39 of 44) also present (Figure 2G). In older animals, tumoural cysts in 16-week or older CA-AhR males. Mucosal cysts, as well adhered to and compressed adjacent organs such as the liver as penetrating tumours in the muscularis propria, were and pancreas (Figure 2H, I). However, no penetration into found at a slightly younger age with a higher frequency in these organs could be demonstrated and no metastases were male mice compared to females (Figure 3A, B). found. Thus, under the influence of the CA-AhR, In 10-week-old CA-AhR males, mucosal cysts were found histologically seemingly normal glands of the gastric mucosa more often in mice of the A3 founder line (80%) compared to formed tumours that penetrated all muscle layers of the males of the Y8 line (25%, Figure 3C). At that age, tumours stomach, without giving rise to invasion in adjacent organs, penetrating the stomach wall were observed in the Y8 line but to regional or to distant metastasis. not in the A3 line (Figure 3D). However, at 16 weeks, tumours were seen in all A3 males but only in 50% of the Y8 males Histochemical characterization of the tumoural cysts. The tall (Figure 3D). The stomachs of A3 males showed increased cylindrical tumour cells stained positive for PAS (Figure relative weight from 13 weeks of age, and later compared to 2M), demonstrating the presence of neutral that both wild-type and Y8 animals (Figure 3E). Y8 male animals normally are expressed by foveolar mucous cells (16). Acidic showed increased relative stomach weights at older ages than mucins, as demonstrated by the Alcian Blue pH 2.5 stain A3 males (Figure 3E). Increased stomach weight was also (Figure 2J), are normally found in cells of the small intestine found in older female CA-AhR mice (Figure 3F). Thus, there (16) and, when found in the stomach, their expression were differences in onset and disease progression, both indicates intestinal metaplasia. The High Iron Diamide stain between the sexes and the different transgenic founder lines. was negative, indicating a lack of sulphated mucins (16). Reduction of parietal cells. By serendipity, it was found that Moreover, the tall cylindrical tumour cells displayed the use of a streptavidin-horseradish peroxidase autoflourescence when hematoxylin-eosin-stained sections visualisation system on stomach sections resulted in intense were analyzed in a fluorescence microscope (17), thereby staining of parietal cells (Figure 4A-D). Sections from substantiating the presence of neutral mucins. The cardio- CA-AhR animals stained with this surrogate marker pyloral glands in the tumours showed the same demonstrated a substantial decrease in parietal cells staining pattern. Immunohistochemical staining for vimentin compared to wild-type stomachs (Figure 4F and E, indicated accumulation of connective tissue (Figure 2K). The respectively). H+/K+-ATPase is expressed at high levels in cardio-pyloric type glands in the tumours were positive for the H+-secreting parietal cells. Glandular stomachs from Concanavalin A staining (Figure 2L). Thus, although the 16-week-old male A3 and 37-week-old Y8 mice showed tumour cells histologically and histochemically appeared to decreased mRNA expression of the ‚-subunit of this proton retain several histochemical characteristics of normal pump (Figure 5A), thereby substantiating the findings with mucosal cells, the widespread intestinal metaplasia and the the surrogate marker. On well-oriented hematoxylin-eosin- observed squamous metaplasia suggest a phenotypic and stained sections, the parietal/chief cells were further functional transformation of some of these cells. analyzed by quantification of this area using the Scion Image program. The parietal/chief cell area in 16- and Observations in wild-type animals. Occasional cysts were 27-week-old male A3 mice was decreased to approximately observed in the mucosa of wild-type animals. However, the 62% and 27%, respectively, of that found in wild-type dilatations and cysts in CA-AhR animals of the same age animals (Figure 5B). In 52-week-old male and female Y8 were always larger and more numerous. In addition, cysts mice, the parietal/chief cells area was decreased to 54% and displaying a stratified epithelium could occasionally be 66% of that in wild-type animals, respectively (Figure 5B, found close to the limiting ridge. Alcian blue pH 2.5 staining C). Thus, associated with the tumour development, there of acidic mucins revealed occasional positive foveolar cells was a significant decrease of the gastric parietal/chief cell in the mucosa. In 27-week or older wild-type mice, region in the CA-AhR mice.

908 Andersson et al: Gastric Tumours in the CA-AhR Transgenic Mouse Model

Discussion Although levels in the CA-AhR mice are currently being investigated, cellular dysplasia and carcinoma, that are The cells that constituted the stomach tumours in the CA- readily observed in the mucosa of gastrin-overexpressing AhR mice were both histologically and histochemically mice (21), were not found in CA-AhR animals. remarkably well-differentiated, resembling cells of foveolar, CA-AhR mice express a constitutively active mutant of parietal and cardio/pyloral type that are found in the the dioxin/aryl hydrocarbon receptor. The wild-type normal gastric mucosa. However, the frequently found receptor functions as a transcription factor when activated metaplastic cells (both squamous and intestinal metaplasia) by ligands such as the environmental pollutants dioxins and during later stages of tumour expansion indicated that PCBs (12), as well as by some compounds derived from the some functional transformation had also occurred. The diet such as indolo-[3,2-b]-carbazole (25). Interestingly, tumour cells were actively proliferating and penetrated all there are several reports describing the effects of Aryl muscle layers of the stomach. Thus, these well- hydrocarbon receptor ligands in the in differentiated tumour cells appear to have escaped hairless mice (26), rats (27-29) and non-human primates proliferation control and positional instructions from (30-40). Notably, all of the described effects showed the adjacent cells (e.g. in the muscularis mucosa) and same characteristics after exposure to commercial PCB extracellular matrix. Moreover, expansion of the tumours mixtures (Aroclors) (27, 28, 30-33), the dioxin-like PCB was always directed away from the gastric lumen towards 3,4,3’,4’-TCB (34-37), commercial mixtures of and through the muscle layers, as well as into the narrow polybrominated biphenyls (29, 38) or TCDD-like structure called the limiting ridge. This is somewhat dibenzofurans (26, 39, 40). The lesions observed after surprising considering that the relatively loosely organized exposure to these dioxin-like ligands include marked corpus mucosa would more easily allow such expansion. hyperplasia of the surface mucous cells and a substantial The tissue constellation found in restricted areas of the decrease in the number of parietal and chief cells, as well as submucosa and of muscularis propria with mesenchymal intestinal metaplasia (27, 29-33, 35-40). Moreover, gastric differentiation, such as fibroblasts, vessels and lymphatic glands with cystic dilatations were found to penetrate into tissue, is classified in human pathology as a hamartomatous the submucosa and were often associated with foci of tumour. Mice deficient in one allele of the LKB1 (18) or lymphatic cells (27, 30-33, 35, 36, 38-40). In addition, only the Smad4 gene (19) were recently found to develop this the zymogenic region of the stomach was affected; the type of gastric tumour. However, the lesions found in those antrum/pyloric region or other parts of the gastrointestinal models resembled the hamartomatous polyps described in tract showed no alterations. In further analogy to our the Peutz-Jeghers syndrome in humans. In contrast to the findings in the CA-AhR mice (10), rhesus monkeys exposed Peutz-Jeghers hamartomas, which evolve as polypoid to the dioxin-like PCB 3,4,3’,4’-TCB demonstrated lesions growing into the lumen of the organ, CA-AhR proliferating cells throughout practically the entire mucosa, animals neither developed exophytic polyps nor showed whereas, in contrast, control animals showed proliferating epithelial dysplasia. cells only in the narrow isthmus region of the There are other transgenic or knockout mouse models (34). Given that the transgenic expression of a genetically (20-22) that develop pathological lesions in the glandular activated Ah receptor reproduces the phenotype of the stomach, which to some extent resemble the findings in CA- gastric lesions produced by these toxic chemicals, this study AhR mice. Of particular interest are the two mouse models provides strong evidence that these effects are mediated by which overexpress TGF· and gastrin, respectively (20, 21), activation of Ah receptor-dependent signalling pathways. since activation of the endogenous Ah receptor by the Moreover, demonstrating in the present study that a genetic prototypical ligand 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin activation of the Ah receptor reproduced ligand-induced (TCDD) can induce expression of TGF· in human effects in the stomach clearly indicates that activation of Ah keratinocytes (23) and gastrin in rats (24). In a similar way receptor signalling per se is sufficient to produce substantial to the CA-AhR animals, these models showed alterations in homeostasis and control of gastric mucosal hyperplasia and depletion of the parietal cell region. cells. Since no apparent differences between sexes and Moreover, the mice overexpressing gastrin showed intestinal founder lines in transcriptional activity or expression of metaplasia (21). Thus, the stomach lesions observed in the CA-AhR in the glandular stomach were found, the CA-AhR mice could possibly be the result of CA-AhR- influence of sex and founder strain on the tumour induced expression of TGF· and/or gastrin. However, we development is probably caused by other factors. These have, to date, failed to detect any increase in TGF· mRNA differences can thus be used for studying the role of sex expression in the CA-AhR stomachs and, in contrast to the hormones, as well as to identify modifier genes that differ CA-AhR animals, mice overexpressing TGF· did not in expression between the founder strains and which develop tumours that penetrate the stomach wall (20). possibly can be of relevance for human gastric disease.

909 ANTICANCER RESEARCH 25: 903-912 (2005)

There are some questions that arise from this study. 4 Tatematsu M, Yamamoto M, Iwata H, Fukami H, Yuasa H, First, what are the molecular mechanisms leading to the Tezuka N, Masui T and Nakanishi H: Induction of glandular unusual characteristics of these tumours? Considering that stomach cancers in C3H mice treated with N-methyl-N- nitrosourea in the drinking water. Jpn J Cancer Res 84: 1258- both genetic and ligand-activation of the Ah receptor 1264, 1993. produces very similar effects, the answer(s) will most 5 Watanabe H, Uesaka T, Kido S, Ishimura Y, Shiraki K, probably be found among the signalling effects Kuramoto K, Hirata S, Shoji S, Katoh O and Fujimoto N: downstream of the activated Ah receptor. This will Gastric tumor induction by 1,2-dimethylhydrazine in Wistar rats probably include signalling pathways that are of central with intestinal metaplasia caused by X-irradiation. Jpn J Cancer importance for cell differentiation, migration and cell cycle Res 90: 1207-1211, 1999. regulation. Studies of dysregulated genes in the CA-AhR 6 Shimizu N, Inada K, Nakanishi H, Tsukamoto T, Ikehara Y, stomachs are underway in our laboratory and may give Kaminishi M, Kuramoto S, Sugiyama A, Katsuyama T and Tatematsu M: Helicobacter pylori infection enhances glandular insight into these mechanisms. Second, can these stomach carcinogenesis in Mongolian gerbils treated with seemingly, non-dysplastic tumour cells that penetrate chemical carcinogens. Carcinogenesis 20: 669-676, 1999. tissue layers only be induced by an activated Ah receptor 7 Sandmöller A, Halter R, Gomez-La-Hoz E, Gröne HJ, Suske in the gastric mucosa of laboratory animals, or can similar G, Paul D and Beato M: The uteroglobin promoter targets characteristics be found to be associated with expression of the SV40 T antigen to a variety of secretory hamartomatous lesions in other organs? epithelial cells in transgenic mice. Oncogene 9: 2805-2815, In summary, the gastric tumours that developed in the 1994. 8 Koike K, Hinrichs SH, Isselbacher KJ and Jay G: Transgenic CA-AhR mice have several unusual characteristics. Despite mouse model for human gastric carcinoma. Proc Natl Acad Sci an aggressive, multifocal penetration of the proliferating USA 86: 5615-5619, 1989. tumours through all gastric muscle layers and the presence 9 Searle PF, Thomas DP, Faulkner KB and Tinsley JM: Stomach of both squamous and intestinal type of metaplasia, the cancer in transgenic mice expressing human papillomavirus type tumour cells did not metastasize, they were not dysplastic 16 early region genes from a keratin promoter. J Gen Virol 75: and retained several histochemical properties of normal 1125-1137, 1994. mucosal cells. The tumours were strictly confined to the 10 Andersson P, McGuire J, Rubio C, Gradin K, Whitelaw ML, Pettersson S, Hanberg A and Poellinger L: A constitutively corpus part of the stomach and were also associated with a active dioxin/aryl hydrocarbon receptor induces stomach decrease in parietal and chief cells. Closely similar tumors. Proc Natl Acad Sci U S A 99: 9990-9995, 2002. observations have been made after exposure to Ah receptor 11 McGuire J, Okamoto K, Whitelaw ML, Tanaka H and ligands, demonstrating that activation of the Ah receptor Poellinger L: Definition of a dioxin receptor mutant that is a signalling pathway per se can cause these unusual lesions. constitutive activator of transcription: delineation of Thus, it is now critical to identify the relevant Ah receptor- overlapping repression and ligand binding functions within the regulated signalling pathways which produce these effects. PAS domain. J Biol Chem 276: 41841-41849, 2001. The model described here may help to investigate the role 12 Gu YZ, Hogenesch JB and Bradfield CA: The PAS superfamily: sensors of environmental and developmental of certain environmental agents in the disease processes that signals. Ann Rev Pharmacol Toxicol 40: 519-561, 2000. lead to gastric hamartomas and other gastric tumours. 13 Lee ER, Trasler J, Dwivedi S and Leblond CP: Division of the mouse gastric mucosa into zymogenic and mucous regions on Acknowledgements the basis of gland features. Am J Anat 164: 187-207, 1982. 14 Katsuyama T and Spicer SS: Histochemical differentiation of Financial support from the Swedish Cancer Society, the complex carbohydrates with variants of the ConcanavalinA Foundation for Strategic Environmental Research, the Swedish horseradish peroxidase method. J Histochem Cytochem 29: Environmental Protection Agency (the ReproSafe programme) and 233-250, 1978. the Swedish Council for Environment, Agricultural Sciences and 15 Molecular Cloning: A Laboratory Manual (Sambrook J, Fritsch Spatial Planning is gratefully acknowledged. EF and Maniatis T, eds). Cold Spring Harbour Laboratory Press 2 ed., Cold Spring Harbour, NY, 1989. References 16 Jass JR and Roberton AM: Colorectal mucin histochemistry in health and disease: a critical review. Pathol Int 44: 487-504, 1 Sugimura T and Fujimura S: Tumour production in glandular 1994. stomach of rat by N-methyl-N'-nitro-N-nitrosoguanidine. 17 Rubio CA: A simple method to demonstrate duodenal gastric Nature 216: 943-944, 1967. metaplasia. J Clin Pathol 55: 520-523, 2002. 2 Schoental R: Carcinogenic activity of N-methyl-N-nitroso-N'- 18 Miyoshi H, Nakau M, Ishikawa TO, Seldin MF, Oshima M and nitroguanidine. Nature 209: 726-727, 1966. Taketo MM: Gastrointestinal hamartomatous polyposis in Lkb1 3 Schoental R and Bensted JP: Gastro-intestinal tumours in rats heterozygous knockout mice. Cancer Res 62: 2261-2266, 2002. and mice following various routes of administration of N- 19 Takaku K, Miyoshi H, Matsunaga A, Oshima M, Sasaki N and methyl-N-nitroso-N'-nitroguanidine and N-ethyl-N-nitroso-N'- Taketo MM: Gastric and duodenal polyps in Smad4 (Dpc4) nitroguanidine. Br J Cancer 23: 757-764, 1969. knockout mice. Cancer Res 59: 6113-6117, 1999.

910 Andersson et al: Gastric Tumours in the CA-AhR Transgenic Mouse Model

20 Sharp R, Babyatsky MW, Takagi H, Tagerud S, Wang TC, 31 Allen JR, Carstens LA and Barsotti DA: Residual effects of Bockman DE, Brand SJ and Merlino G: Transforming growth short-term, low-level exposure of nonhuman primates to factor alpha disrupts the normal program of cellular polychlorinated biphenyls. Toxicol Appl Pharmacol 30: 440-451, differentiation in the gastric mucosa of transgenic mice. 1974. Development 121: 149-161, 1995. 32 Becker GM, McNulty WP and Bell M: Polychlorinated 21 Wang TC, Dangler CA, Chen D, Goldenring JR, Koh T, biphenyl-induced morphologic changes in the gastric mucosa of Raychowdhury R, Coffey RJ, Ito S, Varro A, Dockray GJ and the rhesus monkey. Lab Invest 40: 373-383, 1979. Fox JG: Synergistic interaction between hypergastrinemia and 33 McConnell EE, Hass JR, Altman N and Moore JA: A Helicobacter infection in a mouse model of gastric cancer. spontaneous outbreak of polycholorinated biphenyl (PCB) Gastroenterology 118: 36-47, 2000. toxicity in rhesus monkeys (Macaca mulatta): toxicopathology. 22 Tebbutt NC, Giraud AS, Inglese M, Jenkins B, Waring P, Clay Lab Anim Sci 29: 666-673, 1979. FJ, Malki S, Alderman BM, Grail D, Hollande F, Heath JK and 34 Becker GM and McNulty WP: Gastric epithelial cell Ernst M: Reciprocal regulation of gastrointestinal homeostasis proliferation in monkeys fed 3,4,3',4'- tetrachlorobiphenyl. J by SHP2 and STAT-mediated trefoil gene activation in gp130 Pathol 143: 267-274, 1984. mutant mice. Nature Med 8: 1089-1097, 2002. 35 McNulty WP, Becker GM and Cory HT: Chronic toxicity of 23 Choi EJ, Toscano DG, Ryan JA, Riedel N and Toscano WA: 3,4,3',4'- and 2,5,2',5'-tetrachlorobiphenyls in rhesus macaques. Dioxin induces transforming growth factor-alpha in human Toxicol Appl Pharmacol 56: 182-190, 1980. keratinocytes. J Biol Chem 266: 9591-9597, 1991. 36 Silverman S, Rosenquist CJ and McNulty WP: Radiographic 24 Theobald HM, Mably TA, Ingall GB and Peterson RE: Role of study of gastric hyperplasia induced by polychlorinated hypergastrinemia in the antiatrophy effect of 2,3,7,8- biphenyls in the rhesus monkey. Invest Radiol 14: 65-69, 1979. tetrachlorodibenzo-p-dioxin on oxyntic gland mucosa of the rat 37 van den Berg KJ, Zurcher C, Brouwer A and van Bekkum DW: stomach. J Biochem Toxicol 5: 259-267, 1990. Chronic toxicity of 3,4,3',4'-tetrachlorobiphenyl in the marmoset 25 Kleman MI, Poellinger L and Gustafsson JA: Regulation of monkey (Callithrix jacchus). Toxicology 48: 209-224, 1988. human dioxin receptor function by indolocarbazoles, receptor 38 Lambrecht LK, Barsotti DA and Allen JR: Responses of ligands of dietary origin. J Biol Chem 269: 5137-5144, 1994. nonhuman primates to a polybrominated biphenyl mixture. 26 Hebert CD, Harris MW, Elwell MR and Birnbaum LS: Relative Environ Health Perspect 23: 139-145, 1978. toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo- 39 Brewster DW, Elwell MR and Birnbaum LS: Toxicity and p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), disposition of 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) in and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice. the rhesus monkey (Macaca mulatta). Toxicol Appl Pharmacol Toxicol Appl Pharmacol 102: 362-377, 1990. 93: 231-246, 1988. 27 Morgan RW, Ward JM and Hartman PE: Aroclor 1254-induced 40 McNulty WP, Pomerantz I and Farrell T: Chronic toxicity of intestinal metaplasia and adenocarcinoma in the glandular 2,3,7,8-tetrachlorodibenzofuran for rhesus macaques. Food stomach of F344 rats. Cancer Res 41: 5052-5059, 1981. Cosmet Toxicol 19: 57-65, 1981. 28 Ward JM: Proliferative lesions of the glandular stomach and liver in F344 rats fed diets containing Aroclor 1254. Environ Health Perspect 60: 89-95, 1985. 29 Gupta BN, McConnell EE, Moore JA and Haseman JK: Effects of a polybrominated biphenyl mixture in the rat and mouse. II. Lifetime study. Toxicol Appl Pharmacol 68: 19-35, 1983. 30 Allen JR and Norback DH: Polychlorinated biphenyl- and Received January 18, 2005 triphenyl-induced gastric mucosal hyperplasia in primates. Revised February 16, 2005 Science 179: 498-499, 1973. Accepted February 21, 2005

911