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We have reported that a lack of RUNX3 function is causally associated with gastric carcinogenesis. We have also presented evidence that loss of Runx3 may be related to the genesis of intestinal metaplasia because expression of RUNX3 is reduced in some intestinal metaplasias, and some Runx3−/–p53−/– gastric epithelial cells differentiate into intestinal type cells in vivo. Recently several reports have indicated that blood cells play important roles in the gastric carcinogenesis. In the present study, we therefore examined whether Runx3−/–p53−/– gastric epithelial cells differentiate autonomously into intestinal type cells, or whether the presence of other cells is necessary for the differentiation in vitro. When Runx3−/–p53−/– gastric epithelial cells were cultured with collagen gels, they did not exhibit any morphogenesis and differentiated poorly. When cultured with fetal mouse gastric mesenchymes, the cells formed glandular structures and differentiated into surface mucous cells, but differentiation of intestinal type cells was never observed. When cultured with Matrigel, the cells formed glandular structures, and some cells differentiated into intestinal type cells in vitro. Reverse transcription–polymerase chain reaction analysis showed that the cells expressed stomach-specific genes, and their levels decreased gradually during the culture. The cells expressed some intestine-specific genes weakly at the start of culture, and their levels were increased with time in culture. We therefore conclude that Runx3−/–p53−/– gastric epithelial cells differentiate into intestinal type cells in combination with Matrigel in the absence of other cell types. Extracellular matrix, not blood cells, may play a role in the genesis of intestinal metaplasia. (Cancer Sci 2008; 99: 671–676)
Gastric cancer is one of the leading causes of cancer deaths worldwide.(1) Gastric adenocarcinoma of the intestinal type is assumed to be formed in a stepwise fashion from chronic gastritis to atrophic gastritis, followed by intestinal metaplasia, leading to dysplasia and carcinoma.(2) Intestinal metaplasia in the stomach represents the conversion of gastric type mucosa to a mucosa that closely resembles the intestinal one,(3) and has been considered to be precancerous for gastric carcinogenesis,(4) although there is controversy over whether intestinal metaplasia is a paraneoplastic change in gastric carcinogenesis.(5) Several environmental factors, such as Helicobacter pylori infection,(6) carcinogen treatment,(7) and X-ray irradiation,(8) have been shown to induce intestinal metaplasia, but little is known about the molecular mechanism how gastric epithelial cells differentiate into intestinal type cells, and whether these factors are essential for the genesis of intestinal metaplasia. Recently, expression of Cdx2 has been demonstrated to be sufficient to induce intestinal metaplasias in the stomach in transgenic mice,(9,10) but it remains to be solved how Cdx2 induces differentiation of intestinal type cells.
We have previously reported that Runx3, a runt domain transcription factor, is a major growth regulator of gastric epithelial cells, and that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.(11) We have also found that expression of RUNX3 is reduced in some intestinal metaplasias in human stomachs,(11) and some Runx3−/–p53−/– gastric epithelial cells differentiate into intestinal type cells in vivo,(12) suggesting that a lack of RUNX3 function is also related to the genesis of intestinal metaplasia. Recently, several reports have indicated that blood cells play important roles in gastric carcinogenesis. Houghton et al. showed that bone marrow-derived cells repopulated the gastric mucosa, and contributed over time to metaplasia, dysplasia, and gastric cancer in bone marrow-transplanted mice.(13) Brenner et al. reported that loss of Runx3 function induced leukocyte infiltration in the gut, and suggested that this might induce a progressive hyperplasia of the gastric mucosa.(14)
To examine whether Runx3−/–p53−/– gastric epithelial cells differentiate autonomously into intestinal type cells, or whether other cells or factors are necessary for the differentiation, we cultured the cells in combination with various mesenchymal components, and their differentiation in vitro was investigated histochemically and molecular biologically. The results indicate that Runx3−/–p53−/– gastric epithelial cells differentiate into intestinal type cells in vitro in the absence of other cell types.
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In the present study, we found that GIF-11 gastric epithelial cells differentiated poorly in combination with collagen gel, but that they formed glandular structures and differentiated into surface mucous cells when combined with fetal gastric mesenchymes, and that some of them differentiated into intestinal type cells when cultured with Matrigel. The results are consistent with a previous report where Runx3−/–p53−/– cells did not exhibit any morphogenetic potencies and differentiated poorly when cultured with collagen gel.(12) RT-PCR analysis of GIF-11 cells combined with Matrigel showed that the cells expressed both gastric-specific (IF, Muc1, and Sox2) and intestine-specific (Muc2 and Cdx2) genes at the start of culture, and that the level of gastric type genes was decreased and the level of intestinal type gene expression was increased during culture, resulting in the differentiation of intestinal type cells. The results are consistent with a previous immunohistochemical analysis that showed that intestinal type cells differentiated from GIF-11 cells in nude mice expressed Cdx2.(12) There are several papers reporting differentiation of intestinal type cells in transgenic mouse stomachs,(9,10) but it is not clear from these previous reports whether intestinal type cells transdifferentiated from gastric type cells, or undifferentiated cells residing in the gastric stem cells exhibited intestinal type differentiation in the mouse. The present study clearly showed that GIF-11 cells expressing gastric-specific genes differentiate into both surface mucous cells and intestinal type cells, depending on the culture conditions. To our knowledge, this is the first study showing that intestinal type cells differentiate from gastric type stem-like cells in vitro. Tatematsu et al. suggested that gastric and intestinal mixed type intestinal metaplasia might be formed by differentiation of stem cells that can produce both gastric and intestinal type cells.(5) Thus, the characteristics of GIF-11 cells are similar to those of the stem cells suggested by Tatematsu et al.(5) GIF-11 cells may be useful for further studies to analyze how intestinal metaplasia is generated in the stomach, and how intestinal metaplasia is related to gastric carcinogenesis.
There are several papers suggesting that bone marrow-derived cells or leukocytes play a role in the genesis of gastric hyperplasia and gastric cancer.(13,14) In the present study, we used GIF-11 cells, which were established from gastric epithelial cells of a Runx3−/–p53−/– mouse fetus using a technique that allows complete separation of epithelial and mesenchymal tissues without cross contamination.(19,20) Thus, the present experiment clearly shows that Runx3−/–p53−/– gastric epithelial cells differentiate into intestinal type cells in the absence of blood cells. Concerning intestinal metaplasia induced by loss of function of Runx3, bone marrow-derived cells and leukocytes do not seem to play a major part.
In the present investigation, we found that GIF-11 Runx3−/–p53−/– cells expressed both gastric type and intestinal type genes at the start of culture when combined with collagen gel or Matrigel. The result indicates that the cells retain intermediate characteristics between gastric and intestinal type cells. We have previously found that the expression of RUNX3 is downregulated in some cases of intestinal metaplasia,(11) and that differentiation of intestinal type cells is found only in Runx3−/–p53−/–, but not p53−/– gastric epithelial cells.(12) We also observed ectopic expression of Cdx2 in the stomach epithelium in Runx3−/– mice while it was never expressed in wild-type stomachs (K. Ito et al., unpublished observation, 2004). All of these results suggest that Runx3 plays an important role in the control of intestinal type genes in gastric epithelial cells. Taniuchi et al. have shown that the Runx protein is important for lineage specification in developing lymphocytes.(21) Runx3 may also play an important role in the specification of gastric epithelial cells, and loss of Runx3 may destabilize the differentiation of gastric epithelial cells. We speculate that during culture with Matrigel, expression of Cdx2 in the destablized cells increased gradually by an unknown mechanism, and its level rose above a threshold to induce differentiation of intestinal type cells after 4 weeks.
Silberg et al. found that ectopic expression of Cdx2 in the gastric epithelium is sufficient to cause transdifferentiation of gastric mucosa into intestinal type cells.(9) They also found that SI was not activated in the transgenic mouse stomach, and speculated that other factors may be necessary for the full differentiation of intestinal epithelial cells. Mutoh et al. reported that all of the gastric mucosal cells except enterochromaffin-like cells were completely replaced by intestinal type cells, including goblet cells and absorptive cells, in another transgenic mouse strain where Cdx2 was expressed in parietal cells under the control of the H+/K+-ATPase promoter.(10) In this case, parietal cells disappeared after approximately 6 weeks, and the pH in the stomach increased from 2 to more than 7. Watanabe et al. repeatedly suggested that elevation of pH in the gastric juice due to the loss of parietal cells in the fundic mucosa induced the development of intestinal metaplasia in the rat.(22,23) Therefore, differentiation of intestinal type cells may be induced not by the expression of Cdx2, but by the loss of parietal cells in the transgenic stomach reported by Mutoh et al.(10) We found in the present investigation that GIF-11 Runx3−/–p53−/– cells expressed SI when cultured with Matrigel. Thus, not only the expression of Cdx2 but also the loss of Runx3 may be essential for the differentiation of intestinal type cells.
In the present study, we found that GIF-11 gastric epithelial cells differentiated into surface mucous cells when cultured with gastric mesenchymes, but that they differentiated into intestinal type cells when combined with Matrigel. It remains to be solved why intestinal type differentiation could not be found in culture with gastric mesenchymes. It is possible that mesenchymes could not survive long culture periods up to 4 weeks when the differentiation of intestinal type cells was first noted, and that epithelial cells died before the differentiation of intestinal type cells in combination with mesenchymes. It is, however, more probable that Matrigel contains factors necessary to induce intestinal type differentiation of GIF-11 cells, and that gastric mesenchymes secrete factors that inhibit intestinal type differentiation and promote gastric type differentiation of the cells. Consistent with this idea, we found that surface mucous cells were differentiated only when GIF-11 cells were combined with gastric mesenchymes but not with Matrigel. Also, Tsukada et al.(24) have reported the importance of gastric mesenchymes in inducing gastric epithelial differentiation in rat fetuses. Matrigel has been reported to contain various extracellular matrix and growth factors, including laminin, type IV collagen, and insulin-like growth factor-1.(25) It is a problem for the future to identify factors that facilitate the differentiation of GIF-11 cells into intestinal type cells.
Intestinal metaplasia is considered to be preneoplastic for gastric carcinogenesis. It is possible that metaplastic tissues are reverted to normal tissues to inhibit carcinogenesis if the deregulated system is normalized. We have previously reported that expression of RUNX3 is often silenced by hypermethylation of its promoter region in human gastric tissues.(11) If the loss of Runx3 function plays an important role in the genesis of intestinal metaplasia, differentiation of metaplastic tissues into normal gastric tissues may be induced by reactivating the silenced gene by using reagents such as demethylating agents or histone deacetylation inhibitors. Further investigations are necessary to identify such chemicals that induce redifferentiation of metaplastic tissues. Our in vitro culture system would be very useful for these purposes.