Copyright ©The Histochemical Society, Inc.

Mixed Gastric- and Intestinal-type Metaplasia Is Formed by Cells with Dual Intestinal and Gastric Differentiation

Toru Niwa, Yuzuru Ikehara, Hayao Nakanishi, Harunari Tanaka, Ken-ichi Inada, Tetsuya Tsukamoto, Masao Ichinose and Masae Tatematsu

Division of Oncological Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan (TN,YI,HN,HT,K-iI,TT,MT) and Second Department of Internal Medicine, Wakayama Medical College, Wakayama City, Japan (TN,MI)

Correspondence to: Yuzuru Ikehara MD, PhD, Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. E-mail: yikehara{at}aichi-cc.jp


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
We have proposed to divide intestinal metaplasia (IM) into two categories, i.e., a mixed gastric and intestinal (GI) type, and a solely intestinal (I) type, based on the residual gastric phenotype cells. The GI-mixed-type IM can be identified by the presence of both cells with either gastric or intestinal phenotypes in a single gland. This study is conducted to elucidate whether cells in the GI-mixed-type IM glands can simultaneously present both gastric and intestinal phenotypes. MUC5AC, MUC2, CD10 and villin expressions were investigated in 20 samples from five gastric cancer cases, directly using either AlexaFluor 488- or 568-labeled specific monoclonal antibodies and observed by fluorescent microscopy and confocal laser-scanning microscopy. GI-mixed IM glands comprise a population expressing MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10. MUC2 and villin expressions were reciprocally increased with decreasing MUC5AC expression, while CD10 expression was limited to cells with only a residual MUC5AC expression or no expression. These results suggest that a heterogeneous cell population with both gastric and intestinal phenotypes would develop into a single intestinal phenotype, as reflected in the progression of intestinal metaplasia from GI-mixed-type- to I-type IM-type glands.

(J Histochem Cytochem 53:75–85, 2005)

Key Words: intestinal metaplasia • MAC5AC • MUC2 • villin • CD10 • human stomach


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
INTESTINAL METAPLASIA (IM) is histologically defined by the presence of intestinal-type cells such as goblet, Paneth, and absorptive cells and is often encountered in chronic and/or atrophic gastritis. IM has long been widely believed to be a premalignant condition associated with a differentiated adenocarcinoma genesis (Morson 1955Go; Stemmermann and Hayashi 1968Go; Sugimura et al. 1982Go; Filipe et al. 1985Go; Correa 1992Go; You et al. 1993Go). As the various histological features of IM glands are well known, efforts have been made to distinguish IM glands morphologically and/or enzyme histochemically to elucidate which typical premalignant aspects might be associated with differentiated adenocarcinomas (Kawachi et al. 1974Go; Teglbjaerg and Nielsen 1978Go; Jass and Filipe 1979Go; Matsukura et al. 1980Go; Segura and Montero 1983Go; Filipe et al. 1988Go; Correa 1992Go; Jass and Walsh 2001Go; Silberg et al. 2002Go). For example, IM has been recognized as either a complete or an incomplete type IM, or as a small- (Type I and Type II) or large- (Type III) intestinal-type IM (Kawachi et al. 1974Go; Matsukura et al. 1980Go; Filipe et al. 1985Go, 1988Go; Matsukuma et al. 1990Go). Though these classifications have been generally accepted, they have overemphasized the characteristics common to cells in the small intestine, while neglecting to take into account the preserved gastric phenotype. In contrast, we have proposed a new classification of IM based on the presence or absence of gastric-type cells in IM glands, which we have subdivided into two major types, i.e., a mixed gastric and intestinal type (GI-mixed-type) and a solely intestinal type (I-type) (Inada et al. 1997Go, 2001Go).

According to this classification, I-type IM glands are solely comprised of intestinal phenotypic cells, whereas GI-mixed-type IM glands also contain gastric phenotypic cells. Interestingly, although the number of gastric cells varies as much as those in a gastric-predominant or an intestinal-predominant type, GI-mixed-type IM glands appear to gradually become I-type IM glands (Inada et al. 1997Go; Tatematsu et al. 2003Go). The exact mechanisms by which these two kinds of phenotype cells or phenotype shifts come to be produced have yet to be determined, but at least two explanations are possible. The first is that aberrantly expressed Cdx1 and/or Cdx2 transcriptional factors, mammalian homologs of the caudal-related homeobox genes, may play an important role in such processes (Silberg et al. 1997Go; Mizoshita et al. 2001Go; Tsukamoto et al. 2003Go; Yuasa 2003Go). The other would be that some genetic alterations such as methylation occur in stem cells leading them to supply such various cell types, especially as seen in GI-mixed-type IM in our classification, resulting in the phenotype change caused by their accumulation (Kang et al. 2001Go,2003aGo,bGo; Kim et al. 2004Go; Lee et al. 2004Go).

Immunohistochemical techniques are widely used to identify intestinal and gastric cell differentiation for the classification of gastric cancers and IM (Inada et al. 1997Go; Reis et al. 1999Go; Inada et al. 2001Go; Jass and Walsh 2001Go; Mizoshita et al. 2001Go; Silva et al. 2002Go; Kawachi et al. 2003Go; Tatematsu et al. 2003Go). To date, a preferred method to evaluate IM and gastric cancers utilizes the anti-mucin core proteins 5AC (MUC5AC) and 6 (MUC6) together with anti-CD10, anti-villin, and anti-MUC2 antibodies. Mucin core proteins comprise an expanded gene family consisting of at least 19 members (Tanaka et al. 1991Go; Gum et al. 2002Go; Chen et al. 2003Go; Ringel and Lohr 2003Go), of which MUC5AC, MUC6 and MUC2 genes are homologous to each other and are localized at chromosome 11p15.5 within a 400-kbp gene span (Pigny et al. 1996Go; Winterford et al. 1999Go). Their expressions might be differentially regulated by the restricted MUC5AC presence on surface epithelial cells (Reis et al. 1997Go), MUC6 on cells in the glandular compartment of pyloric mucosa (Ho et al. 1995Go; Reis et al. 2000Go), and MUC2 in the goblet cells of small and large intestines, and on IM (Jass 2000Go). A secreting endopeptidase, CD10 (Landry et al. 1994Go; Sezaki et al. 2003Go), and one of the actin-binding cytoskeletal proteins, villin (Landry et al. 1994Go; MacLennan et al. 1999Go; Pinto et al. 1999Go), are also observed in intestinal cells, whose expressions indicate absorptive-cell differentiation in IM (Landry et al. 1994Go). Expressions of these molecules are widely used to evaluate gastric cancers whether the differentiation direction is toward gastric or intestinal phenotype.

Using an adaptation of this approach to investigate IM glands, it has been demonstrated that small populations of MUC2-positive cells containing either MUC5AC or MUC6 are present in IM glands (Ho et al. 1995Go; Inada et al. 1997Go,2001Go; Reis et al. 1999Go,2000Go; Tatematsu et al. 2003Go). However, it remains unclear whether these glands, probable GI-mixed IM glands, are composed of dual phenotype cells with intestinal and gastric differentiation. In the present study we evaluated the co-expression of MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10 in GI-mixed-type IM glands using multiple immunofluorescent staining techniques at the single-cell level. The combined use of these markers succeeded in providing evidence of cells with both gastric and intestinal phenotypes in IM.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Tissue Samples
Twenty normal stomach tissue samples were obtained from five patients with gastric cancer who underwent gastrectomy at Aichi Cancer Center Hospital. They were cut from normal areas more than 10 cm away from the cancer, immediately frozen in Tissue-Tek OCT (Optimal Cutting Temperature) Compound (Sakura Finetechnical Co. Ltd.; Tokyo, Japan) with liquid nitrogen and then stored at –80C until use. Four-µm-thick frozen sections prepared with a cryostat were fixed in cold methanol and dried at room temperature for use in immunohistochemical analysis.

Antibodies
Table 1 shows the characteristics of mouse monoclonal antibodies (MAbs) used in this study. To specifically detect the immune reactions with two respective mouse MAbs, we employed Zenon Mouse IgG-labeling kits to directly label the MAbs with either AlexaFluor 488 or AlexaFluor 568 (Molecular Probes; Eugene, OR). Fluor-labeled MAbs were prepared immediately prior to use, according to the suppliers' protocols. The optimal concentrations of primary MAbs were determined empirically, and the final concentrations were 1:100 of anti-MUC5AC, 1:100 of anti-CD10, and 1:5000 of anti-villin MAbs. In all cases, isotype-matched monoclonal antibodies were used as a negative control.


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Table 1

Antibodies used in this study

 
Immunofluorescent Staining
Four µm-thick frozen sections were fixed in cold methanol for 10 min, then air-dried at room temperature for 30 min and rehydrated in PBS for 15 min at room temperature. To reduce nonspecific bindings, the sections were incubated with a blocking reagent (PBS containing 0.2% Triton X-100, 0.2% BSA, and 5% heat-inactivated normal goat serum) for 30 min at room temperature and then reacted with a mixture of two primary antibodies labeled with either AlexaFluor 488 or 568 for 2 hr at room temperature. After washing twice with PBS containing 0.2% Triton X-100 for 15 min, the sections were incubated with 4',6-diamidino-2-phenylindole (DAPI, Molecular Probes) to stain the nucleus for 1 min at a dilution of 1:20000. After washing with PBS, the sections were mounted in Mowiol 4-88 Reagent (CALBIOCHEM; San Diego, CA). To remove O-linked glycosylation on MUC2 for Ccp58 anti-MUC2 antibody to bind the epitope, we performed alkali-catalyzed ß-elimination on the frozen section, the same as previously reported (Hong and Kim 2000Go).

Immunofluorescence and Confocal Laser-scanning Fluorescence Microscopy
Multicolor-stained tissues with AlexaFluor 488, AlexaFluor 568, and DAPI were observed using an Olympus BH5 fluorescence microscope (Olympus; Tokyo, Japan) equipped with a xenon arc lamp and an appropriate filter set. Confocal laser scanning was performed with the Radiance 2100 K-3 system (BioRad; Clinisciences S.A., Montrouge, France) that employs optical fibers both in the illumination source and the detection aperture. This system was equipped with a 50-mW Crypton-Argon laser and filters allowing excitation with both a 488-nm and a 560-nm laser line. Two channels were available for simultaneous data acquisition: Channel 1 (displayed as green) could use a 510–550-nm bandpass filter and either a 515-nm or a 530-nm longpass filter, while Channel 2 (displayed as red) could include either a 550-nm or 590-nm longpass filter.

Image Analysis
All images were recorded by a digital video camera and converted to TIFF files. Merged images were made using Adobe Photoshop software (Adobe Systems; San Jose, CA).


    Results
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 Summary
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Since all of the monoclonal antibodies used in this study were IgG1, we employed a Zenon antibody-labeling kit to detect two antigens simultaneously. This technique makes it possible to individually detect several co-expressed antigens when using the same isotype antibodies. We conjugated anti-MUC2 mAb, anti-villin mAb and anti-CD10 MAb with AlexaFluor 488, while anti-MUC5AC was conjugated with AlexaFluor 568 (anti-CD10 MAb was also conjugated with AlexaFluor 568 in some cases). Their specific bindings (Table 1) were not modified by the Zenon-labeling procedure.

Co-expression of MUC2 and MUC5AC on GI-mixed-type IM
As shown in Figure 1, intestinal and gastric differentiation makers were detected in IM glands. In the first series of experiments using such specimens, we examined the expression of MUC2 as a marker for intestinal goblet cells and that of MUC5AC as a marker for gastric-surface columnar cells. Surface columnar epithelial cells exhibited MUC5AC without MUC2 expression in normal gastric mucosa, while goblet cells of the intestinal or I-type IM exhibited MUC2 without MUC5AC, as demonstrated in previous studies (Figures 2A and 2C) (Inada et al. 1997Go,2001Go; Tatematsu et al. 2003Go). GI-mixed-type IM glands, which were identified by the presence of both MUC5AC and MUC2 in a single gland (Figure 2B), demonstrated both the antigens in glands with a differential positive cell ratio. MUC2-positive cells showed a goblet cell-like feature with MUC5AC expression that included the MUC2 epitope in their cytoplasm of determined GI-mixed-type IM glands (Figure 2B). This result indicates that GI-mixed-type IM glands include cells sharing both gastric and intestinal phenotypes. Interestingly, the co-expressed MUC2 and MUC5AC epitope was not completely colocalized in their cytoplasm. To examine the differential cellular localization between MUC2 and MUC5AC in GI-mixed-type IM cells in more detail, we used a confocal laser-scanning microscope. MUC2 was clearly observed at the center of the cytoplasm, which was surrounded by the MUC5AC-expressing area (Figures 3A–3F), suggesting that MUC2 and MUC5AC are sorted in a differential manner.



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Figure 1

Immunnohistochemical analysis for IM glands with sequential sections. IM glands were detected by the expression of either CD10 or MUC2. Solely I-type IM glands express the intestinal markers without MUC5AC expression (lower panel, x40), while GI-mixed-type IM glands express the intestinal markers with MUC5AC (upper panel, x40).

 


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Figure 2

Localization of MUC5AC and MUC2 mucin core protein in normal gastric glands and IM glands. Frozen sections were stained with two mouse MAbs against MUC5AC directly labeled with AlexaFluor 568 and MUC2 with AlexaFluor 488 as described in Materials and Methods. Nuclei were counter-stained with DAPI (blue) and observed by fluorescence microscopy. MUC5AC (red) and MUC2 (green) mucin core protein expressions were observed in the cytoplasm of foveolar epithelial cells in normal gastric mucosa (A) and in the Golgi apparatus of goblet cells in solely I-type IM glands (C), respectively. Colocalization of MUC5AC and MUC2 on the same cells was demonstrated by merged images (yellow) in GI-mixed-type IM glands (B, x60).

 


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Figure 3

Colocalization of MUC5AC and MUC2 mucin core proteins in GI-mixed-type IM glands. Frozen sections were stained by the double-immunofluorescence method using two mouse MAbs against MUC5AC and MUC2 directly labeled with AlexaFluor 568 or 488. MUC5AC (red) and MUC2 (green) expressions in GI-mixed-type IM glands were observed by confocal laser scanning microscopy (A–C: low-power views; D–F: high-power views). (A,D) MUC5AC was observed in the cytoplasm of almost all columnar epithelial and goblet cells, except for the center of a secretory vesicle of goblet cells. (B,E) MUC2 expressions were observed in the Golgi area in columnar cells (arrowhead) and the secretory vesicles in goblet cells. (C,F) Colocalization of MUC5AC and MUC2 was seen on the periphery of secretory vesicles in the composite images (yellow, arrow). (G,H) ß-elimination increased MUC2 antibody (Ccp58) reactivity in mucous vesicle of solely I-type IM glands. (A–C,G,H x20), (D–F, x60).

 
Furthermore, MUC2 antigen appeared in the supra-nuclear region of goblet cells in I-type IM glands (Figure 2C), whereas its distribution appeared more diffusely in the mucous vesicle of GI-mixed type IM cells (Figure 2B). The alteration in MUC2 distribution seems to arise from the difference between I-type IM and GI-mixed-type IM cells. To elucidate the possibility that higher glycosylation reduced antibody binding in goblet cells of I-type IM the same as in the colonic mucosa previously described (Hong and Kim 2000Go), alkali-catalyzed ß-elimination was performed to remove glycosylation. Following the procedure, increased anti-MUC2 MAb reactivity was observed, which was MUC2 staining similar to that in GI-mixed-type IM glands (Figures 3G and 3H). These results suggest that MUC2 in I-type IM cells is more abundantly glycosylated than in GI-mixed-type IM. The cellular distribution of MUC5AC showed no apparent changes between GI-mixed-type IM cells and normal gastric surface columnar cells.

Co-expression of MUC5AC and Villin/CD10 in GI-mixed-type IM
In a second set of experiments, we examined MUC5AC as a marker for gastric surface columnar cells and either villin or CD10 as the other markers for intestinal cells to verify whether the GI-mixed-type IM glands exhibit both intestinal and gastric phenotypes. Both villin and CD10 have been widely used as specific markers to identify intestinal absorptive cells and are exclusively expressed at the brush borders of cells solely in I-type IM glands but not in gastric glands (Figures 4 and 5). GI-mixed-type IM glands were identified by the presence of MUC5AC and villin in individual glands (Figure 4). Villin was detected on the apical end of MUC5AC-positive cells in GI-mixed-type IM glands, whose expressed cell numbers increased with diminishing MUC5AC expression in the glands. It is noteworthy that CD10 expression was not always detected with villin at the brush border in GI-mixed IM glands (Figure 6) but was also occasionally localized in the cytoplasm (Figure 5). Its inclusion body-like appearance was the same as that reported in familial microvillus-inclusion body disease (Groisman et al. 2002Go). This inclusion body-like CD10 staining pattern is more apparent in GI-mixed-type IM glands with more abundant MUC5AC-expressing cells, but it disappears with declining MUC5AC expression.



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Figure 4

Expression of MUC5AC and villin in normal gastric glands and GI-mixed-type IM glands. Frozen sections were stained by the double-immunofluorescence method using two mouse MAbs against villin and MUC5AC directly labeled with AlexaFluor 488 or 568. Nuclei were counter-stained by DAPI (blue) and observed by fluorescence microscopy. (A–C) Only MUC5AC (red) without villin (green) was seen in gastric columnar epithelial cells. (G–I) Such cells in I-type IM glands exhibited only villin without MUC5AC. (D–F) MUC5AC was observed in the cytoplasm and villin of the micro-villi of the columnar cells in GI-mixed-type IM glands. Composite images demonstrate the absence of any yellow signal in the cells, suggesting their different subcellular localization (F). (A–I, x60).

 


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Figure 5

Cellular localization of CD10 in GI-mixed IM glands. Frozen sections were stained by the double-immunofluorescence method using two mouse MAbs against MUC5AC and CD10 directly labeled with AlexaFluor 568 and 488, respectively. This figure was obtained by fluorescence microscopy. CD10 (green) was expressed in the microvilli of columnar cells without MUC5AC expression (red), while CD10 stained as inclusion body-like signals in MUC5AC-positive cells. Arrowheads indicate CD10-positive inclusion bodies (x60).

 


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Figure 6

Expression of villin and CD10 in GI-mixed IM glands. Frozen sections were stained by the double-immunofluorescent method using two mouse MAbs against villin and CD10 directly labeled with AlexaFluor 488 and 568, respectively. Nuclei were counter-stained by DAPI (blue) and observed by fluorescence microscopy. CD10 (red; A,D,G) and villin (green; B,E,H) were observed to be colocalized in the micro-villi of columnar cells in I-type IM glands and small intestine by composite images (yellow) (F,I). In contrast, compared with villin (B), CD10 expression (A) was faint in GI-mixed-type IM glands, and the colocalization was barely visible (C). (A–I, x20).

 

    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
To date, several immunohistochemical studies have demonstrated an intestinal or a gastric phenotype by the detection of specific molecular expressions in IM glands using serial sections (Ho et al. 1995Go; Inada et al. 1997Go, 2001Go; Reis et al. 1999Go; Jass 2000Go; Shaoul et al. 2000Go; Silva et al. 2002Go; Tatematsu et al. 2003Go). These studies have attempted to demonstrate that some cells are either intestinal or gastric phenotypic markers, suggesting limited possibilities for the same cells to have both gastric- and intestinal-marker antigens (Reis et al. 1999Go, 2000Go; Lopez-Ferrer et al. 2000Go,2001Go; Shaoul et al. 2000Go). In the present study, we were able to overcome such limitations by employing Zenon antibody-labeling technology, thus successfully demonstrating the co-expression of both gastric and intestinal molecular markers in the same cells in GI-mixed-type IM glands, in which MUC5AC clearly existed with MUC2, or villin and/or CD10. These findings suggest that mixed gastric and intestinal type metaplasia is formed by cells with dual differentiation and are consistent with the previously demonstrated evidence that some metaplastic cells have both intestinal and gastric differentiation-specific structures (Goldman and Ming 1968Go).

The cells in GI-mixed-type IM glands exhibited MUC2 with MUC5AC in their cytoplasm (Figures 2 and 3), while the cells in I-type IM glands exhibited MUC2 in the peri-nuclear Golgi apparatus area. The subcellular localization of MUC2 appeared to shift from secretion vesicles to the Golgi apparatus area with a histological alteration from GI-mixed-type IM to I-type IM. A glycosylation change might be one of the probable explanations, since the MUC 2 antibody used in this study could detect only underglycosylated MUC2 core protein for the epitope as discussed in previous studies (Hong and Kim 2000Go; Shaoul et al. 2000Go). In the present study we observed the enhanced anti-MUC2 antibody staining in I-type IM glands by alkali-catalyzed ß-elimination, which was similar to the MUC2 staining on GI-mixed-type IM glands. This result indicates that O-linked glycosylation limited the detection of MUC2 expression in goblet cells in I-type IM glands, suggesting that the difference between the higher glycosylated MUC2 in I-type IM glands and the lower glycosylated MUC2 in the goblet cells in the GI-mixed-type IM glands would be the other maturation indicator for goblet cells in IM glands.

The columnar epithelial cells, similar to the intestinal absorptive cells, were seen in GI-mixed-type IM glands that exhibited MUC5AC and either villin or CD10. Compared with villin-positive cells, CD10 was preferentially found in the faint MUC5AC-preserved columnar cells, suggesting that CD10 exists in cells of the more intestinalized GI-mixed-type IM glands (Figure 6). Analogously, in the development of fetal mouse small intestine, villin appears on the brush border prior to CD10 (Landry et al. 1994Go; Montgomery et al. 1999Go). Villin is an actin-binding cytoskeletal protein essential for brush border formation in normal epithelial cells of the intestine, while CD10 is a brush border-associated neutral peptidase. Adapting these findings to human IM, the structural accomplishment of IMs such as villin expression might precede by functional maturations such as digestive enzyme, CD10 expression. Moreover, it may be reasonable to assume that villin-positive and CD10-negative cells in GI-mixed-type IM are functionally immature absorptive cells, and that the phenotype shift from GI-mixed-type IM to solely I-type IM is a kind of maturation. Furthermore, the expression of CD10 in the cytoplasm of columnar cells in GI-mixed IM (Figure 5) might support this idea, because it has been found in the surface enterocytes of familial microvillus inclusion disease due to the immaturity of CD10 (Groisman et al. 2002Go).

Co-expression ratios of MUC5AC to MUC2, villin, or CD10 varied greatly in terms of establishing a dominant MUC5AC type or a MUC2/villin/CD10 dominant type. Based on these findings, the cells in mixed-type IM glands seem to be about to free themselves from the gastric phenotype, finally becoming solely I-type IM cells. As villin, CD10, and MUC2 expressions progressively increase, MUC5AC expression reciprocally diminishes. Gastric-type cells are gradually reduced and finally replaced by intestinal-type cells, leading to solely I-type IM. These results indicate that GI-mixed-type IM cells are multi-phenotypic, suggesting that the phenotype shift from a GI-mixed type to a solely I-type IM should occur in each cell over time (Figure 7).



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Figure 7

Schematic representation of phenotypic shift of differentiation markers from GI-mixed-type IM to I-type IM glands. Gastric foveolar epithelial cells express only MUC5AC. IM begins from such foveolar epithelial cells, along two different cellular pathways. One begins with an aberrant expression of villin and subsequent CD10 on columnar epithelial cells, resulting in absorptive-like intestinal cells. The other starts from an ectopic expression of MUC2 and then accumulates in mucous vesicles, developing into goblet-like intestinal cells. As villin, CD10, and MUC2 expressions progressively increase, MUC5AC expression reciprocally diminishes. Gastric-type cells are gradually reduced and are finally replaced by intestinal-type cells, leading to solely I-type IM (upper panel, x20).

 
In conclusion, we demonstrated that intestinalization occurs in individual cells with MUC5AC expression in GI-mixed-type IM glands. The cells with an intestinal phenotype in GI-mixed type IM glands are morphologically and functionally less mature than those in I-type IM glands. As they are midway between gastric and intestinal phenotypic cells with varying degrees of differentiation and maturation, the GI-mixed-type IM glands consist of a heterogeneous population of cells. Based on observations in this study, we hypothesize that the cells in GI-mixed-type IM glands remain out of some regulations on intestinal differentiation and subsequent functional maturation toward becoming intestinal type cells. Furthermore, considering that stem cells are also present in GI-mixed-type IM, such unstable phenotypes might be induced at stem cells by either transcriptional factors or DNA methylation. To clarify and confirm these possibilities, further studies based on molecular biological techniques and applying our findings will have to be initiated starting with the identification of GI-mixed IM cells.


    Acknowledgments
 
Supported by a Grant-in-Aid for Scientific Research on Priority Area (16790196) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    Footnotes
 
Received for publication June 15, 2004; accepted September 13, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Chen Y, Zhao YH, Kalaslavadi TB, Hamati E, Nehrke K, Le AD, Ann DK, Wu R (2003) Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues. Am J Respir Cell Mol Biol 30:155–165[CrossRef][Medline]

Correa P (1992) Human gastric carcinogenesis: a multistep and multifactorial process—First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 52:6735–6740[Abstract]

Filipe MI, Barbatis C, Sandey A, Ma J (1988) Expression of intestinal mucin antigens in the gastric epithelium and its relationship with malignancy. Hum Pathol 19:19–26[Medline]

Filipe MI, Potet F, Bogomoletz WV, Dawson PA, Fabiani B, Chauveinc P, Fenzy A, Gazzard B, Goldfain D, Zeegen R (1985) Incomplete sulphomucin-secreting intestinal metaplasia for gastric cancer. Preliminary data from a prospective study from three centres. Gut 26:1319–1326[Abstract]

Goldman H, Ming SC (1968) Fine structure of intestinal metaplasia and adenocarcinoma of the human stomach. Lab Invest 18:203–210[Medline]

Groisman GM, Amar M, Livne E (2002) CD10: a valuable tool for the light microscopic diagnosis of microvillous inclusion disease (familial microvillous atrophy). Am J Surg Pathol 26:902–907[CrossRef][Medline]

Gum JR Jr, Crawley SC, Hicks JW, Szymkowski DE, Kim YS (2002) MUC17, a novel membrane-tethered mucin. Biochem Biophys Res Commun 291:466–475[CrossRef][Medline]

Ho SB, Shekels LL, Toribara NW, Kim YS, Lyftogt C, Cherwitz DL, Niehans GA (1995) Mucin gene expression in normal, preneoplastic, and neoplastic human gastric epithelium. Cancer Res 55:2681–2690[Abstract]

Hong JC, Kim YS (2000) Alkali-catalyzed beta-elimination of periodate-oxidized glycans: a novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections. Glycoconj J 17:691–703[CrossRef][Medline]

Inada K, Nakanishi H, Fujimitsu Y, Shimizu N, Ichinose M, Miki K, Nakamura S, Tatematsu M (1997) Gastric and intestinal mixed and solely intestinal types of intestinal metaplasia in the human stomach. Pathol Int 47:831–841[Medline]

Inada K, Tanaka H, Nakanishi H, Tsukamoto T, Ikehara Y, Tatematsu K, Nakamura S, Porter EM, Tatematsu M (2001) Identification of Paneth cells in pyloric glands associated with gastric and intestinal mixed-type intestinal metaplasia of the human stomach. Virchows Arch 439:14–20[CrossRef][Medline]

Jass JR (2000) Mucin core proteins as differentiation markers in the gastrointestinal tract. Histopathology 37:561–564[CrossRef][Medline]

Jass JR, Filipe MI (1979) A variant of intestinal metaplasia associated with gastric carcinoma: a histochemical study. Histopathology 3:191–199[Medline]

Jass JR, Walsh MD (2001) Altered mucin expression in the gastrointestinal tract: a review. J Cell Mol Med 5:327–351[Medline]

Kang GH, Lee HJ, Hwang KS, Lee S, Kim JH, Kim JS (2003a) Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation. Am J Pathol 163:1551–1556[Abstract/Free Full Text]

Kang GH, Lee S, Kim JS, Jung HY (2003b) Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis. Lab Invest 83:635–641[Medline]

Kang GH, Shim YH, Jung HY, Kim WH, Ro JY, Rhyu MG (2001) CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res 61:2847–2851[Abstract/Free Full Text]

Kawachi H, Takizawa T, Eishi Y, Shimizu S, Kumagai J, Funata N, Koike M (2003) Absence of either gastric or intestinal phenotype in microscopic differentiated gastric carcinomas. J Pathol 199:436–446[CrossRef][Medline]

Kawachi T, Kogure K, Tanaka N, Tokunaga A, Sugimura T (1974) Studies of intestinal metaplasia in the gastric mucosa by detection of disaccharidases with "Tes-Tape". J Natl Cancer Inst 53:19–30[Medline]

Kim TY, Lee HJ, Hwang KS, Lee M, Kim JW, Bang YJ, Kang GH (2004) Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab Invest 84:479–484[CrossRef][Medline]

Landry C, Huet C, Mangeat P, Sahuquet A, Louvard D, Crine P (1994) Comparative analysis of neutral endopeptidase (NEP) and villin gene expression during mouse embryogenesis and enterocyte maturation. Differentiation 56:55–65[CrossRef][Medline]

Lee JH, Park SJ, Abraham SC, Seo JS, Nam JH, Choi C, Juhng SW, Rashid A, Hamilton SR, Wu TT (2004) Frequent CpG island methylation in precursor lesions and early gastric adenocarcinomas. Oncogene 23:4646–4654[CrossRef][Medline]

Lopez-Ferrer A, Barranco C, de Bolos C (2001) Apomucin expression and association with Lewis antigens during gastric development. Appl Immunohistochem Mol Morphol 9:42–48[Medline]

Lopez-Ferrer A, de Bolos C, Barranco C, Garrido M, Isern J, Carlstedt I, Reis CA, Torrado J, Real FX (2000) Role of fucosyltransferases in the association between apomucin and Lewis antigen expression in normal and malignant gastric epithelium. Gut 47:349–356[Abstract/Free Full Text]

MacLennan AJ, Orringer MB, Beer DG (1999) Identification of intestinal-type Barrett's metaplasia by using the intestine-specific protein villin and esophageal brush cytology. Mol Carcinog 24:137–143[CrossRef][Medline]

Matsukuma A, Mori M, Enjoji M (1990) Sulphomucin-secreting intestinal metaplasia in the human gastric mucosa. An association with intestinal-type gastric carcinoma. Cancer 66:689–694[Medline]

Matsukura N, Suzuki K, Kawachi T, Aoyagi M, Sugimura T, Kitaoka H, Numajiri H, Shirota A, Itabashi M, Hirota T (1980) Distribution of marker enzymes and mucin in intestinal metaplasia in human stomach and relation to complete and incomplete types of intestinal metaplasia to minute gastric carcinomas. J Natl Cancer Inst 65:231–240[Medline]

Mizoshita T, Inada K, Tsukamoto T, Kodera Y, Yamamura Y, Hirai T, Kato T, Joh T, Itoh M, Tatematsu M (2001) Expression of Cdx1 and Cdx2 mRNAs and relevance of this expression to differentiation in human gastrointestinal mucosa–with special emphasis on participation in intestinal metaplasia of the human stomach. Gastric Cancer 4:185–191[Medline]

Montgomery RK, Mulberg AE, Grand RJ (1999) Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology 116:702–731[Medline]

Morson BC (1955) Carcinoma arising from areas of intestinal metaplasia in the gastric mucosa. Br J Cancer 9:377–385

Pigny P, Guyonnet-Duperat V, Hill AS, Pratt WS, Galiegue-Zouitina S, d'Hooge MC, Laine A, Van-Seuningen I, Degand P, Gum JR, Kim YS, Swallow DM, Aubert JP, Porchet N (1996) Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes. Genomics 38:340–352[CrossRef][Medline]

Pinto D, Robine S, Jaisser F, El Marjou FE, Louvard D (1999) Cells of small and large intestines. J Biol Chem 274:6476–6482 Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines. J Biol Chem 274:6476–6482[Abstract/Free Full Text]

Reis CA, David L, Carvalho F, Mandel U, de Bolos C, Mirgorodskaya E, Clausen H, Sobrinho-Simoes M (2000) Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas. J Histochem Cytochem 48:377–388[Abstract/Free Full Text]

Reis CA, David L, Correa P, Carneiro F, de Bolos C, Garcia E, Mandel U, Clausen H, Sobrinho-Simoes M (1999) Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression. Cancer Res 59:1003–1007[Abstract/Free Full Text]

Reis CA, David L, Nielsen PA, Clausen H, Mirgorodskaya K, Roepstorff P, Sobrinho-Simoes M (1997) Immunohistochemical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody. Int J Cancer 74:112–121[CrossRef][Medline]

Ringel J, Lohr M (2003) The MUC gene family: their role in diagnosis and early detection of pancreatic cancer. Mol Cancer 2:9[CrossRef][Medline]

Segura DI, Montero C (1983) Histochemical characterization of different types of intestinal metaplasia in gastric mucosa. Cancer 52:498–503[Medline]

Sezaki N, Ishimaru F, Tabayashi T, Kataoka I, Nakase K, Fujii K, Kozuka T, Nakayama H, Harada M, Tanimoto M (2003) The type 1 CD10/neutral endopeptidase 24.11 promoter: functional characterization of the 5'-untranslated region. Br J Haematol 123:177–183[CrossRef][Medline]

Shaoul R, Marcon P, Okada Y, Cutz E, Forstner G (2000) The pathogenesis of duodenal gastric metaplasia: the role of local goblet cell transformation. Gut 46:632–638[Abstract/Free Full Text]

Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG (1997) CDX1 protein expression in normal, metaplastic, and neoplastic human alimentary tract epithelium. Gastroenterology 113:478–486[Medline]

Silberg DG, Sullivan J, Kang E, Swain GP, Moffett J, Sund NJ, Sackett SD, Kaestner KH (2002) Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice. Gastroenterology 122:689–696[CrossRef][Medline]

Silva E, Teixeira A, David L, Carneiro F, Reis CA, Sobrinho-Simoes J, Serpa J, Veerman E, Bolscher J, Sobrinho-Simoes M (2002) Mucins as key molecules for the classification of intestinal metaplasia of the stomach. Virchows Arch 440:311–317[CrossRef][Medline]

Stemmermann GN, Hayashi T (1968) Intestinal metaplasia of the gastric mucosa: a gross and microscopic study of its distribution in various disease states. J Natl Cancer Inst 41:627–634[Medline]

Sugimura T, Matsukura N, Sato S (1982) Intestinal metaplasia of the stomach as a precancerous stage. IARC Sci Publ 39:515–530[Medline]

Tanaka H, Matsui T, Agata A, Tomura M, Kubota I, McFarland KC, Kohr B, Lee A, Phillips HS, Shelton DL (1991) Molecular cloning and expression of a novel adhesion molecule, SC1. Neuron 7:535–545[Medline]

Tatematsu M, Tsukamoto T, Inada K (2003) Stem cells and gastric cancer: role of gastric and intestinal mixed intestinal metaplasia. Cancer Sci 94:135–141[Medline]

Teglbjaerg PS, Nielsen HO (1978) "Small intestinal type" and "colonic type" intestinal metaplasia of the human stomach, and their relationship to the histogenetic types of gastric adenocarcinoma. Acta Pathol Microbiol Scand [A] 86A:351–355[Medline]

Tsukamoto T, Inada K, Tanaka H, Mizoshita T, Mihara M, Ushijima T, Yamamura Y, Nakamura S, Tatematsu M (2003) Down-regulation of a gastric transcription factor, Sox2, and ectopic expression of intestinal homeobox genes, Cdx1 nd Cdx2: inverse correlation during progression from gastric/intestinal-mixed to complete intestinal metaplasia. J Cancer Res Clin Oncol 130:135–145[CrossRef][Medline]

Winterford CM, Walsh MD, Leggett BA, Jass JR (1999) Ultrastructural localization of epithelial mucin core proteins in colorectal tissues. J Histochem Cytochem 47:1063–1074[Abstract/Free Full Text]

You WC, Blot WJ, Li JY, Chang YS, Jin ML, Kneller R, Zhang L, et al. (1993) Precancerous gastric lesions in a population at high risk of stomach cancer. Cancer Res 53:1317–1321[Abstract]

Yuasa Y (2003) Control of gut differentiation and intestinal-type gastric carcinogenesis. Nat Rev Cancer 3:592–600[CrossRef][Medline]





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