Copyright ©The Histochemical Society, Inc.

Expression of Gastric Gland Mucous Cell-Type Mucin in Normal and Neoplastic Human Tissues

Kosei Nakajima, Hiroyoshi Ota, Mu Xia Zhang, Kenji Sano, Takayuki Honda, Keiko Ishii and Jun Nakayama

Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine (KN); Department of Biomedical Laboratory Sciences, School of Health Sciences, School of Medicine, Shinshu University (HO); Department of Laboratory Medicine, School of Medicine, Shinshu University (HO, MXZ,KS,TH,KI,JN); and Department of Pathology, School of Medicine, Shinshu University (JN), Matsumoto, Japan

Correspondence to: Hiroyoshi Ota, Dept. of Biomedical Laboratory Sciences, School of Health Sciences, School of Medicine, Shinshu University, Matsumoto, Nagano 390-8621, Japan. E-mail: hohta{at}gipac.shinshu-u.ac.jp


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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Gastric gland mucous cells produce class III mucin, which is also found in Brunner's glands and mucous glands along the pancreaticobiliary tract, and in metaplasia and adenocarcinomas differentiating towards gastric mucosa. Recently, we showed that class III mucin possesses GlcNAc{alpha}1->4Galß->R, formed by {alpha}1,4-N-acetylglucosaminyltransferase ({alpha}4GnT). Examining the tissue-specific expression of mucin epitopes is useful to clarify cell-lineage differentiation and to identify the site of origin of metastatic carcinomas in histological specimens. Formalin-fixed, paraffin-embedded tissue sections from esophagus, stomach, colon, liver, pancreas, lung, kidney, prostate, breast, and salivary gland resected for carcinoma, as well as salivary gland adenoma, colon adenoma, and metastatic adenocarcinoma of lymph nodes from stomach, pancreas, colon, and breast, were immunostained for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R. These were all expressed in normal, metaplastic, and adenocarcinoma tissues of stomach, pancreas, and bile duct, and in pulmonary mucinous bronchioloalveolar carcinomas. Cells expressing {alpha}4GnT uniformly expressed GlcNAc{alpha}1->4Galß->R. Only MUC6 was expressed in normal salivary glands, pancreas, seminal vesicles, renal tubules, and colon adenomas, and in normal tissue and adenocarcinomas of prostate and breast. No tissues showed immunoreactivity for {alpha}4GnT alone. Immunohistochemistry (IHC) profiles were similar for metastatic carcinomas and primary carcinoma tissues. The IHC profiles for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R may be diagnostically relevant. (J Histochem Cytochem 51:1689–1698, 2003)

Key Words: glycosyltransferase • gastric mucin • immunohistochemistry • mucin core protein


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
IN GASTRIC MUCOSA, two types of mucus-secreting cells exist: the surface mucous cell and the gland mucous cell. The latter, which includes cardiac gland cells, mucous neck cells, and pyloric gland cells, possess class III mucin, as identified by paradoxical concanavalin A staining (Katsuyama and Spicer 1978Go; Suganuma et al. 1981Go; Ota et al. 2001Go). In normal human tissues, class III mucin is expressed in gastric gland mucous cells, Brunner's gland cells, and mucous cells of the accessory glands of the pancreaticobiliary tract (Katsuyama and Spicer 1978Go; Suganuma et al. 1981Go; Ota et al. 1991Go,1998Go; Nakamura et al. 1998Go). Class III mucin is also expressed in gastric metaplasia of the gallbladder (Tsutsumi et al. 1984Go) and mucinous metaplasia of the pancreas (Matsuzawa et al. 1992Go; Ota et al. 2001Go; Zhang et al. 2001Go), and in carcinoma of stomach (Akamatsu and Katsuyama 1990Go; Lesuffleur et al. 1994Go; Fujimori et al. 1995Go; Nakamura et al. 1998Go; Ota et al. 2001Go), bile duct (Kijima et al. 1989Go; Ota et al. 1995Go), gallbladder (Tsutsumi et al. 1984Go), and pancreas (Matsuzawa et al. 1992Go; Nakamura et al. 1998Go; Ota et al. 2001Go), as well as in mucinous bronchioloalveolar cell carcinoma of the lung (Honda et al. 1998Go; Ota et al. 2001Go) and adenoma malignum of the uterine cervix (Ishii et al. 1998Go,1999Go)

Several years ago, Ishihara et al. (1996)Go raised a specific monoclonal antibody (MAb) against gastric mucin, designated HIK1083, which recognizes N-acetylglucosamin{alpha}1->4galactoseß->R (GlcNAc{alpha}1->4Galß->R). We found that the distribution of GlcNAc{alpha}1->4Galß->R is consistent with the distribution of class III mucin in normal vertebrate tissues and in metaplastic and neoplastic human tissues (Nakamura et al. 1998Go; Ota et al. 1998Go,2001Go). More recently, we showed that class III mucin possesses this particular glycan, GlcNAc{alpha}1-> 4Galß->R, which is formed by {alpha}1,4-N-acetylglucosaminyltransferase ({alpha}4GnT) (Nakayama et al. 1999Go), and that two distinct mucin core proteins, MUC5AC and MUC6, present in gastric mucin carry GlcNAc{alpha}1-> 4Galß->R (Zhang et al. 2001Go).

Examining tissue-specific expression of mucin epitopes in human tissues is a useful way to clarify the cell-lineage differentiation of carcinoma cells and to demonstrate the site of origin of metastatic carcinomas in histological specimens.

GlcNAc{alpha}1->4Galß->R is preferentially attached to core2-branched O-glycan (Ishihara et al. 1996Go). The sialyl Lewis X found in O-glycan are also attached to the terminal end of core2-branched structures (Fukuda 1996Go). Therefore, it appears that the expressions of GlcNAc{alpha}1->4Galß->R and sialyl Lewis X may be reciprocally regulated, because these carbohydrates compete for the common precursor oligosaccharide, core2-branched O-glycan. It is well known that sialyl Lewis X serve as preferential ligands for the cell-adhesion molecules E- and P-selectin (Fukushima et al. 1984Go; Itzkowitz et al. 1988Go; Rosen and Bertozzi 1994Go). Recently we demonstrated that, in colorectal and pulmonary cancers, the sialyl Lewis X expressed on core2-branched O-glycans showed a positive correlation with tumor progression (Shimodaira et al. 1997Go; Machida et al. 2001Go).

We designed the present study to explore the IHC expressions of MUC6, {alpha}4GnT, and GlcNAc{alpha}1-> 4Galß->R in human normal, metaplastic, and adenocarcinoma tissues. We also examined the IHC expressions of GlcNAc{alpha}1->4Galß->R and sialyl Lewis X in gastric carcinomas and in metastatic gastric carcinomas of lymph nodes.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Preparation of Tissues
Formalin-fixed, paraffin-embedded sections of human tissues were obtained from the surgical pathology files of the Department of Clinical Laboratory, Shinshu University Hospital, Matsumoto, Japan. Specimens consisted of histologically normal portions of tissues resected for adenoma or carcinoma and various tumor tissues, as well as metastatic adenocarcinomas of lymph nodes from stomach, colon, pancreas, and breast (Tables 1 and 2). This study was performed after written informed consent had been obtained from the patients.


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Table 1

Immunohistochemical expressions of MUC6, a4GnT, and GlcNAc {alpha}1->4Galß->R in non-neoplastic tissuesa

 

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Table 2

Immunohistochemical expression of MUC6, {alpha}4GnT, GlcNAc {alpha}1-4Gal ß-R, and sialyl Lewis X in neoplastic tissues at primary and metastatic lesionsa

 
Histochemistry
Serial paraffin sections of 3-µm thickness were stained with H&E for histological examination or immunostained with anti-MUC6 (Novocastra; Newcastle-upon-Tyne, UK), anti-{alpha}4GnT (Zhang et al. 2001Go), or GlcNAc{alpha}1->4Galß->R (MAb HIK1083; Kanto Chemical, Tokyo, Japan). To facilitate comparison of the distributions of antigens, mirror paraffin sections of 3-µm thickness from gastric adenocarcinomas and metastatic adenocarcinomas of lymph nodes from gastric adenocarcinomas expressing both GlcNAc{alpha}1->4Galß->R and sialyl Lewis X in primary sites were stained with HIK1083 or NCC-ST439 MAb (Kumamoto et al. 1998Go) (Nippon Kayaku; Tokyo, Japan) specific for the sialyl Lewis X attached to O-glycan.

IHC staining was performed using the Envision+ method (DAKO; Carpinteria, CA). Briefly, sections were dewaxed and rehydrated and endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol (30 min). Before immunostaining, antigen retrieval was carried out using a microwave (600 W) for 25 min in 0.01 mol/liter citrate buffer (pH 6.0) for both GlcNAc{alpha}1->4Galß->R and MUC6. For immunostaining with anti-{alpha}4GnT and sialyl Lewis X, antigen retrieval was not carried out. The tissue sections were blocked with 5% normal bovine serum albumin in Tris-buffered saline (TBS; 140 mmol/liter NaCl, 50 mmol/liter Tris-HCl, pH 7.6) and incubated with primary antibodies. After washing in TBS, slides were incubated with peroxidase and second antibody-labeled polymer (DAKO) for 60 min. The reaction was developed with 3,3'-diaminobenzidine (Sigma Chemical; Poole, UK) containig 0.02% H2O2. For immunostaining of seminal vesicles, the Envision+ method for immunoalkaline phosphatase (DAKO) was used. Sections were lightly counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted in synthetic medium.

Negative controls were obtained by omitting the primary antibody. The gastric gland mucous cells and Brunner's gland cells in the specimens were used as internal positive controls for MUC6, anti-{alpha}4GnT, and GlcNAc{alpha}1->4Galß->R. Colon adenocarcinoma tissues were used as positive controls for sialyl Lewis X.

Evaluation of Immunostaining
The degree of staining in tissues examined with specific antibodies was scored semiquantitatively as 0 (negative), 1 (less than one third of the tissue), 2 (more than one third but less than two thirds), or 3 (more than two thirds). Grading of immunoreactivity was carried out by a single observer (KN). To validate the grading method, all specimens were graded twice, on two separate occasions. There was no significant intraobserver variation.

Statistics
The Mann–Whitney U-test was used to compare the scores given for immunoreactivities. Spearman's correlation coefficient by rank was used to analyze the correlations among the immunoreactivity scores given for MUC6, {alpha}4GnT, GlcNAc{alpha}1->4Galß->R, and sialyl Lewis X. Staining scores are nonparametric and are presented as median rather than mean values.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Demonstration of MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in Non-neoplastic Tissues
MUC6 was diffusely expressed in the cytoplasm (Figures 1B, 2B, 3B, 4B, and 5B) , while {alpha}4GnT was expressed in the Golgi area (Figures 1C, 2C, and 3C). GlcNAc{alpha}1->4Galß->R was mainly localized in cytoplasmic mucus granules (Figures 1D, 2D, and 3D), although in some cells it was localized on the apical cytoplasmic membrane.



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Figures 1–5

Figure 1 (A–D) IHC staining for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in normal pyloric mucosa, prepared from serial sections. In pyloric glands (A), MUC6 is diffusely expressed in the cytoplasm (B), {alpha}4GnT is expressed in the Golgi area (C), and GlcNAc{alpha}1->4Galß->R is expressed in cytoplasmic mucus granules (D). H&E staining (A) and immunostaining for MUC6 (B), {alpha}4GnT (C), and GlcNAc{alpha}1->4Galß->R (D). Original magnification x200.

Figure 2 (A–D) IHC staining for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in Brunner's glands, prepared from serial sections. In Brunner's glands (A), MUC6 is diffusely expressed in the cytoplasm (B), {alpha}4GnT is expressed in the Golgi area (C), and GlcNAc{alpha}1->4Galß->R is expressed in cytoplasmic mucus granules (D). H&E staining (A) and immunostaining for MUC6 (B), {alpha}4GnT (C), and GlcNAc{alpha}1->4Galß->R (D). Original magnification x200.

Figure 3 (A–D) IHC staining for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in peribiliary glands, prepared from serial sections. In peribiliary glands (A), MUC6 is diffusely expressed in cytoplasm (B), {alpha}4GnT is expressed in the Golgi area (C), and GlcNAc{alpha}1->4Galß->R is expressed in cytoplasmic mucous granules (D). H&E staining (A) and immunostaining for MUC6 (B), {alpha}4GnT (C), and GlcNAc{alpha}1->4Galß->R (D). Original magnification x400.

Figure 4 IHC staining for MUC6 in normal prostate gland, prepared from serial sections. Some prostate gland cells show diffuse cytoplasmic expression of MUC6. H&E staining (A) and immunostaining for MUC6 (B). Original magnification x250.

Figure 5 IHC staining for MUC6 in normal mammary gland, prepared from serial sections. Some cells of the terminal duct lobular unit of mammary gland show diffuse cytoplasmic expression of MUC6. H&E staining (A) and immunostaining for MUC6 (B). Original magnification x300.

 
MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R were present in discrete cell types to various degrees: gastric gland mucous cells (Figure 1) (cardiac gland cells, mucous neck cells, and pyloric gland cells), Brunner's gland cells (Figure 2), mucous cells of the periductal glands of the pancreaticobiliary tracts (Figure 3), biliary tract epithelial cells, and mucous cells of mucinous metaplasia of the pancreatic ducts. In these tissues, {alpha}4GnT-positive cells always exhibited immunoreactivity for GlcNAc{alpha}1->4Galß->R, as described previously (Zhang et al. 2001Go).

Except in the case of biliary tract epithelial cells, GlcNAc{alpha}1->4Galß->R was found in cytoplasmic mucus granules. The biliary tract epithelial cells exhibited cytoplasmic and apical cytoplasmic membrane staining for GlcNAc{alpha}1->4Galß->R.

MUC6 without {alpha}4GnT and GlcNAc{alpha}1->4Galß->R was expressed in a minority of submandibular gland mucous cells, pancreatic centroacinar cells, renal tubules, prostate glands (Figure 4), and terminal duct lobular units of mammary glands (Figure 5), and was also strongly expressed in seminal vesicle epithelial cells.

MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R were not detected in normal esophagus, colon, hepatocytes, or lungs. The IHC data presented here are summarized in Table 1.

Demonstration of MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in Neoplastic Tissues in Primary Tissues
MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R were detected in carcinoma of the stomach (Figure 6) , pancreas (Figure 7), and intrahepatic bile duct, and in mucinous bronchioloalveolar cell carcinoma (Figure 8). Staining differed quantitatively from case to case (Table 2).



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Figures 6–10

Figure 6 (A–D) IHC staining for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in diffuse type gastric carcinoma, prepared from serial sections. In signet-ring carcinoma cells located in the lower portion (A), MUC6 is diffusely expressed in the cytoplasm (B), {alpha}4GnT is expressed in the Golgi area (C), and GlcNAc{alpha}1->4Galß->R is expressed in the cytoplasmic mucous granules (D). H&E staining (A) and immunostaining for MUC6 (B), {alpha}4GnT (C), and GlcNAc{alpha}1->4Galß->R (D). Original magnification x200.

Figure 7 (A–D) IHC staining for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in pancreatic duct carcinoma, prepared from serial sections. In carcinoma cells (A), MUC6 is diffusely expressed in the cytoplasm (B), {alpha}4GnT is expressed in the Golgi area (C), and GlcNAc{alpha}1->4Galß->R is ex-

pressed in cytoplasmic mucous granules (D). H&E staining (A) and immunostaining for MUC6 (B), {alpha}4GnT (C), and GlcNAc{alpha}1->4Galß->R (D). Original magnification x250.

Figure 8 (A–D) IHC staining for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in pulmonary mucinous bronchioloalveolar cell carcinoma, prepared from serial sections. In carcinoma cells (A), MUC6 is diffusely expressed in the cytoplasm (B), {alpha}4GnT is expressed in the Golgi area (C), and GlcNAc{alpha}1->4Galß->R is expressed in cytoplasmic mucous granules (D). H&E staining (A) and immunostaining for MUC6 (B), {alpha}4GnT (C), and GlcNAc{alpha}1->4Galß->R (D). Original magnification x300.

Figure 9 IHC staining for MUC6 in prostatic adenocarcinoma, prepared from serial sections. Some carcinoma cells show diffuse cytoplasmic expression of MUC6. H&E staining (A) and immunostaining for MUC6 (B). Original magnification x250.

Figure 10 IHC staining for MUC6 in breast carcinoma, prepared from serial sections. Some carcinoma cells show diffuse cytoplasmic expression of MUC6. H&E staining (A) and immunostaining for MUC6 (B). Original magnification x200.

 
In these tumor tissues, MUC6 exhibited a heterogeneous cytoplasmic expression (Figures 6B, 7B, 8B, 9B, and 10B), whereas {alpha}4GnT exhibited Golgi staining (Figures 6C, 7C, and 8C). GlcNAc{alpha}1->4Galß->R was expressed on the luminal surface of anaplastic glands and in secreted mucins, as well as in the cytoplasm of carcinoma cells (Figures 6D, 7D, and 8D). In these tissues, {alpha}4GnT-positive cells uniformly exhibited GlcNAc{alpha}1->4Galß->R.

In gastric carcinomas, the staining scores given for GlcNAc{alpha}1->4Galß->R in diffuse-type carcinomas were greater in early carcinomas than in advanced carcinomas (p<0.05). The staining scores for {alpha}4GnT and MUC6 showed no relation to the depth of invasion of the carcinoma. No significant difference was found among the scores given for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R, irrespective of histological type or depth of wall penetration. The scores given for GlcNAc{alpha}1->4Galß->R showed a significant correlation with those given for {alpha}4GnT (r=0.73 in diffuse-type gastric carcinomas; r=0.86 in intestinal-type carcinomas). There was no correlation between the scores for {alpha}4GnT and MUC6 or between those for MUC6 and GlcNAc{alpha}1->4Galß->R.

In pancreatic carcinomas, there was no significant difference among the scores given for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R expression. The degree of GlcNAc{alpha}1->4Galß->R expression showed a significant correlation with the degree of expression for {alpha}4GnT and for MUC6 (r=0.65 and r=0.65, respectively).

In cholangiocarcinomas, there was no significant difference among the staining scores for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R. The degree of GlcNAc{alpha}1->4Galß->R expression showed a significant correlation with that of {alpha}4GnT expression (r=0.91). There was no correlation between MUC6 expression and {alpha}4GnT expression or between GlcNAc{alpha}1->4Galß->R expression and MUC6 expression.

In mucinous bronchioloalveolar carcinomas, the scores given for GlcNAc{alpha}1->4Galß->R were significantly higher than those for {alpha}4GnT expression (p<0.05). There were no correlations among the scores for MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R.

In a minority of colon adenomas, prostate carcinomas (Figure 9), and breast carcinomas (Figure 10), MUC6 was detected, whereas {alpha}4GnT and GlcNAc{alpha}1->4Galß->R were not. These IHC data are summarized in Table 2.

Demonstration of MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in Metastatic Adenocarcinomas of Lymph Nodes
Gastric carcinomas and pancreatic carcinomas showed immunoreactivity for MUC6, {alpha}4GnT, and GlcNAc{alpha}1-> 4Galß->R. In gastric carcinomas, the degree of GlcNAc{alpha}1->4Galß->R expression showed a significant correlation with the degree of {alpha}4GnT expression in gastric carcinomas (r=0.79). In both gastric carcinomas and pancreatic carcinomas, {alpha}4GnT-positive cells uniformly co-expressed GlcNAc{alpha}1-> 4Galß->R. Breast carcinomas showed immunoreactivity only for MUC6. Colon carcinomas showed immunoreactivity for none of these antigens.

These IHC data are summarized in Table 2.

Demonstration of GlcNAc{alpha}1->4Galß->R and Sialyl Lewis X in Gastric Carcinomas and Metastatic Gastric Carcinomas of Lymph Nodes
The distribution of carcinoma cells reactive for GlcNAc{alpha}1->4Galß->R was completely different from that of carcinoma cells reactive for sialyl Lewis X both in primary (Figure 11) and in metastatic sites, although we found no negative correlation between GlcNAc{alpha}1->4Galß->R and sialyl Lewis X expression. Carcinoma cells expressing both GlcNAc{alpha}1-> 4Galß->R and sialyl Lewis X were not found.



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Figure 11

IHC staining for GlcNAc{alpha}1->4Galß->R and sialyl Lewis X in gastric carcinoma, prepared from mirror sections. The distribution of the carcinoma cells reactive for GlcNAc{alpha}1->4Galß->R was completely different from that of the carcinoma cells reactive for sialyl Lewis X. Immunostaining for GlcNAc{alpha}1->4Galß->R (A) and sialyl Lewis X (B). Original magnification x200.

 
These IHC data are summarized in Table 2.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
In this study we evaluated the IHC expressions of MUC6, {alpha}4GnT, and GlcNAc{alpha}1->4Galß->R in normal, metaplastic, and neoplastic human tissues. The results showed (a) that {alpha}4GnT and GlcNAc{alpha}1-> 4Galß->R were co-located in discrete cell-types (a finding consistent with involvement of {alpha}4GnT in the formation of GlcNAc{alpha}1->4Galß->R in normal, metaplastic, and neoplastic cells) and (b) that both primary carcinoma tissues and metastatic tissues showed similar IHC profiles with regard to the expressions of MUC6, {alpha}4GnT, and GlcNAc{alpha}1-> 4Galß->R.

In this study we have confirmed and extended our knowledge regarding the specific distribution of GlcNAc{alpha}1->4Galß->R in human tissues. GlcNAc{alpha}1-> 4Galß->R has been reported to be present in gastric gland mucous cells (cardiac gland cells, mucous neck cells, and pyloric gland cells), Brunner's gland cells, mucous cells of the periductal glands of the pancreaticobiliary tract, mucinous metaplasia of gallbladder and pancreas, and neoplastic cells expressing gastric mucins [including mucinous tumors of the ovary, adenocarcinomas of stomach, pancreas, gallbladder, lung (mucinous bronchioloalveolar cell carcinoma), and uterine cervix (adenoma malignum) (Nakamura et al. 1998Go; Ota et al. 1998Go,2001Go; Zhang et al. 2001Go) ], but not in normal esophagus, colon, pancreas, ovary, lung, uterine cervix, normal and neoplastic salivary glands, or adenocarcinomas of colon, breast, kidney, thyroid gland, endometrium, liver, or prostate (Ishii et al. 1998Go; Nakamura et al. 1998Go; Ota et al. 2001Go). In addition, we found that GlcNAc{alpha}1->4Galß->R was present in the normal epithelium of the intrahepatic bile ducts and cholangiocarcinoma but not in seminal vesicle epithelium.

In this study, {alpha}4GnT-positive cells uniformly co-expressed GlcNAc{alpha}1->4Galß->R, irrespective of whether the sections were from normal, metaplastic, or neoplastic tissues. The degree of GlcNAc{alpha}1->4Galß->R expression showed a significant correlation with the degree of {alpha}4GnT expression in various adenocarcinoma tissues, as well as in normal and metaplastic cells.

The expressions of {alpha}4GnT and GlcNAc{alpha}1-> 4Galß->R were more restricted than that of MUC6. MUC6 has been demonstrated to be present in human tissues in which both {alpha}4GnT and GlcNAc{alpha}1-> 4Galß->R were positive, such as normal stomach (Ho et al. 1995aGo,bGo; Buisine et al. 2000bGo; Machado et al. 2000Go; Reis et al. 2000Go), duodenum (Bartman et al. 1998Go; Buisine et al. 2000aGo), bile duct (Bartman et al. 1998Go; Buisine et al. 2000aGo), gallbladder (Bartman et al. 1998Go; Buisine et al. 2000aGo), and in adenocarcinomas of the stomach (Ho et al. 1995bGo; Machado et al. 2000Go; Reis et al. 2000Go), bile duct (Bartman et al. 1998Go), gallbladder (Bartman et al. 1998Go; Sasaki et al. 1999Go), and pancreas (Bartman et al. 1998Go; Terada et al. 1996Go). As previously reported and confirmed in this study, MUC6 was distributed in a wide variety of human tissues in which both {alpha}4GnT and GlcNAc{alpha}1-> 4Galß->R were negative. Thus, MUC6 has previously been demonstrated in normal endometrium (Bartman et al. 1998Go) and seminal vesicles (Bartman et al. 1998Go), adenoma of the colon (Bartman et al. 1999Go), and normal and adenocarcinoma of the breast (Bartman et al. 1998Go; Pereira et al. 2001Go).

The distribution of carcinoma cells reactive for GlcNAc{alpha}1->4Galß->R was completely different from that of carcinoma cells reactive for sialyl Lewis X, both in primary (Figure 11) and metastatic sites, although we found no negative correlation between the GlcNAc{alpha}1->4Galß->R and sialyl Lewis X expressions. GlcNAc{alpha}1->4Galß->R is preferentially attached to core2-branched O-glycan (Ishihara et al. 1996Go), and the sialyl Lewis X found in O-glycan is also attached to the terminal end of core2-branched structures (Fukuda 1996Go). Therefore, the expressions of GlcNAc{alpha}1-> 4Galß->R and sialyl Lewis X may be reciprocally regulated because these carbohydrates compete for the common precursor oligosaccharide, core2-branched O-glycan. It is well known that sialyl Lewis X serve as preferential ligands for the cell-adhesion molecules E- and P-selectin (Fukushima et al. 1984Go; Itzkowitz et al. 1988Go; Rosen and Bertozzi 1994Go). Recently, we demonstrated that the sialyl Lewis X expressed on core2-branched O-glycans were positively correlated with tumor progression in both colorectal and pulmonary cancers (Shimodaira et al. 1997Go; Machida et al. 2001Go). Overall, these results suggest that the expression of GlcNAc{alpha}1->4Galß->R in gastric cancer cells may be a favorable predictor of the patient's outcome. Further study will be needed to test this hypothesis.

In summary, primary carcinoma tissues and metastatic tissues showed similar IHC profiles with regard to the expressions of MUC6, {alpha}4GnT, and GlcNAc{alpha}1-> 4Galß->R. Determination of the site of origin of metastatic carcinomas using examination of histological slides continues to present a diagnostic challenge for the pathologist. It is possible that immunostaining for GlcNAc{alpha}1->4Galß->R (with MAb HIK1083), or {alpha}4GnT, in conjunction with immunostaining for MUC6, could be diagnostically relevant because of their specific distributions in human tissues.


    Acknowledgments
 
Supported by Grants-in-Aid for Scientific Research C-15590482 (to HO) and Priority Area 14082201 (to JN) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We thank Professor Tsutomu Katsuyama and Katsunori Sasaki at Shinshu University School of Medicine, and Professor David Y. Graham at Baylor College of Medicine (Houston, TX) for their helpful comments and encouragement.


    Footnotes
 
Received for publication September 23, 2002; accepted July 18, 2003


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

Akamatsu T, Katsuyama T (1990) Histochemical demonstration of mucins in the intramucosal laminated structure of human gastric signet ring cell carcinoma and its relation to submucosal invasion. Histochem J 22:416–425[Medline]

Bartman AE, Buisine MP, Aubert JP, Niehans GA, Toribara NW, Kim YS, Kelly EJ, et al. (1998) The MUC6 secretory mucin gene is expressed in a wide variety of epithelial tissues. J Pathol 186:398–405[Medline]

Bartman AE, Sanderson SJ, Ewing SL, Niehans GA, Wiehr CL, Evans MK, Ho SB (1999) Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps. Int J Cancer 80:210–218[Medline]

Buisine MP, Devisme L, Degand P, Dieu MC, Gosselin B, Copin MC, Aubert JP, et al. (2000a) Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. II. Duodenum and liver, gallbladder, and pancreas. J Histochem Cytochem 48:1667–1676[Abstract/Free Full Text]

Buisine MP, Devisme L, Maunoury V, Deschodt E, Gosselin B, Copin MC, Aubert JP, et al. (2000b) Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. I. Stomach. A relationship to gastric carcinoma. J Histochem Cytochem 48:1657–1666[Abstract/Free Full Text]

Fujimori Y, Akamatsu T, Ota H, Katsuyama T (1995) Proliferative markers in gastric carcinoma and organoid differentiation. Hum Pathol 26:725–734[Medline]

Fukuda M (1996) Possible roles of tumor-associated carbohydrate antigens. Cancer Res 56:2237–2244[Abstract]

Fukushima K, Hirota M, Terasaki PI, Wakisaka A, Togashi H, Chia D, Suyama N, et al. (1984) Characterization of sialosylated Lewisx as a new tumor-associated antigen. Cancer Res 44:5279–5285[Abstract]

Ho SB, Roberton AM, Shekels LL, Lyftogt CT, Niehans GA, Toribara NW (1995a) Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression. Gastroenterology 109:735–747[Medline]

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

Honda T, Ota H, Ishii K, Nakamura N, Kubo K, Katsuyama T (1998) Mucinous bronchioloalveolar carcinoma with organoid differentiation simulating the pyloric mucosa of the stomach: clinicopathologic, histochemical, and immunohistochemical analysis. Am J Clin Pathol 109:423–430[Medline]

Ishihara K, Kurihara M, Goso Y, Urata T, Ota H, Katsuyama T, Hotta K (1996) Peripheral {alpha}-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody. Biochem J 318:409–416[Medline]

Ishii K, Hosaka N, Toki T, Momose M, Hidaka E, Tsuchiya S, Katsuyama T (1998) A new view of the so-called adenoma malignum of the uterine cervix. Virchows Arch 432:315–322[Medline]

Ishii K, Katsuyama T, Ota H, Watanabe T, Matsuyama I, Tsuchiya S, Shiozawa T, et al. (1999) Cytologic and cytochemical features of adenoma malignum of the uterine cervix. Cancer 87:245–253[Medline]

Itzkowitz SH, Yuan M, Fukushi Y, Lee H, Shi ZR, Zurawski V Jr, Hakomori S, et al. (1988) Immunohistochemical comparison of Lea, monosialosyl Lea (CA 19–9), and disialosyl Lea antigens in human colorectal and pancreatic tissues. Cancer Res 48:3834–3842[Abstract]

Katsuyama T, Spicer SS (1978) Histochemical differentiation of complex carbohydrates with variants of the concanavalin A–horseradish peroxidase method. J Histochem Cytochem 26: 233–250[Abstract]

Kijima H, Watanabe H, Iwafuchi M, Ishihara N (1989) Histogenesis of gallbladder carcinoma from investigation of early carcinoma and microcarcinoma. Acta Pathol Jpn 39:235–244[Medline]

Kumamoto K, Mitsuoka C, Izawa M, Kimura N, Otsubo N, Ishida H, Kiso M, et al. (1998) Specific detection of sialyl Lewis X determinant carried on the mucin GlcNAcbeta1->6GalNAcalpha core structure as a tumor-associated antigen. Biochem Biophys Res Commun 247:514–517[Medline]

Lesuffleur T, Zweibaum A, Real FX (1994) Mucins in normal and neoplastic human gastrointestinal tissues. Crit Rev Oncol Hematol 17:153–180[Medline]

Machado JC, Nogueira AM, Carneiro F, Reis CA, Sobrinho–Simoes M (2000) Gastric carcinoma exhibits distinct types of cell differentiation: an immunohistochemical study of trefoil peptides (TFF1 and TFF2) and mucins (MUC1, MUC2, MUC5AC, and MUC6). J Pathol 190:437–443[Medline]

Machida E, Nakayama J, Amano J, Fukuda M (2001) Clinicopathological significance of core 2 ß1,6-N-acetylglucosaminyltransferase messenger RNA expressed in the pulmonary adenocarcinoma determined by in situ hybridization. Cancer Res 61:2226–2231[Abstract/Free Full Text]

Matsuzawa K, Akamatsu T, Katsuyama T (1992) Mucin histochemistry of pancreatic duct cell carcinoma, with special reference to organoid differentiation simulating gastric pyloric mucosa. Hum Pathol 23:925–933[Medline]

Nakamura N, Ota H, Katsuyama T, Akamatsu T, Ishihara K, Kurihara M, Hotta K (1998) Histochemical reactivity of normal, metaplastic, and neoplastic tissues to {alpha}-linked N-acetylglucosamine residue-specific monoclonal antibody HIK1083. J Histochem Cytochem 46:793–801[Abstract/Free Full Text]

Nakayama J, Yeh JC, Misra AK, Ito S, Katsuyama T, Fukuda M (1999) Expression cloning of a human {alpha}1,4-N-acetylglucosaminyltransferase that forms GlcNAc{alpha}1->4Galß->R, a glycan specifically expressed in the gastric gland mucous cell-type mucin. Proc Natl Acad Sci USA 96:8991–8996[Abstract/Free Full Text]

Ota H, Hayama M, Nakayama J, Hidaka H, Honda T, Ishii K, Fukushima M, et al. (2001) Cell lineage specificity of newly raised monoclonal antibodies against gastric mucins in normal, metaplastic, and neoplastic human tissues and their application to pathology diagnosis. Am J Clin Pathol 115:69–79[Medline]

Ota H, Katsuyama T, Akamatsu T, Fujimori Y, Matsuzawa K, Ishii K, Honda T, et al. (1995) Application of mucin histochemistry for pathological diagnosis—expression of gastric phenotypes in metaplastic and neoplastic lesions and its relation to organoid differentiation. Acta Histochem Cytochem 28:43–53

Ota H, Katsuyama T, Ishii K, Nakayama J, Shiozawa T, Tsukahara Y (1991) A dual staining method for identifying mucins of different gastric epithelial mucous cells. Histochem J 23:22–28[Medline]

Ota H, Nakayama J, Momose M, Kurihara M, Ishihara K, Hotta K, Katsuyama T (1998) New monoclonal antibodies against gastric gland mucous cell-type mucins: a comparative immunohistochemical study. Histochem Cell Biol 110:113–119[Medline]

Pereira MB, Dias AJ, Reis CA, Schmitt FC (2001) Immunohistochemical study of the expression of MUC5AC and MUC6 in breast carcinomas and adjacent breast tissues. J Clin Pathol 54:210–213[Abstract/Free Full Text]

Reis CA, David L, Carvalh F, Mandel U, de Bolos C, Mirgorodskaya E, Clausen H, et al. (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]

Rosen SD, Bertozzi CR (1994) The selectins and their ligands. Curr Opin Cell Biol 6:663–673[Medline]

Sasaki M, Yamato T, Nakanuma Y, Ho SB, Kim YS (1999) Expression of MUC2, MUC5AC and MUC6 apomucins in carcinoma, dysplasia and non-dysplastic epithelia of the gallbladder. Pathol Int 49:38–44[Medline]

Shimodaira K, Nakayama J, Nakamura N, Hasebe O, Katsuyama T, Fukuda M (1997) Carcinoma-associated expression of core 2 ß1,6-N-acetylglucosaminyltransferase gene in human colorectal cancer: role of O-glycans in tumor progression. Cancer Res 57:5201–5206[Abstract]

Suganuma T, Katsuyama T, Tsukahara M, Tatematsu M, Sakakura Y, Murata F (1981) Comparative histochemical study of alimentary tracts with special reference to the mucous neck cells of the stomach. Am J Anat 161:219–238[Medline]

Terada T, Ohta T, Sasaki M, Nakanuma Y, Kim YS (1996) Expression of MUC apomucins in normal pancreas and pancreatic tumours. J Pathol 180:160–165[Medline]

Tsutsumi Y, Nagura H, Osamura Y, Watanabe K, Yanaihara N (1984) Histochemical studies of metaplastic lesions in the human gallbladder. Arch Pathol Lab Med 108:917–921[Medline]

Zhang MX, Nakayama J, Hidaka E, Kubota S, Yan J, Ota H, Fukuda M (2001) Immunohistochemical demonstration of {alpha}1,4-N-acetylglucosaminyltransferase that forms GlcNAc{alpha}1,4Galß residues in human gastrointestinal mucosa. J Histochem Cytochem 49:587–596[Abstract/Free Full Text]





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