Expression of Gastric Gland Mucous Cell-Type Mucin in Normal and Neoplastic Human Tissues
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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Summary |
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Key Words: glycosyltransferase gastric mucin immunohistochemistry mucin core protein
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Introduction |
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Several years ago, Ishihara et al. (1996) raised a specific monoclonal antibody (MAb) against gastric mucin, designated HIK1083, which recognizes N-acetylglucosamin
1
4galactoseß
R (GlcNAc
1
4Galß
R). We found that the distribution of GlcNAc
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. 1998
; Ota et al. 1998
,2001
). More recently, we showed that class III mucin possesses this particular glycan, GlcNAc
1
4Galß
R, which is formed by
1,4-N-acetylglucosaminyltransferase (
4GnT) (Nakayama et al. 1999
), and that two distinct mucin core proteins, MUC5AC and MUC6, present in gastric mucin carry GlcNAc
1
4Galß
R (Zhang et al. 2001
).
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.
GlcNAc1
4Galß
R is preferentially attached to core2-branched O-glycan (Ishihara et al. 1996
). The sialyl Lewis X found in O-glycan are also attached to the terminal end of core2-branched structures (Fukuda 1996
). Therefore, it appears that the expressions of GlcNAc
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. 1984
; Itzkowitz et al. 1988
; Rosen and Bertozzi 1994
). 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. 1997
; Machida et al. 2001
).
We designed the present study to explore the IHC expressions of MUC6, 4GnT, and GlcNAc
1
4Galß
R in human normal, metaplastic, and adenocarcinoma tissues. We also examined the IHC expressions of GlcNAc
1
4Galß
R and sialyl Lewis X in gastric carcinomas and in metastatic gastric carcinomas of lymph nodes.
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Materials and Methods |
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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 GlcNAc1
4Galß
R and MUC6. For immunostaining with anti-
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-4GnT, and GlcNAc
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 MannWhitney 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, 4GnT, GlcNAc
1
4Galß
R, and sialyl Lewis X. Staining scores are nonparametric and are presented as median rather than mean values.
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Results |
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Except in the case of biliary tract epithelial cells, GlcNAc1
4Galß
R was found in cytoplasmic mucus granules. The biliary tract epithelial cells exhibited cytoplasmic and apical cytoplasmic membrane staining for GlcNAc
1
4Galß
R.
MUC6 without 4GnT and GlcNAc
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, 4GnT, and GlcNAc
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, 4GnT, and GlcNAc
1
4Galß
R in Neoplastic Tissues in Primary Tissues
MUC6, 4GnT, and GlcNAc
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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In gastric carcinomas, the staining scores given for GlcNAc1
4Galß
R in diffuse-type carcinomas were greater in early carcinomas than in advanced carcinomas (p<0.05). The staining scores for
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,
4GnT, and GlcNAc
1
4Galß
R, irrespective of histological type or depth of wall penetration. The scores given for GlcNAc
1
4Galß
R showed a significant correlation with those given for
4GnT (r=0.73 in diffuse-type gastric carcinomas; r=0.86 in intestinal-type carcinomas). There was no correlation between the scores for
4GnT and MUC6 or between those for MUC6 and GlcNAc
1
4Galß
R.
In pancreatic carcinomas, there was no significant difference among the scores given for MUC6, 4GnT, and GlcNAc
1
4Galß
R expression. The degree of GlcNAc
1
4Galß
R expression showed a significant correlation with the degree of expression for
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, 4GnT, and GlcNAc
1
4Galß
R. The degree of GlcNAc
1
4Galß
R expression showed a significant correlation with that of
4GnT expression (r=0.91). There was no correlation between MUC6 expression and
4GnT expression or between GlcNAc
1
4Galß
R expression and MUC6 expression.
In mucinous bronchioloalveolar carcinomas, the scores given for GlcNAc1
4Galß
R were significantly higher than those for
4GnT expression (p<0.05). There were no correlations among the scores for MUC6,
4GnT, and GlcNAc
1
4Galß
R.
In a minority of colon adenomas, prostate carcinomas (Figure 9), and breast carcinomas (Figure 10), MUC6 was detected, whereas 4GnT and GlcNAc
1
4Galß
R were not. These IHC data are summarized in Table 2.
Demonstration of MUC6, 4GnT, and GlcNAc
1
4Galß
R in Metastatic Adenocarcinomas of Lymph Nodes
Gastric carcinomas and pancreatic carcinomas showed immunoreactivity for MUC6, 4GnT, and GlcNAc
1
4Galß
R. In gastric carcinomas, the degree of GlcNAc
1
4Galß
R expression showed a significant correlation with the degree of
4GnT expression in gastric carcinomas (r=0.79). In both gastric carcinomas and pancreatic carcinomas,
4GnT-positive cells uniformly co-expressed GlcNAc
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 GlcNAc1
4Galß
R and Sialyl Lewis X in Gastric Carcinomas and Metastatic Gastric Carcinomas of Lymph Nodes
The distribution of carcinoma cells reactive for GlcNAc1
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
1
4Galß
R and sialyl Lewis X expression. Carcinoma cells expressing both GlcNAc
1
4Galß
R and sialyl Lewis X were not found.
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Discussion |
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In this study we have confirmed and extended our knowledge regarding the specific distribution of GlcNAc1
4Galß
R in human tissues. GlcNAc
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. 1998
; Ota et al. 1998
,2001
; Zhang et al. 2001
) ], 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. 1998
; Nakamura et al. 1998
; Ota et al. 2001
). In addition, we found that GlcNAc
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, 4GnT-positive cells uniformly co-expressed GlcNAc
1
4Galß
R, irrespective of whether the sections were from normal, metaplastic, or neoplastic tissues. The degree of GlcNAc
1
4Galß
R expression showed a significant correlation with the degree of
4GnT expression in various adenocarcinoma tissues, as well as in normal and metaplastic cells.
The expressions of 4GnT and GlcNAc
1
4Galß
R were more restricted than that of MUC6. MUC6 has been demonstrated to be present in human tissues in which both
4GnT and GlcNAc
1
4Galß
R were positive, such as normal stomach (Ho et al. 1995a
,b
; Buisine et al. 2000b
; Machado et al. 2000
; Reis et al. 2000
), duodenum (Bartman et al. 1998
; Buisine et al. 2000a
), bile duct (Bartman et al. 1998
; Buisine et al. 2000a
), gallbladder (Bartman et al. 1998
; Buisine et al. 2000a
), and in adenocarcinomas of the stomach (Ho et al. 1995b
; Machado et al. 2000
; Reis et al. 2000
), bile duct (Bartman et al. 1998
), gallbladder (Bartman et al. 1998
; Sasaki et al. 1999
), and pancreas (Bartman et al. 1998
; Terada et al. 1996
). As previously reported and confirmed in this study, MUC6 was distributed in a wide variety of human tissues in which both
4GnT and GlcNAc
1
4Galß
R were negative. Thus, MUC6 has previously been demonstrated in normal endometrium (Bartman et al. 1998
) and seminal vesicles (Bartman et al. 1998
), adenoma of the colon (Bartman et al. 1999
), and normal and adenocarcinoma of the breast (Bartman et al. 1998
; Pereira et al. 2001
).
The distribution of carcinoma cells reactive for GlcNAc1
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
1
4Galß
R and sialyl Lewis X expressions. GlcNAc
1
4Galß
R is preferentially attached to core2-branched O-glycan (Ishihara et al. 1996
), and the sialyl Lewis X found in O-glycan is also attached to the terminal end of core2-branched structures (Fukuda 1996
). Therefore, the expressions of GlcNAc
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. 1984
; Itzkowitz et al. 1988
; Rosen and Bertozzi 1994
). 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. 1997
; Machida et al. 2001
). Overall, these results suggest that the expression of GlcNAc
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, 4GnT, and GlcNAc
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
1
4Galß
R (with MAb HIK1083), or
4GnT, in conjunction with immunostaining for MUC6, could be diagnostically relevant because of their specific distributions in human tissues.
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Acknowledgments |
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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.
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Footnotes |
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