ARTICLE |
Correspondence to: Jun Nakayama, Central Clinical Laboratories, Shinshu Univ. Hospital, Asahi 3-1-1, Matsumoto 390-8621, Japan.
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Summary |
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We set out to determine the expression profiles of glycoproteins possessing N-acetyllactosamine, a precursor carbohydrate of sialyl Lex, during colorectal cancer development. We immunohistochemically analyzed the distribution of N-acetyllactosamine as well as of ß4GalT-I, a member of the ß1,4-galactosyltransferase family responsible for N-acetyllactosamine biosynthesis, in normal mucosa and in adenoma and carcinoma of the human colorectum. Using monoclonal antibody H11, N-acetyllactosamine was barely detectable in the normal mucosa. In low-grade adenoma, however, N-acetyllactosamine was weakly but definitely expressed on the cell surface, and its expression level was moderately increased in high-grade adenoma and markedly increased in carcinoma in situ as well as in advanced carcinoma. To detect ß4GalT-I, we used a newly developed polyclonal antibody (designated A18G), which is specific for the stem region of human ß4GalT-I. Faint expression of ß4GalT-I was detectable in normal mucosa, and the expression level was moderately increased in low-grade adenoma and in high-grade adenoma and markedly increased in carcinoma in situ and advanced carcinoma. The expression of N-acetyllactosamine was highly correlated with the expression of ß4GalT-I in these tumor cells. These results indicate that the expression level of ß4GalT-I is apparently enhanced during tumorigenesis in the colorectum and that ß4GalT-I mostly directs the carcinoma-associated expression of N-acetyllactosamine on the colorectal tumor cell surface. (J Histochem Cytochem 47:15931601, 1999)
Key Words: tumor-associated, carbohydrate antigen, glycosyltransferase gene family, immunohistochemistry
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Introduction |
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THE CARBOHYDRATE MOIETIES of glycoconjugates attached to the cell surface membrane of epithelial cells in the gastrointestinal tract play important roles in various pathophysiological events, such as cell differentiation, inflammation, and neoplastic transformation, through cellcell and/or cellmatrix interactions (2
3Galß1
3(Fuc
1
4) GlcNAcß
R) and an isomer of sialyl Lea, i.e., sialyl Lex (NeuNAc
2
3Galß1
4(Fuc
1
3)GlcNAcß
R), are enriched on the cell surface of carcinoma cells, such as those of colorectal cancers (
N-acetyllactosamine (Galß14GlcNAcß
R) is a precursor carbohydrate of sialyl Lex (Figure 1A) (
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In this study, we immunohistochemically analyzed the expression pattern of N-acetyllactosamine during the adenomacarcinoma sequence in the colorectum to determine whether the expression of N-acetyllactosamine actually is enhanced during colorectal cancer development. In parallel, the expression of ß4GalT-I was also evaluated using a newly developed polyclonal antibody, designated A18G, which is specific for ß4GalT-I. The results presented here indicate that the expression level of ß4GalT-I is apparently enhanced during tumorigenesis in the colorectum and that ß4GalT-I mostly directs the carcinoma-associated expression of N-acetyllactosamine on the colorectal tumor cell surface.
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Materials and Methods |
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Production of Polyclonal Antibody Against Human ß4GalT-I
The monospecific polyclonal antibody A18G, directed to the stem region of human ß4GalT-I, was generated against a synthetic peptide, ASSQPRPGGDSSPVVDSG, corresponding to the stem region (amino acids 89106) of human ß4GalT-I (
Construction of Mammalian Expression Vectors Harboring Human ß4GalT-II, -III, -V, or -VI cDNA
It is shown that transcripts for ß4GalT-II, -III, -V, and -VI are expressed in human fetal brain (
Transient Expression of Human ß4GalTs in Transfected COS-1 Cells
Two micrograms of pCAG-GT encoding a cDNA of human ß4GalT-I (
In parallel, COS-1 cells were transfected by pCAG-GT and then subjected to Western blot analysis (
Colorectal Tumor Specimens and Tissue Preparation
Twenty-one polypectomies for colorectal polyp and 17 surgically resected specimens of advanced colorectal adenocarcinoma were selected from the pathology files of Central Clinical Laboratories (Shinshu University Hospital, Matsumoto, Japan). Histopathological examination was performed based on the criteria of the WHO classification (
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Immunohistochemistry
Immunohistochemical staining for N-acetyllactosamine or ß4GalT-I was performed on consecutive sections by an indirect immunoperoxidase method, as described (
Evaluation
To evaluate the results of the immunohistochemical staining for N-acetyllactosamine and ß4GalT-I, a scoring system based on the criteria laid down by
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Results |
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A18G Antibody Specifically Detects Human ß4GalT-I Expressed in Transfected COS-1 Cells
To test the specificity of the A18G antibody, we transfected COS-1 cells with human ß4GalT-I cDNA (pCAG-GT), then immunostained the transfected cells with A18G. Before the transfection, the permeabilized COS-1 cells were not stained with A18G antibody (Figure 2A). By contrast, after COS-1 cells were transfected with pCAG-GT, A18G antibody reacted strongly with the Golgi region of permeabilized COS-1 cells (Figure 2B) or with the cell surface of unpermeabilized COS-1 cells (Figure 2C). On the other hand, A18G did not react with the transfected COS-1 cells expressing ß4GalT-II (Figure 2D), ß4GalT-III (Figure 2E), ß4GalT-IV (Figure 2F), ß4GalT-V (Figure 2G), or ß4GalT-VI (Figure 2H), irrespective of the permeabilization of the COS-1 cells.
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In addition, Western blot analysis revealed that specific bands detected by A18G antibody were found in cell lysates derived from the COS-1 cells transiently transfected by pCAG-GT approximately at 62, 52, 49, and 46 kD (arrows in Figure 3, Lane 1) as well as 122 kD (arrowhead in Figure 3, Lane 1). Moreover, these proteins were also recognized by the other polyclonal antibody raised against the soluble form of ß4GalT-I (Figure 3, Lane 2). By contrast, these bands were not detectable in the parental COS-1 cells by A18G antibody (Figure 3, Lane 3). These results combined indicate that all the multiple bands found in the transfected COS-1 cells represent ß4GalT-I, because they were specifically detected by the two different kinds of antibodies specific for ß4GalT-I.
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These results have established that the A18G antibody used in this study specifically recognizes the human ß4GalT-I but not other ß4GalTs (i.e., ß4GalT-II to ß4GalT-VI). In addition, A18G does not react with the monkey ß4GalT-I that is endogenously expressed in COS-1 cells, which are derived from African green monkey kidney cells.
Expression of N-acetyllactosamine Is Enhanced During Colorectal Tumorigenesis
The expression profile of N-acetyllactosamine in normal mucosa and in adenoma and carcinoma of the colorectum is shown in Table 1. The results showed that N-acetyllactosamine was detectable on the apical surface but not in the Golgi region or other intracellular components of epithelial cells, and its expression level was progressively enhanced during the adenomacarcinoma sequence in the colorectum. N-acetyllactosamine was barely detectable in normal mucosa (Figure 4A). In adenoma, N-acetyllactosamine was weakly expressed along the apical surface of low-grade adenoma cells (not shown) and moderately on the luminal surface of high-grade adenoma cells (Figure 4E). In carcinoma, N-acetyllactosamine was strongly expressed on the apical surface of carcinoma cells in carcinoma in situ (Figure 4G) and in advanced carcinoma (Figure 4I), irrespective of the histological differentiation of the adenocarcinoma cells.
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Expression of ß4GalT-I Is Correlated with That of N-acetyllactosamine During the AdenomaCarcinoma Sequence in Colorectal Tumors
The general trend followed by the ß4GalT-I expression in normal mucosa, adenoma, and carcinoma in the colorectum is shown in Table 1. In general, ß4GalT-I was detectable in the Golgi region of epithelial cells but not on the cell surface, and its expression level was progressively enhanced during the transition from normal mucosa to adenoma and from adenoma to carcinoma. In normal mucosa, the expression of ß4GalT-I was weak (Figure 4B). Moderate expression of ß4GalT-I was found in low-grade adenoma (Figure 4D) and also in high-grade adenoma (Figure 4F). By contrast, ß4GalT-I was strongly expressed in carcinoma in situ, and the expression level appeared to be stronger than in the adenomas (Figure 4H). In advanced carcinoma, ß4GalT-I appeared to be more strongly expressed than in carcinoma in situ, irrespective of the histological differentiation of the adenocarcinoma cells (Figure 4J).
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Discussion |
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In this study we have demonstrated that the expression of N-acetyllactosamine, a precursor carbohydrate of sialyl Lex, is progressively enhanced during development of colorectal cancer. N-Acetyllactosamine is well known as the antigenic determinant for the carbohydrate antigens i [(Galß14GlcNAcß1
3)nGalß1
4 GlcNAcß
R] and I [Galß1
4GlcNAcß1
3(Galß1
4 GlcNAcß1
6)Galß1
4GlcNAcß
R], which are present in fetal and adult erythrocytes, respectively (
Recently, five novel members of the human ß4GalT gene family, ß4GalT-II to ß4GalT-VI, have been cloned by utilizing the expressed-sequence tag database (
Several authors have carried out immunohistochemical studies in various cells, including HeLa cells and bovine and rat epithelial cells, using polyclonal or monoclonal antibodies against ß4GalT-I (
Using the A18G and H11 antibodies, we next examined the co-expression of ß4GalT-I and N-acetyllactosamine in serial sections at various stages in the adenomacarcinoma sequence of colorectal tumors. We found that the expression of ß4GalT-I in the carcinoma cells showed a good correlation with that of N-acetyllactosamine (Figure 4G4J). Previously, we demonstrated that the expression of N-acetyllactosamine in the human colorectum was oncodevelopmentally regulated (
It is reported that increased activity of plasma membrane sialidase of the JB6 mouse epidermal cell is associated with transformation induced by phorbol ester (2,3- or
2,6-linked sialic acid can be attached to the galactose residue of N-acetyllactosamine by sialyltransferase, ST3Gal IV, or ST6Gal I (
It is also noteworthy that in 13 of 19 lesions in low-grade adenoma, some adenoma cells expressing ß4GalT-I were negative for N-acetyllactosamine on the cell surface (Figure 4C and Figure 4D, arrows). In such adenoma cells, it is most likely that N-acetyllactosamine synthesized by ß4GalT-I was subsequently sialylated and eventually could not be detected by the H11 antibody.
It has been reported that ß4GalT-I is expressed on the cell surface of various cells, including sperm, and it was suggested that this cell surface-associated ß4GalT-I could serve as a cell-adhesion molecule by recognizing oligosaccharides either on the cell surface of adjacent cells or in the extracellular matrix, as exemplified in the binding of sperm to the egg coat (
In conclusion, we have demonstrated by the use of H11 and A18G antibodies that the expression of ß4GalT-I in tumor cells increases progressively during colorectal cancer development and that the enhanced activity of ß4GalT-I may, in turn, regulate the cell surface expression on these tumor cells of those carbohydrate antigens having N-acetyllactosamine at their termini. Further studies will be required to determine how the expression of ß4GalT-I is regulated during the development of colorectal cancer.
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research on Priority Area (10178104) and C (09670222) from the Ministry of Education, Science, Sports and Culture of Japan (to JN), and Grant PO1 CA71932 from the NCI, National Institutes of Health (to MF and MNF).
We are grateful to Dr Tsutomu Katsuyama for encouragement and discussion during this study, to Dr Yukiko Matsumoto and Noriko Kubota for their excellent technical assistance, Dr Jiuun Yeh-Chen for providing pcDNA3-ß4GalT-IV, and to Drs Edgar Ong and Mu-Xia Zhang for critical reading of the manuscript.
Received for publication March 9, 1999; accepted July 30, 1999.
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