ARTICLE |
Correspondence to: Celso A. Reis, Inst. of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, 4200 Porto, Portugal.
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Summary |
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To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas. (J Histochem Cytochem 48:377388, 2000)
Key Words: mucin, MUC6, MUC5AC, MUC2, gastric carcinoma, monoclonal antibody
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Introduction |
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Mucins are high molecular weight glycoproteins that are synthesized by secretory epithelial cells as membrane bound or secreted products. Thus far, nine human mucin genes have been identified and designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, and MUC8 in the international nomenclature (for review see
The expression of mucins exhibits organ-, tissue-, and cell-specific patterns (
Quantitative and qualitative alterations of mucin expression have been frequently reported in gastric carcinomas (
Here we report the development of monoclonal antibodies (MAbs) CLH4 and CLH5 against a synthetic peptide based on the MUC6 tandem repeat amino acid sequence. The biochemical and immunohistochemical properties of MAb CLH5 are described in detail. MAb CLH5 was used to study the expression profile of MUC6 in 104 gastric carcinomas to determine if there is any association between the expression of MUC6 and the clinicopathological characteristics of the carcinomas. Furthermore, we aimed to analyze if the expression of MUC6 is related to the expression of another "gastric" mucin, MUC5AC, and/or to that of the "intestinal" mucin MUC2.
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Materials and Methods |
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Synthetic Peptides
The synthetic peptides used in this study (Table 1) were HPLC-purified and the structure confirmed by amino acid analyses and mass spectrometry. Peptides MUC1, MUC2, and MUC5AC were synthesized by Carlbiotech (Copenhagen, Denmark). Peptide MUC7 was synthesized by Neosystem (Strasbourg, France). Peptide MUC3 was a kind gift of Dr. G. Hansson. Peptide MUC6 was a kind gift of Dr. C de Bolós (IMIM, Barcelona). Peptide MUC6 sequence corresponds to an antigenic region within the tandem repeat of the MUC6 mucin and was selected using the PEP polypeptide analysis system version 5.4 (IntelliGenetics; Mountain View, CA). Peptide MUC6 and control peptide MUC5AC were coupled to maleimide-activated keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL) by mixing 1 mg of peptide with 1 mg of KLH, followed by incubation for 1 hr at room temperature (RT). The coupled peptides were dialyzed overnight at 4C against PBS.
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Production of Monoclonal Antibodies
Female 812-week old BALB/c mice were immunized IP three times with the coupled MUC6 peptide mixed with Freund's adjuvant, with an interval of 14 days between immunizations. Blood samples from the immunized mice were collected 7 days after the third immunization and the sera tested by ELISA, immunohistology, and Western blotting. Three days after an IV boost, spleen cells from one mouse were fused with NS-1 cells and hybridomas secreting anti-MUC6 antibodies were cloned by limiting dilution. The screening strategy involved (a) direct ELISA using synthetic peptides, (b) inhibitory ELISA with the MUC6 peptide and irrelevant peptides, (c) immunohistology using normal gastric mucosa, and (d) Western blotting with native and deglycosylated mucin extracts from gastric mucosa. Hybridomas producing MAbs specifically reactive with MUC6 peptides and showing reactivity with gastric mucin samples in Western blots were selected. Two hybridomas, CLH4 and CLH5, were cloned. Immunoglobulin isotypes were determined using subclass-specific goat anti-mouse peroxidase-conjugated antibody (Southern Biotechnology Associates; Birmingham, AL).
Enzyme-linked Immunosorbent Assay
Maxisorp plates (Nunc; Roskilde, Denmark) were coated with peptides diluted in PBS, pH 7.2, overnight at 4C. Plates were blocked with 5% BSA diluted in PBS, followed by incubation for 1 hr with hybridoma supernatants or sera. Plates were then incubated with peroxidase-conjugated rabbit anti-mouse antibody (DAKO; Glostrup, Denmark) for 1 hr and developed using 0.4 mg/ml of 1,2-phenylendiamin (OPD) tablets (DAKO) in 0.1 citric acidphosphate buffer (pH 5.0) and 1 µl of 30% H2O2/ml of development solution. The inhibitory ELISA was carried out as above, except that the primary antibody was incubated with inhibitor for 1 hr before it was added to the coated and blocked ELISA plate.
Gastric Mucin Extracts, SDS-PAGE, and Western Blotting
Gastric mucins were obtained from surgical specimens of patients with gastric carcinoma undergoing surgical resection in Hospital S. João (Porto, Portugal). Samples were collected after informed consent. Mucin samples from antral gastric mucosa adjacent to carcinomas were purified, characterized, and deglycosylated as previously described (
In Vitro Glycosylation of Peptides
Peptide MUC6 and control peptide MUC2 were glycosylated in vitro using a recombinant enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase T2 (GalNAc-T2). The production and characterization of the recombinant enzyme have been described elsewhere (
Determination of Glycopeptide Structure
The different glycoforms of the peptide MUC6 were purified on a microbore HPLC system (ABI 130A; PerkinElmer, Norwalk, CT) using a Brownlee C18 narrow-bore column (30 mm2.1 mm, 7-µm particle size), with a gradient of 080% acetonitrile in 0.1% trifluoroacetic acid (TFA). HPLC fractions were collected manually and analyzed by MALDIMS. MALDI mass spectra of nonglycosylated and glycosylated peptides were acquired on a Voyager-Elite MALDI time-of-flight mass spectrometer (PerSeptive Biosystems; Framingham, MA) equipped with delayed extraction. The MALDI matrix was 2,5-dihydroxybenzoic acid 10 g/liter (Aldrich; Milwaukee, WI) dissolved in 0.1% TFA in water/acetonitrile 2/1 (v/v). Reverse-phase HPLC purified samples were prepared for MALDI analysis by placing 1 µl of the fractions on a probe tip followed by 1 µl of matrix solution. All mass spectra were obtained in the linear mode and calibrated using external calibration. Data processing was carried out using GRAM/386 software.
Immunohistochemistry
Normal tissue samples from several organs were selected from the files of the Department of Pathology, Hospital S. João (Porto, Portugal), using surgical specimens removed from patients with non-neoplastic diseases not affecting the tissues under study (Table 2). Gastric carcinoma specimens (n = 104) were obtained from patients undergoing surgery. All tissue sections were collected after informed consent. Sections were fixed in formalin buffered with PBS and embedded in paraffin. Hematoxylin and eosin-stained sections were used to classify gastric carcinomas according to the classifications of
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Sections from normal tissues and gastric carcinomas were immunostained by the avidinbiotin complex method (
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Co-expression of MUC6, MUC5AC, and MUC2 was analyzed in a group of 20 gastric carcinomas. The MAbs used are listed in Table 3.
Scoring of Immunostaining in Gastric Carcinomas and Statistical Analysis
A semiquantitative approach was used to score the immunostaining: +, few positive cells (<25%); ++ well-defined areas with positive cells (2550%); +++, extensive areas with positive cells (5075%); ++++, most cells stained (>75%). The chi-square test with the Yates correction was used for statistical analysis. Differences were considered to be significant at p<0.05. Fisher's test was applied whenever appropriate.
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Results |
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Production and Characterization of the Anti-MUC6 MAb
Two hybridomas, CLH4 (IgG1) and CLH5 (IgG1), were selected and cloned. Both MAbs reacted specifically with the MUC6 peptide in ELISA and stained mucin extracts from gastric mucosa by Western blotting. MAb CLH5 was used for further immunohistochemical studies because it showed stronger staining with paraffin-embedded sections. Fig 1 shows an ELISA testing MAb CLH5 with several synthetic peptides (MUC1, MUC2, MUC3, MUC5AC, MUC6, and MUC7). MAb CLH5 reacted only with MUC6 peptide. Inhibitory ELISA also showed the specificity of MAb CLH5 for the MUC6 peptide (not shown).
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Western blot analyses showed that MAb CLH5 reacted with mucin extracts from gastric mucosa in the high molecular weight range (Fig 2). Deglycosylation of mucins with TFMSA has been shown to cleave carbohydrate side chains of mucins, leaving some of the innermost carbohydrate residue, GalNAc1-O-Ser/Thr (Tn antigen) (
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To evaluate the interference of GalNAc glycosylation with the epitope recognized by CLH5 in vitro, GalNAc-O-glycosylation of the MUC6 peptide was performed. The exhaustively glycosylated MUC6 peptide incorporated up to 5 mol of GalNAc per mol of peptide (Fig 3). MAb CLH5 showed reactivity in an inhibitory ELISA with the different glycoforms of the MUC6 peptide, as shown in Fig 4.
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MUC6 Distribution in Normal Adult Tissues, Including Gastric Mucosa, Detected with the Novel MAb CLH5
Table 2 summarizes the reactivity of MAb CLH5 with normal gastrointestinal and non-gastrointestinal adult human tissues. In the stomach, MAb CLH5 stained mucopeptic cells, which are characterized by particular histochemical and ultrastructural features of the mucin granules (
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Expression of MUC6 in Gastric Carcinomas
In contrast to normal gastric mucosa, only 31 of 104 gastric carcinomas (29.8%) displayed immunoreactivity for CLH5. The expression of MUC6 mucin in positive cases was heterogeneous, either clustered in focal areas or dispersed in isolated cells, and showed variable scoring (+, 8 cases; ++, 18 cases; +++, 4 cases; ++++, 1 case). Positive cells showed diffuse cytoplasmic staining. The comparison of MUC6 expression in the different histomorphological types of gastric carcinoma disclosed no significant differences. The expression of MUC6 was not associated with other clinicopathological characteristics of gastric carcinomas, such as depth of wall penetration, lymphatic invasion, nodal metastasis, venous invasion, and pathological TNM stage (Table 4).
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Co-expression of Secreted Mucins (MUC6, MUC5AC, and MUC2) in Gastric Carcinomas
Analysis of the co-expression and co-localization of the gastric secreted mucins MUC6 and MUC5AC and the intestinal mucin MUC2 in a group of 20 gastric carcinomas is shown in Table 5. We observed that the majority of gastric carcinoma cases (n = 11; 55%) express more than one of the studied mucins. Co-expression and co-localization of MUC6 with MUC5AC and MUC2 were observed in nine cases and one case, respectively. Co-expression and co-localization of MUC5AC and MUC2 were observed in five cases. Simultaneous expression and co-localization of MUC6, MUC5AC and MUC2 were observed in one case (case 6). Topographic heterogeneity of the mucin expression profile was observed in four cases (cases 4, 6, 9, and 11). Expression of MUC5AC alone was observed in cases 1 and 8, of the diffuse and intestinal type, respectively. Expression of MUC2 alone was observed in five cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification (Table 5; Fig 6).
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The patterns of mucin co-expression were not associated with the histological characteristics nor with the stage of the tumors (Table 5).
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Discussion |
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In this article we describe the production and characterization of two MAbs to MUC6 mucin. These MAbs, CLH4 and CLH5, reacted exclusively with a peptide based on the tandem repeat of MUC6, as well as with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 reacted by immunohistology in agreement with the results of earlier immunohistochemical studies using polyclonal antibodies as well as with the results obtained by in situ hybridization (
As previously described (
In normal gastric mucosa, CLH5 immunoreactivity was observed exclusively in the glands of the antrum and in the mucopeptic cells of the neck zone of the body region of the stomach. This observation indicates that MUC6 appears to be a good marker of mucopeptic differentiation. In this study we evaluated the expression of MUC6 in gastric carcinomas and observed a loss of MUC6 expression in the majority of the cases. Only 30% of gastric carcinomas showed expression of MUC6, and most of them expressed MUC6 in less than 50% of the cells. In agreement with our results,
The present study showed that expression of MUC6 is not associated with the histological type of the gastric carcinomas. This finding is in agreement with the presence of heterogeneous cell populations in gastric carcinomas of different histotypes (
In normal tissues, the type of mucin expressed is closely associated with the cell phenotype: MUC5AC with foveolar cells, MUC6 with mucopeptic cells, and MUC2 with goblet intestinal-type cells. In a previous study we found that, in gastric carcinomas, expression of MUC5AC was significantly associated with diffuse-type carcinomas (
We observed the co-expression of different mucin core proteins in 55% of our series of gastric carcinomas. The presence of more than one cell type in a single tumor has been frequently observed in gastric carcinomas, with the different cell types occurring either in separate areas or mixed in a single area (
In our series, the prominent expression of MUC2 appears to be a hallmark of the mucinous type of gastric carcinoma. MUC2 immunodetection was confirmed using two different MAbs specific for MUC2. The "ectopic" expression and overexpression of MUC2 have been described as common features of mucinous carcinomas of the colon, pancreas, breast, and ovary (
At variance with
In conclusion, using the novel well-characterized CLH5 monoclonal antibody, we observed that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas are independent of histomorphology and staging. Finally, we observed that the intestinal mucin MUC2 is expressed as the most prominent mucin in mucinous type gastric carcinomas.
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Acknowledgments |
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Supported by Fundação para a Ciência e a Tecnologia (PRAXIS XXI, Project SAU/14111/1998), EU Concerted Action BMH4-CT98-3222, and by The Danish Cancer Society.
We thank Ms Lone Malte and Ms Dina Leitão for expert technical assistance.
Received for publication September 7, 1999; accepted November 17, 1999.
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