ARTICLE |
Correspondence to: Uwe Karsten, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str.10, D 13122 Berlin-Buch, Germany.
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Summary |
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In a comprehensive study, we examined the expression of the membrane and secretory mucins MUC1 and MUC3, respectively, in normal and neoplastic gastrointestinal and breast epithelia before and after specific alterations of their glycan structures by neuraminidase, -fucosidase, or carbohydrate-specific periodate oxidation. MUC1 mRNA was also identified in normal colorectal tissues by in situ hybridization. The data revealed that normal colorectal epithelia express both MUC1 mRNA and protein, which were detectable after periodate oxidation with all tested MUC1-specific antibodies. During tumorigenesis in the colon, MUC1 became recognizable without periodate treatment concomitantly with highly dysplastic lesions and the malignant state. In the breast, in which MUC1 is detectable with most antibodies in normal epithelium as well as in carcinomas, staining could be enhanced by pretreatment with periodate and casually by enzyme treatments. MUC3 was detectable in normal and neoplastic colorectal tissues and was more intensely stained after periodate oxidation. It was absent in normal breast even after pretreatment but was expressed in seven of 20 breast carcinomas. Therefore, incomplete glycosylation, abnormal distribution, and ectopic expression of mucins are characteristics of malignancy. Periodate oxidation may be widely applicable to immunohistochemistry for examining changes in glycosylation and for detecting antigens masked by glycans. (J Histochem Cytochem 45:1547-1557, 1997)
Key Words: MUC1, MUC3, immunohistochemistry, in situ hybridization, carbohydrate, periodate oxidation, gastrointestinal tract, breast, tumor
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Introduction |
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Mucins are a family of highly glycosylated, high molecular weight glycoproteins present on the surface of many glandular epithelial cells and in their secretions (
In this study, serial sections from various gastrointestinal (normal stomach, ileum, and colon; colorectal adenomas and carcinomas) and mammary tissues (normal breast and mammary carcinomas) were used to examine the presence of MUC1 mRNA in the different cell types of normal gastrointestine by in situ hybridization and the expression of MUC1 and MUC3 proteins, the latter as an example of a secretory mucin, by immunohistology with MAbs to peptide epitopes before and after glycan-intruding measures such as mild periodic acid oxidation, -fucosidase, or neuraminidase incubation. The results show that MUC1 is indeed expressed in normal colorectal mucosa but is inaccessible to anti-MUC1 peptide-recognizing MAbs. Epitope masking by glycans can be overcome by pretreatment of the tissue sections with periodate.
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Materials and Methods |
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Normal Adult Human Tissues and Tumor Specimens
Two stomach (including corpus and pyloric antrum), one ileum, and four colon specimens were obtained at autopsy from individuals without colon disease. One ileum and 15 colon tissue samples were taken at surgery or colonoscopy from apparently normal parts of the colon wall distant from the tumor. All mucosae showed normal histomorphological and cytomorphological findings. Twenty-three cases of colorectal adenomas and 22 carcinomas were obtained at surgery or colonoscopy. Adenomatous polyps were classified as to histological type and grade of dysplasia according to the criteria of
Twenty mammary carcinomas and 10 normal mammary tissues from patients with cancer were derived from surgical specimens. The tumors were classified according to the criteria of the WHO (1982) and the Bloom-Richardson system (
Two of the normal stomach, ileum, and colon samples and all mammary samples were fixed in 10% buffered formalin and embedded in paraffin. All other tissues were immediately frozen and stored at -80C.
In Situ Hybridization
RNA Probe.
The MUC1 cDNA probe, pum24P (
In Situ Hybridization (ISH). ISH was performed as described (Schaeren-Wiemers and Gerfin-Moset 1993) in two samples each of normal stomach, ileum, and colon. Deparaffinized sections or formalin-fixed cryosections were digested with proteinase K (Boehringer; Mannheim, Germany) [10 µg/ml in PBS, 20 min at room temperature (RT)]. After three rinses in PBS, sections were treated with 0.5% and 1% triethanolamine, pH 8.0, and 0.25% acetic anhydride for 5 min. Prehybridization was done with hybridization buffer [50% formamide, 0.75 M NaCl, 0.075 M sodium citrate, pH 7.0 (5 x SSC), 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.25 mg/ml tRNA, 1.25 mg/ml herring sperm DNA, 4 mM EDTA] at 45C for 3 hr. The hybridization mixture was prepared by adding 1 µl DIG-RNA probe per 50 µl hybridization buffer, heated for 5 min at 85C to denature the probe, and then chilled on ice. Fifty µl of this hybridization mixture was added per slide, which was then covered with a coverslip and sealed with rubber cement. Hybridization was done overnight at 72C. Posthybridization washings were successively performed in SSC solutions of decreasing concentrations (5 x SSC, 50% formamide, 10 min at 50C; 2 x SSC, 25% formamide, three times for 10 min at 42C and twice for 30 min at 42C; 0.2 x SSC, twice for 5 min at RT). Thereafter, the sections were rinsed three times with PBS and incubated with Fab fragments from anti-DIG antibody conjugated to alkaline phosphatase (Boehringer; 1:500) for 1 hr at RT. After three washing steps, the color development was performed with the Fast Red Substrate System (DAKO; Hamburg, Germany). Controls for in situ hybridization consisted of (a) DIG-labeled sense RNA probe, (b) competition with a 20-fold excess of unlabeled anti-sense RNA in the presence of DIG-labeled anti-sense RNA, (c) pretreatment of sections with ribonuclease, and (d) hybridization mixture without RNA probe.
Immunohistochemistry
Monoclonal Antibodies.
The antibody A76-A/C7 (IgG1) was developed in this laboratory (
Tissue Staining. Staining of tissue sections was performed by the avidin-biotin-peroxidase complex (ABC) method with a commercial kit (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA) as follows. Paraffin sections (4 µm) were deparaffinized in xylene and rehydrated through a graded ethanol series. Frozen sections (4 µm) were air-dried at RT and fixed with 10% formalin in PBS for 15 min at RT. Endogenous peroxidase activity was eliminated by treatment with 3% H2O2 in PBS for 30 min at RT. Nonspecific binding sites were blocked with normal rabbit serum. After washing with PBS, sections were incubated with MAbs in appropriate dilutions for 1 hr at RT. The thoroughly washed sections were treated with biotinylated anti-mouse immunoglobulin antiserum for 30 min at RT and thereafter with the ABC complex. Color development during incubation with the peroxidase substrate (diaminobenzidine) was controlled under the microscope. Counterstaining was done with hematoxylin. Negative controls were incubated with comparable dilutions of IgM or IgG paraproteins from mouse plasmocytomas (Sigma; Deisenhofen, Germany) instead of the MAb.
Partial Deglycosylation. One of the following pretreatments was performed on the sections before incubation with anti-MUC1 or MUC3 MAbs.
Neuraminidase. Sections were incubated with neuraminidase from Vibrio cholerae (Serva; Heidelberg, Germany) at a concentration of 0.02 U/ml in PBS containing 0.01 M Ca++ for 1 hr at RT to remove sialic acid residues. After enzyme treatment, sections were rinsed for 5 min in PBS. Positive control of neuraminidase action was achieved by staining of erythrocytes and endotheliocytes of a tissue section by MAb A78-G/A7 (anti-Thomsen-Friedenreich antigen) after the enzyme treatment.
-Fucosidase.
Sections were incubated for 24 hr at 37C with
-fucosidase (bovine kidney, Boehringer; 0.4 U/ml in acetate buffer, 0.2 M, pH 5). Then they were rinsed for 5 min in PBS. Loss of staining of erythrocytes and endotheliocytes in tissue sections from individuals with blood group O with the anti-H2 MAb A46-B/B10 served as control of enzyme action.
Periodate Oxidation.
Sections were treated as described (
Blood Groups and Secretor Status.
Scoring. For normal mucosa, the percentage of positive crypts was counted, whereas in carcinomas the percentage of positive cells in several optical fields (12.5 x lens) was estimated. For further details, see legends to Figure 2 Figure 3 Figure 4 and Figure 6.
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Results |
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Expression of MUC1 mRNA in Normal Gastrointestinal Epithelium
By in situ hybridization, MUC1 mRNA was found to be expressed in surface epithelial cells and in gastric glands of the stomach. In all of two ileum and four colon specimens, the DIG-labeled MUC1 anti-sense RNA probe intensely stained epithelial cells, columnar cells and goblet cells (Figure 1A). The staining corresponded to the cytoplasm of columnar cells and to the perinuclear region of goblet cells. DIG-labeled sense probes did not hybridize to any tissue structure (Figure 1B). The other control experiments also showed no staining.
Expression of Apomucin Along the Normal Gastrointestinal Tract Before and After Partial Deglycosylation
The staining patterns with MUC1 mAbs before and after partial deglycosylation are shown in Figure 1 and Figure 2. Analysis of the stained sections revealed that A76-A/C7 and HMFG-2 did not stain, as expected, any of the following untreated normal tissue sections: two stomach specimens from secretor individuals, two ileum samples from nonsecretor individuals, and 19 colon specimens (including two from ascending colon) from nonsecretor individuals (Figure 1C). SM3 behaved identically except for weak staining of the supranuclear region of columnar cells and goblet cells in one case from colon. In contrast to this, MUC1 MAbs strongly labeled all normal gastrointestinal epithelia after periodate treatment, but in most cases their secretions remained unstained. Enzymatic pretreatment with -fucosidase led to MUC1 staining exclusively in sections of the two stomach specimens, which were both secretors. Neuraminidase was without effect in all normal gastrointestinal tissues examined. Staining patterns after periodate oxidation varied with the antibody employed. A76-A/C7 staining was predominantly distributed at the apical membranes of surface mucous cells of the stomach and of columnar cells and goblet cells of the intestine (Figure 1D), whereas HMFG-2 and SM3 staining was localized in the supranuclear cytoplasm of surface mucous cells of the stomach and in the perinuclear region of columnar cells and goblet cells of the intestine. All MUC1 MAbs diffusely stained gastric glands proper and pyloric glands.
MUC3 positivity in untreated normal tissue sections from the stomach was restricted to occasional cytoplasmic staining of the surface epithelium of stomach and gastric glands. In normal ileum and colon, M3.2 stained the supranuclear cytoplasm of goblet cells and columnar cells, but not secretions (Figure 1E). After periodate oxidation, mucous cells, gastric glands proper, and pyloric glands, as well as secretions in the stomach became positive. Vacuoles of goblet cells and secretions in the ileum and colon were also positive (Figure 1F).
Expression of Apomucin in Colorectal Adenomas and Carcinomas Before and After Partial Deglycosylation
The results of mucin staining in adenomas and carcinomas with and without partial deglycosylation are shown in Figure 1, Figure 3, and Figure 4. For colorectal adenomas and carcinomas, similar results were obtained with all three anti-MUC1 MAbs, A76-A/C7, HMFG-2, and SM3. In Figure 3 and Figure 4, data for A76-A/C7 are shown. The anti-MUC1 MAbs did not stain mildly or moderately dysplastic adenomas, with the exception of a few weak reactions. As expected, staining was greatly enhanced in all cases after periodate treatment. These MAbs, however, reacted with untreated sections of severely dysplastic adenomas; this was enhanced to a certain degree after periodate treatment (Figure 1G and Figure 1H). Adenocarcinomas were generally reactive, but periodate treatment still slightly enhanced the reactivity. After -fucosidase treatment slightly increased staining could be seen. In most cases, secretions in benign lesions did not react with MUC1 MAbs, even after periodate treatment. In contrast, secretions of some carcinomas showed variable (from slight to moderate) reactivity with MUC1 MAbs, and this was increased after periodate treatment. Neura-minidase treatment did not lead to significantly increased staining of MUC1 in gastrointestinal tumor cells and secretions.
Staining of MUC3 apomucin in colorectal benign and malignant lesions and secretions was significantly enhanced after periodate treatment (Figure 1I and Figure 1J). A slightly increased reaction of MUC3 mAb with several severely dysplastic adenomas and carcinomas was observed after -fucosidase treatment. Neuraminidase treatment did not enhance staining of MUC3 in gastrointestinal tumor cells and secretions.
In adenomas with mild or moderate dysplasia, MUC1 and MUC3 (native and deglycosylated) were localized in the supranuclear cytoplasm, at the apical membrane, and/or in the apical cytoplasm of tumor cells (Figure 1E, Figure 1F, Figure 1I, and Figure 1J). In carcinomas and adenomas with severe dysplasia, both mucins were observed either diffusely distributed in the whole cell or at the basolateral membrane.
Figure 5 provides a somewhat simplified overview of the staining results during the stages of colorectal carcinogenesis.
Expression of Apomucin in Normal Mammary Tissues Before and After Partial Deglycosylation
Normal mammary gland sections were reacted with A76-A/C7 and HMFG-2 before and after partial de-glycosylation (data not shown). Staining was observed in all cases; it was restricted to the apical membranes of epithelial cells. Antibody SM3 did not react with normal mammary gland tissues (independent of whether secretors or nonsecretors), and remained nonreactive after neuraminidase or -fucosidase treatment. After periodate treatment, however, SM3 reacted with normal mammary gland tissue at the apical membranes of epithelial cells, similar to the localization seen with A76-A/C7 and HMFG-2.
All normal mammary tissues were negative for MUC3 with or without partial deglycosylation.
Expression of Apomucin in Breast Carcinomas Before and After Partial Deglycosylation
The staining results with anti-mucin MAbs in mammary carcinoma sections with and without partial deglycosylation are shown in Figure 1 and Figure 6. The stainability of MUC1 in mammary carcinoma cells was strongly increased after periodate oxidation (Figure 1M and Figure 1N). Neuraminidase treatment of the sections also distinctly increased the reactivity of MUC1 with the respective MAbs, whereas -fucosidase slightly increased staining. Three patterns of MUC1 localization could be distinguished: (a) the antigen was present at the apical membranes of tumor cells in glandular adenocarcinomas; (b) the antigen was diffusely distributed in the cytoplasm of tumor cells in non-gland-forming areas of tumors; and (c) in some poorly differentiated carcinomas, a proportion of positive cells exhibited distinct granular cytoplasmic staining (focal cytoplasmic staining).
Unexpectedly, five of 20 mammary carcinomas expressed MUC3. Two further cases became positive after periodate oxidation, and in one case the staining increased after this treatment (Figure 1O and Figure 1P). Native and deglycosylated MUC3 was diffusely distributed in the cytoplasm of tumor cells. Neuraminidase and -fucosidase treatments had no influence on the reactivity (except in one case).
Expression of MUC1 and MUC3 apomucins before and after partial deglycosylation was not related to the secretor status of the patients.
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Discussion |
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A common feature of glandular epithelial tissues is the apical (luminal) expression and secretion of mucins. MUC1 is generally assumed to be the major mucin expressed in normal breast, pancreas, stomach, and other glandular epithelia, but not or only sparsely in the intestine (1-O-Ser/Thr), Tn (GalNAc
1-O-Ser/Thr), sialosyl-Tn (NeuAc
2-6G
lNAca1-O-Ser/Thr)] were detectable by immunohistochemistry at an earlier stage than MUC1 itself (
-fucosidase. Therefore, gastric and colorectal MUC1 may differ in their glycosylation and, accordingly, in their staining behavior.
Localization of partially deglycosylated MUC1 (observed after periodate oxidation) was found, as expected for a membrane-bound mucin, at the the apical membrane and in the cytoplasm of surface mucous cells of the stomach and gastric gland cells, and at the apical membrane and in the supranuclear or perinuclear cytoplasm of goblet cells and columnar cells of the intestine; it was absent in secretions. Staining patterns were different between A76-A/C7 and HMFG-2 or SM3. A76-A/C7 reacted primarily with the apical membrane of epithelial cells, whereas HMFG-2 and SM3 stained the apical membrane and the supranuclear cytoplasm. This is interpreted as indicating that A76-A/C7 recognizes a later stage in MUC1 processing, whereas HMFG-2 and SM3 may bind to an earlier stage (in terms of glycosylation or conformation). Such differences have also been frequently observed in immunofluorescence experiments on tumor cells in vitro with other MUC1-specific MAbs (
Compared to most other epithelial tissues, such as mammary gland, salivary gland, esophageal epithelium, pancreas, bile ducts, lung epithelium, distal tubules and collecting ducts of kidney, urothelium, uterus, and rete testis, MUC1 of gastrointestinal epithelial cells appears to be more heavily glycosylated and its peptide backbone therefore not accessible to MAbs such as HMFG-2 or A76-A/C7. One consequence of heavy O-glycosylation is the relative resistance of these regions in glycoproteins towards proteases (
The immunohistological distribution of MUC3 in epithelial cells of the gastrointestinal tract largely confirmed earlier reports (
Alterations of expression of MUC1 and MUC3 in colorectal carcinomas and adenomas of various stages have been studied by several groups (
In normal colonic epithelia, MUC1 is heavily glycosylated, preventing its immunostaining with anti-peptide MUC1-specific MAbs. During tumorigenesis, the apparent expression of native MUC1 showed a gradual increase from mildly through moderately to severely dysplastic adenomas. Periodate treatment greatly enhanced the reactivity of anti-MUC1 MAbs with lesions of a lower grade of dysplasia, but less so with lesions of a higher grade of dysplasia. This reflects the fact that lesions of a higher grade of dysplasia express MUC1 with a lower degree of glycosylation. In malignant lesions, native MUC1 was strongly expressed, and periodate treatment only slightly enhanced the staining. This indicates that, similar to MUC1 in highly dysplastic adenomas, MUC1 in carcinomas is less glycosylated, resulting in exposed peptide epitopes that can be recognized by their corresponding antibodies. It is obvious that the grade of dysplasia of adenomas is closely related to their size. Adenomas of larger size, of a higher grade of dysplasia, or of the villous type have a higher malignant potential (
Normal breast and mammary carcinomas have been examined for the expression of MUC1 and MUC3 in a number of reports (-fucosidase treatment did not expose the SM3 epitope, indicating that sialic acid and fucose do not play major roles in epitope masking in breast epithelium.
For carcinomas of the breast, some general aspects of MUC1 immunoreactivity were found similar to that of colorectal carcinomas, e.g., decreased epitope masking compared to normal epithelial cells, disturbed polar distribution in cancer cells, and heterogeneous reactivity within a given tumor. In addition, some features of apomucin expression specific for mammary carcinomas were observed. The reactivity of MUC1 MAbs with mammary carcinomas could be enhanced by prior neuraminidase treatment to a greater extent than in the case of colorectal carcinomas. This is in accord with a report by
The observed changes of mucin expression and its glycosylation in colorectal and mammary carcinomas may be of biological and clinical significance. For example, the exposed glycan core structures on the surface of cancer cells may lead to or enhance binding to the basal membrane and extracellular matrix (
In conclusion, we have presented evidence that normal gastrointestinal epithelia express both MUC1 mRNA and the protein itself. The immunohistochemical detection of the MUC1 protein is hampered by a qualitatively different type of glycosylation compared to other secretory epithelia (e.g., breast). This can be overcome by carbohydrate-specific pretreatment with periodate, which not only leads to the detection of masked MUC1 but also allows evaluation of the changes in MUC1 glycosylation that occur in neoplastic lesions. During malignant transformation, epithelial mucins experience dramatic (yet tissue-specific) alterations in their glycosylation and cellular localization, and in some cases show ectopic expression. These changes may be significant in tumor progression, and possibly also in the clinical setting. Periodate oxidation as a complementary measure in immunohistochemistry may help us to examine and understand these changes.
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Acknowledgments |
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Supported by Deutsche Forschungsgemeinschaft grant no. Ka 921/1.
We thank Dr D. Swallow (London) for providing the cDNA probe, and especially acknowledge the help of Ms Andrea Schaffrath (Bremen) with the in situ hybridization. Drs G. Pilgrim and W. Fregin are thanked for their help in providing blood group and secretor data for breast cancer patients.
Received for publication March 3, 1997; accepted June 19, 1997.
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