Imperial Cancer Research Fund Breast Cancer Biology Groupand 3Hedley Atkins/ICRFBreast Pathology, Guys Hospital, London SE1 9RT, UK, 4In situ HybridizationService and Histopathology Unit, Imperial Cancer Research Fund,44 Lincolns Inn Fields, London WC2A 3PX, UK, 5Department of Oral Diagnostics,School of Dentistry, University of Copenhagen, DK2200 Copenhagen,Denmark
Received on January 23, 1999. revisedon April 26, 1999; accepted on May 28, 1999.
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Abstract |
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Introduction |
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Mucin-type O-linked glycosylation is initiated in the Golgi apparatus(17Rottger et al., 1998)by the addition of N-acetylgalactosamine to the hydroxylgroup of serines and threonines. The oligosaccharide side chainsare then built up by the sequential addition of individual sugars,via various core structures, each reaction being catalyzed by specificglycosyltransferases (2
Brockhausen, 1996).Thus the final structure of the O-glycans is determined by the activityof individual glycosyltransferases and by their position relativeto each other in the Golgi pathway.
In the MUC1 mucin, galactose is added to the initial GalNAc toform the core 1 structure, which in normal breast epithelial cellsis then converted to core 2 by the addition of N-acetylglucosamine;the reaction being catalysed by the enzyme core 2 ß1,6N-acetylglucosaminyltransferase (C2GnT). Core 2 is then extendedby the addition of polylactosamine units (11Hanisch et al., 1989; 14
Lloyd et al., 1996). However, in breast carcinomas the core1 to core 2 conversion is reduced, resulting in the O-glycans onthe tumour associated MUC1 being shorter and less complex (12
Hull et al., 1989; 14
Lloyd et al., 1996). ST3GalI, which catalyzes the addition of sialic acid in an
2,3linkage to Gal ß13 GalNAc, terminatingchain extension, uses the same substrate (core 1) as C2GnT. Thus,changes in the expression and activity of these enzymes could leadto changes in the structure and length of the O-glycans attachedto MUC1 and to the exposure of peptide epitopes such as that recognisedby SM3. We have previously shown that, when compared to SM3 negativenormal mammary epithelial cell lines, SM3 positive breast cancercell lines have an 810 fold elevation in the enzymic activity responsiblefor transferring sialic acid in
2,3linkage to the core 1 substrate (3
Brockhausen et al., 1995). In contrast, C2GnT activityis absent or decreased in the tumor cell lines.
To determine whether similar changes in the expression of glycosyltransferasesare also seen in primary breast cancers we have used insitu hybridization to detect levels of mRNA encoding a specificsialyltransferase, ST3Gal I. Our results show that ST3Gal I mRNAis indeed more abundant in carcinoma than benign breast epitheliumand the level correlates with the intensity of staining with themonoclonal antibody SM3.
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Results |
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Elevated level of ST3Gal I is correlated with thestaining intensity of SM3
The monoclonal antibody SM3 was raised to MUC1 largely strippedof its carbohydrate by exposure to hydrogen fluoride (5Burchell et al., 1987). This antibody reacts witha peptide epitope (6
Burchell etal., 1989) which is selectively exposed in carcinomas(9
Girling et al., 1989).Sections parallel to those used for in situ hybridizationwere stained with SM3 by indirect immunoperoxidase (Figure 1C,G) and the staining intensity scored 0 to +++ withoutknowledge of the ST3Gal I results. Table
showsthe staining intensity observed with SM3 in comparison to the levelof expression of ST3Gal I. To analyze if the apparent correlationwas indeed statistically significant, a Spearmans Correlationtest was performed. This gave a positive correlation with a p valueof 0.0053, indicating that there is a statistically highly significantcorrelation between ST3Gal I mRNA expression and the intensity ofstaining of the monoclonal antibody SM3.
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Discussion |
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In normal breast epithelium, the core 1 glycan is acted uponby C2GnT which initiates chain extension involving the formation of polylactosamineside-chains (11Hanisch et al.,1989; 14
Lloyd et al.,1996). Thus, C2GnT could compete with ST3Gal I for thecore 1 substrate. In the cell lines previously examined, the nonmalignant cellline (MTSV1-7) exhibited reasonable levels of the C2GnT, while twoof the three breast cancer lines studied appeared to have lost expressionof this enzyme at the level of the mRNA. In the third cell line,the message level was higher than in the normal cell line but theactivity was lower (3
Brockhausen et al., 1995), possibly suggesting posttranscriptionalcontrol. Thus, levels of mRNA encoding C2GnT may not reflect theactivity of the enzyme and indeed, in situ hybridizationof the core 2 transferase mRNA in primary breast cancers showedthere was no consistent pattern of reduction in the level of message expressed.An accurate comparison of glycosyltransferase activity would beextremely difficult to achieve in breast tissue as normal specimensconsist mainly of stroma with very little epithelium, in contrastto tumor samples which can contain a very high proportion of epithelialcells. However, by transfecting ST3Gal I into a cell line expressingactive C2GnT, we have shown that overexpression of ST3Gal I, evenin the presence of active core 2 enzyme, can result in shorter sugarside-chains being found on MUC1 (21
Whitehouse et al., 1997). Furthermore, by transfectionof the core 2 enzyme into the breast cancer cell line T47D, we haveshown that the SM3 epitope is masked by the core 2 branch (20
Whitehouse, 1998). Thus the positive correlation ofSM3 staining intensity with ST3Gal I expression levels suggeststhat in primary breast carcinomas, overexpression of ST3Gal I allowsthis glycosyltransferase to compete with C2GnT for the core 1 substrate,resulting in sialylation of core 1, inhibiting further chain extension.Monoclonal antibodies to ST3Gal I are now being developed whichwill permit the analysis of the larger number of specimens requiredto confirm the correlation with SM3 binding, and to confirm theassociation of ST3Gal I with increased grade of ductal carcinomas.
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Materials and methods |
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The template for synthesis of the ST3Gal I riboprobe was SacI linearized pcDNA1 plasmid containing the fullcoding region of human ST3Gal I (7Chang et al., 1995), generously provide by Dr. JosephLau. The riboprobe was produced with Sp6 RNA polymerase and contained942 bases complementary to ST3Gal I mRNA. For human core 2 N-acetylglucosaminyltransferase(C2GnT) (1
Bierhuizen and Fukuda, 1992), HaeII linearized pBluescript plasmidcontaining a 1 kb HindIII fragment of C2GnT, obtained by PCR fromthe breast cancer cell line MCF-7, was used with T7 RNA polymeraseto generate a riboprobe containing 965 bases complementary C2GnTtransferase mRNA. Hybridization was essentially as described by 18
Senior et al. (1988),for formalin-fixed paraffin-embedded tissue.
The presence of hybridizable mRNA in all compartments of thetissues studied was established in near serial sections using anantisense ß-actin probe generated withSP6 RNA polymerase and DraI linearizedphßA-10, prepared by subcloning a ~450bp region of a human ß-actin cDNA intopSP73.
Autoradiography was at 4°C (twoexposures per section; 11 d and 14 d for the enzyme mRNA targetsand 11d for ß-actin mRNA), before developingin Kodak D19 and counterstaining by Giemsas method. Sectionswere examined under conventional or reflected light dark-field conditions(Olympus BH2 with epi-illumination) that allowed individual autoradiographicsilver grains to be seen as bright objects on a dark background.
Evaluation of RNA expression was carried out by assessing the intensityof the silver grains giving a score of 0 for negative, 1+ forweak, 2++ for moderate, and 3+++ forstrong intensity. When a section contained areas of morphologicallydefined malignant and benign tissue, these areas were scored separately.
Histochemistry
Dewaxed and rehydrated 3 µm sectionsfrom the primary tumors of the selected patients were incubatedin tissue culture supernatant containing the mouse monoclonal antibodySM3 for 1 h at room temperature and the binding detected by incubation with aperoxidase conjugated rabbit antimouse and a standard streptavidinbiotincomplex method (DAKO Denmark). Staining was visualized with diaminobenzidine(Sigma UK) and lightly counterstained with hematoxylin. The primaryantibody was omitted and replaced with Tris buffer pH 7.6 on sectionsused as negative controls.
Evaluation of SM3 immunostaining was carried out by assessingthe intensity of apical and cytoplasmic staining, giving a scoreof 0 for negative, 1+ for weak, 2++ formoderate, and 3+++ for strong staining.
The binding of Maackia amurensis was determinedby incubating dewaxed and rehydrated sections with biotinylated Maackia amurensis (Vector Laboratories, UK), followedby streptavidinbiotin horseradish peroxidase. Stainingwas visualized with diaminobenzidine (Sigma UK) and thesections lightly counterstained with hematoxylin. The scoring system wasthe same as that used for SM3. Due to specimen availability, only19 sections were analyzed for Maackia amurensis staining.
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Acknowledgments |
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Footnotes |
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b Presentaddress: Cancer Genetics Laboratory, 8th Floor, Guys Tower, Guys Hospital, London SE1 9RT, UK
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References |
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