Expression of M-N#1, a histo-blood group B–like antigen, is strongly up-regulated in nonapoptosing mammary epithelial cells during rat mammary gland involution

Jörg Mengwasser and Jonathan P. Sleeman1

Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, PO Box 3640, D-76021 Karlsruhe, Germany

Received on September 13, 2000; revised on January 5, 2001; accepted on January 11, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Antibodies against the histo-blood group B–like antigen M-N#1 efficiently block the growth in vivo of rat mammary carcinoma cells that bear the antigen (Sleeman et al., 1999Go, Oncogene 18, 4485–4494). To try to understand the function of the M-N#1 antigen, we investigated when and where the antigen is expressed during the normal function of the rat mammary gland. Expression was virtually only seen during mammary gland involution. Here, strong expression of the antigen was observed in mammary epithelial cells, beginning around 2 days postweaning and lasting throughout the involution process. Dexamethasone treatment of animals postlactation inhibited alveolar collapse and remodeling in the mammary gland but inhibited neither the apoptosis of mammary epithelial cells nor the expression of the M-N#1 antigen. We show that up-regulation of carbohydrate antigens is not a general phenomenon during mammary gland involution, and thus that M-N#1 antigen expression is specifically regulated. Up-regulation of {alpha}(1,2)fucosyltransferase A, an enzyme required for M-N#1 antigen synthesis, is at least partly responsible for regulated M-N#1 antigen expression postlactation. Most significantly, we observed that the M-N#1 antigen is virtually exclusively expressed on nonapoptosing epithelial cells in the involuting mammary gland. These data suggest that M-N#1 antigen expression might either provide a survival function and/or be expressed in epithelial cells that are destined to grow and remodel mammary duct structures.

Key words: ABH histo-blood group antigen/apoptosis/mammary gland/involution/tissue remodeling


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ABH antigens are N- or O-linked sugar modifications of glycoproteins and glycosphingolipids or exist as free oligosaccharides (Clausen and Hakomori, 1989Go; King, 1994Go). They are expressed on glandular epithelia, on neurones, and in exocrine secretions. Only humans and certain primates carry ABH antigens on endothelial cells and on erythrocytes (Oriol et al., 1994Go). In the latter case they have a histocompatibility function, constituting the major blood group ABO allo-antigen system.

Synthesis of ABH antigens requires several glycosyltransferases, which act on precursor oligonucleotides. ABH antigens can be built on four main disaccharide precursors, giving four different ABH antigen subtypes. The {alpha}(1,2)fucosyltransferases add fucose in {alpha}(1,2) to the terminal sugar residue of these precursors to generate the H antigen (Rajan et al., 1989Go; Rouquier et al., 1995Go). Further modification of the H antigen by {alpha}3-GalNAc and {alpha}3-Gal transferases produces A and B antigens, respectively (see Figure 1). These transferases require {alpha}(1,2)fucose modification of the precursor disaccharides, and thus A and B antigens can only be synthesized from H antigen (Oriol et al., 1992Go). In humans, H antigen synthesis is under the control of at least two polymorphic genes, each encoding a distinct {alpha}(1,2)fucosyltransferase (Rajan et al., 1989Go; Rouquier et al., 1995Go). The A and B transferases are encoded by the ABO genetic polymorphism. The A and B genes differ by only four amino acids, whereas O individuals possess a nonfunctional gene due to a frame shift and premature stop codon (Yamamoto et al., 1990Go).



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Fig. 1. Diagram showing the sugar structures of ABH antigens and their different subtypes.

 
Cell adhesive functions have been attributed to blood group antigens, such as Lex, sialyl-Lex, and sialyl-Lea (reviewed in Hakomori, 1996Go), but the physiological and pathological function of ABH blood group antigens remains obscure. In human cancers, changes in ABH antigen expression are often observed, and in some cases these changes can be prognostically significant (reviewed in Dabelsteen, 1996Go). Recent evidence suggests that H antigen expression protects tumor cells from apoptosis-inducing signals (Goupille et al., 2000Go).

We have previously generated an antibody called M-N#1, which binds to an N-linked carbohydrate modification of proteins found on metastasizing rat mammary tumor cells (Sleeman et al., 1999Go). This antibody efficiently inhibits the growth in vivo of tumors derived from these cells. Using synthetic oligosaccharides, we were able to define the carbohydrate structures to which the M-N#1 antibody binds, namely, B antigen, subtypes 2, 3, and 4. Out of a large panel of other synthetic oligosaccharides, the only other reactivity observed was weak binding to A antigen, subtype 2 oligosaccharides (Sleeman et al., 1999Go). No binding was observed to "linear B" oligosaccharides. In a wide range of assays, the antibody acts as a classical anti–blood group B antigen, for example, binding very strongly to human erythrocytes of B blood group but not to blood group A erythrocytes, including A subtype 2 (Sleeman et al., 1999Go). Cross-reactivity with A antigen was only detectable when the latter antigen was present in large amounts on the pyloric surface epithelium of human A secretors. Thus, an antibody against essentially B antigen determinants is able to inhibit the growth of tumors expressing these antigens. We refer to the M-N#1 antigen as "B-like antigen" rather than "B antigen" due to the lack of reactivity of the M-N#1 antibody with B subtype 1 and its weak reactivity with A antigen subtype 2.

Analysis of the chemical structures to which the M-N#1 antibody binds and does not bind permits some conclusions to be made about the chemistry of its epitope. Antibody binding requires fucose, as the M-N#1 antibody combining site is destroyed by fucosidase (Sleeman et al., 1999Go). Furthermore, antibody binding requires the addition of {alpha}-D-GalNAc or {alpha}-D-Gal to the H antigen structure, as the antibody does not bind to H antigens or linear B-antigens (Sleeman et al., 1999Go). However, of the A antigen subtypes, the antibody only binds weakly to A subtype 2. It can therefore be concluded that the core of the M-N#1 epitope is made up of {alpha}-L-fucose linked in 1,2 to ß-D-Gal, and at least part of the {alpha}-D-GalNAc or {alpha}-D-Gal sugar (sugar X in Figure 1) linked in 1,3 to the ß-D-Gal. The blood group and subtype sensitivity of the antibody is critically dependent on the stereochemistry and linkage of oxygen groups at the 3' and 4' positions of sugar Y. Because the M-N#1 antibody only binds weakly to A antigen subtype type 2 while binding strongly to B antigens subtypes 2, 3, and 4, presumably the M-N#1 antibody requires a hydoxyl group at the 2' position of sugar X (Figure 1), but when the ß-D-Gal sugar is linked in 1,4 to sugar Y as in the case of subtype 2 antigens, then an -NH- at the 2' position of sugar X suffices to give weak antibody binding.

Our aim in the study presented in this article was to identify normal roles for the M-N#1 antigen and thereby gain some insight into the possible function of the antigen on tumor cells. As the M-N#1 antigen was identified on mammary carcinoma cells, we focused on its expression in nonneoplastic mammary glands. Prior to pregnancy and lactation, mammary glands contain branching networks of ducts formed by mammary epithelial cells (reviewed in Vonderhaar, 1985Go). During pregnancy and lactation, lateral buds form along the ducts and subsequently develop into alveoli. These alveoli are comprised of differentiated mammary epithelial cells that secrete milk during lactation. After weaning, the mammary glands regress in a process called involution. During involution the mammary gland is restructured through the coordinated processes of apoptosis of luminal epithelial, myoeptithelial, and endothelial cells and lobular-alveolar remodeling (Pitelka, 1988Go; Lund et al., 1996Go; Strange et al., 1992Go). Loss of suckling leads to accumulation of milk in the alvoeli and a fall in the levels of systemic lactogenic hormones. These events trigger the involution process (Feng et al., 1995Go; Marti et al., 1997Go). There are two distinct phases to mammary gland involution. The first is reversible and controlled by local factors. Alveolar cells apoptose, but no remodeling of the lobular-alveolar structure occurs (Li et al., 1997Go). In the second phase, proteases degrade extracellular matrix and basement membrane components, and the lobular-alveolar structures collapse (Lund et al., 1996Go). Continued apoptosis, replacement of most of the epithelial component with adipose tissue, and reestablishment of the resting mammary gland ductal structures leads to remodeling of the gland. The involution process is completed within 10–15 days (Lascelles and Lee, 1978Go).

Here we show that the M-N#1 antigen is specifically and strongly up-regulated during mammary gland involution, virtually exclusively on nonapoptosing mammary epithelial cells. Thus, M-N#1 expression might either protect against apoptosis induction and/or be involved in promoting the growth of mammary epithelial cells destined to be involved in tissue remodeling. Similar functions on tumor cells would be expected to promote their tumorigenic properties, and blockade of these functions by the M-N#1 antibody would therefore inhibit tumor growth.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The M-N#1 antigen was identified by raising antibodies against mammary carcinoma cells. Thus, we reasoned that the M-N#1 antigen may play a physiological role during the function of the mammary gland and that the identification of such a physiological function may give an idea as to the function of the antigen on tumor cells. We therefore investigated M-N#1 antigen expression in rat mammary tissue. In virgin mammary tissues, only occasional ductal epithelial cells showed weak staining with the M-N#1 antibody. No staining was observed in mammary glands during pregnancy or lactation. However, within 2 days of cessation of lactation and the onset of mammary gland regression, the M-N#1 antigen was dramatically up-regulated and remained highly expressed on mammary epithelial cells during the involution process (Figure 2).



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Fig. 2. Immunohistochemical staining of paraffin wax–embedded sections of female rat mammary tissue with the M-N#1 antibody (red/brown stain). (A) Normal mammary gland. While the vast majority of the tissue was not stained, occasional ductal cells showed weak staining with the antibody (arrows). (B) Mammary gland from a rat pregnant for 12 days. (C) Mammary gland from a rat lactating for 3 days. (DH) Mammary glands undergoing involution after lactation. Pups were withdrawn from the rats at day 0. Strong M-N#1 staining can be seen in the involuting ducts. (D) Two days after pup withdrawal. (E) Four days after pup withdrawal. (F, G) Six days after pup withdrawal. (H) Ten days after pup withdrawal. Bar: 20 µm (AE, G), 40 µm (H), 160 µm (F).

 
Mammary involution occurs in two stages: a first, reversible stage controlled by local factors in which alveolar structure remains intact; and a second stage dependent on systemic hormone levels, which is characterized by alveolar collapse and tissue remodeling and which begins around 4 days postweaning (Lund et al., 1996Go; Li et al., 1997Go). The M-N#1 antigen was observed to be expressed during both stages of involution, but most strongly during the second stage (Figure 2, Table I). Glucocorticoid hormones have been reported to be able to inhibit the second stage of involution in postlactating animals (Lund et al., 1996Go; Li et al., 1997Go). To determine whether M-N#1 antigen expression is inhibited if the second phase of involution is blocked, we examined M-N#1 expression in the mammary glands of animals treated with dexamethasone. Morphologically, dexamethasone-treated glands remained swollen and full of milk even 8 days after weaning. Histological examination showed that dexamethasone treatment dramatically inhibited the onset of alveolar collapse and tissue remodeling, yet the M-N#1 antigen was still expressed throughout this time (Figure 3). Quantification of the M-N#1 staining (Table I) showed that dexamethasone treatment indeed moderately increased the number of cells which were M-N#1 antigen-positive. Apoptosis was not inhibited by dexamethasone treatment (Figure 3, Table I). The data in Figures 2 and 3 and Table I therefore suggest that although strongest up-regulation of M-N#1 antigen expression occurs during the second phase of mammary gland involution, blockade of the onset of the second phase of involution does not inhibit this up-regulation.


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Table I. Influence of dexamethasone treatment on the number of M-N#1-positive cells and apoptotic cells in involuting rat mammary glands
 


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Fig. 3. Sections of mammary glands taken from animals 2 days (A, E), 4 days (B, F), 6 days (C, G), and 8 days (D, H) postweaning. The animals were either treated (EH) or not treated (AD) with dexamethasone. Sections were immunostained with either M-N#1 antibody (main panels), or stained with Apoptag® to show apoptotic bodies (insets). In both cases, positive staining is red/brown and the hematoxylin counterstain is blue. Bar: main panels, 50 µm; insets, 32 µm.

 
The M-N#1 antibody binds strongly to subtypes 2, 3, and 4 of B antigen and weakly to A antigen subtype 2 (Sleeman et al., 1999Go). To determine to what extent changes in expression of blood group antigens is a general feature during mammary gland involution, or whether A or B antigens are specifically up-regulated, we immunohistologically stained involuting mammary tissue with a panel of anti–blood group antigen antibodies. As can be seen in Table II, expression of blood group antigens in regressing mammary epithelium was limited to A and B antigens, both components of the M-N#1 antigen. These data suggest that the M-N#1 antigen is specifically up-regulated during mammary gland involution.


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Table II. Immunohistochemical analysis of carbohydrate antigen expression in the rat mammary gland during involution
 
A and B antigens are complex carbohydrate structures that require a number of different enzymes for their synthesis. We reasoned that up-regulation of one or other of these enzymes would be responsible for the up-regulated M-N#1 expression we observed in regressing mammary epithelium; therefore, we investigated the expression of these enzymes during mammary gland involution. In the rat, the transferases that create A and B antigens from H antigen precursors have not been cloned. However, two rat {alpha}(1,2)fucosyltransferases have been described, which are necessary for the synthesis of H antigen. We therefore performed Northern blot analysis to investigate expression of these two enzymes, {alpha}(1,2)fucosyltransferases A (FTA; homolog of human FUT1) and B (FTB; homolog of human FUT2). We also analyzed expression of rat {alpha}(1,3)galactosyltransferase, an enzyme that transfers galactose to ABH antigen disaccharide precursors in the absense of {alpha}(1,2)fucose. In principle this enzyme could also be involved in M-N#1 antigen synthesis, because in the rat it remains to be determined whether this transferase can also transfer galactose onto the H antigen to create the B antigen. As a positive control, we also analyzed the expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase), an enzyme that O-links N-acetylgalactosamine to mucin glycoproteins in milk and that would be predicted to be down-regulated during mammary gland involution. Figure 4 shows that although FTB is not expressed, FTA is up-regulated during mammary gland involution and thus is likely to be involved in regulated expression of M-N#1 antigen expression during mammary regression. Interestingly, we also observed up-regulation of {alpha}(1,3)galactosyltransferase during involution. As expected, ppGaNTase was strongly down-regulated. Note that the RNA samples for the Northern blots in Figure 4 were loaded to equalize glyceraldehyde phosphate dehydrogenase (GAPDH) expression between the samples. The corresponding ethidium-stained gels used for the Northern blots show that much more RNA from lactating tissue just prior to involution had to be loaded. These data demonstrate that GAPDH is also strongly up-regulated during mammary gland regression.



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Fig. 4. Northern blot analysis of transferase expression during mammary gland involution. Poly (A+) RNA from mammary glands taken 0, 2, 4, 6, and 8 days postweaning was hybridized with probes for FTA, FTB, UDP-GalNAc: ppGaNTase (ppGANTase), and {alpha}(1,3)galactosyltransferase (GaTase). As positive controls for these probes, poly (A+) RNA from rat colon (C) or stomach (S) was used. The sizes of the New England Biolabs RNA markers (M) and of the hybridization signals obtained with the different probes are indicated. All lanes were loaded with 5 µg RNA, except for the 0 days postweaning lanes, where the amount of RNA loaded was increased to obtain a hybridization signal with the loading control GAPDH, which was approximately equivalent to that obtained with the other samples. Ethidium-stained gels are shown to demonstrate the relative amounts of RNA loaded per lane.

 
Mammary gland regression is typified by apoptosis and tissue remodeling. To determine whether the M-N#1 antigen is expressed on actively apoptosing cells, we double-stained sections of involuting rat mammary glands to localize both M-N#1 antigen expression and cells undergoing active DNA fragmentation. Figure 5 shows representative fields of view from sections of involuting mammary glands taken 2, 4, and 6 days after weaning and demonstrates that M-N#1 expression (blue stain) is distinct from cells undergoing active DNA fragmention (red stain). Quantification of M-N#1-positive cells and apoptosing cells in mammary tissue 4 days after weaning showed that on average 283 cells/mm2 were positive for M-N#1 antigen and 156 cells/mm2 were apoptotic, yet an average of only 1 cell/mm2 was double positive. Thus, throughout mammary gland involution, M-N#1 staining is inversely correlated with apoptosis.



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Fig. 5. Sections of mammary glands taken from animals 2 days (A), 4 days (B), and 6 days (C) postweaning. Sections were double-stained with M-N#1 (blue staining) and Apoptag® to show apoptotic bodies (red stain). Counterstaining was omitted for clarity. Bar: 50 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We have previously demonstrated that an antibody designated M-N#1 is efficiently able to block the growth of a rat mammary tumor in vivo (Sleeman et al., 1999Go). Here we show that during the life cycle of the mammary gland the antigen bound by the M-N#1 antibody is virtually exclusively expressed postlactation. The strong up-regulation of the M-N#1 antigen during mammary gland involution connects the expression of subtypes of A and B antigens with cellular events occuring during mammary gland involution. The striking lack of coincidence between M-N#1 expression and apoptotic cells is suggestive of a role for M-N#1 expression in the survival or growth of mammary epithelial cells involved in tissue remodeling. These findings cast light on the so far elusive physiological role of the ABH histo-blood group antigens.

Changes in the expression of a number of different genes during mammary gland involution have been described, including decreased expression of milk protein genes and up-regulated expression of bcl-2 family members, interleukin-1ß converting enzyme, sulfated glycoprotein-2, and TIMP-1 (Lund et al., 1996Go; Li et al., 1996Go). During the second phase of involution the serine protease uPA and the matrix metalloproteases stromelysin 1, stromelysin 3, and gelatinase A are additionally up-regulated (Lund et al., 1996Go and references therein). Here we report that expression of the FTA, {alpha}(1,3)galactosyltransferase, and GAPDH genes is also up-regulated during involution, and that the ppGaNTase gene is down-regulated. Changes in gene expression during involution are accompanied by changes in the levels of a number of transcription factors, including up-regulation of c-jun, junB, junD, c-fos, and c-myc; activation of Stat3; and decreased activity of Stat5a and Stat5b (reviewed in Marti et al., 1999Go). These or other transcription factors are likely to be responsible for regulated gene expression during mammary gland involution. Regulated expression of FTA is clearly at least partly responsible for synthesis of the M-N#1 antigen during mammary gland involution. Presumably the additional transferases required for A and B antigen synthesis are either constitutively active or are also up-regulated, as we observed no H antigen staining in the involuting mammary gland (Table I). It remains to be seen whether the up-regulation of the rat {alpha}(1,3)galactosyltransferase gene also plays a role in regulated M-N#1 antigen expression, as the activity of this transferase has not been fully investigated.

There are conflicting data in the literature concerning the precise effect of glucocorticoids on mammary gland involution. Dexamethasone slow-release pellets have been reported to inhibit the second phase of involution and also to suppress apoptosis (Feng et al., 1995Go). On the other hand, subcutaneous injection of hydrocortisone was observed to inhibit the second phase on involution without affecting apoptosis induction (Lund et al., 1996Go; Li et al., 1996Go). Our results with dexamethasone treatment are consistent with the conclusion that glucocorticoids inhibit the second phase of involution but not apoptosis. This is also in agreement with other data that shows that systemic hormones preserve lobular-alveolar structure without blocking apoptosis (Li et al., 1996Go). Up-regulation of the M-N#1 antigen was not inhibited by dexamethasone treatment, suggesting that the regulated expression of the antigen during mammary gland involution is determined by factors other than changes in systemic glucocorticoid hormone levels.

An outstanding question concerns the molecule(s) bearing the M-N#1 antigen in mammary epithelial cells of the involuting mammary gland. ABH antigens can be present on both glycoproteins and glycolipids. In the case of the mammary tumor cell line MT-450, we were able to use immunoprecipitation to show that two proteins from these cells are M-N#1-modified (Sleeman et al., 1999Go). An obvious possibility is that the same proteins are modified in nonneoplastic mammary epithelial cells.

The striking absence of M-N#1 antigen expression on apoptotic cells in the involuting mammary gland speaks strongly for an involvement of this antigen in cell proliferation or programmed cell death. Members of the ABH antigens have previously been implicated in apoptosis regulation. A and H antigens are down-regulated on apoptotic cells (Rapoport and Le Pendu, 1999Go). Ectopic expression of fucosyltransferases in colonic carcinoma cells led to H antigen expression and concomitant resistance to apoptosis induced by serum deprivation (Goupille et al., 2000Go). In another cell line that consitutively expresses H antigen, anti-sense expression of fucosyltransferase cDNA decreased H antigen expression and made the cells more sensitive to apoptosis induction (Goupille et al., 2000Go).

How might ABH antigens be involved in the regulation of apoptosis or growth of cells? In recent years, a large family of endogenous mammalian lectins called galectins has been identified. The evidence to date connects many galectin family members with the regulation of growth and apoptosis (reviewed in Rabinovich, 1999Go). At least one of these family members, galectin-3, binds to ABH antigens (Sato and Hughes, 1992Go). It is therefore intriguing to note that galectin 3 has antiapoptotic functions (Yang et al., 1996Go; Akahani et al., 1997Go; Kim et al., 1999Go), stimulates cell proliferation (Inohara et al., 1998Go), and promotes cell invasiveness (Le Marer and Hughes, 1996Go). One possibility we are currently investigating is whether galectin-3 or a similar molecule mediates its growth or survival-promoting activities through binding to the M-N#1 antigen. Blockade of such binding by the M-N#1 antibody might then explain why the M-N#1 antibody is able to inhibit the growth of M-N#1 antigen-expressing tumors.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Mammary gland preparation
Female BDX rats were maintained and treated according to German regulations. For virgin mammary glands, 8-week-old rats were used. Pregnancies were timed by plug analysis. Lactating rats were separated from their pups after 22 days lactation. Dexamethasone-treated animals were injected subcutaneously daily with 2 mg/kg dexamethasone dissolved in a volume of 200 µl benzyl benzoate:castor oil (1:2.5). At different times after weaning, rats were sacrificed and the no. 4 mammary glands removed for histology or RNA analysis. All experiments were repeated independently at least twice.

Immunohistochemistry
Mammary glands were fixed in 4% paraformaldehyde then embedded in paraffin wax and sectioned. The sections were subsequently immunostained as described (Dall et al., 1995Go), using AEC for color development. Antibodies used in this study were: M-N#1 (Sleeman et al., 1999Go); anti-blood group A antigen subtypes 1 and 2 (Dako); anti-blood group B antigen (Dako); anti-blood group H antigen, subtype2 (Dako); anti-blood group B antigen subtype 2 (Signet); anti-blood group H antigen, nonreactive with subtype 2 (Signet), subtype 1 precursor (Signet); anti-Lea (Signet); anti-Leb (Signet); anti-Lex (Signet); and anti-Ley (Signet). The activity of each antibody was ensured by using rat colon sections as a positive control, the exception being anti-Lex where rat kidney sections were used instead. For double staining experiments, sections were stained first with M-N#1 using alkaline phosphatase standard (Vector Laboratories) and the alkaline phosphatase Substrate Kit III (blue color, Vector Laboratories) for color development. Subsequent staining of apoptotic cells was performed using an Apoptag® kit (Oncor) according to the manufacturer’s instructions, except that alkaline phosphate Substrate Kit I (red color, Vector Laboratories) was used for color development. Quantification of staining was performed by taking photographs of stained sections and counting the number of stained cells in five independent 1-mm square fields of view.

Northern blots
RNA was prepared from snap-frozen mammary gland, colon and stomach tissue using peqGOLD RNA Pure (Peqlab) according to the manufacturer’s instructions. Poly (A)+ RNA was subsequently purified from the total RNA using standard protocols, and 5-µg aliquots were size-fractionated on 1.0 % formaldehyde-agarose gels and blotted onto Hybond N+ membrane (Amersham). The membranes were then cross-linked (UV Stratalinker 2400, Stratagene) and hybridized at 65°C in QuickHyb® (Stratagene). Probes were generated by 32P-labeling of cDNA fragments (ReadyPrime, Amersham). Unincorporated label was removed prior to hybridization using an Elutip (Schleicher & Schüll) according to the manufacturer’s specifications. After hybridization with the labeled probes, membranes were washed twice in 2x SSC, 0.1% SDS, and twice in 1x SSC, 0.1% SDS at 64°C, after which they were exposed to film. Rat {alpha}(1,2)fucosyltransferase FTA (GenBank accession number AF131237) and {alpha}(1,2)fucosyltransferase FTB (GenBank accession number AF131238) cDNA probes were a kind gift from Dr. Jacques Le Pendu. The rat UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase cDNA probe (GenBank accession number RNU35890) was a kind gift from Dr. Fred Hagen. The rat {alpha}(1,3)galactosyltransferase EST probe (GenBank accession number AA819687) was obtained from Research Genetics.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Anja Steffen for excellent technical assistance, Norma Howells and Jonathan Ward for animal husbandry, and Jacques Le Pendu for valuable discussions. This work was supported by the Association for International Cancer Research.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
FTA, {alpha}(1,2)fucosyltransferase A; FTB, {alpha}(1,2)fucosyltransferase B; GAPDH, ; ppGaNTase, polypeptide N-acetylgalactosaminyltransferase.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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