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.
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Abstract |
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Key words: ABH histo-blood group antigen/apoptosis/mammary gland/involution/tissue remodeling
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Introduction |
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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 (1,2)fucosyltransferases add fucose in
(1,2) to the terminal sugar residue of these precursors to generate the H antigen (Rajan et al., 1989
; Rouquier et al., 1995
). Further modification of the H antigen by
3-GalNAc and
3-Gal transferases produces A and B antigens, respectively (see Figure 1). These transferases require
(1,2)fucose modification of the precursor disaccharides, and thus A and B antigens can only be synthesized from H antigen (Oriol et al., 1992
). In humans, H antigen synthesis is under the control of at least two polymorphic genes, each encoding a distinct
(1,2)fucosyltransferase (Rajan et al., 1989
; Rouquier et al., 1995
). 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., 1990
).
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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., 1999). 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., 1999
). No binding was observed to "linear B" oligosaccharides. In a wide range of assays, the antibody acts as a classical antiblood 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., 1999
). 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., 1999). Furthermore, antibody binding requires the addition of
-D-GalNAc or
-D-Gal to the H antigen structure, as the antibody does not bind to H antigens or linear B-antigens (Sleeman et al., 1999
). 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
-L-fucose linked in 1,2 to ß-D-Gal, and at least part of the
-D-GalNAc or
-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, 1985). 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, 1988
; Lund et al., 1996
; Strange et al., 1992
). 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., 1995
; Marti et al., 1997
). 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., 1997
). In the second phase, proteases degrade extracellular matrix and basement membrane components, and the lobular-alveolar structures collapse (Lund et al., 1996
). 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 1015 days (Lascelles and Lee, 1978
).
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.
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Results |
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Discussion |
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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., 1996; Li et al., 1996
). 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., 1996
and references therein). Here we report that expression of the FTA,
(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., 1999
). 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
(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., 1995). On the other hand, subcutaneous injection of hydrocortisone was observed to inhibit the second phase on involution without affecting apoptosis induction (Lund et al., 1996
; Li et al., 1996
). 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., 1996
). 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., 1999). 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, 1999). 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., 2000
). 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., 2000
).
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, 1999). At least one of these family members, galectin-3, binds to ABH antigens (Sato and Hughes, 1992
). It is therefore intriguing to note that galectin 3 has antiapoptotic functions (Yang et al., 1996
; Akahani et al., 1997
; Kim et al., 1999
), stimulates cell proliferation (Inohara et al., 1998
), and promotes cell invasiveness (Le Marer and Hughes, 1996
). 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.
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Materials and methods |
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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., 1995), using AEC for color development. Antibodies used in this study were: M-N#1 (Sleeman et al., 1999
); 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 manufacturers 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 manufacturers 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 manufacturers 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 (1,2)fucosyltransferase FTA (GenBank accession number AF131237) and
(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
(1,3)galactosyltransferase EST probe (GenBank accession number AA819687) was obtained from Research Genetics.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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