From the
Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, 466-0065, the
Department of Applied Bio-organic Chemistry, Gifu University, Gifu, 501-1193, ¶Central Laboratories for Key Technology, Kirin Brewery Co., Kanazawa-ku, Yokohama 236-0004, and the ||Program of Molecular Pathology, Aichi Cancer Center, Research Institute, Nagoya 464-8681, Japan
Received for publication, October 29, 2002 , and in revised form, March 17, 2003.
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ABSTRACT |
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INTRODUCTION |
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Monosialyl Lewis a (Lea)1 has been suggested to be a representative tumor-associated antigen in cancers of the pancreas and colon. This antigen is recognized by monoclonal antibody (mAb) 19-9 (5) and has been utilized as an indicator of the tumor size in those tumor patients. In fact, the majority of colon cancer tissues express monosialyl Lea, whereas normal mucosa rarely expresses the antigen (6). On the other hand, both normal and malignant pancreatic tissues express sialyl Lea, although it can be used as a tumor-associated antigen when measured for its serum levels.
In addition to monosialyl Lea antigen, disialyl Lea was identified in the disialoganglioside fraction of human adenocarcinoma of the colon (7). Serum levels of disialyl Lea antigen were measured using mAb FH7, and they turned out to be increased in patients with colonic and pancreatic cancers (8). These reports suggested that this antigen is also applicable as a tumor-associated antigen. Moreover, immunohistochemical analysis of Lea, monosialyl Lea, and disialyl Lea antigens in human colorectal and pancreatic tissues revealed that disialyl Lea might affect the expression mode of sialyl Lea antigen (i.e. an oncofetal antigen) by competing with each other in the biosynthesis of individual structures (9). The synthesis and expression of disialyl Lea might result in masking the expression of sialyl Lea. However, little has been known about the biosynthetic pathway of disialyl Lea or about the sialyltransferase responsible for the transfer of a sialic acid onto GlcNAc with 2,6 linkage.
To identify the 2,6-sialyltransferase that catalyzes the synthesis of disialyl Lea from monosialyl Lea or the synthesis of disialyl lactotetraosylceramide (disialyl Lc4) from monosialyl Lc4 as a precursor for the synthesis of disialyl Lea, a sialyltransferase assay was performed using previously cloned
2,6-sialyltransferases (10, 11). We demonstrated here that ST6GalNAc V/VI, which were cloned as the sialyltransferases responsible for the synthesis of
-series gangliosides, could significantly synthesize disialyl Lc4, indicating the main synthetic pathway of disialyl Lea. Based on the analyses of expression levels of the transferase genes and resulting antigens in human colon cancer cell lines, ST6GalNAc VI appears most likely to be a key enzyme in the synthesis of disialyl Lea structure.
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MATERIALS AND METHODS |
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Cell CultureMouse fibroblast L cells, human colon cancer cell lines (CACO-2 cells, Colo320, DLD-1, HT-29, Lovo, SW1080, and SW1116), and COS1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum.
Construction of the Expression VectorThe expression vector pcDNA3.1-ST6GalNAc VI was prepared by inserting an XbaI and XhoI fragment from pMIKneo-ST6GalNAc VI (11) into XbaI and XhoI sites of pcDNA3.1(+) vector.
Preparation of Membrane FractionL cells (3 x 106) were plated in 10-cm dishes at least 48 h prior to transfection. Cells were transiently transfected with an expression plasmid (4 µg) by the DEAE-dextran method (18). After 48 h of culture in Dulbecco's modified Eagle's medium containing 7.5% fetal calf serum, the cells were harvested by trypsinization. Cells were pelleted, washed with phosphate-buffered saline (PBS), and lysed in ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride using a nitrogen cavitation apparatus (Parr Instrument Co., Moline, IL) at 400 p.s.i. for 30 min. Nuclei were removed by low speed centrifugation, and supernatant was centrifuged at 100,000 x g for 1 h at 4 °C. The pellet was resuspended in ice-cold 100 mM sodium cacodylate buffer, pH 7.0, and used as an enzyme source for the fucosyltransferase assay as described below.
Fucosyltransferase AssayThe fucosyltransferase assay was performed in a mixture containing 100 mM sodium cacodylate buffer, pH 7.0, 10 mM MgCl2, 0.25% Triton X-100, 5 mM CDP-choline (Sigma), 0.1 mM GDP-fucose (Sigma), 25,000 dpm/nmol GDP-[14C]fucose (Amersham Biosciences), 50 µg of a membrane fraction, and 10 µg of acceptors in a total volume of 50 µl. The reaction mixture was incubated at 37 °C for 1 h, and the enzyme products were isolated and analyzed by TLC as described above.
Preparation of Soluble Forms of ST6GalNAc V and VIAs described previously (10, 11), soluble forms of ST6GalNAc V and VI as fusion proteins with protein A were prepared for the sialyltransferase assay.
Sialyltransferase AssayThe sialyltransferase assay was performed in a mixture containing 100 mM sodium cacodylate buffer, pH 6.0, 10 mM MgCl2, 0.3% Triton CF-54, 0.66 mM CMP-NeuAc (Sigma), 6,000 dpm/µl CMP-[14C]NeuAc (Amersham Biosciences), the enzyme solution, and 10 µg of acceptors in a total volume of 50 µl. The reaction mixture was incubated at 37 °C for 2 h. The products were isolated using a C18 Sep-Pak cartridge (Waters, Milford, MA) and analyzed by TLC with a solvent system of chloroform/methanol/0.2% CaCl2 (55:45: 10). The radioactivity on each plate was visualized with a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan). For kinetic analysis, incubation was performed using various concentrations of acceptor substrates, 00.2 mM GM1b2 or sialyl Lc4.
TLC-ImmunostainingTLC-immunostaining was performed as described previously (19). Disialyl Lc4 was detected using anti-disialyl Lc4 mAb FH9 (kindly provided by S. Hakomori (Pacific Northwest Research Institute, University of Washington, Seattle)) (8) at a 1:3 dilution as a primary antibody. Briefly, after chromatography of the glycolipids, the TLC plate was heat-blotted onto a polyvinylidene difluoride membrane. The membrane was incubated with mAb for 1 h, washed, and incubated with biotinylated horse anti-mouse IgG at a 1:200 dilution for 1 h. The antibody binding was visualized with an ABC-PO kit (Vector, Burlingame, CA) and HRP-1000 (Konica, Tokyo, Japan).
Transfection for Flow Cytometric Analysis and Immunofluorescence AssayCOS1 cells and colon cancer cell lines in a 6-cm dish (Falcon) were transiently transfected with pcDNA3.1, pcDNA3.1ST6GalNAc VI, and/or pcXN2-FUT-3 (provided by S. Nishihara and H. Narimatsu (Soka University, Tokyo)) (1 µg/µl) by the DEAE-dextran method and cultured for 48 h in Dulbecco's modified Eagle's medium containing 7.5% fetal calf serum before observation.
Flow Cytometric AnalysisThe cell surface expression of disialyl Lea and sialyl Lea was analyzed using the transfectant cells after transient transfection of expression vectors. Two days after transfection, cells were trypsinized and washed twice with PBS and then used for flow cytometric analysis using anti-disialyl Lea mAb FH7, anti-sialyl Lea mAb 1H4 (Seikagaku Corp.), anti-sialyl Lea/sialyl Lc4 mAb 2D3 (Seikagaku Corp.), anti-GD1 mAb KA-17 (presented by Y. Hirabayashi (RIKEN Brain Science Institute, Wako, Japan)) (20), and anti-GQ1b
mAb GGR41 (presented by T. Tai (Tokyo Metropolitan Institute of Medical Science)) (21) on FACScalibur with Cell QuestTM version 3.1f software (Becton Dickinson). Fluorescein isothiocyanate-conjugated anti-mouse IgG (whole) antibody (ICN/Cappel) or anti-mouse IgM antibody (ZYMED) was used as second antibodies.
The expression of disialyl Lea, sialyl Lea, GD1, and GQ1b
on various human colon cancer cell lines was detected with mAb FH7 (hybridoma supernatant) at a 1:3 dilution, mAb 1H4 (1 mg/ml) at a 1:200 dilution, mAb 2D3 (1 mg/ml) at a 1:200 dilution, mAb KA-17 (ascites) at a 1:100 dilution, and mAb GGR41 (supernatant) at a 1:2 dilution.
Immunofluorescence AssayCOS1 cells were cultured on cover glasses in 24-well plates and incubated at 37 °C for 24 h and transiently transfected with expression vectors as described above. The cells were fixed with cold acetone for 10 min or with 3.6% paraformaldehyde in PBS for 5 min. In the latter case, they were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Then they were processed for indirect immunofluorescence analysis as described previously (11). The staining was observed using the µ RadianceTM confocal imaging system (Bio-Rad) and also with ORCA-ER-1394 imaging systems (Hamamatsu Photonics, Hamamatsu, Japan).
Analysis of ST6GalNAc V/VI Gene ExpressionThe expression levels of the ST6GalNAc V/VI gene in human colon cancer cell lines were determined by RT-PCR and Northern blotting. RT-PCRs were performed with the ST6GalNAc V and VI gene-specific primers, 5'-GGTCTGGCAGTGTGTTTAGC-3' (nucleotides 2241 in the coding sequence) and 5'-AACTGGGCACGGACATTCAA-3' (nucleotides 916935) for ST6GalNAc V, 5'-CAGACGCCGGAGAGAAATGA-3' (nucleotides 6079 in the coding sequence) and 5'-GCCCCCAGAAGATGAACACG3' (nucleotides 526507) for ST6GalNAc VI. For Northern blot analysis, total RNA was prepared using TRIZOL ReagentTM (Invitrogen) according to the manufacturer's instructions. Fifteen micrograms each of total RNA was electrophoresed and blotted onto a nylon membrane (GeneScreen Plus) (PerkinElmer Life Sciences). They were hybridized with [32P]dCTP-labeled ST6GalNAc V or ST6GalNAc VI cDNA probes as previously described (11).
Analysis of FUT-3, 3Ga-T5, and ST3Gal IV Gene ExpressionThe expression levels of the FUT-3,
3Gal-T5, and ST3Ga IV genes in human colon cancer lines were determined by RT-PCR. RT-PCRs were performed with FUT-3,
3Gal-T5 and ST3Gal IV gene-specific primers, 5'-GCAGCGACTCCGACATCTTC-3' (nucleotides 479498 in the coding sequence) and 5'-CGTAGTTGCTTCTGCTGGGG-3' (nucleotides 844825) for FUT-3, 5'-CAGACACCTCCCTTCCTCGT-3' (nucleotides 593612 in the coding sequence) and 5'-AAAAGGTCGGCTGGGAGTGG-3' (nucleotides 12341215) for
3Gal-T5, and 5'-GCTCCTCCATCCCCAAGAAC-3' (nucleotides 329348 in the coding sequence) and 5'-GACATTATGGCCTGACCCCG-3' (nucleotides 948929) for ST3Gal IV. The intensities of the amplified bands were quantified by scanning picture of gels using the public domain NIH Image program.3 The band intensity of each transferase gene was normalized with that of
-actin.
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RESULTS |
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To further confirm that the enzyme products are disialyl Lc4, TLC-immunostaining of the products using an anti-disialyl Lc4 mAb FH9 was performed. As shown in Fig. 1B, the products with ST6GalNAc V and VI clearly showed bands stained with mAb FH9. Anti-disialyl Lc4 mAb FH9 stained the chemically synthesized disialyl Lc4 and a band with the same migration in the products of ST6GalNAc VI (Fig. 2). Thus, the products were confirmed to be disialyl Lc4. Then, we determined their kinetic parameters using various concentrations of two acceptor substrates (i.e. GM1b and sialyl Lc4). The apparent Km showed no marked differences, but Vmax values were fairly low for sialyl Lc4, resulting in lower Vmax/Km, compared with GM1b in both enzymes (Table II).
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Synthetic Pathway of Disialyl LeaFUT-3 exhibits both 1,3- and
1,4-fucosyltransferase activity and is the only enzyme that is responsible for the synthesis of type I antigens, such as Lea, Leb, and sialyl Lea (15). Sialyl Lc4, which could be fucosylated by FUT-3, is a precursor of sialyl Lea known as a cancer-associated antigen (22). Disialyl Lea was also suggested to be one of the cancer-associated antigens (7), but its synthetic pathway and function are not clear. To clarify the order of
2,6-sialylation and
1,4-fucosylation in the synthesis of disialyl Lea, substrate specificity of FUT-3 toward Lc4, sialyl Lc4, and disialyl Lc4 was examined using GDP-[14C]fucose as a donor. FUT-3 mainly acted on Lc4 and sialyl Lc4 and produced Lea and sialyl Lea, respectively (Fig. 3A). It also fucosylated disialyl Lc4, resulting in the new bands, probably disialyl Lea. The enzyme product synthesized from disialyl Lc4 was stained with anti-disialyl Lea mAb FH7 (Fig. 3B), showing its identity to be disialyl Lea. Furthermore, ST6GalNAc V and VI were able to sialylate sialyl Lc4 but not sialyl Lea (Fig. 3C). These results suggest that FUT-3 acted to complete the synthesis of disialyl Lea at the final step and competed with ST6GalNAc V or VI for the common substrate, sialyl Lc4 (Fig. 4).
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Synthesis of Disialyl Lea in Cultured CellsTo explore the function of the ST6GalNAc VI gene in vivo, we transiently transfected the expression vectors for ST6GalNAc VI and/or FUT-3 into COS1 cells, which have sialyl Lc4 as a precursor of sialyl Lea and disialyl Lc4, while having no inherent FUT-3 (15). Expression vectors pcDNA3.1-ST6GalNAc VI and pcXN2-FUT-3 were used. The flow cytometric analysis of the transfectant cells with ST6GalNAc VI alone exhibited no expression of sialyl Lea or disialyl Lea antigen as the mock transfectants (Fig. 5, A and B (a, b, e, and f)). When FUT-3 was transfected, not only sialyl Lea but disialyl Lea was expressed, suggesting the inherent ST6GalNAc VI gene expression (Fig. 5, A and B (c and g)). When ST6GalNAc VI was co-transfected with FUT-3, the expression level of disialyl Lea increased, whereas that of sialyl Lea decreased (Fig. 5, A and B (d and h)). The results of immunocytostaining revealed that disialyl Lea antigen mainly localized in the cytoplasm (Fig. 5B).
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Expression of ST6GalNAc VI in Human Colon Cancer Cell LinesTo determine the expression pattern of ST6GalNAc V/VI and FUT-3 mRNA, RT-PCR analysis and Northern blot analysis were performed with seven human colon cancer cell lines (CACO-2, Colo320, DLD-1, HT-29, Lovo, SW1080, and SW1116). RT-PCR was also performed for 3Gal-T5 and ST3GalIV genes. To analyze the correlation between the expression levels of disialyl Lea and those of glycosyltransferase genes involved in the synthesis of disialyl Lea, RT-PCR analysis was conducted using the primers based on the human cDNA of ST6GalNAc V/VI, FUT-3,
3Gal-T5, and ST3Gal IV. As shown in Fig. 6A, a PCR product of ST6GalNAc VI with 446 base pairs was detected in all human colon cancer cell lines, although the intensity of the bands varied. Lovo cells expressed the ST6GalNAc VI gene at the highest level, whereas SW1080 cells showed a very weak band. In contrast, no bands were detected for the ST6GalNAc V gene in any of them, although the band as positive control was clearly detected with 912 base pairs. Northern blot analysis (Fig. 6B) also indicated the expression of ST6GalNAc VI mRNA with proportional levels to those in RT-PCR but not of V mRNA as shown in Fig. 6A. The FUT-3 gene was expressed strongly in DLD-1 and SW1116, moderately in HT29, and weakly in Colo320 and Lovo. ST3Gal IV was broadly expressed, and
3Gal-T5 was expressed highly in DLD-1 and Lovo and weakly in CACO-2 and HT-29 (Fig. 6C).
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To investigate the expression of final enzyme products (i.e. cancer-associated antigens and -series ganglioside antigens in these cell lines), flow cytometric analysis and immunofluorescence assay were carried out. Table III summarizes the expression patterns of the ST6GalNAc V/VI mRNA, cancer-associated antigens, and
-series ganglioside antigens in these cell lines. It was found that
-series ganglioside antigens (GD1
and GQ1b
) were minimal or zero in these cell lines, indicating that the main products of ST6GalNAc VI enzyme might be those of type I lacto-series glycolipids in human colon tissues. The expression levels of these antigens on the cell surface appeared to not necessarily correlate with the expression levels of inherent ST6GalNAc VI gene. Two lines with high levels of ST6GalNAc VI gene showed almost null expression of disialyl Lea. But the expression of sialyl Lea was also low in these two lines. Sialyl Lea is also at low levels, suggesting that these two lines lack precursors. On the other hand, cell lines expressing very low levels of ST6GalNAc VI did not express high levels of disialyl Lea except for SW1116. Consequently, there were no critical controversial points in these results to take ST6GalNAc VI as a responsible enzyme for the synthesis of disialyl Lea if we consider other factors such as low precursor levels or low activity of FUT-3.
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To inquire about the correlation between the levels of glycosyltransferase genes and the levels of disialyl Lea expression, we further evaluated the expression of responsible genes (3Gal-T5, ST3GalIV, and FUT-3) and that of relevant antigens in these cell lines. Fig. 7A showed the expression pattern of these genes, and Fig. 7B is a summary of the antigen expression. The expression levels of FUT-3 correlated rather well with those of disialyl Lea. Otherwise, there was no clear correlation between the expression levels of glycosyltransferase genes and relevant sialyl compounds.
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Substrate Competition between ST6GalNAc VI and FUT-3As we did for COS1 (Fig. 5), we transiently transfected the ST6GalNAc VI and/or FUT-3 expression vectors into six colon cancer cell lines, which originally expressed both genes more or less. Flow cytometric analysis with mAbs FH7 and 1H4 revealed that the overexpression of ST6GalNAc VI cDNA resulted in the elevation of disialyl Lea expression and suppression of sialyl Lea expression in many cell lines (DLD-1, Lovo, and SW1116). Two lines (CACO-2 and Colo320) showed no change with transfection of any genes. On the other hand, the transfection of FUT-3 cDNA enhanced the expression levels not only of sialyl Lea but also of disialyl Lea (DLD-1, Lovo, and SW1080). When ST6GalNA VI and FUT-3 cDNA were co-transfected, disialyl Lea levels generally increased compared with the single gene transfection, and sialyl Lea levels were higher than those with the transfection of ST6GalNA VI and lower than those with FUT-3 alone (Fig 8, A and B). Thus, it was confirmed that both ST6GalNAc VI and FUT-3 contribute in the synthesis and expression of disialyl Lea, and they partly compete to share sialyl Lc4 as a common acceptor substrate.
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DISCUSSION |
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In this study, we clearly demonstrated, for the first time, the biosynthetic pathway of disialyl Lea and the possibility of competition of ST6GalNAc VI with FUT-3 (i.e. synthesis of monosialyl Lea and that of disialyl Lc4 (and disialyl Lea)). The synthesis of disialyl Lea was achieved in COS1 cells by transfection of both ST6GalNAc VI and FUT-3 or FUT-3 alone. The expression rate of disialyl Lea was not so high as expected, partly because the efficiency of co-transfection might be not so high. Sialyl Lea was not converted to disialyl Lea with ST6GalNAc VI. Taken together, sialyl Lc4 is only one substrate examined so far leading to the synthesis of disialyl Lea. Therefore, synthesis of sialyl Lea and disialyl Lc4 (and disialyl Lea) should compete with each other sharing a common substrate, monosialyl Lc4. Transfection of the expression vector of ST6GalNAc VI into a sialyl Lea-expressing cell line could induce a mild reduction in the expression level of sialyl Lea in addition to new expression of disialyl Lea. These results really suggested the possibility that overexpression of disialyl Lea, based on the action of ST6GalNAc VI, might result in the suppression of the expression level of sialyl Lea. If this is the case, the expression level of ST6GalNAc VI gene might be low in fetal and colon cancers but high in normal colonic mucosa, corresponding with an onco-fetal nature of sialyl Lea. This issue is now under investigation in our laboratory.
Among ST6GalNAc families (IVI), ST6GalNAc I and II preferentially act on nonsialylated substrates. ST6GalNAc III was poorly active for a type I structure (14), and a human homolog has not yet been reported. ST6GalNAc IV was a protein-dominant (O-glycan) enzyme (14). Consequently, we concentrated our efforts on the function of ST6GalNAc V and VI. The results obtained in this study elucidated that these sialyltransferases could act on multiple substrate structures including ganglioseries and lacto-series. Thus, we have defined a novel substrate specificity of previously cloned sialyltransferases.
Whether these sialyltransferases actually exert a catalytic activity in the tissues is a critical point to be clarified. Analyses with seven human colon cancer cell lines showed that ST6GalNAc VI but not V was expressed at various levels as analyzed with RT-PCR. Northern blotting with these cell lines also demonstrated similar results. The intensity in flow cytometry or immunocytostaining of disialyl Lea did not correlate well with the expression levels of ST6GalNAc VI gene. However, two cell lines with poor expression of the ST6GalNAc VI gene also showed only low level expression of disialyl Lea. Two cell lines with high levels of the ST6GalNAc VI gene (CACO-2 and Colo320) scarcely showed disialyl Lea expression. However, they expressed the lowest levels of sialyl Lea among the cell lines examined, suggesting that FUT-3 level and/or sialyl Lc4 level are very low, resulting in inefficient synthesis of disialyl Lea despite high levels of ST6GalNAc VI expression. -Series gangliosides such as GD1
and GQ1b
were very poor in all of these cell lines. Taken together, it appears quite likely that ST6GalNAc VI is really active and contributes to the synthesis of disialyl Lc4 and disialyl Lea in human colon tissues.
As for the correlation between sialyl Lea/disialyl Lea and FUT3/ST6GalNA VI, it seemed difficult to find a simple competitive relation between these two enzymes by comparing the expression patterns of these genes and their products among cell lines as shown in Fig. 7. This is because the intra-Golgi localization of the ST6GalNA VI and FUT-3 might bias one product over another, resulting in the deviated efficiency of the utilization of the common substrate. Furthermore, the expression levels of other glycosyltransferase genes involved in the synthesis of sialyl Lc4 were various, depending on the cell lines, probably forming different situations in the individual lines. On the other hand, when either one or both of these transferase genes were transfected, we could find the reasonable effects of the expression of individual genes on the expression of sialyl Lea and disialyl Lea as shown in COS1 (Fig. 5) and colon cancer cell lines (Fig. 8). Namely, ST6GalNAc VI is essential for the expression of disialyl Lea, and it partly competes with FUT-3 for the acceptor substrate, sialyl Lc4. However, it simultaneously needs the help of FUT-3 to generate disialyl Lea. The importance of FUT-3 in the synthesis of disialyl Lea was indicated by the fact that FUT-3 expression levels fairly well correlated with disialyl Lea levels (Fig. 7A). Our results of transient expression as shown in Fig. 8 demonstrate well the dual aspects of FUT-3 in the synthesis of disialyl Lea.
Beyond the result obtained in this study, the regulatory mechanisms for the expression of disialyl Lea in normal/cancer tissues and the biological roles of the structure in the normal tissues and in colonic malignant cells remain to be investigated. New findings obtained in this study will contribute to promote those studies.
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FOOTNOTES |
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** To whom correspondence should be addressed. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp.
1 The abbreviations used are: Lea, Lewis a (Gal1,3(Fuc
1,4)Glc-NAc
1,3Gal
1,4Glc
1-Cer); Lc4, lactotetraosylceramide (Gal
1,3Glc-NAc
1,3Gal
1,4Glc
1-Cer); nLc4, neolactotetraosylceramide (Gal-
1,4GlcNAc
1,3Gal
1,4Glc
1-Cer); mAb, monoclonal antibody; PBS, phosphate-buffered saline; RT, reverse transcriptase; ST6GalNAc, sialyltransferase as defined in Ref. 25.
2 The nomenclature of gangliosides is based on that of Svennerholm (24). The abbreviated nomenclature for cloned sialyltransferases follows Tsuji et al. (25).
3 The NIH Image program was developed at the National Institutes of Health and is available through the Internet by anonymous FTP from zippy nimb.gov or on a floppy disk from the National Technical Information Service (Springfield, VA) (part no. PB95-500195GED).
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ACKNOWLEDGMENTS |
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REFERENCES |
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