Tumor-related expression of {alpha}1,2fucosylated antigens on colorectal carcinoma cells and its suppression by cell-mediated priming using sugar acceptors for {alpha}1,2fucosyltransferase

Shin Yazawa1,2, Toyo Nishimura2, Munenori Ide3, Takayuki Asao3, Akihiko Okamura3, Susumu Tanaka2, Izumi Takai2, Yuko Yagihashi2, Abby R. Saniabadi2 and Naohisa Kochibe4

2 Japan Immunoresearch Laboratories, 351-1 Nishiyokote-cho, Takasaki 370-0021, Japan; 3 1st Department of Surgery, Gunma University School of Medicine, Maebashi, Japan; and 4 Department Biology, Faculty of Education, Gunma University, Maebashi, Japan

Received on February 12, 2002; revised on April 29, 2002; accepted on May 28, 2002


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
The accumulation of {alpha}1,2fucosylated antigens, such as Y (Fuc{alpha}1,2Galß1,4 [Fuc{alpha}1,3]GlcNAcß), Leb (Fuc{alpha}1,2Galß1,3-[Fuc{alpha}1,4]GlcNAcß), and H type 2 (Fuc{alpha}1,2 Galß1,4GlcNAcß) occurs specifically within human colorectal tumor tissues and can be detected by an antifucosylated antigen antibody, such as the YB-2 antibody. In the present investigation, we found that the expression of these antigens bearing an {alpha}1,2-linked fucose correlated with the resistance of the tumor cells to anticancer treatments. Addition of an exogenous sugar acceptor for {alpha}1,2fucosyltransferase to the cell medium resulted in suppression of {alpha}1,2fucosylated antigen expression on the tumor cells and increased susceptibility to anticancer treatment. The increased susceptibility may be attributed to cancer cell–mediated priming by sugar acceptors for {alpha}1,2fucosyltransferase added to the medium.

Key words: {alpha}1,2fucosylated antigens/anticancer treatment/human colorectal cancer cells/priming of acceptors for {alpha}1,2fucosyltransferase/tumorigenicity


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Changes of glycosylation in various glycoconjugates have been widely observed on the surface of tumor tissue cells. Accordingly, monoclonal antibodies raised to such glycoconjugates have been used as clinically useful reagents (Hakomori, 1985Go; Sakamoto et al., 1989Go; Le Pendu et al., 2001Go). Furthermore, accumulation of several fucosylated glycoconjugates, including certain blood group and related antigens, has been reported in various tumor tissues (¯rntoft et al., 1991Go). Similarly, we have reported accumulation of {alpha}1,2fucosylated antigens, including Y (Fuc{alpha}1,2Galß1,4[Fuc{alpha}1,3]GlcNAcß), Leb (Fuc{alpha}1,2Galß1,3[Fuc{alpha}1,4]GlcNAcß), and H type 2 (Fuc{alpha}1,2Galß1,4 GlcNAcß) in human colorectal and hepatocellular carcinoma cells together with characterization of {alpha}1,2fucosyltransferase (Yazawa et al., 1993aGo,b; Naitoh et al., 1994aGo,b; Nakamura et al., 1997aGo,b).

The expression of tumor-associated fucosylated antigens in colorectal carcinoma cells has been specifically determined by using the monoclonal antibody YB-2, developed in our laboratory, which reacts equally with Y, Leb, and H type 2 antigens. YB-2 was found to have useful application for the diagnosis of colorectal cancer (Yazawa et al., 1993aGo; Naitoh et al., 1994aGo,b). Furthermore, the expression of {alpha}1,2fucosyltransferase and the extremely elevated activities of {alpha}1,3 and {alpha}1,4 fucosyltransferase in colorectal tumors and cell lines were also seen (Yazawa et al., 1993bGo; Nakamura et al., 1997aGo,b). These observations led to the notion that {alpha}1,2fucosyltransferase with aberrant substrate specificities that were not seen in normal colorectal tissues has an important role in the synthesis of the cancer-associated YB-2 antigen in colorectal tissues (Yazawa et al., 1993bGo; Nakamura et al., 1997aGo).

An elevated level of {alpha}1,2fucosyltransferase, which may result in an increased expression of H type 2 antigen in the rat colorectal carcinoma cells, is known to be involved in the increased tumorigenicity and resistance to apoptosis and treatment with 5-fluorouracil (5-FU) (Labarriére et al., 1994Go; Goupille et al., 1997Go, 2000; Hallouin et al., 1999Go; Cordel et al., 2000Go). Therefore, it is likely that Fuc{alpha}1,2Galß1,3(4) ± [Fuc{alpha}1,4(3)]- GlcNAcßR structures when abnormally expressed in human colorectal tumor tissues could influence tumorigenicity and resistance to anticancer treatments. These understandings are relevant for developing new therapeutic agents against human colorectal cancer based on the strategy for the induction of suppressed expression of such structures on the surface of colorectal cancer cells.

In this study, we successively transfected the human FUT1 and FUT3 genes into mouse colon26 cells; the surfaces of these cells do not express the YB-2 antigen. With these transfectants, human colorectal carcinoma cells bearing the YB-2-related antigens were investigated to see if the expression of the YB-2 antigen on the cell surface was involved in the resistance to anticancer treatments. The FUT gene–transfected colon26 cells and human colorectal cancer cells showed the resistance against anticancer treatments in parallel with the expression of the YB-2 antigen on their cell surface. The suppression of the YB-2 antigen expression was also demonstrated in colorectal carcinoma cells via self-mediated priming of sugar substrates added in the medium as an acceptor for {alpha}1,2fucosyltransferase. This caused an increased susceptibility to anticancer treatment.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Immunohistochemical staining of colorectal tissues with YB-2
Sections of colorectal tumors and normal colon tissues (N = 95) were examined with mouse monoclonal YB-2 antibody, which reacts equally with Y, Leb, and H type 2 antigens (YB-2 antigen, Table I). A fourfold higher rate of positive staining was seen in colorectal tumors compared to normal tissues, 86.3% versus 21.1% (P < 0.0001). As shown in Table I, when the immunostaining was done after the colorectal tissues had been subdivided into proximal and distal regions, no positive staining was observed in the normal distal colon, but an extremely high positive staining for YB-2 was seen in the distal colon of the tumor tissue (P < 0.001). Furthermore, no significant difference betwen normal and tumors was seen in the proximal colon (P = 0.37).


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Table I. Immunohistochemical staining of human colorectal tumors and normal tissues with YB-2 antibody
 
Expression of YB-2 antigen on colorectal cell lines
The expression of YB-2 antigen on five different human colorectal cancer cell lines was investigated by fluorescence-activated cell sorting (FACS) (Figure 1). All of the colorectal carcinoma cells showed a high expression of the YB-2 antigen and the YB-2 antigen structure, Fuc{alpha}1,2Galß (which could be detected by the YB-3 antibody) was also found to be highly expressed in these cell lines (data not shown). Also, the YB-2 antigen found in these cell lines was found to comprise Y, Leb, and H type 2 antigens; the expression of these fucosylated antigens could be demonstrated by BM-1, TT42, and OSK16 antibodies, which react with individual antigens (data not shown). These fucosylated Y, Leb, and H type 2 antigens were hardly expressed on colon26 mouse colorectal carcinoma cells.



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Fig. 1. Expression of the YB-2 antigen on human colorectal carcinoma cell lines and the FUT gene–transfected mouse colorectal carcinoma cells. Each bar represents fluorescence intensity.

 
After the transfection of the human FUT1 or FUT1 and FUT3 genes, the expression of {alpha}1,2fucosyltransferase and both {alpha}1,2- and {alpha}1,3/4fucosyltransferases was found in colon26/FUT1 and colon26/FUT1/FUT3 cells, respectively, but none were found in their mock transfectants (colon26/vect1 and colon26/vect1/vect2 cells), except very weak {alpha}1,3fucosyltransferase activities was detected in the wild and mock transfectants (Figure 2). Consequently, YB-2-related antigens were expressed in both colon26/FUT1 and colon26/FUT1/FUT3 cells (Figure 1). Fucosylated antigens expressed on these transfectants were also found to consist of H type 2 antigen and H type 2 and Y antigens, respectively, but the expression of Leb antigen was hardly observed in either of the transfected cells (data not shown).



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Fig. 2. {alpha}-Fucosyltransferase activities in colon26 cells transfected with human FUT genes. {alpha}1,2- (gray shaded bars), {alpha}1,3- (black bars), and {alpha}1,4fucosyltransferase (striped bars) activities were determined in cell lysates.

 
Inhibition of cell proliferation
The effect of 5-FU on the proliferation of human and mouse colorectal carcinoma cells was examined in the presence of 5-FU (2 µg/ml, Table II). Cell proliferation inhibition rates for mouse colon26 cells were significantly higher compared to human colorectal carcinoma cells (P < 0.001). However, the FUT genes–transfected colon26 cells (colon26/FUT1 and colon26/FUT1/FUT3 cells) were found to have acquired a significant (P < 0.001) resistance to 5-FU, in spite of their mock transfectants showing almost the same susceptibility to 5-FU as the wild-type cells. When the effect of 5-FU on the cell proliferation rates was investigated in the DLD-1- and 5HFU-resistant DLD-1 (DLD-1/5-FU-R) cells, it was found that the latter cells had acquired a strong resistance to 5-FU, and accordingly, the cell proliferation inhibition rate was significantly (P < 0.001) reduced (Table II and Figure 3a). The expression of fucosylated antigens and fucosyltransferases was also determined in these cell lines (Figure 3b and c); elevated expression of fucosylated antigens was found in the resistant cells together with a more than twofold higher reactivities with YB-2 and YB-3 antibodies in these cells. Likewise, marked increases of {alpha}1,2- and {alpha}1,3fucosyltransferase activities were also determined in the resistant cells. In contrast, {alpha}1,4fucosyltransferase activity was found to be reduced in these cells.


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Table II. Cell proliferation inhibition of colorectal carcinoma cells by 5-FU and UV irradiation
 


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Fig. 3. Establishment of 5-FU-resistant DLD-1 (DLD-1/5-FU-R) cells and the altered expression of fucosylated antigens and fucosyltransferases. (a) Cell proliferation inhibition rates in the presence of various amounts of 5-FU in DLD-1 and DLD-1/5-FU-R cells. Each bar presents the mean ± SD (N = 8). (b) Demonstration of antigens on DLD-1 (closed bars) and DLD-1/5-FU-R (open bars) cells with antifucosylated antigen antibodies and lectins. (c) {alpha}1,2-, {alpha}1,3-, and {alpha}1,4fucosyltransferase activities in DLD-1 (closed bars) and DLD-1/5-FU-R (open bars) cells. Each bar represents the mean ± SD (N = 4).

 
Cell proliferation inhibition was also induced with UV radiation. Significant (P <0 .001) differences in cell proliferation inhibition rates were seen between human colorectal carcinoma cells and colon26 cells, between DLD-1 and DLD-1/5-FU-R cells, and between the FUT gene–transfected colon26 cells and other colon26 cells (Table II). The ladder formation of DNAs extracted from colon26 wild and mock transfectants was detected on the agarose gel after the cells had been exposed to UV but was hardly observed in the FUT gene–transfected cells (Figure 4). The apoptotic cell death induced by UV radiation was also assayed by measuring DNA fragments in colon26 wild and transfectant cells. The amount of cellular DNA fragments in the FUT gene–transfected colon26 cells was markedly lower compared with wild and mock transfectants (data not shown).



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Fig. 4. A 2% agarose gel electrophoresis of DNAs isolated from colon26 and transfectant cells after UV radiation. colon26/wild (1), colon26/FUT1 (2), colon26/FUT1/FUT3 (3), colon26/vect1(4), colon26/vect1/vect2 (5).

 
Cell-mediated priming of sugar substrates
Colo201 cells were cultured in the presence of phenyl ß-galactoside (1 mM) as a primer and [3H]fucose as a tracer. After a 3-day incubation, most of the radioactivity was found in the spent medium (data not shown). When the spent medium was applied to a Glyco-Pak N column, a single major peak corresponding to the authentic Fuc{alpha}1,2Galßphenyl was detected (Figure 5). More than 95% of the radioactivity corresponding to this peak was abolished by treatment with {alpha}1,2fucosidase but not by treatment with {alpha}1,3/4- or {alpha}1,6fucosidase (data not shown). Less than 2% and 6% of the radioactivity of the spent medium and cell extract, respectively, were detected at the elution position corresponding to the authentic phenyl ß-galactoside. The expression of YB-2 antigen on colo201 cells was investigated in the presence of phenyl ß-galactosides at different concentrations (0.1–10 mM, Figure 6). The decrease of the expression appeared gradually in line with the concentrations of phenyl ß-galactosides and more than 70% of the expression was suppressed at 5 mM phenyl ß-galactosides. No marked difference in the growth and cell morphology was observed in the presence of up to 10 mM primer alone.



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Fig. 5. Normal phase HPLC of [3H]fucosylated phenyl ß-galactoside on a Glyco-Pak N column. Elution positions of phenyl ß-galactoside (A) and Fuc{alpha}1,2Galßphenyl (B). Radioactivities of culture medium (circles) and cells (squares).

 


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Fig. 6. Expression of the YB-2 antigen on colo201 cells after priming with various concentrations of phenyl ß-galactoside. Solid histograms represent the fluorescence intensity without priming. Concentrations of phenyl ß-galactoside added were 0.1 mM (a), 0.5 mM (b), 1 mM (c), 5 mM (d), and 10 mM (e).

 
Relationship between YB-2 antigen expression and susceptibility to anticancer treatment
The priming of phenyl ß-galactoside by the colo201 cells was found to be strongly associated with a dramatic decrease in YB-2 antigen expression and resistance to anticancer treatments as reported. Therefore, it was thought that the suppression of YB-2 antigen expression by cell-mediated priming of sugar substrates induces susceptibility to anticancer treatment. As shown in Table III, the expression of YB-2 antigen in these three cell lines was inhibited via priming of phenyl ß-galactoside by the cells, and this had resulted in an increase of cell proliferation inhibition rate with 5-FU or UV. The suppression of the YB-2 antigen in these cells was shown to induce a significant (P < 0.001) susceptibility to UV radiation.. As shown in Table III, an increase of cell proliferation inhibition with 5-FU was also seen in the two human cell lines (P < 0.001) and colon26/FUT1/FUT3 cells (P < 0.05).


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Table III. Changes of YB-2 antigen expression and cell proliferation inhibition rates in colorectal cancer cells treated with phenyl ß-galactoside
 
Effects of galactosyl primers on cell proliferation and YB-2 antigen expression
Several synthetic {alpha}- and ß-galactosyl derivatives with hydrophobic aglycones as primers were separately added to the medium (5 mM final concentration) in which colo201 cells were incubated, and cell proliferation inhibition rate together with the expression of YB-2 antigen were investigated. The results are presented in Table IV and show that all three ß-galactoside derivatives were effective as suppressor of YB-2 antigen expression, which was reflected in an increase of susceptibility to treatment with 5-FU or UV. The suppression of YB-2 antigen expression was stronger with o-nitrophenyl ß- and phenyl ß-galactosides compared with methyl ß-galactoside. When {alpha}1,2fucosyltransferase, prepared from the lysates of colo201 cells, was assayed with these ß-galactosides as sugar substrates and GDP-fucose as a sugar donor, both o-nitrophenyl ß- and phenyl ß-galactosides showed higher acceptor activities compared with methyl ß-galactoside (data not shown).


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Table IV. Effects of galactosyl derivatives as primers on the cell proliferation inhibition rate and the expression of YB-2 antigen in colo201 cells
 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
The accumulation of {alpha}1,2fucosylated antigens such as Y, Leb, and H type 2 antigens occurs specifically within human colorectal tumor tissues and can be detected by an antifucosylated antigen antibody including our YB-2 antibody. This understanding has stimulated an interest in research with the hope of elucidating factors that promote cell proliferation in cancer tissues. Likewise, an understanding of cell proliferation inducers or suppressors should contribute toward finding specific and sensitive targets for antitumor drug therapy. Accordingly, in the present investigation, we found that the expression of {alpha}1,2fucosylated antigens on colorectal cancer cells had a major role in the resistance to anticancer treatment. In contrast, the suppression of the {alpha}1,2fucoylated antigen expression on the cells was associated with a dramatic increase in susceptibility to anticancer treatment. The increased susceptibility to anticancer treatment could be attributed to cancer cell–mediated priming of sugar acceptors for {alpha}1,2fucosyltransferase.

In our previous investigations on immunohistochemical staining of human colorectal tumor tissues, the accumulation of fucosylated antigens was specifically demonstrated in tumors by establishing and using the YB-2 antibody (Yazawa et al., 1993aGo; Naitoh et al., 1994aGo,b). In this study, with the availability of an increased number of specimens from colorectal tumors and by subdividing the colorectal tissues into proximal and distal regions, we could further our understanding on the YB-2 antigen as a tumor-specific antigen in the distal colon. A detailed immunohistochemical analyses of colorectal tumor tissues along with enzymatic analyses of their fucosyltransferases will be published elsewhere (Yazawa et al., in preparation).

Analyses of a series of colorectal carcinoma cells by flow cytometry revealed a high expression of the YB-2 antigen not only on human colorectal carcinoma cells but also on the FUT gene–transfected mouse colorectal carcinoma cells. From an enzymatic analysis of the FUT gene–transfected cells, it became clear that both {alpha}1,2- and {alpha}1,3/4fucosyltransferases were stably expressed on the colon26/FUT1/FUT3 cells; in particular, the level of {alpha}1,4fucosyltransferase activity in these cells was strikingly high, which is in line with our earlier report (Yazawa et al., 1995Go). Because little expression of the Leb active structure synthesized by both {alpha}1,2- and {alpha}1,4fucosyltransferases was seen on the colon26/FUT1/FUT3 cells, it is likely that colon26 cells lack the type 1 precursor structure (Galß1,3GlcNAcßR) on their surface. Indeed, {alpha}1,2- and {alpha}1,3fucosylated antigens found on the colon26/FUT1 and colon26/FUT1/FUT3 cells were carried mainly on the glycolipids consisting of the type 2 chain (Galß1,4GlcNAcßR) (unpublished data). In contrast, a high expression of the Leb antigen as well as the Y antigen was observed on human colorectal carcinoma cells (Yazawa et al., 1993aGo). As already described, different susceptibility to anticancer treatments demonstrated between human and mouse colorectal carcinoma cells might reflect the presence or absence of the Leb antigen on the cells.

Recently, an acquisition of resistance to 5-FU was demonstrated in a rat colorectal cell line after continuous exposure of the cells to 5-FU following the expression of blood group H type 2 antigen (Cordel et al., 2000Go). Furthermore, cell lines resistant to 5-FU have been established, and the mechanisms of their resistance to the drug have also been investigated (Lesuffleur et al., 1991Go; Inaba et al., 1998Go; Cordel et al., 2000Go; Murakami et al., 2000Go). Similarly, in the present study, 5-FU resistant human colorectal cancer cells (DLD1/5-FU-R), were established after incubation with 5-FU; consequently, the cell proliferation inhibition rate with 5-FU was dramatically reduced. Interestingly, the expression of fucosylated antigens on the cell surface was found to be altered markedly in the resistant cells. Additionally, the YB-2 as well as the YB-3 antigen was highly expressed in these cells. The altered expression of fucosylated antigens in the resistant cells might reflect changes in activities of their corresponding fucosyltransferases. Accordingly, further work in this line should increase our understanding of the mechanism(s) involved in tumor cell proliferation.

Apoptosis is well characterized by DNA fragmentation, which can also be induced by UV radiation. The extent of resistance to UV radiation observed in colorectal carcinoma cells seemed to correlate with the amount of YB-2 antigen expressed on the surface of these cells. The results indicated that the alterations of glycoconjugates on the cell surface altered the sensitivity of the cells to apoptosis. In an earlier study (Akamatsu et al., 1996Go), elevation of {alpha}1,3fucosyltransferase activity was found to correlate with apoptosis in the human colorectal carcinoma cells HT-29. Additionally, a strong increase in the expression of the Lex antigen on the surface of the apoptotic cells was demonstrated and was attributed to an increase in the activity of {alpha}1,3fucosyltransferase encoded by the FUT4 gene. Alteration of glycoconjugates on the apoptotic cell surface attributable to {alpha}1,2fucosyltransferase activity has also been reported previously (Dini et al., 1992Go; Falasca et al., 1996Go; Russell et al., 1996Go; Goupille et al., 2000Go). Considered together, it would appear that {alpha}1,2- and {alpha}1,3/4fucosylation on the cell surface of colorectal cancer cells impact differently on the apoptotic process.

Tumor cell–mediated priming of mono- and oligosaccharides has been observed previously (Okayama et al., 1973Go; Kuan et al., 1989Go; Zhuang et al., 1991Go; Kojima et al., 1992Go; Freeze et al., 1993Go), and the structures and processes in living cells that are involved in the priming of cell surface saccharides have been described recently (Sarkar et al., 1995Go, 1997; Laferté et al., 2000Go). In particular, the intracellular glycosylation in certain cells was shown to be inhibited when an appropriate compound(s) was added to the cells as a primer. The disaccharide of the type 1 precursor with some hydrophobic aglycones could be used as enzyme-specific and site-directed inhibitors for the synthesis of sialyl Lex antigen, which resulted in suppression of the sialyl Lex antigen expression on HL-60 and mouse embryonal carcinoma cells (Sarkar et al., 1997Go). The primed compounds were also found to be sialylated and fucosylated through intracellular glycosylation pathways, and a sialyl Lex analog (NeuAc{alpha}2,3Galß1,4[Fuc{alpha}1,3]GlcNAcßR) was secreted, which caused a reduction in E-selectin-dependent cell adhesion.

Phenyl ß-galactoside is one of the common and specific substrates for detecting {alpha}1,2fucosyltransferase (Chester et al., 1976Go). In this study, we found that phenyl ß-galactoside when added to the culture medium of colo201 was fucosylated and secreted as Fuc{alpha}1,2Galßphenyl. Interestingly, at the same time, the expression of YB-2 antigen was found to be strongly suppressed. As previously reported, a series of ß-galactoside derivatives with some hydrophobic aglycones at their reducing end could be used as acceptors for {alpha}1,2fucosyltransferase, but no acceptor activities for {alpha}1,2fucosyltransferase were found in {alpha}-galactoside derivatives (Chester et al., 1976Go). Furthermore, suppression of the YB-2 antigen expression in colo201 cells was observed only when a ß-galactoside was added as a primer. Based on these observations, it was suggested that substrates for {alpha}1,2fucosyltransferase could be primed by colo201 cells, and, conversely, the intracellular {alpha}1,2fucosylation was specifically inhibited, which was reflected as suppression of YB-2 antigen expression on the surface of colo201 cells. Because acceptor activities of ß-galactosides (such as o-nitrophenyl and phenyl derivatives for {alpha}1,2fucosyltransferase from colo201 cells) were higher compared with the methyl derivative, it was thought that the extent of suppression might depend on the structure of primers acting as acceptors for intracellular {alpha}1,2fucosyltransferase.

Suppression of the YB-2 antigen expression was seen by cancer cell–mediated priming of sugar substrates for {alpha}1,2fucosyltransferase involved in an increased susceptibility of the cancer cells to 5-FU or UV. Tumorigenicity, as well as resistance to anticancer treatment in certain colorectal carcinoma cells, has been reported to be associated with the presence of {alpha}1,2fucosyl residues on the cell surface (Hallouin et al., 1999Go; Cordel et al., 2000Go). However, a suppressed expression of {alpha}1,2fucosyltransferase found in human pancreatic cancer cells showed reduced adhesive and metastatic capacity when injected into nude mice after transfection of the cells with the FUT1 gene (Aubert et al., 2000Go). Nude mice injected with the FUT1– and the FUT3 gene–transfected colon26 cells were found to acquire a significant increase in tumor growth compared to mice injected with their wild-type cells (Yazawa et al., 2001Go). However, the tumorigenicity was reduced when the FUT gene–transfected cells were pretreated with phenyl ß-galactoside, which caused suppression of the expression of {alpha}1,2fucosylated antigens on the surface of the injected cells (unpublished data).

In conclusion, the tumor cell–mediated priming of substrates for {alpha}1,2fucosyltransferase was associated with suppression of the colorectal tumor cell–specific YB-2 antigen expression and a corresponding increase in tumor cell susceptibility to 5-FU or UV. These findings should further understanding of factors that contribute to tumor cell proliferation and propagation as well as help toward finding specific and sensitive targets for antitumor drug therapy. A number of chemically synthesized oligosaccharides are currently under evaluation to find effective and selective primers that can effectively suppress the expression of tumor-associated fucosylated antigens including the YB-2.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Materials
GDP-[3H]fucose (85.1 GBq/mmol), 5-[6-3H]fluorouracil (0.185 TBq/mmol) and Aquaxol-2 were purchased from DuPont (Boston, MA.). GDP-fucose, phenyl ß-D-galactoside, 5-FU, and RPMI 1640 medium were from Sigma (St. Louis, MO). Sep-Pak plus C18 reverse-phase cartridges and a Glyco-Pak N column were obtained from Waters (Milford, CT). Synsorb H disaccharide was obtained from Chembiomed (Edmonton, Alberta, Canada). Lipofectin, fetal calf serum (FCS), trypsin, Geneticin, ethylenediamine tetra-acetic acid, penicillin, and streptomycin were obtained from GibcoBRL (Gaithersburg, MD). The cell counting kit was from Dojin (Tokyo). pcDNA4/TO, pRC-CMV, and Zeocin were obtained from Invitrogen (Carlsbad, CA). YB-2 and YB-3 mouse monoclonal antibodies were prepared as described elsewhere (Yazawa et al., 1993aGo) and purified by affinity chromatography using a column of Synsorb H-disaccharide. {alpha}1,2Fucosidase was prepared and purified from Bacillus fulminans (Kochibe, 1973Go). {alpha}1,2 (Xanthomonas sp.), {alpha}1,3/4 (Xanthomonas sp.), and {alpha}1,6fucosidase (Chryseobacterium meningosepticum) preparations were obtained from Calbiochem (Darmstadt, Germany). Human colorectal carcinoma cells, colo201 and DLD-1, and mouse colorectal carcinoma cells (colon26) were obtained from American Type Culture Collection and cultured in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. DLD-1/5-FU-R cells were established as a 5-FU-resistant subline of DLD-1 cells by repeating continuous exposure to 5-FU with stepwise increases of the concentrations up to 100 µM. The human FUT1 gene inserted into the pRC/CMV vector was kindly supplied by Prof. H. Kimura, Kurume University, Kurume, Japan. Colorectal tissue specimens (N = 95) were obtained from colorectal cancer patients following written informed consent.

{alpha}-Fucosyltransferase assay
{alpha}1,2-, {alpha}1,3 and {alpha}1,4fucosyltransferases were assayed as described previously using phenyl ß-galactoside, Galß1,3[Fuc{alpha}1,4]GlcNAcßBn, Galß1,4[Fuc{alpha}1,3]GlcNAc ßBn (Yazawa et al., 1993bGo), Fuc{alpha}1,2Galß1,4GlcNAcßBn (Nakamura et al., 1997aGo), and 2'Omethyllacto-N-biose I ßBn (Yazawa et al., 1990Go) as an acceptor, respectively. The incubation mixture in a final volume of 100 µl contained 4 µmol HEPES-NaOH, pH 7.0; 1 µmol of MnCl2; 0.5 µmol of ATP; 500 µg of Triton X-100; 10 µl of enzyme preparation; 5 nmol of GDP-[3H]fucose (85.1GBq/mmol); and 20 nmol of each acceptor. After incubation at 37°C for 4 h, the reaction mixture was terminated by the addition of an equal volume of ethanol. The enzyme activities of the supernatant were measured by using a Sep-Pak plus C18 cartridge described elsewhere (Yazawa et al., 1992Go). Activity was expressed as pmol fucose transferred to the acceptor per h per mg enzyme preparation.

Determination of protein
Protein was determined with a DC protein assay kit (Bio-Rad, Rochmond, CA) using bovine serum albumin as a standard.

Immunohistochemical analysis of tissues
The deparaffinized sections were stained by the immunoperoxidase method using antifucosylated antigen antibodies (YB-2 and YB-3), biotinylated anti-mouse IgM, and the Vectastain ABC kit (Vector Laboratories) as describe elsewhere (Naitoh et al., 1994aGo). The epitopes of each antibody was determined as Y, Leb and H type 2 (YB-2), and H disaccharide (YB-3) as describe (Yazawa et al., 1993aGo). Positive and negative stainings were classified as previously reported (Naitoh et al., 1994aGo).

FACS analysis
Fucosylated antigens on the cell surface were analyzed by a Coulter EPICS-PROFILE II flow cytometer (Yazawa et al., 1993aGo; Nakamura et al., 1997aGo) using either biotin-labeled lectins such as Ulex europaeus and Lotus tetragonolobus (Honen) and fluorescein isothiocyanate (FITC)-labeled avidin (Sigma) or antifucosylated antigen monoclonal antibodies and FITC-labeled anti-mouse IgM (Vector Labs).

Transfection and selection of colon26/FUT1 and colon26/FUT1/FUT3 cells
Colon26 cells were transfected with the human FUT1 gene inserted in the pRc/CMV vector (Koda et al., 1997Go) and selection was initiated by addition of Geneticin (G418) to the medium to establish the colon26/FUT1 cells. These cells were then transfected with the human FUT3 gene inserted in the pcDNA4/TO (Nakamura et al., 1997aGo); selection was also initiated by the addition of both Geneticin and Zeocin in the medium to establish the colon26/FUT1/FUT3 cells. In each case, control cells transfected with pRc/CMV only or with pRc/CMV and pcDNA4/TO were prepared, and selections were done under the same condition. The control cells were called colon26/vect1 and colon26/vect1/vect2 cells, respectively.

Cell proliferation and proliferation inhibition assays
The proliferation capacity of each cell type was measured in a 96-well culture plate (Falcon) with the aid of a cell counting kit according to the manufacturer’s instruction. Fifty microliters of a cell suspension (1 x 104 cells) were seeded into each well of a 96-well microtiter plate and incubated at 37°C overnight. Fifty microliters of each medium with and without 5-FU were added to each well, and the plate was incubated at 37°C for 2 days. To investigate the effect of UV light on each cell type, cells were exposed to UV for 30 s with the aid of a UV transilluminator (312 nm), then a 50 µl of the medium was added and the plate was incubated. Cell proliferation rate was then determined by measuring the absorbance of the well at 450 nm with the reference wavelength at 600 nm. Cell proliferation inhibition rate (%) was calculated thus: (1 – OD of the treated cells / OD of the untreated cells) x 100. The Student t-test was used for the statistical analysis of the cell proliferation inhibition data.

Establishment of anticancer drug–resistant cells
Human colorectal carcinoma cells (DLD-1) were cultured in the presence and absence of 5-FU with a stepwise increase to 100 µM for 5 months. Then the resistant cells (DLD-1/5-FU-R cells) were cultured in the absence of 5-FU.

DNA fragmentation assay
Cells (1 x 107) were prepared for the extraction of the DNA in the cells after exposure to UV for 30 s. The DNA was extracted by using a DNA extraction kit (Stratagene) according to the manufacturer’s instruction. Twenty micrograms of DNA extracted from the cells were analyzed on 2% agarose gel stained with ethidium bromide.

Priming of galactosyl derivatives by colorectal carcinoma cells
Galactosyl derivatives with phenyl, methyl, and o-nitrophenyl were added to the medium (5 mM final concentration) in which colorectal carcinoma cells (approximately 5 x 105 cells/dish) were cultured. After incubation at 37°C for 3 days, FACS was done using the corresponding antibodies and lectins. Effects of the priming of sugar substrates (mediated by cancer cells) on the cell proliferation inhibition rate (attributable to anticancer treatment) were also determined as described.

Analysis of primed sugars
After incubation of cells with 5 mM phenyl ß-galactoside and [3H]fucose (200,000 dpm) for 16 h, the culture medium was centrifuged and the spent medium together with cells were recovered. Both spent medium and cell extracts treated with 2% Triton X-100 were applied on a Sep-Pak C18 reverse phase cartridge. The bound materials were eluted with methanol, and the eluate was dried by evaporating the methanol. Then the bound materials were dissolved in a small volume of solution containing CH3CN/H2O (80/20, v/v). The radioactive products were separated by high-performance liquid chromatography (HPLC) using a normal phase Glyco-Pak N column (7.8 x 300 mm) (Waters). The radioactivity in each fraction was measured with a liquid scintillation spectrometer after homogenization with an Amersham NCS-II solubilizer (Yazawa et al., 1984Go). Fuc{alpha}1,2Galßphenyl was prepared as a standard (Yazawa and Furukawa, 1980Go).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
5-FU, 5-fluorouracil; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HPLC, high-performance liquid chromatography.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: syazawa{at}po.sphere.ne.jp Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
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