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
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Abstract |
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Key words: 1,2fucosylated antigens/anticancer treatment/human colorectal cancer cells/priming of acceptors for
1,2fucosyltransferase/tumorigenicity
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Introduction |
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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., 1993a; Naitoh et al., 1994a
,b). Furthermore, the expression of
1,2fucosyltransferase and the extremely elevated activities of
1,3 and
1,4 fucosyltransferase in colorectal tumors and cell lines were also seen (Yazawa et al., 1993b
; Nakamura et al., 1997a
,b). These observations led to the notion that
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., 1993b
; Nakamura et al., 1997a
).
An elevated level of 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., 1994
; Goupille et al., 1997
, 2000; Hallouin et al., 1999
; Cordel et al., 2000
). Therefore, it is likely that Fuc
1,2Galß1,3(4) ± [Fuc
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 genetransfected 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 1,2fucosyltransferase. This caused an increased susceptibility to anticancer treatment.
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Results |
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Discussion |
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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., 1993a; Naitoh et al., 1994a
,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 genetransfected mouse colorectal carcinoma cells. From an enzymatic analysis of the FUT genetransfected cells, it became clear that both 1,2- and
1,3/4fucosyltransferases were stably expressed on the colon26/FUT1/FUT3 cells; in particular, the level of
1,4fucosyltransferase activity in these cells was strikingly high, which is in line with our earlier report (Yazawa et al., 1995
). Because little expression of the Leb active structure synthesized by both
1,2- and
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,
1,2- and
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., 1993a
). 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., 2000). 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., 1991
; Inaba et al., 1998
; Cordel et al., 2000
; Murakami et al., 2000
). 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., 1996), elevation of
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
1,3fucosyltransferase encoded by the FUT4 gene. Alteration of glycoconjugates on the apoptotic cell surface attributable to
1,2fucosyltransferase activity has also been reported previously (Dini et al., 1992
; Falasca et al., 1996
; Russell et al., 1996
; Goupille et al., 2000
). Considered together, it would appear that
1,2- and
1,3/4fucosylation on the cell surface of colorectal cancer cells impact differently on the apoptotic process.
Tumor cellmediated priming of mono- and oligosaccharides has been observed previously (Okayama et al., 1973; Kuan et al., 1989
; Zhuang et al., 1991
; Kojima et al., 1992
; Freeze et al., 1993
), 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., 1995
, 1997; Laferté et al., 2000
). 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., 1997
). The primed compounds were also found to be sialylated and fucosylated through intracellular glycosylation pathways, and a sialyl Lex analog (NeuAc
2,3Galß1,4[Fuc
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 1,2fucosyltransferase (Chester et al., 1976
). In this study, we found that phenyl ß-galactoside when added to the culture medium of colo201 was fucosylated and secreted as Fuc
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
1,2fucosyltransferase, but no acceptor activities for
1,2fucosyltransferase were found in
-galactoside derivatives (Chester et al., 1976
). 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
1,2fucosyltransferase could be primed by colo201 cells, and, conversely, the intracellular
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
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
1,2fucosyltransferase.
Suppression of the YB-2 antigen expression was seen by cancer cellmediated priming of sugar substrates for 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
1,2fucosyl residues on the cell surface (Hallouin et al., 1999
; Cordel et al., 2000
). However, a suppressed expression of
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., 2000
). Nude mice injected with the FUT1 and the FUT3 genetransfected colon26 cells were found to acquire a significant increase in tumor growth compared to mice injected with their wild-type cells (Yazawa et al., 2001
). However, the tumorigenicity was reduced when the FUT genetransfected cells were pretreated with phenyl ß-galactoside, which caused suppression of the expression of
1,2fucosylated antigens on the surface of the injected cells (unpublished data).
In conclusion, the tumor cellmediated priming of substrates for 1,2fucosyltransferase was associated with suppression of the colorectal tumor cellspecific 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.
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Materials and methods |
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-Fucosyltransferase assay
1,2-,
1,3 and
1,4fucosyltransferases were assayed as described previously using phenyl ß-galactoside, Galß1,3[Fuc
1,4]GlcNAcßBn, Galß1,4[Fuc
1,3]GlcNAc ßBn (Yazawa et al., 1993b
), Fuc
1,2Galß1,4GlcNAcßBn (Nakamura et al., 1997a
), and 2'Omethyllacto-N-biose I ßBn (Yazawa et al., 1990
) 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., 1992
). 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., 1994a). 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., 1993a
). Positive and negative stainings were classified as previously reported (Naitoh et al., 1994a
).
FACS analysis
Fucosylated antigens on the cell surface were analyzed by a Coulter EPICS-PROFILE II flow cytometer (Yazawa et al., 1993a; Nakamura et al., 1997a
) 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., 1997) 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., 1997a
); 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 manufacturers 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 drugresistant 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 manufacturers 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., 1984). Fuc
1,2Galßphenyl was prepared as a standard (Yazawa and Furukawa, 1980
).
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Abbreviations |
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Footnotes |
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References |
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