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Tumor Biology |
1-6 Fucosyltransferase in Hepatoma Cells Suppresses Intrahepatic Metastasis after Splenic Injection in Athymic Mice1
Departments of Biochemistry [E. M., K. N., J. H. K., A. E., T. K., N. V., Y. I., N. T.] and Internal Medicine and Therapeutics [E. M., K. N., Y. S., N. H., M. H.], Osaka University Medical School, and Department of Pathology, Allied Health Science, Osaka University Faculty of Medicine [N. M.], Osaka 565-0871, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6 fucosyltransferase (1-6FucT) catalyzes the transfer of fucose to the innermost GlcNAc in N-glycans. Although 1-6FucT is barely detected in normal liver, it is enhanced during rat hepatocarcinogenesis and in human hepatoma. To understand the biological meaning of the 1-6FucT in hepatoma, especially in terms of metastasis, we established human hepatoma cell lines, which express high levels of 1-6FucT by transfection of the 1-6FucT gene and investigated intrahepatic metastasis after splenic injection to athymic mice. Tumor formation in the liver was dramatically suppressed in the 1-6FucT transfectants (1 of 9 and 1 of 10 in 1-6FucT transfectants versus 6 of 9 and 6 of 9 in controls). Although there were no differences in terms of cell invasiveness to a Matrigel or in terms of cytotoxicity to interleukin 2-treated lymphocytes between 1-6FucT transfectants and control cells, cell adhesion to mice hepatocytes and nonparenchymal liver cells in culture was significantly inhibited in 1-6FucT transfectants, compared to the controls. Attachment of 1-6FucT transfectants to a fibronectin-coated dish was decreased compared to controls because 5ß1 integrin was more strongly 1-6 fucosylated in the 1-6FucT transfectants. Two-dimensional electrophoresis followed by lectin blot showed that certain glycoproteins (Mr 50,000–150,000, pI 4.8–5.5) were 1-6 fucosylated and might be linked to suppression of intrahepatic metastasis. This is the first demonstration of the biological significance of 1-6 fucosylation on N-glycans in hepatoma cells under in vivo conditions. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6 fucosylation of AFP3
in patients with hepatoma has a diagnostic significance (3, 4, 5)
. Recently, we were successful in the purification and cDNA cloning of 1-6FucT, which catalyzes the addition of an 1-6 fucose to the innermost GlcNAc (1-6 fucosylation) on N-glycans, as shown in Fig. 1
(6
, 7)
. The level of expression of 1-6FucT mRNA was enhanced in hepatic tumor lesions but not in their adjacent tissues, in the case of a rodent hepatocarcinogenic model, the LEC rat (8)
, suggesting that it might represent a promising marker for the early diagnosis of hepatoma. Hutchinson et al. (9)
reported that levels of 1-6FucT activities were increased in hepatoma tissues of patients with HCC, as compared with their surrounding tissues. However, when the expression of 1-6FucT mRNA in 12 human HCC tissues was investigated by Northern blot, using our cloned 1-6FucT cDNA, the 1-6FucT mRNA was expressed in both HCC tissues and their surrounding tissues but not in normal liver (10)
. Some cases of HCC expressed high levels of 1-6FucT mRNA, whereas others did not. In addition, it is reported that the 1-6 fucosylation of AFP was also a predictor of the prognosis in patients with hepatoma after an operation (11)
. The authors concluded that the appearance of 1-6 fucosylated AFP in the serum was an indicator of poor prognosis because portal invasion and recurrence of hepatoma were frequently observed in such patients.
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1-6 fucosylation on N-glycans. Stubbs et al. (14)
suggested that 1-6 fucosylation greatly influences the conformation and flexibility of mannose--1,6-mannose antenna of the biantennary oligosaccharide from porcine fibrinogen. This study supported the reports that 1-6 fucosylation is essential for polysialylation of neural cell adhesion molecule, which is catalyzed by a specific polysialic acid synthase (15)
and is involved in the regulation of de-N-glycosylation by mammalian peptide N-glycosidase (16)
. These reports suggested that 1-6 fucosylation might modify the interactions of proteins and carbohydrates. However, until recently, no studies have been reported on the biological function of 1-6 fucosylation of N-glycans using in vivo experimental procedures.
Here, we established hepatoma cells that expressed high levels of 1-6FucT by transfection of the 1-6FucT gene and investigated intrahepatic metastasis after splenic injection to athymic mice, as compared with their parental cells. The data show that overexpression of 1-6FucT in hepatoma cells dramatically suppressed intrahepatic metastasis by inhibiting cell adhesion to both parenchymal and nonparenchymal hepatic cells. We also found that some glycoproteins were modified in the 1-6FucT transfectants, as judged by two-dimensional electrophoresis, followed by lectin blot experiments.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6FucT-transfected Hep3B Cells.1-6FucT cDNA (6)
was inserted into a mammalian expression pCAGGS, which is regulated by the ß-actin promoter (provided by Dr. K. Yamamura, Kumamoto University Medical School, Kumamoto, Japan), and 20 µg of the 1-6FucT expression vector and 1 µg of pSVneo (Health Science Research Resources Bank, Tokyo, Japan) were cotransfected into Hep3B cells by Lipofectamine (GIBCO-BRL, Rockville, MD). Selection was performed via the addition of 600 µg/ml G418 (Sigma). Six positive clones and 5 negative clones were randomly selected. Two positive clones (Hep3B-FT1 and Hep3B-FT2) and one negative clone (Mock) were used for the experiments that are described herein, but the results using other clones were very similar. Cell growth was determined by trypan blue staining after treatment with trypsin via microscopy.
Expression of 1-6FucT Activity and mRNA.
1-6FucT activity was analyzed by a fluorescent assay method, involving high-performance liquid chromatography with a minor modification of the original method, which has been reported previously by Uozumi et al. (17)
. A 4-(2-pyridylamino)butylamine-labeled sugar chain was used as a substrate, and 15 µl of sonicated-cell lysates containing PBS were used as an enzyme source. Protein concentrations were determined by a bicinchoninic acid kit (Pierce) using BSA as the standard. Total RNA was prepared from various cell lines according to the method reported by Chomczynski and Sacchi (18)
. Twenty µg of RNAs were electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde and transferred onto a Zeta-probe membrane (Bio-Rad, Hercules, CA) by capillary action. The membrane filter was prehybridized in a prehybridization buffer for 3 h and then hybridized with a [32P]1-6FucT cDNA fragment for 12 h at 42°C in a hybridization buffer (19)
. Detailed procedures have been described elsewhere (8)
.
Lectin Blot and Western Blot.
Lectin blot analysis was performed as described previously (20)
. Briefly, 10 µg of proteins extracted from Hep3B cells and mock- and 1-6FucT-transfected Hep3B cells (1-6FucT transfectants) were electrophoresed on a 10% polyacrylamide gel and then transferred onto a PVDF membrane (Millipore). After blocking with PBS containing 3% BSA overnight at room temperature, the filter was incubated with 1 µg/ml biotinylated LCA (Seikagaku Corp., Tokyo, Japan) which preferentially recognizes 1-6 fucose on the innermost GlcNAc in N-glycans (21)
for 1 h. The washing and developing procedures have been described previously (20)
. To verify that the total proteins were equally loaded, we stained the gel with Coomassie blue. For Western blotting of E-cadherin, 10 µg of proteins extracted from mock and 1-6FucT transfectants were electrophoresed on an 8% polyacrylamide gel. After blotting onto a PVDF membrane, the membrane filter was incubated with antihuman E-cadherin, HECD-1 (Takara Shuzou, Kyoto, Japan), diluted 1:1000 for 2 h at room temperature. The filter was washed three times with TBS containing 0.05% Tween 20 for 10 min each and then incubated with TBS containing peroxidase-conjugated goat antibody to mouse IgG (Promega) diluted 1:2500 for 1 h. After the membrane was washed three times with TBS containing 0.05% Tween 20 for 10 min each, it was developed by an enhanced chemiluminescence system (ECL; Amersham), according to the manufacturer’s protocol.
Experimental Metastasis.
To evaluate hepatic tumor formation in the 1-6FucT transfectants, we injected 1 x 106 1-6FucT transfectants into athymic nude mice (6-week-old male BALB/c mice; Charles River Japan Inc., Kanagawa, Japan) by percutaneous trans-splenic methods (22)
. Briefly, 1 day prior to the experiments, 5 x 106 cells were plated on a 10-cm dish in normal conditioned medium. After treatment with PBS containing 1 mM EDTA, the cells were suspended to a single-cell level with Hank’s buffer. 1 x 106 cells were percutaneously injected into the spleen of athymic mice. At 1 month after the injection, the mice were sacrificed under anesthesia. Tumor formation in the liver and the spleen was macroscopically counted and also histologically examined after H&E staining. These mice were maintained in temperature-controlled rooms at the Institute of Experimental Animal Science, Osaka University Medical School, and were treated according to NIH guidelines.
Invasion Assay.
The invasiveness of 1-6FucT transfectants and control cells was evaluated by a Matrigel assay using Boyden chambers (Biocoat Matrigel; Collaborative Biomedical Products-Becton Dickinson, Bedford, MA; Ref. 23
). Invasion to the Matrigel was evaluated at 24 and 48 h after starting the experiments. Details of these procedures have been described previously (13)
.
Cell Adhesion Assay.
Mice parenchymal hepatocytes and nonparenchymal cells were isolated by the two-step collagenase perfusion method described by Seglen et al. (24)
with minor modifications. The livers were perfused at 30 ml/min in situ by way of a canalicula inserted into the portal vein, first with 10 ml of Ca2+-free, Mg2+-free HEPES buffer containing 0.5 mM ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH 7.4) and then with 40 ml of Mg2+-free HEPES buffer containing 5 mM CaCl2, 0.5% BSA, 0.05% collagenase, and 0.1 mM phenylmethylsulfonyl fluoride. The isolated cells were separated into parenchymal cells and nonparenchymal cells by centrifugation (24)
. Cells (2.5 x 105 hepatocytes per well and 1 x 106 nonparenchymal cells per well on 24-well collagen-coated dishes; Iwaki, Kyoto, Japan) were cultured in 500 µl of Eagle medium (Nikken Kagaku, Kyoto, Japan) supplemented with 5% FCS, 10-6 M dexamethasone, 10-5 M insulin, and 100 µg/ml kanamycin. After 2 days, 2.5 x 104 1-6FucT transfectants and control cells were labeled with [3H]thymidine overnight and then cocultured with mice primary hepatocytes and nonparenchymal cells in culture. After 30 and 60 min and 2 and 4 h, cells were gently washed with ice-cold PBS four times, washed once with ice-cold trichloroacetic acid, and then solubilized in 1 N NaOH. The radioactivity incorporated into the cells was measured by a scintillation counter. Adhesion rate was calculated as the radioactivity from the cocultured cells per total cell labeling counts. Cell adhesion to collagen, laminin, and fibronectin was assayed by crystal violet (Sigma) staining, as described previously (13)
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Cytotoxic Assay.
The susceptibility of 1-6FucT transfectants and control cells to human lymphocytes activated by IL-2 (Otsuka, Tokushima, Japan) was assayed by a standard 4-h 51Cr-releasing assay. Human lymphocytes were prepared from heparinized peripheral blood of four healthy individuals by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway) and cultured in RPMI 1640 containing 10% FCS with 500 units/ml IL-2 for 72 h. These cells were used as effector cells for cytotoxic assay. After labeling with 51Cr, they were incubated with the target cells, 1-6FucT transfectants and control cells at various E:T ratios in 96-well dishes in triplicate. Cytotoxicity was determined as percentage lysis of the target cells. Detailed procedures have been described previously (25)
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Two-Dimensional Gel Electrophoresis.
High-resolution two-dimensional gel electrophoresis was carried out using the ATTO SJ-1060D system (ATTO Corp., Tokyo, Japan), according to the manufacturer’s protocol. Briefly, 200 µg of total cellular proteins were dissolved in 9.5 M urea, 2% Triton X-100, 2% ampholine, and 5% mercaptoethanol were applied to a tube gel containing 2% ampholine (1.6% pH 5–8, 0.4% pH 3.5–10). Isoelectric focusing was performed at 400 V for 12 h and then at 800 V for 2 h. The second dimension-gel electrophoresis was run at 20 mA per gel using SDS-11–12% polyacrylamide gradient gel. After blotting onto a PVDF membrane (Millipore), LCA blot analysis was performed as described above. The gels, after blotting, were stained with 2D-silver stain II "Daiichi" (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan) After dehybridization, the membrane filter was used for LCA blot, as described above.
Analysis of Oligosaccharide Structures of Integrins and Inhibition of Cell Adhesion to Fibronectin.
Two hundred µg of proteins that were extracted from mock and 1-6FucT transfectants were immunoprecipitated with antihuman 02ß1 (Dako, Kyoto, Japan), 3ß1 (Chemicon International Inc., Temecula, CA), 5ß1 (Dako), and vß3 (Santa Cruz Biotechnology, Santa Cruz, CA) integrins and antihuman CD44 (Santa Cruz Biotechnology). These anti-integrins recognize the subunit of each integrin. Immunoprecipitates were analyzed by LCA blot as described above.
To determine the involvement of 5ß1 integrin with cell attachment to fibronectin, we used the anti-5ß1 integrin antibody (Dako) for inhibiting cell adhesion.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6FucT Transfectants.1-6FucT was investigated in six human hepatoma cell lines, Hep3B showed the lowest expression of 1-6FucT (data not shown). We then transfected the 1-6FucT gene into this cell and established 1-6FucT transfectants. All positive clones showed >10 times the normal levels of 1-6FucT activity, and Hep3B-FT1 and Hep3B-FT2 showed 22 and 14 times the normal levels of 1-6FucT activity, respectively (Table 1)
. Levels of other glycosyltransferases, such as GnT-III and -V, which are involved in tumor metastasis, were not changed by 1-6FucT gene transfection. Northern blot analysis showed a high level of expression of 1-6FucT mRNA that is consistent with the observed enzyme activity (Fig. 2)
, indicating that high levels of 1-6FucT activity in Hep3B-FT1 and -FT2 were due to the high transcriptional levels. To better understand the changes in oligosaccharide structures in 1-6FucT transfectants, lectin blot analysis of total cellular proteins was performed, using LCA, which recognizes the 1-6 fucose residue on the innermost N-glycans. The 1-6FucT transfectants showed numerous bands, 
50–150 kDa in molecular mass, which strongly bound to LCA (Fig. 3A)
. SDS-PAGE analysis showed no changes in total cellular proteins (Fig. 3B)
. These results suggest that the overexpression of 1-6FucT in Hep3B cells leads to an increase in 1-6 fucose residue on the N-glycans of their glycoproteins.
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1-6FucT transfectants, we counted tumor formation in the liver after splenic injection to athymic mice. Although large tumor formation in the liver was observed in both parental cells and mock cells, it was not detected in the 1-6FucT transfectants (Fig. 4A)
. Histological examination of the tumor showed moderately differentiated HCC (Fig. 4B)
. The results of all experiments are summarized in Table 2
. The incidence of hepatic tumor and intrahepatic metastasis was dramatically suppressed in the 1-6FucT transfectants, and the size of the hepatic tumor was much larger in control and mock cells than it was in 1-6FucT transfectants. No difference in tumor formation in the spleen among control, mock, and 1-6FucT transfectants was detected, and splenic tumors were not correlated with hepatic tumor formation. When these cells were injected into the foot pads of athymic mice, no changes were observed (data not shown). These results indicate that the suppression of tumor formation in the 1-6FucT transfectants occurred specifically in the liver.
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1-6FucT transfectants, invasion, cell adhesion, and cytotoxic assays were performed. As shown in Fig. 5
, no differences in tumor invasion into a Matrigel were evident at either 24 or 48 h, suggesting that modification of oligosaccharides by 1-6FucT did not affect the invasive ability of Hep3B cells. The degree of invasion, however, was not so high in comparison with other tumor cells such as melanoma and glioma (data not shown). In contrast, cell adhesion to hepatocytes and nonparenchymal cells of athymic mice liver in culture was inhibited in 1-6FucT transfectants compared to the control and mock cells (Fig. 6, A and B)
. This inhibition of cell adhesion was more obvious in nonparenchymal cells than in parenchymal hepatocytes. These results suggest that 1-6FucT is capable of modifying certain glycoproteins that might be adhesion molecules. When cell adhesion was evaluated by attachment to extracellular matrix, the attachment to fibronectin and laminin was significantly delayed for 1-6FucT transfectants than for the control cells (Fig. 7)
, but attachment to collagen was not significantly changed among these cells. In general, immune systems reject implanted tumor cells, although they attach to some organs. To understand the involvement of the immune system in athymic mice, we investigated the cytotoxicity of 1-6FucT transfectants to lymphokine-activated killer cells (IL-2-activated lymphocytes; Fig. 8
). Although 15% of the cells were killed at an E:T ratio of 50 in this assay, no difference was observed between 1-6FucT transfectants and control cells.
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1-6FucT transfection (Fig. 3)
, some of which might be involved in cell adhesion. To determine the types of proteins which were modified, we performed two-dimensional electrophoresis followed by LCA blot (Fig. 9)
. Silver staining of the gels showed that the patterns of total cellular proteins were similar in mock cells and 1-6FucT transfectants but that a few bands appeared in the case of the transfectants, suggesting that molecular weight and pI of some proteins were changed by 1-6FucT gene transfection. A LCA blot showed that the density of some bands with a molecular mass of 50–150 kDa and pI 4.8–5.5 was more increased in the 1-6FucT transfectants as compared with mock cells, suggesting that the amounts of 1-6 fucosylation of those proteins were increased. However, these bands were scarcely detected in silver staining, indicating that the amounts of these proteins were quite low and difficult to identify.
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1-6FucT transfectants as compared to that to other extracellular matrix (Fig. 7)
and certain proteins with molecular mass of 100,000 were highly fucosylated (Fig. 9)
, oligosaccharide structures on 2ß1, 3ß1, 5ß1, and vß3 integrins and CD44 were investigated by immunoprecipitation followed by LCA blot. As shown in Fig. 10A
, the 2ß1 3ß1 integrins were scarcely expressed or fucosylated in either mock or 1-6FucT transfectants. Increases in LCA binding to 5ß1 integrin and CD44 were observed in 1-6FucT transfectants compared to mock cells. 1-6 fucosylation of CD44 minor bands was more prominent than that of the major band, although its biological meaning is unknown. In contrast, vß3 integrin was highly fucosylated in both mock and 1-6FucT transfectants. To determine the involvement of 5ß1 integrins in the adhesion to fibronectin, we added anti-5ß1 integrin antibody to inhibit cell attachment (Fig. 10B)
. Decreases in cell adhesion to fibronectin were observed in both mock and 1-6FucT transfectants. When high amounts of the antibody were added, the cell adhesion approached the same level, suggesting that the difference in cell adhesion between mock and 1-6FucT transfectants was due to 5ß1 integrin.
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1-6FucT Transfectant.1-6FucT transfectants, protein levels of E-cadherin were increased in 1-6FucT transfectants as compared with mock cells (Fig. 11)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6FucT in hepatoma cells suppresses intrahepatic metastasis via the splenic vein by inhibiting adhesion to the liver. Oligosaccharide structures of many glycoproteins were greatly changed, as shown by LCA blotting using total cellular proteins (Fig. 3)
. It is important to identify the likely candidate glycoproteins that are modified by 1-6FucT. When 1-6FucT transfectants were analyzed by two-dimensional analysis followed by LCA blot, >20 spots were enhanced in 1-6FucT transfectants in comparison with controls (Fig. 8)
. Because the avidin-biotin system enhanced their expression, protein levels of these spots were extremely low because they could not be detected by silver staining. Some of these could be involved in cell adhesion to the liver, especially nonparenchymal cells. As shown in Fig. 10A
, 5ß1 integrin (5 chain) was highly fucosylated in the 1-6FucT transfectant, suggesting that this modification might affect the ability of fibronectin to adhere. Other integrin family proteins were selectively fucosylated. Because in the 2ß1 and 3ß1 integrins were expressed in these cells at extremely low levels, 5ß1 integrin was thought to be a major ligand to fibronectin. The biological significance of 1-6 fucosylated CD44 must be clarified with reference to tumor metastasis; this is a future project.
E-Cadherin, an adhesion molecule linked to tumor metastasis, was increased in the 1-6FucT transfectant (Fig. 11)
. This increase did not result from transcriptional levels. A similar phenomenon was observed in GnT-III-transfected melanoma cells (13)
. However, E-cadherin in 1-6FucT transfectants is not 1-6 fucosylated because immunoprecipitated E-cadherin did not bind to LCA, suggesting that the increase in E-cadherin expression in 1-6FucT transfectants is due to unknown mechanisms. Osada et al. (30)
reported that overexpression of E-cadherin promoted intrahepatic metastasis of an HCC cell line, Li7. In their experiments, E-cadherin transfectants formed a compact colony, but the parental cells that did not express E-cadherin were loosely connected. In contrast, in our experiments, both parental Hep3B cells and 1-6FucT-transfected Hep3B cells formed similar compact colonies.
When GnT-III was introduced into melanoma cells, lung metastasis was dramatically suppressed (13)
. In this case, GnT-III transfectants displayed both decreased invasion to Matrigel and inhibition of cell attachment to laminin and collagen. When GnT-III was introduced into a leukemia cell line K562, susceptibility to cytolysis by natural killer cells was decreased (25)
. A bisecting GlcNAc, a product of GnT-III, inhibited further processing of N-glycans, resulting in dramatic changes in total oligosaccharides (31)
. Therefore, the transfection of GnT-III unexpectedly resulted in a variety of biological modifications to tumor cells (32)
. In the case of 1-6FucT, invasiveness and resistance to the immune system remained unchanged. As shown in Fig. 7
, 1-6FucT transfectants showed different patterns of delayed cell adhesion to extracellular matrix, as compared to GnT-III transfectants (13)
. To deny another artificial modification as the result of overexpression of 1-6FucT, for example, consumption of GDP fucose, we examined levels of sialyl-LeX antigen in 1-6FucT transfectants by immunostaining or Western blot analysis. No differences in sialyl-LeX expression levels in controls and 1-6FucT transfectants were observed, suggesting that inhibition of intrahepatic metastasis is not due to sialyl-LeX levels (data not shown). In contrast, overexpression of GnT-V in lung epithelial cells resulted in malignant transformation (33)
, and overexpression of 1–2 fucosyltransferase enhanced tumorigenicity of colon cancer cells (34)
. These results indicate that modification of N-glycans by the transfection of the glycosyltransferase gene could bring about various pathophysiological phenomena in tumor cells.
When we started the present experiments, we expected that overexpression of 1-6FucT might promote intrahepatic metastasis. This is because the appearance of 1-6 fucosylation of AFP in the serum of a patient with HCC indicates a poor prognosis after the operation (11)
. However, the result was completely different. 1-6 fucosylation on AFP seen in the serum of patients with hepatoma is not due only to the up-regulation of 1-6FucT because high expression of 1-6FucT in the liver was observed in noncancerous surrounding tissues as well as in hepatoma tissues (10)
. If AFP is produced in noncancerous hepatocytes with a high expression of 1-6FucT, 1-6 fucosylation on AFP should be expected in only patients with liver cirrhosis. However, it was not detected in patients with chronic liver disease without hepatoma, suggesting that certain mechanisms for inhibiting secretion of fucosylated AFP might exist in noncancerous hepatocytes. The mechanisms by which 1-6 fucosylated AFP is a predictor of poor prognosis of hepatomas remain unknown. Abnormal secretion of 1-6 fucosylated AFP might, however, be involved. Expression levels of 1-6FucT in 12 hepatoma tissues were different in each case. One case of a patient with hepatoma displaying high expression of 1-6FucT survived >5 years without recurrence after the operation (data not shown). Remodeling of oligosaccharides by 1-6FucT might alter the phenotypes of hepatomas that have biologically low-malignant characteristics. This is the first demonstration of the biological significance of 1-6 fucosylation on N-glycans in hepatoma cells under in vivo conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant-in-Aid 10178105 for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan. 
2 To whom requests for reprints should be addressed, at Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3420; Fax: 81-6-6879-3429; E-mail: proftani{at}biochem.med.osaka-u.ac.jp
3 The abbreviations used are: AFP, -fetoprotein; 1-6FucT, 1-6 fucosyltransferase; GlcNAc: N-acetylglucosamine; HCC, hepatocellular carcinoma; PVDF, polyvinylidene difluoride; LCA, Lens culinaris agglutinin; IL-2, interleukin 2; GnT, N-acetylglucosaminyltransferase.
Received 10/14/98. Accepted 3/ 4/99.
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REFERENCES |
|---|
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-fetoprotein in patients with primary hepatocellular carcinoma. Biochim. Biophys. Acta, 830: 217-223, 1985.[Medline]
-fetoproteins in early detection of hepatocellular carcinoma. Cancer Res., 53: 5419-5423, 1993.-fetoprotein: characteristics of N-linked sugar chains of glycoproteins produced in human liver and hepatocellular carcinomas. Cancer Res., 53: 2970-2975, 1993.-D-glucosaminide 1-6 fucosyltransferase. J. Biol. Chem., 271: 27810-27817, 1996.-D-glucosaminide:1-6 fucosyltransferase (1-6FucT) from human gastric cancer MKN45 cells. J. Biochem., 121: 626-633, 1997.1-6 fucosyltransferase during rat hepatocarcinogenesis. Int. J. Cancer, 75: 444-450, 1998.[Medline]
1-6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of fetoprotein. Hepatology, 28: 944-952, 1998.[Medline]
-fetoprotein in small hepatocellular carcinomas. Gastroenterology, 111: 996-1001, 1996.[Medline]
-D-glucosaminide 1-6 fucosyltransferase activity, involving high performance liquid chromatography. J. Biochem., 120: 385-392, 1996.(1–2) fucosyltransferase gene transfection. Glycobiology, 7: 221-229, 1997.This article has been cited by other articles:
|
|
 |
|
  S. Watanabe, M. Misawa, T. Matsuzaki, T. Sakurai, T. Muramatsu, T.-a. Yokomine, and M. Sato Production and Characterization of Transgenic Mice Systemically Expressing Endo- -Galactosidase C Glycobiology, January 1, 2008; 18(1): 9 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ihara, Y. Ikeda, S. Toma, X. Wang, T. Suzuki, J. Gu, E. Miyoshi, T. Tsukihara, K. Honke, A. Matsumoto, et al. Crystal structure of mammalian {alpha}1,6-fucosyltransferase, FUT8 Glycobiology, May 1, 2007; 17(5): 455 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Isaji, Y. Sato, Y. Zhao, E. Miyoshi, Y. Wada, N. Taniguchi, and J. Gu N-Glycosylation of the beta-Propeller Domain of the Integrin {alpha}5 Subunit Is Essential for {alpha}5beta1 Heterodimerization, Expression on the Cell Surface, and Its Biological Function J. Biol. Chem., November 3, 2006; 281(44): 33258 - 33267. [Abstract] [Full Text] [PDF]
|
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H. Ihara, Y. Ikeda, and N. Taniguchi Reaction mechanism and substrate specificity for nucleotide sugar of mammalian {alpha}1,6-fucosyltransferase--a large-scale preparation and characterization of recombinant human FUT8 Glycobiology, April 1, 2006; 16(4): 333 - 342. [Abstract] [Full Text] [PDF]
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X. Wang, J. Gu, H. Ihara, E. Miyoshi, K. Honke, and N. Taniguchi Core Fucosylation Regulates Epidermal Growth Factor Receptor-mediated Intracellular Signaling J. Biol. Chem., February 3, 2006; 281(5): 2572 - 2577. [Abstract] [Full Text] [PDF]
|
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J. Gu, Y. Zhao, T. Isaji, Y. Shibukawa, H. Ihara, M. Takahashi, Y. Ikeda, E. Miyoshi, K. Honke, and N. Taniguchi {beta}1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells Glycobiology, February 1, 2004; 14(2): 177 - 186. [Abstract] [Full Text] [PDF]
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 |
|
 S. Nakahara, E. Miyoshi, K. Noda, S. Ihara, J. Gu, K. Honke, H. Inohara, T. Kubo, and N. Taniguchi Involvement of oligosaccharide changes in {alpha}5{beta}1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line Mol. Cancer Ther., November 1, 2003; 2(11): 1207 - 1214. [Abstract] [Full Text] [PDF]
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 K. Noda, E. Miyoshi, J. Gu, C.-X. Gao, S. Nakahara, T. Kitada, K. Honke, K. Suzuki, H. Yoshihara, K. Yoshikawa, et al. Relationship between Elevated FX Expression and Increased Production of GDP-L-Fucose, a Common Donor Substrate for Fucosylation in Human Hepatocellular Carcinoma and Hepatoma Cell Lines Cancer Res., October 1, 2003; 63(19): 6282 - 6289. [Abstract] [Full Text] [PDF]
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Y. Yamaguchi, Y. Ikeda, T. Takahashi, H. Ihara, T. Tanaka, C. Sasho, N. Uozumi, S. Yanagidani, S. Inoue, J. Fujii, et al. Genomic structure and promoter analysis of the human {alpha}1,6-fucosyltransferase gene (FUT8) Glycobiology, June 1, 2000; 10(6): 637 - 643. [Abstract] [Full Text] [PDF]
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) and 48 (
) h was evaluated using a Matrigel-coated Boyden chamber. Results are expressed as relative units of absorbance compared to absorbance of Hep3B cells as a control (mean ± SD). Columns, means of three independent experiments; bars, SD.
), mock (
), Hep3B-FT1 (•), and Hep3B-FT2 (
