Masao Iwamori

Alterations in the glycolipid composition and cellular properties of ovarian carcinoma-derived RMG-1 cells on transfection of the α1,2-fucosyltransferase gene

Transfection of the mouse Fut1 and Fut2, and human FUT1 genes into human ovarian carcinoma‐derived RMG‐1 cells resulted in 20–30‐fold increases in cellular α1,2‐fucosyltransferase activity, and in alteration of the glycolipid composition, including not only fucosylated products, but also precursor glycolipids. Although globo‐series glycolipids were not significantly affected by the transfection, the major glycolipids belonging to the lacto‐series type 1 chain family in RMG‐1 cells and the transfectants were the Lc4Cer, Lewis a (Le)a and Leb, and H‐1 glycolipids, respectively, suggesting that fucosylation of Lc4Cer to the H‐1 glycolipid prevents the further modification of Lc4Cer to Lea and Leb in the transfectants. Also, the lacto‐series type 2 chains in RMG‐1 cells were LeX, NeuAc‐nLc4Cer and NeuAc‐LeX, and those in the transfectants were LeX and LeY, indicating that the sialylation of nLc4Cer and LeX is restricted by increased fucosylation of LeX. As a result, the amount of sialic acid released by sialidase from the transfectants decreased to 70% of that from RMG‐1 cells, and several membrane‐mediated phenomena, such as the cell‐to‐cell interaction between cancer cells and mesothelial cells, and the cell viability in the presence of an anticancer drug, 5‐fluorouracil, for the transfectants was found to be increased in comparison to that for RMG‐1 cells. These findings indicate that cell surface carbohydrates are involved in the biological properties, including cell‐to‐cell adhesion and drug resistance, of cancer cells. (Cancer Sci 2005; 96: 26 –31)

Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues.

Enhancement of the adhesive and spreading potentials of ovarian carcinoma RMG-1 cells due to increased expression of integrin α5β1 with the Lewis Y-structure on transfection of the α1,2-fucosyltransferase gene

Le(Y) antigen is known to be associated with malignant properties including metastasis and a poor prognosis of ovarian carcinomas. To clarify the mechanisms underling these properties, we established ovarian carcinoma-derived cells exhibiting enhanced expression of Le(Y) by transfection with alpha1,2-fucosyltransferase and compared their cellular properties with those of the original cells. So the human alpha1,2-fucosyltransferase gene was transfected into ovarian carcinoma-derived RMG-1 cells, which are known to contain Le(X), a precursor of Le(Y), and RMG-1-hFUT cells exhibiting enhanced expression of Le(Y) were established by selection with anti-Le(Y) antibodies, and their adhesive and spreading potentials on fibronectin-coated plates were compared with those of RMG-1 cells. Results showed that the relative expression of Le(Y) in RMG-1-hFUT cells was about 20-fold that in RMG-1 cells, and that of integrin alpha5beta1 and an integrin-mediated signal transduction molecule, focal adhesion kinase, was also increased in RMG-1-hFUT cells. Interestingly, anti-Le(Y) antibodies were revealed to immunoprecipitate integrin alpha5beta1, indicating that its oligosaccharides are composed of Le(Y), the amounts of which was substantially elevated in RMG-1-hFUT cells. The adhesion and spreading potentials on fibronectin-coated plates of RMG-1-hFUT cells were significantly enhanced in comparison to those of RMG-1 cells, and were greatly suppressed by anti-Le(Y) antibodies, indicating that Le(Y) is involved in the integrin-fibronectin interaction. These results suggested that transfection of the alpha1,2-fucosyltransferase gene into ovarian carcinoma-derived cells brought about elevated expression of integrin alpha5beta1 with Le(Y), resulting in enhancement of the adhesion and spreading potentials of cells through the integrin-fibronection interaction, which was inhibited by anti-Le(Y) antibodies. Thus, Le(Y) in integrin alpha5beta1 was thought to be involved in the enhanced cell adhesion properties of malignant ovarian carcinomas.

Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway

BackgroundLewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumors pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells.MethodsWe constructed a plasmid encoding α1,2-fucosyltransferase (α1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after α-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.ResultsOur results showed that the levels of α1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of α-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of α1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor LY294002.ConclusionsIncreased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.

GDP-fucose: β-galactoside α1,2-fucosyltransferase, MFUT-II, and not MFUT-I or -III, is induced in a restricted region of the digestive tract of germ-free mice by host-microbe interactions and cycloheximide

A shift from sialylation to fucosylation of mucosal glycoconjugates occurred in the mammalian digestive tract in the weaning period, but mice under germ-free conditions were found to express both fucosyl GM1 (FGM1) and fucosyl asialo GM1 (FGA1) in the stomach, cecum and colon, but not in the small intestine. By host-microbe interactions and administration of cycloheximide, FGA1 was quickly induced in the small intestine, but the concentrations of fucosylated glycolipids in the other regions were not altered significantly. Their expression coincided with the activity of GDP-fucose:GA1 alpha(1, 2)-fucosyltransferase (alpha1,2-FT), and we isolated a cDNA with an open reading frame encoding the murine alpha1,2-FT (MFUT-II) of 347 amino acids with a predicted molecular mass of 39.21 kDa. The intraperitoneal injection of cycloheximide induced the mRNA and activity of alpha1,2-FT (MFUT-II) in the small intestine of germ-free mice, whereas no change in the mRNA or activity was observed in the stomach, cecum and colon, indicating that expression of FGA1 in response to microbial colonization or cycloheximide is transcriptionally regulated in a restricted region of the murine digestive tract. At 24 h after the administration of cycloheximide, FGA1 was preferentially produced in the upper half of the duodenal microvilli.

Lewis (y) antigen overexpression increases the expression of MMP-2 and MMP-9 and invasion of human ovarian cancer cells.

Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion.

Characteristic expression of globotriaosyl ceramide in human ovarian carcinoma‐derived cells with anticancer drug resistance

The transporter protein genes and lipids in human ovarian carcinoma‐derived KF28 cells with anticancer‐drug‐sensitive properties were compared with those in resistant cells, taxol‐resistant KF28TX, cisplatin‐resistant KFr13, and taxol‐ and cisplatin‐resistant KFr13TX, to identify the molecules required for anticancer‐drug resistance. In accordance with previous reports, taxol and cisplatin resistance was closely correlated with expression of the multidrug resistance 1 and bile acid export pump, and multidrug resistance‐associated protein 2 genes, respectively. In addition, we found a distinct difference in glycosphingolipids between the sensitive and resistant cells. Although GlcCer was the major glycolipid (83.0%) in sensitive cells, GalCer, LacCer and, particularly, Gb3Cer were characteristically increased in all resistant cells, irrespective of whether the resistance was to taxol or cisplatin, and comprised 65–84% of total glycosphingolipids. GM3, which was present at 0.04 µg/mg dry weight in the sensitive cells, showed a twofold increase in the taxol‐resistant cells, but was absent in the cisplatin‐resistant cells. The altered glycolipid composition was proven to be due to enhanced or suppressed expression of the respective sugar transferase genes. In addition, the ceramide moiety of ceramide monohexoside in the sensitive cells constituted 83% of non‐hydroxy fatty acids, but that in the resistant cells comprised 67–74% of α‐hydroxy fatty acids. Thus, cells containing Gb3Cer with α‐hydroxy fatty acids were found to survive selectively in the presence of taxol and cisplatin, and modification of the glycolipid structure was revealed to occur in association with anticancer‐drug resistance. (Cancer Sci 2006; 97: 1321–1326)

Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways

Objective To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells. Methods mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot. Results Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression. Conclusion Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.

Changes in the glycolipid composition and characteristic activation of GM3 synthase in the thymus of mouse after administration of dexamethasone

AbstractGlycolipids in the thymus of mice after administration of dexamethasone were compared with those in control mice. In parallel with a decrease in the tissue weight due to the disappearance of immature thymocytes in the cortex, the amounts of GlcCer, Gg4Cer and GM1 decreased from 18 h after intraperitoneal administration of dexamethasone, but those of Gb4Cer and Forssman glycolipid did not change, indicating the differential distribution of ganglio- and globo-series glycolipids in the thymus, GlcCer, Gg4Cer and GM1 being on dexamethasone-sensitive cortical thymocytes, and Gb4Cer and Forssman glycolipid on dexamethasone-resistant cells including thymic stromal cells, respectively. At the same time, a characteristic increase in GM3, whose amount per thymus and concentration per mg of thymus were increased 4-fold and 13-fold compared to those in the control mice, respectively, was observed at the onset of the decrease in tissue weight and was due to the increased activity of LacCer sialyltransferase with the enhanced expression of its gene and the concomitant decrease in cytosolic sialidase activity. One can suggest that endogenous accumulation of GM3 is involved in the dexamethasone-induced apoptosis of cortical thymocytes. On radiolabeling of the thymus with CMP-[14C]-NeuAc, the incorporation of radioactivity into GM3 was preferentially observed in the thymuses of dexamethasone-administered mice, but not in those of control mice, suggesting the possible involvement of plasma membrane-associated sialytransferase in GM3 synthesis in the thymuses of dexamethasone-administered mice. Published in 2005.

Estrogen sulfotransferase and sulfatase: Roles in the regulation of estrogen activity in human uterine endometrial carcinomas

The regulation of estrogen activity through the formation and cleavage of sulfoconjugates of estrogens is known to be related to the progression and metastasis of estrogen‐dependent breast carcinomas, but the involvement of sulfoconjugates in the steroid stimulation of endometrial functions and the progression of endometrial adenocarcinomas is not clearly understood yet. Estrogen sulfotransferase (EST) in the uterine endometria during the follicular phase was more active than during the luteal phase, but estrogen sulfate (ES) sulfatase exhibited lower activity during the follicular phase than during the luteal phase. However, ES sulfatase activities in cancerous tissues were lower than those in normal endometria and endometrial adenocarcinoma‐derived cells, among which the activity was exceedingly high in Ishikawa cells, suggesting that ES sulfatase in Ishikawa cells contributes to the estrogen‐dependent growth of these cells. EST activities higher than that in Ishikawa cells were found in only 3 of 24 cancerous tissues. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of the EST and ES sulfatase genes in carcinoma‐derived cells demonstrated the extensive expression of both genes in Ishikawa cells. The isolated EST gene was transfected into Ishikawa cells with a mammalian expression vector to establish cell clones with enhanced EST activity, and the estrogen‐dependent cell growth of the resultant cell clones was found to be abolished, due to the enhanced sulfoconjugation of estrogen. Since ES sulfatase activity in cancerous tissues was significantly lower than that in Ishikawa cells, it might be not involved in the enhancement of estrogen activity associated with the pathogenesis of endometrial adenocarcinoma tissues.