Yuriko Iwamori

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)

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.

Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera

Because anti-glycolipid antibodies are involved in the onset of several neurological diseases, the reactivity of glycolipids on erythrocytes and the probability of generating the antibodies were determined to clarify the contribution of glycolipids as antigens. Anti-erythrocyte antisera reacted with the following glycolipids in a species-specific manner, i.e. blood group A-active glycolipid for man, Forssman glycolipid for sheep, Gg3Cer for guinea pig, and Gg4Cer and fucosyl GM1 for rat, and the hemolytic activities of the anti-erythrocyte antisera were attenuated by absorption of the antisera with liposomes prepared from the lipids of erythrocytes to the following levels, 94.5% for man, 24.5% for sheep, 17.5% for guinea pig, and 54.5% for rat. These species-specific glycolipids on erythrocytes reacted well with the respective anti-glycolipid antisera, but Gb4Cer in man and GM1 in rat were shown to be cryptic on immunization with erythrocytes, indicating that the contribution of glycolipids as erythrocyte antigens differs among animal species.

Inhibitory activity of sulphoglycolipid derivatives towards pancreatic trypsin.

Amphipathic sulpholipids have been shown to inhibit pancreatic serine proteases due to their detergent-like properties. To evaluate the structural requirement for this inhibitory activity, we examined the effects of various derivatives of sulphoglycolipids, some of which were prepared by deacylation with sphingolipid ceramide N-deacylase, followed by acylation with acyl chloride, on the activity of pancreatic trypsin. Both deacylated sulphatides and seminolipids exhibited inhibitory activity towards trypsin without any requirement for solubilisation and preincubation. On the other hand, stronger inhibition was observed for acylated sulphatides than for deacylated ones, but increasing the chain length of the fatty acid moiety resulted in the need for a solubilisation agent and preincubation in order to achieve maximal inhibitory activity. The structural isomers of sulphoglycolipids, such as I6SO3-GalCer, and phytosphingosine- and diglyceride-containing sulphoglycolipids, showed similar inhibitory activity, indicating the involvement of sulphate and hydrophobic groups, irrespective of the fine structure, in the inhibition. Among the sulphoglycolipids examined, II3SO3-LacCer was found to exhibit the highest inhibitory activity.

Selective Reduction in α‐Hydroxypalmitic Acid‐containing Sphingomyelin and Concurrent Increase in Hydroxylated Ceramides in Murine Skin Tumors Induced by an Initiation‐promotion Regimen

The sphingomyelin cycle is activated to accumulate ceramides in the process of epidermal differentiation. We found that sphingomyelin in the epidermis of 4 different murine strains gave three bands on TLC, the lower band containing α‐hydroxypalmitic acid (C16h:0(α)). However, in the papillomas induced in the skin of SENCAR and SSIN mice by initiation with 7,12‐dimethylbenz[α] anthracene followed by promotion with 12‐O‐tetradecanoylphorbol acetate, the concentration of C16h:0(α)‐ containing sphingomyelin was selectively diminished with a concomitant increase in the concentrations of the ceramides containing α‐hydroxy fatty acids. These findings indicate a possible involvement of the selective hydrolysis of α‐hydroxy fatty acid‐containing sphingomyelin in the process of tumorigenesis in mouse skin.

Shedding of sulfated lipids into gastric fluid and inhibition of pancreatic DNase I by cholesterol sulfate in concert with bile acids.

Cholesterol sulfate (CS) and sulfatides in the epithelium of the digestive tract were found in the 1000xg supernatants of digestive fluid, particularly in gastric juices containing the duodenal contents and bile acids, there being 14-131 microg of CS and 3-54 microg of sulfatides per mg of protein in the fluid, respectively. CS and sulfatides dissolved in detergents including bile acids inactivated pancreatic trypsin to the same level as by DMSO-solubilized sulfated lipids at 37 degrees C. Similarly, pancreatic DNase I was inhibited by CS solubilized with DMSO or bile acids, but not by sulfatides or other membrane lipids at 37 degrees C. Both the sulfate group and the hydrophobic side chain of CS were indispensable structures for the inhibition of DNase I. Also, the optimum molar ratio of bile acids to CS was important for expression of the inhibitory activity of CS toward DNase I, it being 0.18 of the optimum ratio for sodium taurocholate, and the molar ratio of CS to DNase I for complete inhibition was 342:1. Thus, CS was shown to play a role as an epithelial inhibitor of DNase I in concert with bile acids.

Establishment of cells exhibiting mutated glycolipid synthesis from mouse thymus by immortalization with SV-40 virus

Immortalization with simian virus-40 and cloning of immortalized cells from mouse thymus were performed to establish cell lines for characterization of the mode of glycolipid expression in the thymic cells. Among the 25 cell lines obtained, three lines with different morphologies were established, that is, epithelial (IMTH-E), fibroblastic (IMTH-F), and asterisk-like (IMTH-I) cells, and their glycolipids, together with those in the thymus, were determined systematically. The major glycolipids in mouse thymus were the globo- and ganglio-series, both of which, were co-expressed in the three cell lines established. However, the mode of modification of the globo- and ganglio-series was distinct for each cell line. As to the globo-series, the structures with the longest carbohydrate chain for IMTH-E, -F, and -I cells were Gb3Cer, Gb4Cer, and Forssman antigen, respectively, having stepwise shorter carbohydrates at the nonreducing termini. Although the acidic glycolipids in IMTH-E cells comprised GM3 and GM2, and their sulfated isomers, IMTH-F and -I cells expressed GMlb and GDlc for the α-pathway, and up to GDI a for the a-pathway of ganglio-series glycolipids. GMlb-GalNAc present in the thymus was not detected in IMTH-F and -I cells, probably due to the lower synthetic activity for the metabolic intermediate Gg4Cer. The results indicate that the immortalization technique is useful for obtaining individual cells having unique glycolipid profiles for analysis of the functional significance and metabolism of glycolipids in the thymus.