Ganglioside GD3/GD2 are over-expressed in various neuroectoderm-derived tumors. Previous studies indicated that GD3 is involved in the enhancement of cancer properties such as rapid growth and increased invasiveness. However, little is known about the functions of GD3/GD2 in glioma cells and glioma microenvironments. To clarify the functions of GD3/GD2 in gliomas, we used a mouse glioma model based on the RCAS/Gtv-a system. At first, we compared the gliomas size between wild-type (WT) and GD3 synthase (GD3S) knockout (KO) mice, showing a less malignant histology and slower tumor growth in GD3S-KO mice than in WT mice. Immunohistochemistry of glioma sections from WT and GD3S-KO mice revealed that reactive microglia/macrophages showed different localization patterns between the two genetic types of mice. CD68+ cells were more frequently stained inside glioma tissues of GD3S-KO mice, while they were stained mainly around glioma tissues in WT mice. The number of CD68+ cells markedly increased in tumor tissues of GD3S-KO mice at 2 weeks after injection of transfectant DF-1 cells. Furthermore, CD68+ cells in GD3S(-/-) glioma tissues expressed higher levels of inducible nitric oxide synthase. We observed higher expression levels of pro-inflammatory cytokine genes in primary-cultured glioma cells of WT mice than in GD3S-KO mice. DNA microarray data also revealed differential expression levels of various cytokines and chemokines in glioma tissues between WT and GD3S-KO mice. These results suggest that expression of GD3S allows glioma cells to promote polarization of microglia/macrophages towards M2-like phenotypes by modulating the expression levels of chemokines and cytokines.
GalNAc-disialyl Lc4 (GalNAc-DSLc4) was reported as a novel antigen that associated with malignant features of renal cell cancers (RCCs). To clarify roles of GalNAc-DSLc4 in malignant properties of RCCs, we identified B4GalNAc-T2 as a responsible gene for the synthesis of GalNAc-DSLc4, and prepared stable transfectants of GalNAc-T2 cDNA using VMRC-RCW cells, resulting in the establishment of high expressants of GalNAc-DSLc4. They showed increased proliferation and invasion, and specific adhesion to laminin. In the transfectants, PI3K/Akt signals were highly activated by serum stimulation or adhesion to laminin. GalNAc-DSLc4 was co-localized in lipid rafts with integrin β1 and caveolin-1 in both immunoblotting of fractionated detergent extracts and immunocytostaining, particularly when stimulated with serum. Masking of GalNAc-DSLc4 with antibodies as well as PI3K inhibitor suppressed malignant properties of the transfectants. These results suggested that GalNAc-DSLc4 is involved in malignant properties of RCCs by forming a molecular complex with integrins in lipid rafts.
The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (α1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed α1,4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.
Since cDNAs of glycosyltransferase genes were isolated, and become applicable for genetic engineering of glycosylation patterns, biological functions of glycolsphingolipids have been largely elucidated via glyco-remodeling cells and animals. The progress in these glycobiology techniques has enabled us to understand the roles of "tumor-specific" carbohydrates during these 3 decades. Tumor antigens recognized by host immune systems of cancer patients were classified into three classes based on "autologous typing", that is, class 1: individual antigens present only in the patient's tumors, class 2: shared antigens among some group of cancers, but not in normal cells, class 3: universal antigens that are present in not only some cancers but also in normal cells. Many of class 2 antigens have been elucidated to be carbohydrate antigens, and are considered to be differentiation antigens. Many of them have been used for cancer diagnosis and treatment. Functional analyses of those cancer-associated glycosphingolipids based on the genetic engineering of glyco-genes have revealed that disialylated glycolipids generally enhance malignant properties such as cell growth, invasion, and motility. On the other hand, monosialylated glycolipids rather suppress those phenotypes. As a regulatory platform for cell signaling, cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been proposed. Interestingly, cancer-associated glycosphingolipids play critical roles in the composition of GEM/rafts, and in the regulation of signal transduction. Molecular complex formation of glycolipids with membrane molecules that are defined by enzyme-mediated activation of radical sources/mass spectrometry should be a key factor to regulate cell signals and to determine the cell fates. They are also expected to be targets of the cancer treatment.
