Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine

AbstractA novel O-acetylated GM3 containing 3-O-acetyl 4-sphingenine was isolated with one having a non-acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one- and two-dimensional proton nuclear magnetic resonance, and fast atom bombardmentmass spectrometries. In addition, the O-acetyl showed higher immunological activity toward anti-melanoma antibody in the presence of non-acetylated GM3 in complement-dependent liposome lysis than did non-acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O-acetyl GM3

DNA diagnosis in the NF2 gene

Neurofibromatosis 2 (NF2) is a genetic disorder characterized by the develop-ment of multiple tumors in the central nervous system. Recently, the NF2 gene has been cloned and found to encode a novel member of the protein 4.1 family which is thought to link integral membrane proteins to the cytoskeleton. The identification of the NF2 tumor suppresser gene has allowed us to screen for pathological mutations in the gene. We have studied germline mutations in the NF2 gene by direct sequence analysis of genomic DNA from blood samples of NF2 patients. In the present report, we demonstrate a novel pathological missense mutation in a patient with NF2, which reveals that the variant observed may affect important functional regions or alter the protein on a larger scale by affecting conformation or degradation

Biochemical Analysis of Cytokine Effect on the Glycosphingolipid Expression of Human Glioma Cell Lines

We have already found that a human glioma cell line U118MG could modu-late glycosphingolipid (GSL) expression via certain cytokines, though this effect is changeable and very difficult to reproduce even under consistent culture condi-tions. Consecutive analysis of cytokine effect using other 6 glioma (A172, T98G, KG-1C, U87MG, U138MG and U373MG) and one melanoma cell line (Mewo) failed to detect any GSL modulation. A total of ten cytokines were used in this experiment : IL-1beta, IL-2, IL-4, IL-6, IL-8, TNF-alpha, IFN-alpha, IFN-beta, IFN-gamma, and G-CSF, for treating KG-1C mixed glioma cells and 8 cytokines out of those for treating T98G glioblastoma cells. The rest of the cell lines were cultured with IFN-gamma and IL-4 because those two had once shown dramatic effect on GSL expression on U118MG cells. The fact that none of the 6 glioma cell lines responded to cytokines in terms of GSL modulation implies that cytokines do not play a significant role in regulating GSL expression in the glial cell lineage

Cell Density Regulates Antibody Accessibility and Metabolic Turnover of Gangliosides in Human Glioma Cells

The effects of cell density on the gangliosides of 4 human glioma cell lines were studied. The cell lines used were KG-1C (GM3-dominant) , A172 and H4 (GM2-dominant), and Hs683 (GM3, GM2-co-dominant) cells. All these cell lines showed higher immunofluorescence with anti-ganglioside antibodies in FACS analysis at sparse density than at confluent density. Steric hindrance from cell surface proteins had been removed by the pretreatment of the cells with trypsin. The chemical content of gangliosides was consistent throughout the time of cell growth. The mechanisms of crypticity of gangliosides at confluent culture were under investigation. We first evaluated the metabolic turnover rate of gang-liosides at different cell densities. The results clearly showed a more rapid turn-over of gangliosides at sparse density from approximately 2 to 4 fold in terms of radioactivities of incorporated tritium into gangliosides. The profiles of labeled gangliosides were also different between the sparse and confluent cultures. We speculate that better accessibilities of antibodies toward gangliosides should be facilitated by the same mechanism which should, in turn, provide easier access of carbohydrate-hydrolysis enzymes to gangliosides at sparse cell density in order to keep an enhanced turnover rate

Reduction of glycolipids with D-PDMP, a glucosylceramide synthetase inhibitor, caused cell growth inhibition, enhanced cell adhesion, and facilitated cell motility in human glioma cells

Glycolipid synthesis inhibitor, D-PDMP, not only inhibited the production of glycolipid but also inhibited cell growth in human glioma cell line KG-1C in a cell cycle non-dependent manner. The reduction of glycolipid from the cell membrane allowed us to study the biological functions of glycolipids. The ability of cells to adhere to collagen was enhanced by the reduction of glycoli-pids, and random cell migration was also activated by the effect of D-PDMP. The results supported our speculation that glycolipids might function in cell growth, adherence and locomotion

Modulatory Effect of Cytokines on Glycolipid Expression of Human Glioma Cell Line U118MG : Changeable response and gradual loss of sensitivity to cytokines of glioma cells in culture

The effect of immune cytokines on the composition of neutral and acidic glycolipids was analyzed. Untreated U118MG cells expressed four kinds of neu-tral glycolipid molecules (CMH, CDH, CTH, and Gb?Cer), and two kinds of acidic glycolipid (GM3 and GM2) as its major components. Effect of immune cytokines on the composition of the glycolipid composition was analyzed. U118MG cells were grown subconfiuently before a panel of cytokines (IL-lbeta, 100U/ml ; IL-2, 4000JRU/ml ; IL-4, 100U/ml ; IL-6, 100 ng/ml ; IL-8, 100 ng/ ml ; IFN-alpha, 1000U/ml ; IFN-beta, 1000U/ml ; IFN-gamma, 1000U/ml ; TNF- alpha 10U/ml, and G-CSF 2 ng/ml) were pulsed for 48 hrs. In the neutral glycolipids new bands supposed to be subspecies of CMH and CDH with different fatty acid chains were recognized in the cells treated with IFN-alpha, IL-4, IL-6 and IL-8, although no constitutional change of sugar moieties was noted. In the acidic ones, IFN-gamma and IL-4 treatment brought about remarkable changes in the glycolipid profile enhancing sulfatides, GM1, GD1a, GD1b, and GT1b of the acidc lipids, with or without GM2 and GM3 expression. TNF-alpha also induced GM1. Repeated experiments with the same culture conditions revealed different responses to cytokines in the expression of subspecies of CMH and CDH, and significant changes in the acidic glycolipids were noted in IL-2, G-CSF, TNF-alpha and IL-4 treatment. The 3rd series of repeated experiments using TNF-alpha, IFN-gamma, IL-4 and IL-6 showed only induction of subspecies of CMH by IFN-gamma ; no alteration of acidic glycolipid profile was noted. The 4th series of the same experiments in the same conditions resulted in no response. Raising the dose of cytokines, change of the passage of the cells, addition of DMSO (a potent differentiation factor) did not restore the sensitivity to the cyto-kines. Similar experiments using 6 other glioma cell lines and one melanoma cell line did not induce any glycolipid modulation. We believe that glioma cells have the potential to respond to certain cytokines to modulate their glycolipid expression under undetermined special conditions, and they are biologically unsta-ble in terms of cytokine sensitivity in culture