ganglioside--gd1b and Melanoma

Gangliosides are expressed in neuroectoderm-derived tumors, and seemed to play roles in the regulation of cancer properties. To examine the behavior and roles of individual gangliosides, GM1/GD1b/GA1 synthase cDNA was introduced into the melanoma cell line SK-MEL-37, and changes in tumor phenotypes were analyzed. The transfectant cells showed neo-expression of GD1b, GT1b, and GM1, and reduced expression of GM3, GM2, GD2, and GD3. Function analyses revealed that the transfectant cells had definite reduction in cell growth and invasion. Tyrosine-phosphorylation levels of proteins such as p130Cas and paxillin were also reduced in the transfectants. These results suggested that the expression of GM1/GD1b/GA1 synthase resulted in the suppression of tumor properties. In the analyses of the floating patterns of gangliosides using fractions from sucrose density gradient ultracentrifugation of TritonX-100 extracts, the majority of gangliosides were found in glycolipid-enriched microdomain (GEM)/raft fractions, while GD3, GD1b, and GT1b in the transfectant cells tended to disperse to non-GEM/raft fractions. Furthermore, GD3, GD1b, and GT1b in non-GEM/raft dominantly had unsaturated fatty acids, while those in GEM/rafts contained more saturated forms than in non-GEM/rafts. This might be a mechanism for the decreased tumor properties in the transfectants of GM1/GD1b/GA1 synthase cDNA.

Topics: Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chromatography, Thin Layer; Crk-Associated Substrate Protein; Flow Cytometry; Ganglioside Galactosyltransferase; Gangliosides; Humans; Intracellular Space; Melanoma; Membrane Microdomains; Paxillin; Phosphorylation; Transfection

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD(1b) but not to any other gangliosides (GM(1), GM(2), GM(3), GD(1a), GD(2), GD(3), and GT(1b)). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the sugar moiety of the ganglioside recognized by IL-2 and establishes the dosimetry of the ganglioside-IL-2 interaction. Since rIL-2 is administered systematically into stage IV melanoma patients, we have examined 45 tumor biopsies for GD(1b) content. The incidence of GD(1b) in tumor biopsies is 51%. We postulate that GD(1b) associated on the tumor or in the circulation of cancer patients may bind to rIL-2 and prevent the availability of rIL-2 to augment antitumor-immune response.

Topics: Binding Sites; Gangliosides; Humans; Immunoassay; In Vitro Techniques; Interleukin-2; Kinetics; Melanoma; Protein Binding; Recombinant Proteins

We studied the effects of various gangliosides on in vitro growth of human metastatic melanoma WM266-4. GD1b, GT1b, and GQ1b inhibited 3H-thymidine uptake and growth rate of WM266-4 whereas the other gangliosides were ineffective. The growth inhibition by GD1b, GT1b, and GQ1b was counteracted by interleukin-8 but not by the other growth factors. The growth inhibition by gangliosides was not detected in the presence of anti-interleukin-8 antibody. GD1b, GT1b, and GQ1b reduced the constitutive interleukin-8 secretion and mRNA levels in WM266-4. Transient transfection showed that GD1b, GT1b, and GQ1b inhibited the constitutive chloramphenicol acetyltransferase expression driven by interleukin-8 promoter in WM266-4. Transfection with a series of 5'-deleted mutants demonstrated that the sequences between -98 and -62 bp on interleukin-8 promoter may be involved in the transcriptional repression by these gangliosides. Cyclic AMP analog dibutyryl cAMP counteracted GD1b, GT1b, and GQ1b-induced inhibition of interleukin-8 production at the levels of protein secretion, mRNA expression, and promoter activity. GD1b, GT1b, and GQ1b reduced cAMP level and protein kinase A activity in WM266-4. These gangliosides suppressed adenylate cyclase activity without altering that of cyclic nucleotide phosphodiesterase in WM266-4. The data indicate that GD1b, GT1b, and GQ1b may suppress the growth of melanoma by inhibiting interleukin-8 production via the inhibition of adenylate cyclase.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclase Inhibitors; Cell Division; Chloramphenicol O-Acetyltransferase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Female; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Melanoma; Promoter Regions, Genetic; RNA, Messenger; Skin Neoplasms; Transcription, Genetic; Tumor Cells, Cultured