Functions of Complex Glycosphingolipids in the Cell Proliferation, Differentiation, and Cell Death Controlled at the Gene Level of Their Synthesizing Enzymes, and Their Medical Applications

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(1)In this project, on the basis of our first success in isolating and molecularly characterizing a human relevant gene which encodes a key glycosyltransferase, ganglioside GM3 synthase (sialyltransferase-1:ST3 GalV), we next isolated by the homology cloning technique murine ST3GalV gene (located at chromosome 6C, spanning about 58 kb), which is responsible for GM3 biosynthesis. Three kinds of transcripts (L-,B1- and B2-type) of the gene were detected in mice by 5'-RACE analyses, a single transcript detectable in human organs on the other hand, whereas human GM3 synthase gene spans approximately 56 kb in the human genome, mapped near the centromere of the short arm of chromosome 2(2p11.2), consisting of seven exons and six introns, with a number of cis-acting elements for transcription in the 5'-flanking region, 3 Sp1 binding sites in the GC-rich region being critical positive regulatory element in cell-specific expression. Murine tissue-specific expressions were clarified : L-type tra … More nscript was specifically expressed in liver. (2)Transfection of this enzyme cDNA into human colon carcinoma HCT116 cells or murine invasive bladder cancer cells induced apoptosis, resulting in the remarkable suppression of cell proliferation, motility, invasiveness, and tumorigenesis in vivo whereas the ST3GalV cDNA transfection into ganglioside-deficient mouse lung carcinoma 3LL cells was interestingly shown to induce cell growth and the characteristic shedding of GM3-rich membrane microdomains (we call them ‘gangliosomes') into the medium. This indicates that biological functions of the enforced expression of ganglioside GM3 might be cell-type/organ specific. (3)We recently found that insulin resistance induced by TNF-α in adipocytes was accompanied by elevating GM3 synthase activity and its mRNA, suggesting that the increased synthesis of cellular GM3 might participate in the pathological conditions of insulin resistance in type 2 diabetes, and that monocyte-derived dendritic cells and macrophages in vitro expressed high levels of GM3 synthase, indicating that at least part of excessive GM3 in atherosclerotic intima is locally synthesized by cells overexpressing GM3 synthase. (4)Lastly we have succeeded in producing the GM3 synthase-deficient mice, which were born as apparently normal offspring, and of which tissues and organs were completely deficient in ganglioside GM3 and remarkably deficient in a- and b-series higher gangliosides. (5)Additionally in this project, we succeeded in cloning a type la membrane glycoprotein, mKirre/NEPH2,which is involved in the hematopoietic supportive capacity of OP9 mouse stromal cells, on the basis of a retrovirus-based signal sequence trap(SST) method. Less