GLYCOLIPID METABOLISM IN TUMOR AND TRANSFORMED CELLS

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The immediate objectives of the proposed research are sixfold: 1. Separation, characterization, and purification of GalNAcT-2(UDP-GalNAc:GbOse3Cer(Betal-3)N-acetylgalactosaminyltransferase) and GalNAcT-3(UDP-GalNAc:GbOse4Cer(Alphal-3)N-acetyl-galactosaminyltransferase) present in the detergent solubilized supernatants from guinea pig tumor cell lines 104Cl (tumorigenic) and 106B(nontumorigenic), respectively. 2. Determination of the exact chemical linkages in the Forssman biosynthetic products of the 104Cl and 106B GalNAcT-3-catalyzed reactions. 3. Purification and kinetic studies of GalNAcT-1(UDP-GalNAc:GM3 or Lac-Cer(Betal-4)N-acetyl-galactosaminyltransferase) from guinea pig bone marrow. 4. Determination of the effects of tunicamycin and insulin on GalNAcT-2 and GalNAcT-3 activities in 104Cl and 106B guinea pig tumor cells. 5. Biosynthesis in vitro of the tumor-specific antigens sialosylAlpha2-3Le-x, sialosylAlpha2-3Le-a in neuroblastoma IMR-32 cells and GD3 and Fuc-GM1 in rat prostate PA-I/II cells. 6. Characterization of GalT-5(UDP-Gal:nLcOse4Cer(Alphal-3)galactosyltransferase) from neuroblastoma IMR-32 cells and study of its activity under various growth conditions during the cell cycle and in the presence of differentiating agent ((But)2cAMP and HMBA). Our overall goal is to develop an understanding of the specificities of glycolipid:glycosyltransferases in three tumor cell lines of guinea pig and human origin. Tunicamycin inhibits glycoconjugate biosynthesis. However, inhibition of glycosylation of glycosyltransferase is a novel observation. At the end of this project an attempt will be made to establish a correlation between inhibition of a glycosyltransferase (GalNAcT-2) by tunicamycin and the change of a tumor-specific antigen (Forssman GSL) on the surface of 104Cl and 106B cells using 125I-labeled Helix pomatia and Dolichos biflorus lectins and Siga toxin. Screening of the mouse genomic library, prepared from a partial EcoRI digestion of AJ mouse genomic DNA (Shotgun in Charon 4a) with a 32P-labeled cDNA insert of GalNAcT-2 will be performed. Using 125I-mono and polyclonal antibodies the translated products for particular 104Cl GalNAcT-2 DNA sequences will also be screened.