GLYCOLIPID METABOLISM IN TUMOR AND TRANSFORMED CELLS

拟议研究的近期目标有六个方面： 1.的分离、表征和纯化 GalNAcT-2（UDP-GalNAc：GbOse 3Cer（Betal-3）N-乙酰氨基半乳糖转移酶） 和 GalNAcT-3（UDP-GalNAc：GbOse 4Cer（Alphal-3）N-乙酰氨基半乳糖转移酶） 存在于豚鼠肿瘤的洗涤剂溶解上清液中 细胞系104 Cl（致瘤性）和106 B（非致瘤性）。 2. Forssman中精确化学键的确定 104 Cl和106 B GalNAcT-3催化反应的生物合成产物。 3. GalNAcT-1（UDP-GalNAc：GM 3或GM 4）的纯化和动力学研究 来自豚鼠骨的Lac-Cer（Betal-4）N-乙酰氨基半乳糖转移酶 骨髓 4.测定衣霉素和胰岛素对GalNAcT-2和GalNAcT-3的影响。 104 Cl和106 B豚鼠肿瘤细胞中的GalNAcT-3活性。 5.肿瘤特异性抗原的体外生物合成 神经母细胞瘤IMR-32细胞中的唾液酸基α 2 -3Le-x、唾液酸基α 2 -3Le-a GD 3和Fuc-GM 1在大鼠前列腺PA-Ⅰ/Ⅱ细胞中的表达。 6.表征 GalT-5（UDP-Gal：nLcOse 4Cer（Alphal-3）半乳糖基转移酶），来自 神经母细胞瘤IMR-32细胞及其在不同生长条件下活性的研究 在细胞周期期间和存在分化的条件下， （But）2cAMP和HMBA）。 我们的总体目标是了解 糖脂：糖基转移酶在豚鼠三种肿瘤细胞系中的表达， 人类起源 衣霉素抑制糖缀合物的生物合成。 然而，在这方面， 抑制糖基转移酶的糖基化是一个新的观察结果。 在本项目结束时，将尝试建立一个 糖基转移酶（GalNAcT-2）的抑制与 衣霉素和肿瘤特异性抗原（Forssman GSL）的变化 104 Cl和106 B细胞的表面，使用125 I标记的苹果酸， 双花扁豆凝集素和Siga毒素。 小鼠基因组的筛选 从AJ小鼠基因组DNA的部分EcoRI消化制备的文库 （Charon 4a中的鸟枪）与GalNAcT-2的32 P标记的cDNA插入物将被 执行。 使用125 I-单克隆和多克隆抗体， 还将筛选特定104 Cl GalNAcT-2 DNA序列的产物。

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.