GLYCOSYLATION IN EXPERIMENTAL LIVER CARCINOGENESIS

本研究提案的长期目标是深入了解 N-糖基化在致癌过程中的相关性， 新的抗体和基因探针不仅有助于更好地了解 癌症的诊断和预后。朝向 为此，我们已经证明肝结节表达一种独特的 N-乙酰葡糖胺基转移酶III（GnT-III）参与了 在癌症中检测到二等分N-乙酰葡糖胺（Gn）的生物合成 衍生的糖蛋白，而不是在正常的肝脏。的具体目标 目前的建议是：（a）完成GnT-III的纯化 （B）制备多克隆抗体， 针对纯化的酶的单克隆抗体，（c）克隆基因 对于酶和（d）研究其在实验性肝脏中的表达， 致癌过程酶将从肝结节中纯化 通过乳清酸模型使用1，2-二甲基肼作为 雄性Fischer 344大鼠中的起始剂。初步净化将是 使用我们开发的新程序完成，该程序基于 磷脂酶C处理的微粒体膜的活性。 最终纯化将在柱色谱法上实现，包括 亲和色谱法，使用一种新的亲和色谱吸附剂， 其中UDP-N-乙酰葡糖胺已在100 ℃与硫丙基-琼脂糖连接， 通过5-汞巯基键连接尿嘧啶的5位。抗体与 酶将通过标准方法制备。用于克隆 GnT-III基因，肝结节cDNA表达文库， 构建了将采用两种互补的方法。在第一种方法中 通过筛选大肠杆菌的cDNA表达文库， 结节和大鼠肾脏与抗体。在第二种方法中 寡核苷酸将基于实验确定的浓度合成。 GnT-III的氨基酸序列。寡核苷酸探针将用于 筛选相同的cDNA表达文库。克隆的cDNA将 限制性酶切图谱和测序，最终得到GnT-III的基因组DNA 将被克隆。

The long term objective of this research proposal is to gain insight on the relevance of N-glycosylation in carcinogenic process with a view to develop newer antibodies and gene probes useful not only for a better understanding of cancer but also in the diagnosis and prognosis of human cancer. Towards this end we have demonstrated that hepatic nodules express an unique N-acetylglucosaminyltransferase III (GnT-III) implicated in the biosynthesis of bisecting N-acetylglucosamine (Gn) detected in cancer derived glycoproteins and not in normal liver. The specific aims of the current proposal will be (a) to complete the purification of the GnT-Ill initiated during the past grant period, (b) to prepare polyclonal and monoclonal antibodies against the purified enzyme, (c) to clone the gene for the enzyme and (d) to study its expression during experimental liver carcinogenic process. The enzyme will be purified from hepatic nodules generated by orotic acid model using 1,2-dimethylhydrazine as the initiating agent in male Fischer 344 rats. Initial purification will be done using a novel procedure developed by us which is based on the release of the activity from the microsomal membrane by phospholipase C treatment. Final purification will be achieved on column chromatography including affinity chromatography using a new affinity chromatography absorbant in which UDP-N-acetylglucosamine has been linked to thiopropyl-sepharose at the 5 position of the uracil via a 5-mercurimercaptide bond. Antibody to the enzyme will be prepared by standard procedures. For the cloning of GnT-III gene, cDNA expression library for hepatic nodule will be constructed. Two complementary methods will be adopted. In the first method the gene will be cloned by screening the cDNA expression libraries of the nodules and rat kidney with the antibody. In the second method oligonucleotide will be synthesized based on the experimentally determined amino acid sequence of GnT-III. The oligonucleotide probe will be used to screen the same cDNA expression libraries. The cloned cDNA will be restriction mapped and sequenced and ultimately genomic DNA for GnT-III will be cloned.