GLYCOSYLATION IN EXPERIMENTAL CARCINOGENESIS

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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.