GLYCOSYLTRANSFERASE GENE EXPRESSION IN TRANSFORMED CELLS

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Most cell surface proteins of mammalian cells are posttranslationally modified to express branched oligosaccharides. One of the characteristic changes in cellular pathology during neoplastic transformation is the increase in the branching of ASN-linked oligosaccharides. Furthermore, these changes in branching patterns have been correlated with metastatic potential of malignant cells. For example, a 2-fold increase in [GlcNAcbeta(1 ,6)Man] sequences occurs when fibroblasts are transformed by tumor viruses or by the ras oncogene alone. The addition of these branches is performed by the sequential action of specific glycosyltransferases. The regulation and expression of the specific glycosyltransferases. the regulation and expression of the genes encoding these enzymes in both normal and malignant cells are not well characterized. A better understanding of the control of these genes is necessary to understand, in detail, their role in regulating oncologic pathology and the consequences of the cell surface changes which they cause. The goals of this project are to identify and isolate by molecular cloning the genes and cDNAs encoding N-acetylglucosaminyltransferases I and V to be used as probes for the analysis of the expression of these genes. We describe preliminary data to show that we can generate, identify and isolate revertants of mutant, enzyme-deficient cell lines using DNA- mediated transformation with human genomic DNA combined with screening by differential lectin binding. We are now cloning the human DNA sequences from these revertants to determine whether they contain the human glycosyltransferase gene. We also will use a complementary approach to directly clone the cDNAs for these genes using eucaryotic expression vectors and a transient, panning selection scheme which will be much faster. After these sequences have been cloned, characterized and sequenced, they will be used as probes to study the expression of these genes during Neoplastic transformation and during the in vitro differentiation of HL60 cells. These probe will also be used to determine the molecular basis of the lesions in the mutant cells lines.