Glycosylation of Prostate Cancer Cell Lines and Glycosyltransferase Characterization and Inhibition
Biochemistry , Glycosyltransferases
Altered glycosylation is a hallmark of cancer and can be used to distinguish cancer cells from normal cells. This thesis shows a comprehensive study of glycosylation on a variety of human prostate cell lines. Cell surface glycans were analyzed using lectins and antibodies. Glycans were also released from glycoproteins, purified and analyzed by tandem mass spectrometry. The enzymatic activities and expression levels of glycosyltransferases responsible for the syntheses of N- and O-glycans have been determined and correlated with glycan structures. We found that prostate cells are capable of synthesizing complex N-glycans and core 1 based O-glycans, and that each cell line has characteristic glycosylation pathways. Elevated levels of truncated O-glycans including sialyl-T and di-sialyl-T antigens were detected in prostate cancer cell lines. Complex O-glycans without sialic acid residues constituted the major O-glycans in normal prostate cells, but sialyl-T antigens were prevalent in the most aggressive prostate cancer cells derived from bone metastasis (PC-3). The expression and activity of sialyltransferase ST3Gal-I, responsible for the synthesis of sialyl-T antigens, were up-regulated in PC-3 cells, providing a mechanism for sialyl-T expression. Forced down-regulation of ST3Gal-I by RNA interfering technology as well as neuraminidase treatments of cells were employed to modify cellular glycosylation, and indicated a critical role of sialic acid in the regulation of apoptosis. These observations facilitate the identification of prostate cancer markers and help to understand the importance of glycans in cancer progression. To elucidate mechanisms of glycosylation and develop glycosyltransferase inhibitors, we characterized recombinant soluble human Gal- and GlcNAc-transferases that synthesize O-glycan cores 1 to 4, critical for the overall structures of O-glycans. The biochemical properties and substrate specificities of these enzymes were determined using synthetic acceptor substrate analogs. These collective results were used to develop novel glycosyltransferase inhibitors, including acceptor substrate analogs and bivalent imidazolium salts. These compounds inhibited selected glycosyltransferases, and thus have the potential to modify cellular glycans for functional studies. Glycosyltransferase inhibitors could also be used in biotechnology and in therapeutic applications to increase the apoptotic potential of prostate cancer cells.