Identification and characterization of nucleoside and nucleotide kinases : Pharmacological activation of anti-cancer and anti-viral nucleoside analogs

Abstract: Cells require deoxyribonucleotides to synthesize DNA. Structurally modified precursors of deoxyribonucleotides, i.e. nucleoside analogs, are used as anti-viral and anti-cancer compounds. The deoxyribonucleosides and the nucleoside analogs are phosphorylated to the triphosphate form in three subsequent phosphorylation steps, that are catalyzed by nucleoside kinases, nucleoside monophosphate kinases and nucleoside diphosphate kinases. Five of these enzymes have been cloned and characterized in this thesis. Deoxyribonucleoside kinases catalyze the phosphorylation of deoxyribonucleosides to deoxyribonucleoside monophosphates. We have cloned and expressed the cDNA of the Drosophila melanogaster deoxyribonucleoside kinase. This enzyme showed a broader substrate specificity and higher catalytic rate compared to the human enzymes and the herpes simplex virus type-1 thymidine kinase. The enzyme phosphorylated all four natural deoxyribonucleosides as well as several nucleoside analogs. The broad substrate specificity and high catalytic rate of Dm-dNK makes this enzyme an interesting candidate for suicide gene therapy. Two human uridine-cytidine kinases (UCK) were cloned and shown to catalyze the phosphorylation of uridine and cytidine to their monophosphate forms. UCK1 mRNA was ubiquitously expressed in twelve different human tissues, whereas human UCK2 mRNA was detected only in placenta. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and lq22-lq23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs and several of these were shown to be substrates of the enzymes. UCK1 and UCK2 were overexpressed in a Chinese hamster ovary cancer cell line to determine the importance of UCK mediated nucleoside analog phosphorylation. The transfected cells showed a nuclear and cytosolic location for UCK1 and UCK2, respectively. The UCK1 and UCK2 overexpressed cell line exhibited increased sensitivity to several analogs. Our data imply that UCK1 and UCK2 activity are ratelimiting for the efficacy of some ribonucleoside analogs in the cell line investigated. Nucleoside monophosphate kinases catalyze the second phosphorylation step, from the monophosphate to the diphosphate form. A novel human adenylate kinase, AK5, was cloned and shown to be predominantly expressed in brain. The enzyme phosphorylated AMP and dAMP with ATP as phosphate donor. The gene that encoded this cytosolic enzyme was localized to chromosome 1p3 1. The cytosolic human UMPCMP kinase was cloned and characterized. The UMP-CMP kinase phosphorylated CMP, dCMP, UMP and dUMP and also the nucleoside analogs dFdC-MP, AraC-MP and ddC-MP. UMP-CMP kinase mRNA was ubiquitously present in human tissues. The human UMP-CMP kinase gene was localized to chromosome 1p34 1-1p33. The cloning, recombinant expression and characterization of these five nucleoside and nucleotide kinases will allow the pathways of nucleoside and nucleoside analog metabolism to be outlined in more detail. Knowledge of the metabolic pathways will be important for the development of novel ribonucleoside- and deoxyribonucleoside analogs and for the characterization of their pharmacological activation in different tissues.