Cloning and characterization of human deoxyribonucleoside kinases : Phosphorylation of anti-cancer and anti-viral nucleoside analogs

Abstract: Deoxyribonucleoside kinases catalyze the phosphorylation of deoxyribonucleosidesto deoxyribonucleoside monophosphates. There are four major deoxyribonucleoside kinasesin human cells: thymidine kinase 1 (TKl), deoxycytidine kinase (dCK), deoxyguanosinekinase (dGK), and thymidine kinase 2 (TK2). The deoxyribonucleoside kinases phosphorylateseveral clinically important anti-cancer and anti-viral nucleoside analogs. The nucleosideanalogs are inactive prodrugs that are dependent on intracellular phosphorylationfor pharmacological activity. We have cloned and expressed the cDNAs of human dGK and TK2. The predicted primarystructures of human dCK, dGK and TK2 are = 30% similar, and these enzymes are sequence-relatedto both viral and prokaryotic deoxyribonucleoside kinases. The cloning of the deoxyribonucleosidekinases provides the basis for studies on their structures, substrate specificity,and activation of novel therapeutic nucleoside analogs. The genes that encode human dGK and TK2 are located at chromosomes 2pl3 and 16q22,respectively. The chromosome regions are associated with several human diseases,and the regions show allelic rearrangements or deletions in certain malignant tumours.There are, however, no obvious phenotypic features that suggest involvement of thedeoxyribonucleoside kinases in the pathogenesis of these disorders. Toxicity of dCK phosphorylated nucleoside analogs differ between human and mousecells. We have identified differences in kinetic properties of recombinant humanand mouse dCK for both deoxyribonucleosides, nucleoside analogs, and phosphate donorsthat may be determinants of the species differences observed in vivo. We cloned the complete mouse dCK gene and determined its structure to enable futurestudies on the physiological importance of dCK by gene targeting experiments in transgenicmice. To study the transcription regulation of dCK, we cloned and determined theDNA sequences of = 1.7 kbp upstream of both the human and mouse dCK genes. This regioncontained five major interspecies sequence conserved regions that included the transcriptionstart site and an SP1 binding site. Reporter gene assays in transiently transfectedhuman cell lines showed, however, no transcription regulatory properties of the otherthree identified regions. Human TK1 and dCK are described as cytosolic enzymes in the literature, whereasdGK and TK2 are believed to be located in the mitochondria. We have determined thesubcellular locations of the deoxyribonucleoside kinases and showed that "cytosolic"dCK is located in the cell nucleus. Human dGK was, as expected, located in the mitochondriaand both TKl and TK2 were predominantly located in the cytosol. Nuclear import ofdCK was mediated by a signal sequence in the N-terminal region. The nuclear locationof dCK was, however, not required for cytotoxicity of dCK phosphorylated nucleosideanalogs. Key words: deoxyribonucleoside kinase, nucleoside analog, deoxycytidinekinase, deoxyguanosine kinase, thymidine kinase 1, thymidine kinase 2, deoxyribonucleosidephosphorylation, deoxyribonucleotides, anticancer therapy, antiviral therapy 1997 Magnus Johansson ISBN 91-628-2696-4