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

Abstract

Deoxyribonucleoside kinases catalyze the phosphorylation of deoxyribonucleosides to 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 phosphorylate several clinically important anti-cancer and anti-viral nucleoside analogs. The nucleoside analogs are inactive prodrugs that are dependent on intracellular phosphorylation for pharmacological activity.

We have cloned and expressed the cDNAs of human dGK and TK2. The predicted primary structures of human dCK, dGK and TK2 are = 30% similar, and these enzymes are sequence-related to 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 the deoxyribonucleoside kinases in the pathogenesis of these disorders.

Toxicity of dCK phosphorylated nucleoside analogs differ between human and mouse cells. We have identified differences in kinetic properties of recombinant human and mouse dCK for both deoxyribonucleosides, nucleoside analogs, and phosphate donors that may be determinants of the species differences observed in vivo.

We cloned the complete mouse dCK gene and determined its structure to enable future studies on the physiological importance of dCK by gene targeting experiments in transgenic mice. To study the transcription regulation of dCK, we cloned and determined the DNA sequences of = 1.7 kbp upstream of both the human and mouse dCK genes. This region contained five major interspecies sequence conserved regions that included the transcription start site and an SP1 binding site. Reporter gene assays in transiently transfected human cell lines showed, however, no transcription regulatory properties of the other three identified regions.

Human TK1 and dCK are described as cytosolic enzymes in the literature, whereas dGK and TK2 are believed to be located in the mitochondria. We have determined the subcellular locations of the deoxyribonucleoside kinases and showed that "cytosolic" dCK is located in the cell nucleus. Human dGK was, as expected, located in the mitochondria and both TKl and TK2 were predominantly located in the cytosol. Nuclear import of dCK was mediated by a signal sequence in the N-terminal region. The nuclear location of dCK was, however, not required for cytotoxicity of dCK phosphorylated nucleoside analogs.