Deoxyribonucleoside kinases in nuclear and mitochondrial DNA precursor synthesis : phosphorylation of anti-cancer nucleoside analogs in different subcellular compartments

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Immunology, Microbiology, Pathology and Infectious Diseases

Sammanfattning: Nucleoside analogs have been developed as anticancer and antiviral agents. The main mechanism of action of nucleoside analogs is the incorporation of the corresponding analog triphosphate into DNA where it interferes with DNA replication. The phosphorylation to the triphosphate form by cellular or viral enzymes is therefore required for the activation of nucleoside analogs. The first step of phosphorylation, catalyzed by nucleoside kinases, is regarded as being rate-limiting for most nucleoside analogs. DNA replication occurs both in the nucleus and in the mitochondria. Thus, both nuclear and mitochondrial DNA can be targeted by nucleoside analogs. My research mainly addresses questions regarding nucleoside analog phosphorylation in the mitochondria. First, one of the mitochondrial deoxyribonucleoside kinases, deoxyguanosine kinase (dGK), was characterized with regard to its kinetic properties for natural substrates as well as for clinically important nucleoside analogs. We showed that dGK could efficiently phosphorylate the nucleoside analogs araG, CdA and dFdG. Overexpression of dGK in the mitochondria of pancreatic cancer cell lines enhanced the sensitivity to dGK phosphorylated nucleoside analogs. These data suggested that this mitochondrial dcoxyribonucleoside kinase plays a role for nucleoside analog phosphorylation. Furthermore, we created a cell model by targeting genetically engineered dCK to the different subcellular compartments of a dCK deficient cell line. The expression of dCK in the nucleus, the cytosol or the mitochondria restored the sensitivity to dCK phosphorylated nucleoside analogs. By using autoradiography, we showed that nucleoside analogs phosphorylated by dCK in the mitochondria were predominantly incorporated into mitochondrial DNA, while nucleoside analogs phosphorylated in the nucleus or cytosol were incorporated into nuclear DNA. We concluded that incorporation of nucleoside analogs into nuclear or mitochondrial DNA was determined by the intracellular phosphorylation site. We also showed that nucleoside analogs phosphorylated in the mitochondria could initiate apoptosis similar to nucleoside analogs phosphorylated in the nucleus or the cytosol. The same cell model was used to study the cytotoxicity of nucleoside analogs in combination with the ribonucleotide reductase inhibitor hydroxyurea. The results showed that the combination of nucleoside analogs and hydroxyurea resulted in synergistic effects when the nucleoside analogs were phosphorylated in the nucleus or the cytosol but not when phosphorylated in the mitochondria. We have also shown by autoradiography that araG was predominantly incorporated into mitochondrial DNA while araC was incorporated into nuclear DNA. This finding may contribute to explain the selective cytotoxicity of araG in T-lymphocytes. In summary, our results showed that mitochondrial kinases play a role for micleoside analog activation. We believe that our findings are important in the development of improved therapeutic strategies involving nucleoside analogs.

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