Nucleoside analog toxicity and nucleoside kinase deficiency : Effects on mitochondrial DNA

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Sammanfattning: Nucleoside analogs are modified nucleosides used in treatment of cancer and viral infections. They are dependent on intracellular phosphorylation to be pharmacologically active. Deoxyribonucleoside kinases catalyze the rate-limiting step in the phosphorylation of many clinically used nucleoside analogs. Human cells contain four distinct deoxyribonucleoside kinases that have partially overlapping substrate specificities for both naturally occurring deoxyribonucleosides as well as nucleoside analogs. The deoxycytidine analog 2 ,3-dideoxycytidine (ddC) has been used for treatment of human immunodeficiency virus (HIV) infections. ddC causes delayed toxicity, due to mitochondrial DNA (mtDNA) depletion, when cells are exposed to the drug at low concentration for prolonged periods of time. CEM T-lymphoblast cell lines resistant to the delayed toxicity of ddC were generated and studied. Although the cells were resistant to mtDNA depletion they had a retained anti-HIV activity of ddC. The ddC resistant cells were shown to have slightly increased levels of mtDNA and decreased mRNA expression of deoxycytidine kinase and thymidine kinase 2. Mitochondria are the main providers of energy for cells and these organelles have a separate genome distinct from the nuclear genome. The mitochondrial genome is dependent on the mitochondrial deoxyribonucleoside kinases for normal DNA replication. Inherited deficiency of either of the two mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), causes mtDNA depletion in patients. In order to understand the molecular mechanism of mtDNA depletion due to TK2 deficiency, a Tk2 deficient mouse strain was generated and characterized. The Tk2 deficient mice showed growth retardation, severe hypothermia and a reduced life span. They also exhibited mtDNA depletion in multiple organs. It was concluded that TK2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for the loss of this enzyme. TK2 phosphorylates several nucleoside analogs that are also substrates of other deoxyribonucleoside kinases. To elucidate the role of TK2 mediated phosphorylation in the cytotoxic effects of nucleoside analogs, a human fibroblast cell line with partial TK2 deficiency was studied. A recombinant enzyme with the same mutation found in the TK2-deficient cells was also investigated in order to obtain further insights into the mechanisms of TK2 deficiency. It was concluded that the loss of TK2 activity did not alter the cells sensitivity to nucleoside analogs and that TK2 phosphorylation may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds. The deoxyribonucleoside kinase of the fruit fly Drosophila melanogaster (Dm-dNK) is a multisubstrate enzyme that phosphorylates pyrimidine and purine deoxyribonucleosides as well as several anticancer and antiviral nucleoside analogs. The broad substrate specificity together with the high catalytic rate makes it an interesting candidate gene for suicide gene therapy. In order to understand the determinants of the substrate specificity of the enzyme and to find Dm-dNK mutants with improved kinetic properties for application in suicide gene therapy, we performed site directed mutagenesis on the enzyme. It was shown that the mutants showed partial or complete loss of activity compared with the wild type enzyme in terms of thymidine phosphorylation. This decreased phosphorylation efficiency supports their importance for substrate binding.

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