Mitochondrial expression of the Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.
The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) is studied as a candidate suicide gene for applications in combined gene/chemotherapy of cancer. We have created an engineered Dm-dNK nucleoside kinase that is targeted to the mitochondrial matrix. The enzyme was expressed in a thymidine kinase 1-deficient osteosarcoma cell line, and the sensitivity of the cells to cytotoxic nucleoside analogs was determined when the enzyme was targeted to either the nucleus or the mitochondrial matrix. Although the total deoxythymidine (dThd) phosphorylation activity was similar in cells expressing Dm-dNK in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria showed higher sensitivity to the antiproliferative activity of several pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine. Labeling studies using [3H]dThd showed that the cells expressing the mitochondrial enzyme had an increased incorporation of [3H]dThd into DNA, shown to be due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which Dm-dNK was targeted to the mitochondria. The difference in the specific activity of the dTTP pool is a result of different contributions of the de novo and the salvage pathways for the dTTP synthesis in transduced cells. In summary, these findings suggest that mitochondrial targeting of Dm-dNK facilitates nucleoside and nucleoside analog phosphorylation and could be used as a strategy to enhance the efficacy of nucleoside analog phosphorylation and concomitantly their cytostatic potential.
Solaroli
Nicola
N
Karolinska Institute, Department of Laboratory Medicine, Division of Clinical Virology F68, S-14186 Stockholm, Sweden. nicola.solaroli@ki.se
Zheng
Xinyu
X
Johansson
Magnus
M
Balzarini
Jan
J
Karlsson
Anna
A
D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000096:biosynthesis* / Q000235:genetics; D004331:Drosophila melanogaster; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology
D029721:Drosophila Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases