Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster.
The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.
Bertoli
Ada
A
Karolinska Institute, Department of Laboratory Medicine, Karolinska University Hospital/Huddinge, Stockholm, Sweden.
Franco
Maribel
M
Balzarini
Jan
J
Johansson
Magnus
M
Karlsson
Anna
A
D000818:Animals; D002461:Cell Line, Transformed; D004261:DNA Replication / Q000502:physiology; D004272:DNA, Mitochondrial / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D004331:Drosophila melanogaster / Q000201:enzymology*; D006801:Humans; D010770:Phosphotransferases / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology; D012313:RNA / Q000096:biosynthesis; D013379:Substrate Specificity / Q000235:genetics; D013601:T-Lymphocytes / Q000201:enzymology*; D014161:Transduction, Genetic
D004272:DNA, Mitochondrial; D003854:Deoxyribonucleotides; D029721:Drosophila Proteins; D012313:RNA; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase