Enhanced toxicity of purine nucleoside analogs in cells expressing Drosophila melanogaster nucleoside kinase mutants.
The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is investigated for possible use as a suicide gene in combined gene/chemotherapy of cancer. The enzyme has broader substrate specificity and higher catalytic rate compared to herpes simplex type 1 thymidine kinase and other known dNKs. Although the enzyme has broad substrate specificity, it has a preference for pyrimidine nucleosides and nucleoside analogs. We have evaluated the substrate specificity and kinetic properties of Dm-dNK proteins containing M88R, V84A+M88R or V84A+M88R+A110D mutations in the amino-acid sequence. These engineered enzymes showed a relative increase in phosphorylation of purine nucleoside analogs such as ganciclovir, 9-beta-D-arabinofuranosylguanine and 2',2'-difluorodeoxyguanosine compared to the wild-type enzyme. The mutant enzymes were expressed in an osteosarcoma thymidine kinase-deficient cell line and the sensitivity of the cell line to nucleoside analogs was determined. The cells expressing the M88R mutant enzyme showed the highest increased sensitivity to purine nucleoside analogs with 8- to 80-fold decreased inhibition constant IC(50) compared to untransduced control cells or cells expressing the wild-type nucleoside kinase. In summary, our data show that enzyme engineering can be used to shift the substrate specificity of the Dm-dNK to selectively increase the sensitivity of cells expressing the enzyme to purine nucleoside analogs.
Solaroli
N
N
Department of Laboratory Medicine, Division of Metabolic Diseases, Karolinska Institute, Stockholm, Sweden. nicola.solaroli@ki.se
Johansson
M
M
Balzarini
J
J
Karlsson
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000627:therapeutic use*; D001087:Arabinonucleosides / Q000627:therapeutic use; D045744:Cell Line, Tumor; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000627:therapeutic use; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D015774:Ganciclovir / Q000627:therapeutic use; D015870:Gene Expression; D041722:Genes, Transgenic, Suicide; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000201:enzymology / Q000628:therapy*; D010770:Phosphotransferases / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage; D016415:Sequence Alignment; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods
D000970:Antineoplastic Agents; D001087:Arabinonucleosides; C096208:LY 223592; C028771:9-arabinofuranosylguanine; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase; D003849:Deoxyguanosine; D015774:Ganciclovir