Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs
Författare
Summary, in English
Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell lines in the presence of the NAs fludarabine (F-AraA), cladribine (CdA), vidarabine and cytarabine were compared to the parental cell lines. The sensitivity of 143B cells was increased by 470-fold in the presence of CdA and of U-87M-G cells by 435fold in the presence of F-AraA. We also show that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution.
Avdelning/ar
Publiceringsår
2007
Språk
Engelska
Sidor
1278-1286
Publikation/Tidskrift/Serie
Gene Therapy
Volym
14
Issue
17
Dokumenttyp
Artikel i tidskrift
Förlag
Nature Publishing Group
Ämne
- Biological Sciences
Nyckelord
- purines
- nucleoside analogs
- directed evolution
- deoxyribonucleoside kinase
- cancer gene therapy
Status
Published
ISBN/ISSN/Övrigt
- ISSN: 0969-7128