Transfection of wild-type deoxycytidine kinase (dck) cDNA into an AraC- and DAC-resistant rat leukemic cell line of clonal origin fully restores drug sensitivity.
The AraC-resistant rat leukemic cell line RO/1-A has been shown to have a typical deoxycytidine kinase (DCK)-deficient phenotype and cannot metabolize the antileukemic drugs cytarabine (AraC) and decitabine (DAC). To investigate the relative contribution of mutations in the dck gene to the development of in vitro-induced AraC-resistance, a neomycin selectable plasmid construct harboring the wild-type dck coding region was transfected into RO/1-A. Polymerase chain reaction analysis confirmed the presence of vector DNA in the target cells (RO/1-ADCK) that were stably transfected and monitored over a period of 14 weeks. Northern and Western blot analysis showed restoration of dck mRNA and protein expression. Initial rate measurements of DCK activity showed that Km values for dck were only slightly altered as a result of transfection, whereas strongly increased Vmax values were observed, resulting in a 12-fold increased phosphorylation efficiency for both dC and AraC, compared with the AraC-sensitive parental cell line RO/1 from which the RO/1-A was originally derived. In vitro sensitivity to AraC- and DAC-mediated cytotoxicity was fully restored in RO/1-ADCK. The data pinpoint acquired DCK deficiency caused by mutations of the dck gene as the major cause of AraC resistance in this model.
Stegmann
A P
AP
Department of Hematology, University Hospital Leiden, The Netherlands.
Honders
W H
WH
Willemze
R
R
Ruiz van Haperen
V W
VW
Landegent
J E
JE
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D003561:Cytarabine / Q000378:metabolism / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D000077209:Decitabine; D003842:Deoxycytidine Kinase / Q000235:genetics* / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D007942:Leukemia, Experimental / Q000473:pathology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D003561:Cytarabine; D000077209:Decitabine; D003842:Deoxycytidine Kinase; D001374:Azacitidine