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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
AP Stegmann, WH Honders, R Willemze, VW Ruiz van Haperen and JE Landegent
Department of Hematology, University Hospital Leiden, The Netherlands.
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.
Volume 85,
Issue 5,
pp. 1188-1194,
03/01/1995
Copyright © 1995 by The American Society of Hematology
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