Mjt. Veuger et al., A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells, LEUKEMIA, 14(9), 2000, pp. 1678-1684
In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a
crucial role in the mechanism of resistance to cytarabine (AraC). The resis
tant phenotype in vitro is always a result of mutational inactivation of dC
K, leading to defects in the metabolic pathways of AraC. Although inactivat
ion of dCK has shown to be one of the major mechanism of resistance to AraC
in vitro, limited in vivo data are available. To improve research concerni
ng the involvement of dCK inactivation in patients with acute myeloid leuke
mia (AML), we have set up a protocol that allows direct assessment of dCK e
xpression and activity in primary human cells. In this protein activity tru
ncation assay (PAT assay), the complete coding region of dCK is amplified b
y RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream o
f the coding region in a nested PCR reaction. After in vitro transcription-
translation dCK proteins are analyzed for their molecular weight and phosph
orylating capacities. We show that this relatively quick method can be used
in purified, primary human leukemic blasts. In addition, inactivation of d
CK by point mutations, deletions or genomic rearrangements can easily be de
tected in AraC-resistant cell lines. This novel assay may contribute to fur
ther elucidate the mechanism of AraC resistance in vivo.