A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells

Citation
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
Citations number
27
Categorie Soggetti
Onconogenesis & Cancer Research
Journal title
LEUKEMIA
ISSN journal
08876924 → ACNP
Volume
14
Issue
9
Year of publication
2000
Pages
1678 - 1684
Database
ISI
SICI code
0887-6924(200009)14:9<1678:ANRPAT>2.0.ZU;2-D
Abstract
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.