title: A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells.
abstract: In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation 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 concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.
authors.affiliation: Department of Hematology, Leiden University Medical Center, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Veuger
authors.forename: M W
authors.initials: MW
authors.lastname: Honders
authors.forename: J E
authors.initials: JE
authors.lastname: Landegent
authors.forename: R
authors.initials: R
authors.lastname: Willemze
authors.forename: R M
authors.initials: RM
authors.lastname: Barge
mesh_terms: D000208:Acute Disease; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000201:enzymology; D003561:Cytarabine / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007951:Leukemia, Myeloid / Q000201:enzymology* / Q000473:pathology; D010835:Phytohemagglutinins / Q000494:pharmacology; D014176:Protein Biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes / Q000201:enzymology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D010835:Phytohemagglutinins; D003561:Cytarabine; D003842:Deoxycytidine Kinase