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.

Determinants of sensitivity and resistance to gemcitabine: The roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non‐small cell lung cancer

“…Error bars indicate SDs. tween mutational inactivation of dCK and resistance of nucleoside analogues, [20][21][22][23] so we sequenced dCK mRNA. Most of the coding regions of dCK were amplified and sequenced directly.…”

“…In addition, we did not detect any mutation in dCK mRNA in the lung cancer cell lines or the gemcitabine-resistant sublines (data not shown), even though previous studies showed that resistance to nucleotide analogues was mediated by mutation in dCK. [20][21][22][23] In this connection, it is noteworthy that mutational inactivation of dCK in patients with refractory or relapsed acute myeloid leukemia is rarely observed. 33,34) Though the mechanisms of dCK inhibition remain to be fully elucidated, we consider that decreased gemcitabine metabolism to its active form by dCK inactivation is one of the mechanisms of development of acquired resistance.…”

Functional role of alternatively spliced deoxycytidine kinase in sensitivity to cytarabine of acute myeloid leukemic cells

“…An AraC-resistant cell line was derived after limiting dilution of an ex vivo-generated AraC-resistant leukemic cell line, designated K7 in this article. 24 In this cell line AraC resistance was caused by the deletion of dCK.…”

“…24 Briefly, total cellular RNA was isolated from 10 6 cells by using TRIzol (Gibco BRL, Life Technologies, Gaithersburg, MD) according to the manufacturer's protocol. Two micrograms total RNA was reverse transcribed into single-strand cDNA.…”

Absence of mutations in the deoxycytidine kinase (dCK) gene in patients with relapsed and/or refractory acute myeloid leukemia (AML)

“…Recently, Veuger et al 7 described AML patients in whom the resistant phenotype was not associated with dCK mutations, but with the expression of alternative splice variants in addition to the wild-type dCK. The alternatively spliced transcripts, in which one or more exons were deleted, were shown to code for enzymic inactive proteins in vitro.…”