Facile method for determination of deoxycytidine kinase activity in biological milieus

Abstract

A new analytical method for determining deoxycytidine kinase (dCK) activity in biological milieus using luminescence is reported here. This method, based on utilizing adenosine triphosphate (ATP) as the sole phosphate donor in the kinase reaction and monitoring ATP consumption via a luciferase-based chemiluminescence reaction, is capable of detecting dCK activity without the use of specific substrates or radioisotope techniques. Comparing with the reverse-phase high-performance liquid chromatography method, this new method is suggested to be efficient and sensitive. Further, application of the proposed method for profiling dCK activity in cultured cancer cells revealed that a cervix cell line exhibited the highest dCK activity to gemcitabine metabolism.

Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

“…To be able to assess the enzymatic activity of dCK in cells, we have developed an in-house assay based on the enrichment of dCK from cell lysate through anion exchange chromatography (Hao et al 2014 ) combined with a commercially available luminescence release assay (Fig. 1 ) that reflects residual ATP concentration in the reaction medium (expressed as RLUs), and thereby dCK enzymatic activity.…”

“…Cells (5 × 10 6 collected from 10 wells of 24-well plates) were lysed using CytoBuster™ Protein Extraction Reagent (Merck KGaA, Germany) following manufacturer’s protocol. Enrichment of dCK from cell lysates was performed essentially as described (Hao et al 2014 ) with some modifications. Briefly, 0.5 ml cell lysate were incubated in a 1.5 ml vial for 5 min with Q Sepharose ® anionic exchange beads (1:3 volume of lysate; Merck KGaA, Germany) pre-equilibrated with 0.01 M Tris-HCl buffer (pH 7.0).…”

Fludarabine resistance mediated by aminoglycoside‐3'phosphotransferase‐IIa and the structurally related eukaryotic cAMP‐dependent protein kinase

“…Indeed, the effectiveness of fludarabine depends on the intracellular concentration of F‐Ara‐ATP (28). Related to this, the level of expression of dCK has been associated with the response to fludarabine (29) and nelarabine (30). Our results indicate that a dephosphorylation of F‐Ara‐ATP could be the resistance mechanism mediated by APH(3')‐IIa.…”