Stimulation of Deoxycytidine Kinase Results in Prolonged Maintenance of the Enzyme Activity

A number of genotoxic and antiproliferative agents such as 2‐chlorodeoxyadenosine (Cladribine; CdA) and aphidicolin (APC) have been shown to stimulate the activity of deoxycytidine kinase, the main deoxynucleoside salvage enzyme in lymphocytes. Here we show that enzyme activation could be prevented by treating cells with the membrane‐permeant calcium chelator BAPTA‐AM. Long‐term incubations demonstrated that CdA and APC not only stimulated but also sustained deoxycytidine kinase activity in the cellular context, as compared to the control and BAPTA‐AM treated enzyme activities.     

Effects of intracellular calcium chelation and pifithrin-alpha on deoxynucleotide metabolism in human lymphocytes

Previously, we have found that activation of deoxycytidine kinase elicited by various DNA-damaging chemical agents could be prevented by BAPTA-AM, a cell-permeable calcium chelator or by pifithrin-alpha, a pharmacological inhibitor of p53. Here, we show that stimulation of deoxycytidine kinase by UV-light also is calcium-dependent and pifithrin-alpha-sensitive in tonsillar lymphocytes, while thymidine kinase 1 activity is stabilised in the presence of BAPTA-AM. Importantly, both UV-irradiation and calcium chelation decreased the incorporation of labelled deoxycytidine and thymidine into DNA. Pifithrin-alpha dramatically reduced the labelling of both the nucleotide and DNA fractions, possibly due to inhibition of transmembrane nucleoside transport.     

Selective increase of dATP pools upon activation of deoxycytidine kinase in lymphocytes: implications in apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction--presumably by supplying dATP or its analogues for the apoptosome formation--deserves consideration. Two-hour exposure of human tonsillar lymphocytes to 2-chloro-deoxyadenosine (CdA) led to a two-fold activation of dCK. This activation process was inhibited by pifithrin-alpha, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide-treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.     

Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay using reversed-phase high performance liquid chromatography; Identification of a novel metabolite of 2-chlorodeoxyadenosine

A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5'-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 microg/ml in the assay. An amount of 8 x 10(3) cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.     

Effects of Intracellular Calcium Chelation and Pifithrin-α on Deoxynucleotide Metabolism in Human Lymphocytes

Previously, we have found that activation of deoxycytidine kinase elicited by various DNA-damaging chemical agents could be prevented by BAPTA-AM, a cell-permeable calcium chelator or by pifithrin-α, a pharmacological inhibitor of p53. Here, we show that stimulation of deoxycytidine kinase by UV-light also is calcium-dependent and pifithrin-α-sensitive in tonsillar lymphocytes, while thymidine kinase 1 activity is stabilised in the presence of BAPTA-AM. Importantly, both UV-irradiation and calcium chelation decreased the incorporation of labelled deoxycytidine and thymidine into DNA. Pifithrin-alpha dramatically reduced the labelling of both the nucleotide and DNA fractions, possibly due to inhibition of transmembrane nucleoside transport.     

Effects of 2‐Chloro‐2′‐Deoxyadenosine on the Cell Cycle in the Human Leukemia EHEB Cell Line

To explain why 2‐chloro‐2′‐deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA‐resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA‐induced increase in G1 to S phase progression.     

Selective Increase of dATP Pools upon Activation of Deoxycytidine Kinase in Lymphocytes: Implications in Apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction—presumably by supplying dATP or its analogues for the apoptosome formation—deserves consideration. Two‐hour exposure of human tonsillar lymphocytes to 2‐chloro‐deoxyadenosine (CdA) led to a two‐fold activation of dCK. This activation process was inhibited by pifithrin‐α, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide‐treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.     

RNAi Depletion of Deoxycytidine and Deoxyguanosine Kinase in Human Leukemic CEM Cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.     

RNAi depletion of deoxycytidine and deoxyguanosine kinase in human leukemic CEM cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.     

Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in the human leukemia EHEB cell line

To explain why 2-chloro-2'-deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA-resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA-induced increase in G1 to S phase progression.     

Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues.

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.     

Study of the efficacy of a pronucleotide of 2-chloro-2'-deoxyadenosine in deoxycytidine kinase-deficient lymphoma cells

2-Chloro-2 '-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 '-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.     

2-Chloro-2'-deoxyadenosine synergistically enhances azidothymidine cytotoxicity in azidothymidine resistant T-lymphoid cells

This report presents quantitative analysis of the synergistic interaction of azidothymidine (AZT) and cladribine (CdA) in human H9-lymphoid cell lines sensitive and resistant to AZT (H9-araC cells). H9-araC cells obtained by cultivation of H9 cells in the presence of 0.5 microM arabinosyl-cytosine (araC) had lower deoxycytidine kinase and thymidine kinase (TK) activities and expressed cross-resistance to araC and AZT. The IC(50) values of AZT and CdA were calculated by using median-effect analysis and CalcuSyn software. The IC(50) values were 0.44 and 0.82 microM for CdA and 67.8 and 30,310 microM for AZT in H9 and H9-araC cells, respectively. However, when the drugs were used in combination the IC(50) values of CdA and AZT were reduced to 0.12 and 15.5 microM in H9 cells and to 0.19 and 24.9 microM in H9-araC cells, respectively. Calculation of dose reduction index (DRI) indicated that at 50-90% growth inhibition level, the combination of the drugs caused 3.6-5.8- and 4.1-11.5-fold reduction in the dose of CdA and 4.4-37.6- and > 1000-fold reduction in the dose of AZT in H9 and H9-araC cells, respectively. The combination index (CI) values simulated from these data suggested synergistic to very strong synergistic lymphocytotoxic effects of AZT combined with CdA. These findings suggest the potential usefulness of a double-targeted approach for designing efficacious therapeutics for the kinase deficient drug resistant tumors.     

Elevation of hyperoxia of thymidine kinase activity in hypertrophic V79 lung fibroblasts

Exposure of V79 cells to hyperoxia (80% O2) for 30 h increased the level of thymidine kinase, a deoxynucleoside salvage enzyme, by approximately 3-fold as compared to cells exposed to room air, but did not cause any significant change in deoxycytidine kinase, the other known deoxynucleoside salvage enzyme. Exposure of cells to anoxia, on the other hand, produced only a slight reduction in thymidine kinase activity. Perturbation in cellular metabolism following exposure to hyperoxia was indicated by marked inhibition of cellular growth and the presence of cellular hypertrophy. Although growth was also inhibited by anoxia, the cell size distribution was minimally altered. The effect of hyperoxia on thymidine kinase suggests that (1) this enzyme may play a role in the modulation of cellular hypertrophy and function following exposure to hyperoxia, and (2) analysis of relative levels of thymidine kinase and deoxycytidine kinase activities may be of value in differentiating between cellular hypertrophy and hyperplasia under some circumstances.     

Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography

A highly efficient new affinity medium for deoxycytidine kinase, deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose), has been constructed. A dCp4-Sepharose column effects a one-step, 19,000-fold, purification to homogeneity of dCyd kinase from the ammonium sulfate fraction of Lactobacillus acidophilus R-26 extract, with 60% recovery. dCTP, a potent end-product inhibitor, is used as an eluent, and it also stabilizes the extremely labile purified enzyme. A noncompeting deoxyadenosine kinase activity accompanies the deoxycytidine kinase activity eluted. Native polyacrylamide gel electrophoresis shows a single protein band, which coincides with both deoxycytidine kinase and deoxyadenosine kinase activities at several gel concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide band of 26,000 daltons. Since the native enzyme is known to have an Mr of 50,000, it appears that the enzyme is composed of two subunits of similar size. Sequence analysis of the intact protein from the N-terminus reveals but a single amino acid species per residue up to the 17th residue; at the 18th, 21st, 26th, and 27th residue positions of the sequence, however, there appear to be two different amino acids in almost equal amounts. This may indicate that the enzyme is composed of two nonidentical subunits having the same amino acid sequence near the N-terminus. Residues 6-13 contain the highly conserved Gly-X-X-Gly-X-Gly-Lys sequence found at the active sites of kinases and other nucleotide-binding proteins.     

Study of the Efficacy of a Pronucleotide of 2-Chloro-2′-Deoxyadenosine in Deoxycytidine Kinase-Deficient Lymphoma Cells

2-Chloro-2 ′-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 ′-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.     

Docking simulation with a purine nucleoside specific homology model of deoxycytidine kinase, a target enzyme for anticancer and antiviral therapy

5'-Phosphorylation, catalyzed by human deoxycytidine kinase (dCK), is a crucial step in the metabolic activation of anticancer and antiviral nucleoside antimetabolites, such as cytarabine (AraC), gemcitabine, cladribine (CdA), and lamivudine. Recently, crystal structures of dCK (dCKc) with various pyrimidine nucleosides as substrates have been reported. However, there is no crystal structure of dCK with a bound purine nucleoside, although purines are good substrates for dCK. We have developed a model of dCK (dCKm) specific for purine nucleosides based on the crystal structure of purine nucleoside bound deoxyguanosine kinase (dGKc) as the template. dCKm is essential for computer aided molecular design (CAMD) of novel anticancer and antiviral drugs that are based on purine nucleosides since these did not bind to dCKc in our docking experiments. The active site of dCKm was larger than that of dCKc and the amino acid (aa) residues of dCKm and dCKc, in particular Y86, Q97, D133, R104, R128, and E197, were not in identical positions. Comparative docking simulations of deoxycytidine (dC), cytidine (Cyd), AraC, CdA, deoxyadenosine (dA), and deoxyguanosine (dG) with dCKm and dCKc were carried out using the FlexX docking program. Only dC (pyrimidine nucleoside) docked into the active site of dCKc but not the purine nucleosides dG and dA. As expected, the active site of dCKm appeared to be more adapted to bind purine nucleosides than the pyrimidine nucleosides. While water molecules were essential for docking experiments using dCKc, the absence of water molecules in dCKm did not affect the ability to correctly dock various purine nucleosides.     

Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.     

Deoxycytidine kinase from human leukemic spleen: preparation and characteristics of homogeneous enzyme

Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.     

Nucleoside kinase activities of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells and appropriate drug-resistant mutants derived from them have been analyzed for nucleoside kinase activities relevant to the phosphorylation of adenosine, deoxyadenosine, deoxyguanosine and deoxycytidine and for resistance to a variety of nucleoside analogs. Fractionation of extracts by DEAE-cellulose chromatography revealed three major peaks of activity. Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20), the first to elute from the column is responsible for the majority of the deoxyadenosine phosphorylation in cell extracts and, according to resistance data, appears to phosphorylate most adenosine analogs tested, including 9-beta-D-arabinosyladenine (ara-A). A deoxyguanosine kinase, the second enzyme to elute from the column, was responsible for the majority of deoxyguanosine and deoxyinosine phosphorylation in cell extracts. The function of this enzyme in cell metabolism is unclear. 2-Chlorodeoxyadenosine, on the other hand, appeared from resistance data to be phosphorylated, at least in part, by deoxycytidine kinase (ATP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74), which in cell extracts could also phosphorylate deoxyguanosine and deoxyadenosine, though much less efficiently than deoxycytidine.