Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

While gemcitabine (2'-2'-difluoro-2'-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.     

Potential of ribozymes against deoxycytidine kinase to confer drug resistance to cytosine nucleoside analogs

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs.     

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.     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn²⁺-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.     

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.     

Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA

The nucleoside analogs 9-β-D-arabinofuranosylguanine (araG) and 1-β-d-arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ⁰ cells (lacking mtDNA). The mRNA levels and activities of deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p < 0.01) in the ρ⁰ cells, whereas the TK1 mRNA level was lower (p < 0.05). The enzyme activity of the S-phase restricted TK1 was also lower (p < 0.05) in the MOLT-4 ρ⁰ cells, whereas the activities of dGK, dCK and TK2 were similar in MOLT-4 wt and ρ⁰ cell lines. The sensitivities to different cytotoxic nucleoside analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells.     

Down-regulation of mitochondrial thymidine kinase 2 and deoxyguanosine kinase by didanosine: Implication for mitochondrial toxicities of anti-HIV nucleoside analogs

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.     

Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line

In order to study nucleoside analog activation in the CEM cell line, a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation. Hematopoetic cell lines can be difficult to transfect with traditional lipid-based transfection, so the electroporation technique was used. Field strength, pulse length, temperature, electroporation media, siRNA concentration, among other conditions were tested in order to obtain approximately 70–80% mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase (dCK), necessary for nucleoside analog activation. Downregulation was assessed at mRNA and enzyme activity levels. After optimizing the protocol, a microarray analysis was performed in order to investigate whether the downregulation was specific. Additionally two genes were differentially expressed besides the downregulation of dCK. These were however of unknown function. The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs. Three of these pools were increased compared to untreated, unelectroporated cells. The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected. The multidrug resistance to other drugs, as seen in nucleoside analog-induced resistant cells, was not seen with this model.     

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k_cat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.     

Incorporation of nucleoside analogs into nuclear or mitochondrial DNA is determined by the intracellular phosphorylation site

Nucleoside analogs used in cancer chemotherapy and in treatment of virus infections are phosphorylated in cells by nucleoside and nucleotide kinases to their pharmacologically active form. The phosphorylated nucleoside analogs are incorporated into DNA and cause cell death or inhibit viral replication. Cellular DNA is replicated both in the nucleus and in the mitochondria, and nucleoside analogs may interfere with DNA replication in both these subcellular locations. In the present study we created a cell model system where nucleoside analogs were phosphorylated, and thereby pharmacologically activated, in either the nucleus, cytosol, or mitochondria of cancer cells. The system was based on the reconstitution of deoxycytidine kinase (dCK)-deficient Chinese hamster ovary cells with genetically engineered dCK targeted to the different subcellular compartments. The nucleoside analogs phosphorylated by dCK in the mitochondria were predominantly incorporated into mitochondrial DNA, whereas the nucleoside analogs phosphorylated in the nucleus or cytosol were incorporated into nuclear DNA. We further show that the nucleoside analogs phosphorylated in the mitochondria induced cell death by an apoptotic program. These data showed that the subcellular site of nucleoside analog phosphorylation is an important determinant for incorporation of nucleoside analogs into nuclear or mitochondrial DNA.     

Bystander effects of nucleoside analogs phosphorylated in the cytosol or mitochondria

The efficiency of nucleoside kinase suicide gene therapy for cancer is highly dependent on "bystander" cell killing, i.e., the transfer of cytotoxic phosphorylated nucleoside analogs to cells adjacent to those expressing the suicide enzyme. We have recently studied the possible use of mitochondrial nucleoside kinases as suicide genes. In the present study, we investigated if nucleoside analogs phosphorylated in the mitochondrial matrix cause bystander killing. We used deoxycytidine kinase-deficient Chinese hamster ovary cells reconstituted with deoxycytidine kinase targeted to either the cytosol or mitochondria matrix and determined the bystander cell killing when these cells were incubated with the nucleoside analogs 1-beta-D-arabinofuranosylcytosine and 2',2'-difluorodeoxycytidine. A bystander effect occurred when nucleoside analogs were phosphorylated in the cytosol, but not when these compounds were phosphorylated in the mitochondria. These findings suggest that nucleoside kinases targeted to the mitochondrial matrix have limited use in suicide gene therapy when efficient bystander cell killing is required.     

The subcellular location of nucleoside analog phosphorylation is a determinant of synergistic effects of hydroxyurea

The ribonucleotide reductase inhibitor hydroxyurea exhibits synergistic pharmacological activity with several nucleoside analogs used in antiviral and anticancer chemotherapy. We have used a cell model system where a deoxycytidine kinase (dCK)-deficient cell line was reconstituted with genetically engineered dCK targeted to the cytosol, the nucleus, or the mitochondria to investigate how the subcellular location of nucleoside analog phosphorylation affected the synergistic effects of a ribonucleotide reductase inhibitor. Hydroxyurea showed synergistic cytotoxicity with the nucleoside analogs 1-beta-d-arabinofuranosylcytosine and 2-chloro-2'-deoxyadenosine when dCK was expressed in the cytosol or in the nucleus, but not when dCK was expressed in the mitochondria. These data indicate that the synergistic effect of ribonucleotide reductase inhibition is limited to nucleoside analogs phosphorylated in the cytosol or the cell nucleus.     

Mechanisms of cross-resistance between nucleoside analogues and vincristine or daunorubicin in leukemic cells

The aim of this study was to clarify the biochemical and molecular mechanisms behind the cross-resistance to nucleoside analogues (NAs) in four erythroleukemic cell lines with acquired resistance to the anthracycline daunorubicin and to the vinca alkaloid vincristine, expressing high levels of p-glycoprotein (P-gp, MDR1). All resistant strains exhibited cross-resistance to NA (cladribine and cytosine arabinoside)-induced apoptosis, assessed by caspase-3-like activation and were less sensitive to NA cytotoxicity in MTT assay. Real-time PCR and enzyme activity analysis showed reduced amounts of deoxycytidine kinase (35-80%) and elevated levels of 5'-nucleotidases (50-100%). The ratio 5'-nucleotidase to deoxycytidine kinase increased between 2.5- and 7.5-folds in resistant cells. This is in agreement with the observation that 5'-nucleotidase/deoxycytidine kinase ratio might be an important factor in predicting resistance to NAs. Implications of this finding for combining anthracyclines or vinca alkaloids with NAs toward leukemic cells are discussed.     

Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase

Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.     

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.     

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.     

The purification and characterization of deoxycytidine kinase from calf thymus

A facile and fast approach for the purification of deoxycytidine kinase (dCK) from calf thymus was developed using a fast performance liquid chromatography system. A 73-fold enrichment of the enzyme was noted compared to unfractionated dCK. Characterization studies demonstrated that dCK had a molecular mass of 31 kDa using SDS–PAGE, an optimum pH of 7.0 and the enzyme maintained stability between 30 and 40°C. The rapid preparation of dCK demonstrated here will be valuable in the synthesis of nucleotide analogs.     

Cytostatic and cytotoxic effects of (E)-2'-deoxy-2'-(fluoromethylene)-cytidine on a solid tumor and a leukemia cell line

(E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), a deoxycytidine analog displaying a very high toxicity toward a variety of solid tumor cell lines and xenografts, is activated intracellularly by deoxycytidine kinase (dCK). We have compared cytotoxicity of FMdC towards a human promyeolocytic leukemia line HL-60 and a human colorectal carcinoma line COLO-205. Despite dCK activity being by far the highest in cells of lymphoid origin, the effects of FMdC were detectable at the lowest drug concentration only in a solid tumor cell line, and at higher concentrations they were qualitatively similar in the two tumor lines (increased cell protein content, cell cycle block and apoptosis). Apparently, low dCK activity in solid tumor cells sufficiently activates FMdC to yield cytotoxic effects, while high dCK activity in leukemia cells does not increase its cytotoxicity.     

Differential incorporation of 1-beta-D-arabinofuranosylcytosine and 9-beta-D-arabinofuranosylguanine into nuclear and mitochondrial DNA

The anti-leukemic nucleoside analogs 1-beta-D-arabinofuranosylcytosine (araC) and 9-beta-D-arabinofuranosylguanine (araG) are dependent on intracellular phosphorylation for pharmacological activity. AraC is efficiently phosphorylated by deoxycytidine kinase (dCK). Although araG is phosphorylated by dCK in vitro, it is a preferred substrate of mitochondrial deoxyguanosine kinase. We have used autoradiography to show that araC was incorporated into nuclear DNA in Molt-4 and CEM T-lymphoblastoid cells as well as in Chinese hamster ovary cells. In contrast, araG was predominantly incorporated into mitochondrial DNA in the investigated cell lines, without detectable incorporation into nuclear DNA. These data suggest that the molecular targets of araG and araC may differ.