Deoxycytidine Kinase Activity Research Articles

Enhanced Activity of Deoxycytidine Kinase After Pulsed Low Dose Rate and Single Dose Gamma Irradiation

In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK protein expression was observed that indicates a post-translational regulation. The benefit of this increased activity induced by irradiation should be further investigated in combination with deoxynucleoside analogues activated by this enzyme.

Deoxycytidine Kinase is Reversibly Phosphorylated in Normal Human Lymphocytes

The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions.

The relation between deoxycytidine kinase activity and the radiosensitising effect of gemcitabine in eight different human tumour cell lines

BackgroundGemcitabine (dFdC) is an active antitumour agent with radiosensitising properties, shown both in preclinical and clinical studies. In the present study, the relation between deoxycytidine kinase (dCK) activity and the radiosensitising effect of gemcitabine was investigated in eight different human tumour cell lines.MethodsTumour cells were treated with dFdC (0–100 nM) for 24 h prior to radiotherapy (RT) (γ-Co60, 0–6 Gy, room temperature). Cell survival was determined 7, 8, or 9 days after RT by the sulforhodamine B test. dCK activity of the cells was determined by an enzyme activity assay.ResultsA clear concentration-dependent radiosensitising effect of dFdC was observed in all cell lines. The degree of radiosensitisation was also cell line dependent and seemed to correlate with the sensitivity of the cell line to the cytotoxic effect of dFdC. The dCK activity of our cell lines varied considerably and differed up to three fold from 5 to 15 pmol/h/mg protein between the tested cell lines. In this range dCK activity was only weakly related to radiosensitisation (correlation coefficient 0.62, p = 0.11).ConclusionGemcitabine needs to be metabolised to the active nucleotide in order to radiosensitise the cells. Since dFdCTP accumulation and incorporation into DNA are concentration dependent, the degree of radiosensitisation seems to be related to the extent of dFdCTP incorporated into DNA required to inhibit DNA repair. The activity of dCK does not seem to be the most important factor, but is clearly a major factor. Other partners of the intracellular metabolism of gemcitabine in relation to the cell cycle effects and DNA repair could be more responsible for the radiosensitising effect than dCK activity.

Development of Forodesine Hydrochloride (FH), an Inhibitor of Purine Nucleoside Phosphorylase, for Patients with Chronic Lymphocytic Leukemia (CLL).

Mammalian purine nucleoside phosphorylase (PNP) catalyzes the cleavage of inosine, deoxyinosine, guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. PNP deficiency in humans produces a relatively selective depletion of T cells without much effect on normal B cells. FH, a potent inhibitor of PNP, was designed based on the transition-state analog structure stabilized by the enzyme. Previous studies demonstrated that FH in the presence of dGuo inhibits the proliferation of T-lymphocytes (Kicska et al. PNAS 2001). Based on these observations, we conducted a phase I clinical trial of FH in patients with advanced T-cell malignancies. Significant antileukemic activity was correlated with an increase in plasma FH (median 5 μM) and dGuo (median 14 μM), and an accumulation of intracellular dGuo triphosphate (dGTP) (Gandhi et al, Blood, in press, 2005). High accumulation of dGTP in T-cells may be dependant on activity of deoxynucleoside kinases. Because B-CLL cells have high activity of deoxycytidine kinase, we hypothesized that they would be sensitive to FH. This postulate was tested in primary CLL lymphocytes during in vitro investigations. Lymphocytes from patients with CLL were incubated in vitro with FH (2 μM) in the presence of 10 μM dGuo. Lymphocytes from 3 of 4 patients showed an elevation in the intracellular dGTP levels to a median 30-fold at 8 hr, without any effect on other deoxynucleotides. This increase in dGTP was associated with phosphorylation of p53 at ser15, stabilization of p53, and an increase in p21 protein. The dGTP accumulation was related to induction of apoptosis measured by activation of caspase 8, 9, and 3 and cleavage of PARP. Incubation with either FH or dGuo alone did not result in dGTP accumulation or cell death suggesting that PNP inhibition by FH and phosphorylation of dGuo to dGTP are essential for CLL cell death. Based on these encouraging results, availability of oral formulation and to validate these in vitro data during clinical trial, a phase II study of FH in patients with advanced, fludarabine-refractory CLL has been initiated. FH is administered orally at a dose of 200 mg/day for 7 days each week for 4 weeks (cycle 1). Patients are evaluated after 1 full cycle of therapy and in the absence of serious side effects, or disease progression, they continue the treatment for up to 5 more cycles. Laboratory endpoints such as level of FH and dGuo in plasma, PNP activity, dGTP levels in cells, will be measured and correlated with cytoreduction. It is postulated that a high kinase/low nucleotidase activity leading to the accumulation of intracellular dGTP in target CLL cells and apoptosis (akin to what has been seen in T-cells) will result in response to therapy. This is the first trial of a PNP inhibitor for treatment of B-CLL.

Cyclopentenyl cytosine-induced activation of deoxycytidine kinase increases gemcitabine anabolism and cytotoxicity in neuroblastoma

The effect of the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) on the metabolism and cytotoxicity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) and the expression and activity of deoxycytidine kinase (dCK) was studied in human neuroblastoma cell lines. The cytotoxicity of dFdC and CPEC was studied in a panel of MYCN-amplified and MYCN-single-copy neuroblastoma cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-assays. dFdC-metabolism was studied in SK-N-BE(2)c cells using [3H]-radiolabeled dFdC. dCK activity and expression were measured using enzyme assays, immunoblot and quantitative PCR, respectively. Both MYCN-amplified and MYCN-single-copy neuroblastoma cell lines were highly sensitive to dFdC, with concentration of the drug resulting in 50% effect when compared to untreated controls (ED50) values in the nanomolar range after a 3-h exposure to dFdC. There was no correlation of the observed ED50 with the dCK activity. Treatment with dFdC induced cell death in MYCN-amplified cells whereas MYCN-single-copy-cell lines underwent neuronal differentiation. Pre-incubation with CPEC significantly increased dFdC-cytotoxicity from 1.3 to 5.3-fold in 13 out of 15 cell lines. [3H]dFdC-anabolism increased 6-44 fold in SK-N-BE(2)c cells after incubation with CPEC and was paralleled by a significant increase in expression and activity of dCK. In conclusion, the combination of dFdC and CPEC is highly toxic to neuroblastoma in vitro.

Pharmacology of 5-Aza-2′-deoxycytidine (decitabine)

The preclinical pharmacology of 5-aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is reviewed. 5AZA-CdR, an analogue of deoxycytidine, is a prodrug that requires metabolic activation by deoxycytidine kinase. The active inhibitor in the cell is its triphosphate form (5AZA-dCTP), which incorporates very readily into DNA to produce an inhibition of DNA methyltransferase. The mechanism responsible for the antileukemic action of 5AZA-CdR is related to its reversal of epigenetic silencing by aberrant DNA methylation of genes that suppress leukemiogenesis. 5AZA-CdR is an S-phase-specific agent. At concentrations in the range of micromolars this analogue can induce terminal differentiation and loss of clonogenicity of human leukemic cells. Drug resistance to 5AZA-CdR occurs primarily by reduction in deoxycytidine kinase activity or increase in the activity of cytidine deaminase, the enzyme that inactivates this analogue. 5AZA-CdR is a very potent antileukemic agent in animal models, more effective than the related antileukemic drug, cytosine arabinoside. In humans, 5AZA-CdR has a short half-life of 15 to 25 minutes due to rapid inactivation by liver cytidine deaminase. The major toxicity produced by 5AZA-CdR is myelosuppression. Preliminary clinical studies in patients with hematologic malignancies indicate that 5AZA-CdR is an active chemotherapeutic agent. The optimal dose-schedule for this interesting epigenetic agent with a novel mechanism of action remains to be determined. Translation of the pharmacology of 5AZA-CdR into therapeutic regimens based on scientific rationale can be used to obtain this objective.

Cytosine arabinoside affects multiple cellular factors and induces drug resistance in human lymphoid cells

Continuous in vitro cultivation of human lymphoid H9 cells in the presence of 0.5 μM arabinosyl-cytosine (araC) resulted in cell variant, H9-araC cells, that was >600-fold resistant to the drug and cross resistant to its analogs and other unrelated nucleosides, e.g. dideoxycytidine (5-fold), thiacytidine (2-fold), 2-fluoro-adenine arabinoside (8.3-fold), and 2-chloro-deoxyadenosine (2.1-fold). Compared to the parental cell line, the resistant cells accumulated <1% araCTP, and had reduced deoxycytidine kinase (dCK) activity (31.4%) and equilibrative nucleoside transporter 1 (ENT1) protein. The expression of the dCK gene in araC resistant cells was reduced to 60% of H9 cells, which correlated with lower dCK protein and activity. Whereas, there was no difference in the expression of ENT1 mRNA between the cell lines, ENT1 protein content was much lower in the resistant cells than in H9 cells. The concentrative nucleoside transporter (CNT3) was slightly increased in H9-araC cells, but CNT2, and MDR1 remained unaffected. Although a definitive correlation remains to be established, the amount of Sp1 protein, a transcription factor, that regulates the expressions of dCK, nucleoside transporters and other cellular proteins, was found reduced in H9-araC cells. Like ENT1, the Sp1 mRNA levels remained unaffected in H9-araC whereas protein contents were reduced. These observations are indicative of differences in the production and/or turnover of ENT1 and Sp1 proteins in H9-araC cells. Since nucleoside transporters and dCK play an important role in the activity of potential antiviral and anticancer deoxynucleoside analogs, understanding of their regulation is important. These studies show that the exposure of cells to araC, in vitro, is capable of simultaneously affecting more than one target site to confer resistance. The importance of this observation in the clinical use of araC remains to be determined.

Antagonistic effects of sequential administration of BL1521, a histone deacetylase inhibitor, and gemcitabine to neuroblastoma cells

Additive to synergistic induction of apoptosis has been reported as a result of sequential incubation of cancer cells with a histone deacetylase inhibitor (HDACi) and gemcitabine (dFdC), a deoxycytidine analogue with proven anti-tumour activity. This study shows that sequential treatment of two neuroblastoma cell lines with BL1521, an HDACi, and dFdC resulted in strong antagonism despite a minor increase of dFdCTP incorporation into the DNA of one cell line. Furthermore, no difference in the deoxycytidine kinase activity was observed in response to BL1521. In conclusion, cancer cells that respond to HDACi with a cell cycle arrest and differentiation may no longer be sensitive to dFdC.

Sensitivity to Gemcitabine and Its Metabolizing Enzymes in Neuroblastoma

We examined the activity of gemcitabine against neuroblastoma in vitro and in vivo. In addition, we investigated the cellular mechanisms of high sensitivity to the agent in neuroblastoma cells. We examined 11 neuroblastoma cell lines for sensitivity to gemcitabine and other chemotherapeutic agents used clinically for neuroblastoma. The in vivo sensitivity of neuroblastoma to gemcitabine was determined in xenograft models. Furthermore, the major metabolic enzymes of gemcitabine were assessed and compared in leukemia and carcinoma cells. Apoptosis and mitochondrial membrane potentials were also evaluated. The IC50s for gemcitabine in 11 neuroblastoma lines ranged between 3 nmol/L and 4 micromol/L. The high activity of gemcitabine against neuroblastoma was confirmed in animal models. Interestingly, enzymes in neuroblastoma cells involved in the metabolism of deoxycytidine analogue have unique characteristics among solid tumors. The median of deoxycytidine kinase activity in neuroblastoma lines was similar to that in leukemia lines, which have low IC50s for cytarabine. Cytidine deaminase (CDA) activity in neuroblastoma was hardly detectable and significantly lower than that in carcinoma. The defect of CDA activity was associated with negative expression of mRNA. Furthermore, gemcitabine-induced apoptosis was observed irrespective of the caspase-8 status of neuroblastoma cells, which indicates that apoptosis depends on the mitochondrial pathway. Neuroblastoma is highly sensitive to gemcitabine. Although the cellular mechanism involved in sensitivity to gemcitabine is multifactorial, low CDA activity may contribute high sensitivity in neuroblastoma cells. These results suggest that clinical application of gemcitabine to the treatment of neuroblastoma is warranted.

Possible antitumor activity of 1‐(3‐C‐ethynyl‐β‐d‐ribo‐pentofuranosyl)cytosine (ECyd, TAS‐106) against an established gemcitabine (dFdCyd)‐resistant human pancreatic cancer cell line

We established a variant of MIAPaCa-2 human pancreatic cancer cells that is resistant to 2',2'-difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa-2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd-resistance in these cells. We also evaluated 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa-2/dFdCyd cells were 2541-fold more resistant to dFdCyd than parental MIAPaCa-2 cells, and the major mechanism of the dFdCyd-resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5'-nucleotidase mRNA expression in the MIAPaCa-2/dFdCyd cells. The cytotoxicity of TAS-106 as an antitumor nucleoside analog was similar in both parental and dFdCyd-resistant cells, with IC(50) values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS-106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS-106 against MIAPaCa-2 and dFdCyd-resistant MIAPaCa-2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS-106 (7 mg/kg, iv) against parental and dFdCyd-resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS-106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd-based chemotherapy has failed.

Thymidine kinase and deoxycytidine kinase activity in mononuclear cells from antiretroviral-naive HIV-infected patients

To evaluate whether an inter-individual variability in the activity of thymidine kinase (TK) and deoxycytidine kinase (dCK), which are involved in the first step of phosphorylation of some nucleoside analogues, exists in antiretroviral-naive, HIV-seropositive patients. Forty-five randomly selected antiretroviral-naive HIV-infected patients were recruited, together with 26 healthy volunteers with no concurrent infection and under no pharmacological treatment. Peripheral blood mononuclear cells (PBMC) were isolated from venous blood and their TK and dCK activities evaluated. CD4 T cells and HIV-RNA were measured in HIV-infected patients, too. There was a broad range of variability in TK activity in HIV-infected individuals. Furthermore, the activity in PBMC was significantly higher in HIV-infected individuals than in healthy volunteers. dCK activity in seropositive patients was significantly lower than in healthy volunteers. A marked inter-individual variability in dCK levels was observed in the HIV-infected group. No correlations were found between TK or dCK activities and plasma viral load, CD4 cell count, sex or age of patients. A marked range of inter-individual variability of TK and dCK activities in PBMC exists in HIV-infected individuals but not in healthy volunteers, indicating that the activity of enzymes with key roles in drug activation could vary greatly from one patient to another. Furthermore, TK expression is greater in HIV-infected individuals than in healthy volunteers. Better understanding of the viral or cellular factors that contribute to this variability, as well as their effect on responses to antiretroviral treatment, may aid optimization of the management of HIV-infected patients.

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.

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.

New Evidences for a Regulation of Deoxycytidine Kinase Activity by Reversible Phosphorylation

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post-translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK-293 cells as a His-tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.

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.

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.

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.

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.

New Evidences for a Regulation of Deoxycytidine Kinase Activity by Reversible Phosphorylation

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post‐translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK‐293 cells as a His‐tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP λ) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.

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.