Deoxycytidine Kinase Activity Research Articles

Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK

BackgroundPreviously, our investigations have underscored the potential of hyperthermia to improve the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC). Nonetheless, the precise underlying mechanisms remain elusive. MethodsWe engineered two GEM-resistant PC cell lines (BxPC-3/GEM and PANC-1/GEM) and treated them with GEM alongside hyperthermia. The impact of hyperthermia on the therapeutic potency of GEM was ascertained through MTT assay, assessment of the concentration of its active metabolite dFdCTP, and evaluation of deoxycytidine kinase (dCK) activity. Lentivirus-mediated dCK silencing was further employed to validate its involvement in mediating the GEM-sensitizing effect of hyperthermia. The mechanism underlying hyperthermia-mediated dCK activation was explored using bioinformatics analyses. The interplay between hyperthermia and the ephrin A4 (EFNA4)/β-catenin/dCK axis was investigated, and their roles in GEM resistance was further explored via the establishment of xenograft tumor models in nude mice. ResultsHyperthermia restored dCK expression in GEM-resistant cell lines, concurrently enhancing GEM sensitivity and fostering DNA damage and cell death. These observed effects were negated by dCK silencing. Regarding the mechanism, hyperthermia activated dCK by downregulating EFNA4 expression and mitigating β-catenin activation. Overexpression of EFNA4 activated the β-catenin while suppressing dCK, thus diminishing cellular GEM sensitivity—a phenomenon remediated by the β-catenin antagonist MSAB. Consistently, in vivo, hyperthermia augmented the therapeutic efficacy of GEM on xenograft tumors through modulation of the ephrin A4/β-catenin/dCK axis. ConclusionThis study delineates the role of hyperthermia in enhancing GEM sensitivity of PC cells, primarily mediated through the suppression of the EFNA4/β-catenin axis and activation of dCK.

Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-associated autoimmunity.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

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

Deoxycytidine kinase (dCK) and 5’ deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA.Graphical

Molecular Imaging of Deoxycytidine Kinase Activity Using Deoxycytidine-Enhanced CEST MRI.

Deoxycytidine kinase (DCK) is a key enzyme for the activation of a broad spectrum of nucleoside-based chemotherapy drugs (e.g., gemcitabine); low DCK activity is one of the most important causes of cancer drug-resistance. Noninvasive imaging methods that can quantify DCK activity are invaluable for assessing tumor resistance and predicting treatment efficacy. Here we developed a "natural" MRI approach to detect DCK activity using its natural substrate deoxycytidine (dC) as the imaging probe, which can be detected directly by chemical exchange saturation transfer (CEST) MRI without any synthetic labeling. CEST MRI contrast of dC and its phosphorylated form, dCTP, successfully discriminated DCK activity in two mouse leukemia cell lines with different DCK expression. This dC-enhanced CEST MRI in xenograft leukemic cancer mouse models demonstrated that DCK(+) tumors have a distinctive dynamic CEST contrast enhancement and a significantly higher CEST contrast than DCK(-) tumors (AUC0-60 min = 0.47 ± 0.25 and 0.20 ± 0.13, respectively; P = 0.026, paired Student t test, n = 4) at 1 hour after the injection of dC. dC-enhanced CEST contrast also correlated well with tumor responses to gemcitabine treatment. This study demonstrates a novel MR molecular imaging approach for predicting cancer resistance using natural, nonradioactive, nonmetallic, and clinically available agents. This method has great potential for pursuing personalized chemotherapy by stratifying patients with different DCK activity. SIGNIFICANCE: A new molecular MRI method that detects deoxycytidine kinase activity using its natural substrate deoxycytidine has great translational potential for clinical assessment of tumor resistance and prediction of treatment efficacy.

Nucleobase and Nucleoside Analogues: Resistance and Re-Sensitisation at the Level of Pharmacokinetics, Pharmacodynamics and Metabolism.

Antimetabolites, in particular nucleobase and nucleoside analogues, are cytotoxic drugs that, starting from the small field of paediatric oncology, in combination with other chemotherapeutics, have revolutionised clinical oncology and transformed cancer into a curable disease. However, even though combination chemotherapy, together with radiation, surgery and immunotherapy, can nowadays cure almost all types of cancer, we still fail to achieve this for a substantial proportion of patients. The understanding of differences in metabolism, pharmacokinetics, pharmacodynamics, and tumour biology between patients that can be cured and patients that cannot, builds the scientific basis for rational therapy improvements. Here, we summarise current knowledge of how tumour-specific and patient-specific factors can dictate resistance to nucleobase/nucleoside analogues, and which strategies of re-sensitisation exist. We revisit well-established hurdles to treatment efficacy, like the blood-brain barrier and reduced deoxycytidine kinase activity, but will also discuss the role of novel resistance factors, such as SAMHD1. A comprehensive appreciation of the complex mechanisms that underpin the failure of chemotherapy will hopefully inform future strategies of personalised medicine.

Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression.

Cytosine arabinoside (Ara‐C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara‐C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara‐CTP) as an active form. In the first step of the metabolic pathway, Ara‐C is phosphorylated to Ara‐CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara‐C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B‐cell precursor ALL (BCP‐ALL) to Ara‐C. Higher DCK expression was associated with higher Ara‐C sensitivity. DCK knockout by genome editing with a CRISPR‐Cas9 system in an Ara‐C‐sensitive‐ALL cell line induced marked resistance to Ara‐C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara‐C sensitivity of BCP‐ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara‐C sensitivity. Clofarabine is a second‐generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP‐ALL cell lines was approximately 20 times lower than that of Ara‐C. In contrast to Ara‐C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP‐ALL that shows relative Ara‐C resistance due to low DCK expression.

18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

A crucial role for ATR in the regulation of deoxycytidine kinase activity

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme for salvage of deoxynucleosides and activation of numerous anticancer and antiviral nucleoside analogs. dCK activity is enhanced in response to several genotoxic treatments, which has been correlated with an increase of dCK phosphorylation at Ser-74. ATM was recently identified as the kinase responsible for Ser-74 phosphorylation and dCK activation after ionizing radiation (IR). Here, we investigated the role of ATM and the related kinase ATR in dCK activation induced by other types of DNA damage. Using ATM-deficient cells or the ATM inhibitor KU-60019, we found that ATM was not required for dCK activation caused by UV light, aphidicolin, cladribine, and unexpectedly also IR. On the other hand, the selective ATR inhibitor VE-821 significantly reduced up-regulation of dCK activity induced by these genotoxic agents, though not IR, and also down-regulated basal dCK activity. A role for ATR in the control of dCK activity was confirmed by using ATR siRNA and ATR-Seckel cells. ATR was also found to directly phosphorylate dCK at Ser-74 in vitro. Further studies revealed that ATR, which is also activated in response to IR, although later than ATM, was responsible for IR-induced dCK activation in ATM-deficient cells or in the presence of KU-60019. Overall, our results demonstrate that ATR controls basal dCK activity and dCK activation in response to replication stress and indicate that ATR can activate dCK after IR if ATM is lacking or inhibited.

Abstract 5473: Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene

Abstract Gemcitabine (GEM) has shown clinical activity in several solid tumors such as pancreatic and biliary tract cancers, urinary cancer, and non-small cell lung cancers, but a substantial number of patients acquire GEM chemoresistance. GEM functions after phosphorylation by deoxycytidine kinase (DCK) and its inactivation is one of the important mechanisms for acquisition of gemcitabine resistance. We established eight GEM resistant cell lines including four pancreatic, two biliary tract, and two gastrointestinal cancers and investigated mechanisms for acquisition of GEM resistances; six GEM resistant cell lines harbored complete inactivating mutations of DCK by protein-truncating mutation or complete or partial loss of the gene, and one gastric cancer cell line harbored a missense mutation that inactivate DCK activity. One pancreatic cancer cell line lost GEM sensitivity by unknown mechanism. Our present results strongly suggest that genetic alteration of DCK can be the major cause of acquisition of GEM resistance. Further studies analyzing primary tumors will give us (1) the fully understanding of acquisition of GEM resistance, and (2) desirable clues to overcome the acquisition of GEM resistance. Citation Format: Akira Horii, Yuriko Saiki, Tomohiro Nakano, Chiharu Kudo, Makoto Sunamura. Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2015-5473

Modulation of deoxycytidine kinase (dCK) and glycogen synthase kinase (GSK-3β) by anti-CD20 (rituximab) and 2-chlorodeoxyadenosine (2-CdA) in human lymphoid malignancies.

BackgroundThe combination of rituximab and 2-CdA is an effective therapy for B-cell tumors. However, the molecular mechanisms and enzymatic pathways involved in the interaction between the two agents are not fully understood. In this study, we provide molecular evidence for positive interaction between these two agents with resultant therapeutic benefit.MethodsEfficacy of the R-2CdA regimen was evaluated in thirteen patients with B-cell tumors (9 CLL; 3 WM and 1 FL), in vitro against 3 lymphoma cell lines and in a xenograft mouse model. Treatment-induced changes involving phenotype, kinase activity and protein expression were assessed in vitro and in the mouse xenograft tumors. The interaction between RTX and 2-CdA was analyzed using the multiple comparison method, Tukey’s honestly significant difference (HSD). For the clinical and animal data, survival functions were estimated using the Kaplan-Meier method and compared by the log-rank test. P-values <0.05 were considered statistically significant. All statistical analyses were evaluated using GraphPad Prism 4 (San Diego, CA).Results9 of 12 (75%) evaluable patients responded to the R-2-CdA regimen with median duration of response of 34 months. Median survival of patients from diagnosis and from completion of R-2-CdA treatment was 13.3 and 7.9 years, respectively. In vitro, the combination was effective in all 3 cell lines of lymphomas but with higher sensitivity in the follicular lymphoma cell line. The combination was also effective in the WSU-WM-SCID xenograft model with dose-dependent response and synergistic benefit. All animals were tumor-free for up to 120 days post 2 cycles of this regimen. Rituximab induced activation of deoxycytidine kinase (dCK), p38 mitogen activated protein kinase (p38MAPK) and glycogen synthase kinase-3β (GSK-3β) in the xenograft WSU-WM tumors. Chemical inhibition of p38MAPK led to inhibition of the GSK-3β phosphorylation suggesting that GSK-3β is regulated by p38MAPK in this model.ConclusionCollectively, our studies show concordance between the activity of R-2-CdA in vitro, in human and in WSU-WM xenograft model attesting to the validity of this model in predicting clinical response. Modulation of dCK and GSK-3β by rituximab may contribute to the positive therapeutic interaction between rituximab and 2-CdA.

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.

Facile method for determination of deoxycytidine kinase activity in biological milieus

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.

Mechanistic studies on the synergistic cytotoxicity of the nucleoside analogs gemcitabine and clofarabine in multiple myeloma: Relevance of p53 and its clinical implications

Hematopoietic stem cell transplantation (HSCT) is an established treatment for multiple myeloma (MM), a plasma cell malignancy. To identify an improved pretransplant conditioning regimen, we investigated the cytotoxicity of gemcitabine (Gem) and clofarabine (Clo) combinations toward MM cell lines and patient cell samples. A strong synergism of the two nucleoside analogs, when combined at their approximate IC10 concentrations, was observed. This synergism could be partly due to the observed Gem-mediated phosphorylation and activation of deoxycytidine kinase, resulting in enhanced phosphorylation of Gem and Clo. Their cytotoxicity correlated with a robust activation of the DNA damage response pathway. [Gem+Clo] decreased the mitochondrial membrane potential with a concomitant release of proapoptotic factors into the cytoplasm and nucleus and the activation of apoptosis. Exposure of MM cells to [Gem+Clo] also decreased the level of ribosomal RNA (rRNA), which might have resulted in nucleolar stress, as reported previously, and caused a p53-dependent cell death. A reduction by approximately 50% in the cytotoxicity of Gem and Clo was observed in the presence of pifithrin α, a p53 inhibitor. Furthermore, MM cell lines with mutant p53 exhibited greater resistance to Gem and Clo, supporting a role for the p53 protein in these cytotoxic responses. Our results provide a rationale for clinical trials incorporating [Gem+Clo] combinations as part of conditioning therapy for high-risk patients with MM undergoing HSCT.

The Apoptotic Effects of Toosendanin Are Partially Mediated by Activation of Deoxycytidine Kinase in HL-60 Cells

Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development.

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37°C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R2 values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.

Isolation and Characterization of a Murine P388 Leukemia Line Resistant to Thiarabine

A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was sensitive as was the parental P388/0 line to 5-fluorouracil, gemcitabine, cyclophosphamide, cisplatin, melphalan, BCNU, mitomycin C, doxorubicin, mitoxantrone, etoposide, irinotecan, vincristine, and paclitaxel, but was cross resistant (at least marginally) to three antimetabolites: palmO-ara-C, fludarabine phosphate, and methotrexate. The deoxycytidine kinase activity in the subline was comparable to that for P388/0, whereas the dCMP deaminase activity was 43% of that for P388/0. No deoxycytidine deaminase activity was detected in either of the leukemias. There appeared to be little, if any, difference in the metabolism of deoxycytidine, cytidine, or thiarabine in the two leukemias.

Isolation and Characterization of a Murine P388 Leukemia Line Resistant to Clofarabine

A murine P388 leukemia line fully resistant to clofarabine was obtained after only two courses of intraperitoneal treatment (three times a day for nine consecutive days). The resistance was stable for at least 13 weeks without treatment. The subline was as sensitive to 5-fluorouracil, methotrexate, cyclophosphamide, cisplatin, melphalan, BCNU, doxorubicin, etoposide, irinotecan, vincristine, and docetaxel as was the parental P388/0 line but was cross-resistant to five antimetabolites [palmO-ara-C, 4′-thio-ara-C, fludarabine phosphate, cladribine, and gemcitabine—all of which require deoxycytidine kinase for activation] and paclitaxel. The subline had less than 1% of the deoxycytidine kinase activity in comparison to P388/0.

Cladribine inhibits cytokine secretion by T cells independently of deoxycytidine kinase activity

Cladribine (2-chloro-2′-deoxyadenosine) is a purine nucleoside analogue (PNA) which causes targeted and sustained reduction of peripheral lymphocyte counts. Cladribine tablets produced significant treatment benefit for patients with relapsing–remitting multiple sclerosis in the phase 3 CLARITY study. In addition to the well-characterised cell-specific phosphorylation of PNAs responsible for lymphocyte reduction, the mode of action of cladribine may encompass distinct activities contributing to its overall effects on the immune system. Here we demonstrate that clinically relevant concentrations of cladribine also inhibit cytokine secretion by human peripheral blood T cells in vitro through mechanisms independent of the induction of lymphocyte death.

The Deaminated Metabolite of Gemcitabine, 2′,2′-Difluorodeoxyuridine, Modulates the Rate of Gemcitabine Transport and Intracellular Phosphorylation via Deoxycytidine Kinase

Gemcitabine (dFdC) is a chemotherapeutic nucleoside analog that undergoes uptake via equilibrative nucleoside transporters (hENT) followed by sequential phosphorylation to the active triphosphate moiety (dFdCTP). Its deaminated metabolite, 2',2'-difluorodeoxyuridine (dFdU), competes with the parent compound for cellular entry via hENTs, but over time dFdU increases the net intracellular accumulation of dFdC by a currently unknown mechanism. In this study, we investigated whether dFdU affects intracellular phosphorylation of gemcitabine by modulating the activity of deoxycytidine kinase (dCK). We report here that coincubation of dFdU with dFdC significantly increases intracellular levels of dFdCTP. dFdCTP was not identified as a substrate for hENTs, suggesting that dFdU affects the formation rather than elimination of the triphosphate. To further characterize the disposition of dFdC in the presence of dFdU, the net intracellular radioactivity of [5-(3)H]dFdC and corresponding metabolic profile were evaluated in HeLa cells transfected with dCK-targeting small interfering RNA. Intracellular radioactivity significantly decreased in cells with compromised intracellular phosphorylation, which was mainly due to a loss in dFdCTP. Although dFdU increased the net intracellular radioactivity of [5-(3)H]dFdC at 24 h in control cells, this increase was abolished in the absence of dCK activity, strongly suggesting that the interaction between dFdU and dFdC occurs via modulation of both transport and metabolism. In conclusion, we have demonstrated that the intracellular distribution of dFdC is dependent on both transport and metabolic processes, and that by affecting the rate at which dFdC enters the cell, the presence of dFdU may be altering the metabolic fate of the parent compound (dFdC).