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1.
Arch Toxicol ; 91(6): 2315-2330, 2017 Jun.
Article in English | MEDLINE | ID: mdl-27942788

ABSTRACT

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.


Subject(s)
Copper/toxicity , Cytokines/metabolism , Laboratories/standards , Metal Nanoparticles/toxicity , Silver/toxicity , Toxicity Tests/standards , Biological Assay/methods , Biological Assay/standards , Cell Line, Tumor , Cell Survival/drug effects , Copper/chemistry , Europe , Humans , Metal Nanoparticles/chemistry , Particle Size , Reproducibility of Results , Silver/chemistry , Surface Properties , Toxicity Tests/methods
2.
Oncotarget ; 7(25): 38367-38379, 2016 Jun 21.
Article in English | MEDLINE | ID: mdl-27223263

ABSTRACT

Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit their clinical use. Here, we report that the DNA polymerase inhibitor aphidicolin, which displayed negligible cytotoxicity as a single agent in primary CLL cells, markedly synergizes with fludarabine and cladribine via enhanced apoptosis. Importantly, synergy was recorded regardless of CLL prognostic markers. At the molecular level, aphidicolin enhanced purine analog-induced phosphorylation of p53 and accumulation of γH2AX, consistent with increase in DNA damage. In addition, aphidicolin delayed γH2AX disappearance that arises after removal of purine analogs, suggesting that aphidicolin causes an increase in DNA damage by impeding DNA damage repair. Similarly, aphidicolin inhibited UV-induced DNA repair known to occur primarily through the nucleotide excision repair (NER) pathway. Finally, we showed that fludarabine induced nuclear import of XPA, an indispensable factor for NER, and that XPA silencing sensitized cell lines to undergo apoptosis in response to fludarabine. Together, our data indicate that aphidicolin potentiates the cytotoxicity of purine analogs by inhibiting a DNA repair pathway that involves DNA polymerases, most likely NER, and provide a rationale for manipulating it to therapeutic advantage.


Subject(s)
Aphidicolin/pharmacology , Cladribine/pharmacology , DNA Repair , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Vidarabine/analogs & derivatives , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA Damage , Drug Synergism , Enzyme Inhibitors/pharmacology , Humans , Vidarabine/pharmacology
3.
FEBS Lett ; 588(5): 727-32, 2014 Mar 03.
Article in English | MEDLINE | ID: mdl-24462681

ABSTRACT

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, Mn(2+)-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.


Subject(s)
Deoxycytidine Kinase/metabolism , Phosphoprotein Phosphatases/physiology , Protein Processing, Post-Translational , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Okadaic Acid/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Protein Phosphatase 2C , Serine/metabolism
4.
Nucleic Acids Res ; 40(19): 9621-32, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22850745

ABSTRACT

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia-telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.


Subject(s)
CDC2 Protein Kinase/metabolism , DNA Damage , Deoxycytidine Kinase/metabolism , G2 Phase Cell Cycle Checkpoints , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Deoxycytidine Kinase/chemistry , Deoxycytidine Kinase/physiology , HeLa Cells , Humans , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Radiation, Ionizing , Serine/metabolism , Tumor Suppressor Proteins/metabolism
5.
Biochem Pharmacol ; 84(1): 43-51, 2012 Jul 01.
Article in English | MEDLINE | ID: mdl-22490700

ABSTRACT

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.


Subject(s)
Antineoplastic Agents/metabolism , Deoxycytidine Kinase/metabolism , Purine Nucleosides/metabolism , Pyrimidine Nucleosides/metabolism , Serine/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Aphidicolin/pharmacology , Biotransformation , Cell Line, Tumor , Cell Survival/drug effects , Cladribine/chemistry , Cladribine/metabolism , Cladribine/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/chemistry , Deoxycytidine/metabolism , Deoxycytidine/pharmacology , Deoxycytidine Kinase/antagonists & inhibitors , Deoxycytidine Kinase/genetics , Enzyme Activation , HCT116 Cells , HT29 Cells , Humans , Kinetics , Mutation , Phosphorylation , Purine Nucleosides/chemistry , Purine Nucleosides/pharmacology , Pyrimidine Nucleosides/chemistry , Pyrimidine Nucleosides/pharmacology , Serine/genetics , Structure-Activity Relationship , Substrate Specificity , Vidarabine/analogs & derivatives , Vidarabine/chemistry , Vidarabine/metabolism , Vidarabine/pharmacology , Gemcitabine
6.
Arch Biochem Biophys ; 502(1): 44-52, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20637175

ABSTRACT

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.


Subject(s)
Casein Kinase Idelta/metabolism , Deoxycytidine Kinase/metabolism , Amino Acid Sequence , Amino Acid Substitution , Casein Kinase Idelta/antagonists & inhibitors , Casein Kinase Idelta/genetics , Cell Line , Deoxycytidine Kinase/chemistry , Deoxycytidine Kinase/genetics , Enzyme Activation , Humans , In Vitro Techniques , Kinetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational , RNA Interference , RNA, Small Interfering/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine/chemistry
7.
Br J Haematol ; 147(5): 641-52, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19764992

ABSTRACT

The functional evaluation of ataxia telangiectasia mutated (ATM) and p53 was recently developed in B-cell chronic lymphocytic leukaemia (B-CLL), a disease in which the response to DNA damage is frequently altered. We identified a novel biomarker of chemosensitivity based on the induction of DNA damage by the purine nucleoside analogues (PNA) fludarabine and 2-chlorodeoxyadenosine (CdA). Using genome-wide expression profiling, it was observed that, in chemosensitive samples, PNA predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly activated at early time points. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using a quantitative real time polymerase chain reaction, we confirmed that PNA dose- and time-dependently increased PLK2 expression in chemosensitive but not chemoresistant B-CLL samples. Analysis of a larger cohort of B-CLL patients showed that cytotoxicity induced by PNA correlated well with PLK2 mRNA induction. Interestingly, we observed that failure to up-regulate PLK2 following PNA and chemoresistance were not strictly correlated with structural alterations in the TP53 gene. In conclusion, we propose that testing PLK2 activation after a 24-h incubation with PNA could be used to investigate the functional integrity of DNA damage-response pathways in B-CLL cells, and predict clinical sensitivity to these drugs.


Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/biosynthesis , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Protein Serine-Threonine Kinases/biosynthesis , Up-Regulation/drug effects , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Cell Death/drug effects , Cladribine/pharmacology , Cohort Studies , DNA Damage , DNA, Neoplasm/genetics , Drug Resistance, Neoplasm , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Enzymologic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Male , Middle Aged , Oligonucleotide Array Sequence Analysis/methods , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Cells, Cultured , Vidarabine/analogs & derivatives , Vidarabine/pharmacology
8.
Blood ; 114(8): 1563-75, 2009 Aug 20.
Article in English | MEDLINE | ID: mdl-19541822

ABSTRACT

Chronic lymphocytic leukemia (CLL) is an incurable disease derived from the monoclonal expansion of CD5(+) B lymphocytes. High expression levels of ZAP-70 or CD38 and deletions of 17p13 (TP53) and 11q22-q23 (ATM) are associated with poorer overall survival and shorter time to disease progression. DNA damage and p53 play a pivotal role in apoptosis induction in response to conventional chemotherapy, because deletions of ATM or p53 identify CLL patients with resistance to treatment. Forodesine is a transition-state inhibitor of the purine nucleoside phosphorylase with antileukemic activity. We show that forodesine is highly cytotoxic as single agent or in combination with bendamustine and rituximab in primary leukemic cells from CLL patients regardless of CD38/ZAP-70 expression and p53 or ATM deletion. Forodesine activates the mitochondrial apoptotic pathway by decreasing the levels of antiapoptotic MCL-1 protein and induction of proapoptotic BIM protein. Forodesine induces transcriptional up-regulation of p73, a p53-related protein able to overcome the resistance to apoptosis of CLL cells lacking functional p53. Remarkably, no differences in these apoptotic markers were observed based on p53 or ATM status. In conclusion, forodesine induces apoptosis of CLL cells bypassing the DNA-damage/ATM/p53 pathway and might represent a novel chemotherapeutic approach that deserves clinical investigation.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/drug effects , DNA-Binding Proteins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Membrane Proteins/genetics , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Purine Nucleosides/pharmacology , Pyrimidinones/pharmacology , Tumor Suppressor Protein p53/physiology , Tumor Suppressor Proteins/genetics , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Murine-Derived , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/physiology , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Bendamustine Hydrochloride , Cyclophosphamide/administration & dosage , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondria/physiology , Nitrogen Mustard Compounds/administration & dosage , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Purine Nucleosides/administration & dosage , Purine Nucleosides/therapeutic use , Pyrimidinones/administration & dosage , Pyrimidinones/therapeutic use , Rituximab , Tumor Cells, Cultured , Tumor Protein p73 , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , Vidarabine/administration & dosage , Vidarabine/analogs & derivatives
9.
Biochem Pharmacol ; 75(7): 1451-60, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18242582

ABSTRACT

2-chloroadenosine (2-CAdo) is an adenosine deaminase-resistant analogue of adenosine, widely used as an adenosine receptor agonist. This compound has been shown to induce apoptosis in several cell types either via activation of adenosine receptors or via intracellular metabolism. However, the molecular mechanisms of 2-CAdo-induced apoptosis are unclear. Here, we analyzed the effects of 2-CAdo in the leukemia cell line EHEB. 2-CAdo was found to induce apoptosis in EHEB cells, as shown by caspase-3 activation, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage and phosphatidylserine exposure. Cytotoxicity of 2-CAdo was completely suppressed by 5-iodotubercidin, an adenosine kinase inhibitor, indicating that apoptosis induced by 2-CAdo was the result of its intracellular metabolism. Accordingly, we found that 2-CAdo was efficiently converted into 2-chloroATP. In parallel, a decrease of intracellular ATP concentration as well as a general inhibition of macromolecular synthesis, involving DNA, RNA and protein synthesis, was observed. Moreover, 2-CAdo induced cytochrome c release into the cytosol, indicating activation of the intrinsic pathway of apoptosis. This was found associated with a decline in Mcl-1 protein level and p53-independent. Inhibition of AMP deaminase by coformycin markedly prevented ATP depletion, and also significantly reduced 2-CAdo cytotoxicity and caspase-3 activation. In conclusion, our data show that intracellular metabolism of 2-CAdo can lead to activation of the intrinsic pathway of apoptosis and that ATP depletion, in addition to the accumulation of the triphosphate analogue, contributes to 2-CAdo-induced apoptosis.


Subject(s)
2-Chloroadenosine/pharmacology , Leukemia, B-Cell/metabolism , 2-Chloroadenosine/pharmacokinetics , 2-Chloroadenosine/therapeutic use , Apoptosis/drug effects , Apoptosis/physiology , Cell Death/drug effects , Cell Death/physiology , Cells, Cultured , Humans , Leukemia, B-Cell/drug therapy , Leukemia, B-Cell/pathology
10.
Cancer Lett ; 253(1): 68-73, 2007 Aug 08.
Article in English | MEDLINE | ID: mdl-17350163

ABSTRACT

Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues.


Subject(s)
Deoxycytidine Kinase/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Serine/metabolism , Antibodies/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Phosphorylation , Phosphoserine/immunology , Tumor Cells, Cultured
11.
Biochem Pharmacol ; 73(3): 351-8, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-17137556

ABSTRACT

EHEB leukemic cells, which are derived from a patient suffering B-cell chronic lymphocytic leukemia (B-CLL), display intermediate sensitivity to the purine analogue 2-chloro-2'-deoxyadenosine (CdA). Because the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway can rescue cancer cells from apoptotic signals, we investigated MAPK/ERK signaling in EHEB cells in response to CdA. We observed that CdA, at concentrations around its IC50, dose- and time-dependently increased the phosphorylation state of ERK 1/2 (p-ERK), indicating an activation of the MAPK/ERK pathway. This activation required CdA metabolism and de novo protein synthesis, and was independent on caspase activation. Interruption of ERK signaling, using the specific MEK inhibitors U-0126 and PD-98059, significantly enhanced CdA cytotoxicity, evaluated by the MTT assay. Drug interaction analysis showed synergism in the majority of combinations between CdA and MEK inhibitors tested. MEK inhibitors also dramatically increased apoptosis induced by CdA alone, evaluated by caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Collectively, these observations show that ERK 1/2 activation elicited by CdA serves as a cytoprotective function and suggest that inhibitors of this pathway could be combined with CdA in the treatment of selected hematological malignancies.


Subject(s)
Antineoplastic Agents/pharmacology , Cladribine/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor , Drug Interactions , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Phosphorylation
12.
J Biol Chem ; 281(8): 4887-93, 2006 Feb 24.
Article in English | MEDLINE | ID: mdl-16361699

ABSTRACT

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.


Subject(s)
Deoxycytidine Kinase/chemistry , Gene Expression Regulation, Neoplastic , Serine/chemistry , Binding Sites , Cell Line , Cell Line, Tumor , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Immunoblotting , Mass Spectrometry , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Sorbitol/chemistry , Spectrometry, Mass, Electrospray Ionization
13.
Biochem Pharmacol ; 68(1): 95-103, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15183121

ABSTRACT

Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway and in the activation of numerous nucleoside analogues used in cancer and antiviral chemotherapy. Recent studies indicate that dCK activity might be regulated through reversible phosphorylation. Here, we report the effects of a large panel of protein kinase inhibitors on dCK activity in the B-leukemia cell line EHEB, both in basal conditions and in the presence of the nucleoside analogue 2-chloro-2'-deoxyadenosine (CdA) which induces activation of dCK. Except staurosporine and H-7 that significantly reduced the activation of dCK by CdA, no specific protein kinase inhibitor diminished basal dCK activity or its activation by CdA. In contrast, genistein, a general protein tyrosine kinase inhibitor, and AG-490, an inhibitor of JAK2 and JAK3, increased basal dCK activity more than two-fold. Two specific inhibitors of the MAPK/ERK pathway, PD-98059 and U-0126, also enhanced dCK activity. These data suggest that the JAK/MAPK pathway could be involved in the regulation of dCK. Moreover, we show that the activity of dCK, raised by CdA, can return to its initial level by treatment with protein phosphatase-2A (PP2A). Accordingly, dCK activity in intact cells increased upon incubation with okadaic acid (OA) at concentrations that should inhibit PP2A, but not protein phosphatase-1. Activation of dCK by protein kinase inhibitors and OA was also observed in CCRF-CEM cells and in chronic lymphocytic leukemia B-lymphocytes, suggesting a general mechanism of post-translational regulation of dCK, which could be exploited to enhance the activation of antileukemic nucleoside analogues.


Subject(s)
Antineoplastic Agents/pharmacology , Carrier Proteins/pharmacology , Deoxycytidine Kinase/metabolism , Intracellular Signaling Peptides and Proteins , Okadaic Acid/pharmacology , Cladribine/pharmacology , Enzyme Activation/drug effects , Humans , Leukemia/pathology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 1 , Protein Phosphatase 2 , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Tumor Cells, Cultured
14.
Biochem Pharmacol ; 65(4): 573-80, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12566084

ABSTRACT

Deoxycytidine kinase (dCK), a key enzyme of the deoxynucleoside salvage pathway, might have a preponderant role in DNA synthesis in resting chronic lymphocytic leukemia B-lymphocytes. In these cells, two important enzymes in deoxynucleoside triphosphate production, ribonucleotide reductase and thymidine kinase (TK), both cell-cycle regulated, are indeed very weakly expressed. This study investigated the regulation of dCK activity in response to UV-C light, a condition which causes DNA lesions and DNA repair synthesis. We observed that activity of dCK in B-CLL cells was upregulated up to 3-fold, 30 min after irradiation with 30 J/m(2) UV-C, whereas TK activity was unchanged. Activation of dCK by UV-C light was caused neither by a change in concentration of a low molecular weight metabolite nor by an increase in the amount of dCK protein. Activation of dCK by UV-C was mimicked by H(2)O(2), markedly counteracted by N-acetylcysteine, a general antioxidant, and completely abolished by the growth factor receptor inhibitor suramin. Taken together, these results indicate that dCK activity is upregulated by UV-C light through a postranslational modification that may be initiated at the cell surface through oxidative mechanisms. Suramin also suppressed the increase in DNA repair synthesis elicited by UV-C irradiation, suggesting that upregulation of dCK activity could contribute to the normal completion of DNA repair synthesis elicited by UV light.


Subject(s)
B-Lymphocytes/radiation effects , Deoxycytidine Kinase/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Ultraviolet Rays , Antineoplastic Agents/pharmacology , B-Lymphocytes/drug effects , B-Lymphocytes/enzymology , DNA Repair/drug effects , Enzyme Activation/radiation effects , Humans , Suramin/pharmacology , Tumor Cells, Cultured
15.
Chem Commun (Camb) ; (6): 558-9, 2002 Mar 21.
Article in English | MEDLINE | ID: mdl-12120122

ABSTRACT

Driving the equilibrium between selenides and osmium(VIII) reagents with selenoxides and osmium(VI) by a subsequent reaction (rearrangement of allyl selenoxides to allyl alcohols or addition of osmium(VIII) species on C=C double bonds) to one side, allows the transformation of methyl geranyl selenides to linalool and of methyl citronellyl selenoxide to 6,7-dihydroxy citronellyl selenide.

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