Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(S,S)-Isodideoxyadenosine [(S,S)-isoddA] is an anti-HIV active compound discovered in our laboratory. However, its cellular mechanism of action, particularly the critical first stage of phosphorylation, is not understood. IsoddA is not phosphorylated by adenosine kinase. Also, because it is not a substrate for adenosine deaminase, it would not be activated by the pathway taken by ddA, i. e. via 5'-nucleotidase phosphorylation of ddI and conversion of ddIMP to ddAMP. However, we have discovered that human recombinant 2'-deoxycytidine kinase (dCK) phosphorylates (S,S)-isoddA. The enzyme kinetic data revealed that the extent of monophosphorylation of this L-related nucleoside was comparable to that found with ddA. (S,S)-IsoddATP is among the most potent inhibitors of HIV reverse transcriptase known, which suggests that the observed low efficiency of phosphorylation of this compound by dCK is a key factor that limits the capacity of human lymphocytes to make (S,S)-isoddA an exceptionally active anti-HIV agent.
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PMID:Phosphorylation of the anti-HIV compound (S,S)-isodideoxyadenosine by human recombinant deoxycytidine kinase. 1102 Apr 53

The pyrimidine analogue cytosine arabinoside (AraC) is one of the most effective drugs used in the treatment of acute leukaemia. Overexpression of the multidrug resistance (MDR-1) gene and its product, P-glycoprotein (P-gp), is associated with cellular resistance to drugs, such as anthracyclines and vinca alkaloids. This resistance can be reversed by cyclosporine analogues or verapamil (ver). We investigated the in vitro cross-resistance to AraC in a doxorubicin-resistant HL60 cell line, with an elevated expression of the MDR-1 gene. The resistant clone showed an eightfold increased resistance to AraC and a two- to fourfold resistance to the other analogues, as measured by cytotoxicity test. There was no significant increase in the activity of 5'-nucleotidase or in the amount of deoxyribonucleotide pools between cell lines. We could, however, detect a reduction in deoxycytidine kinase (dCK) activity (30%, P = 0.021, using deoxycytidine as substrate) and the level of AraC triphosphates was significantly reduced in the resistant cells (70%, P = 0.009). When the cells were exposed to cyclosporin A (CsA) or the cyclosporine analogue PSC 833 (PSC) in combination with AraC, there was more extensive apoptosis, as measured by formation of oligonucleosomal DNA fragmentation and caspase-3-like activity, than with exposure to AraC alone. We also found an increased retention of AraC in the resistant cells when incubated with AraC in combination with CsA. Ver in combination with AraC, failed to increase apoptosis for the resistant cell line. Our data suggests that the resistance to AraC for the P-gp-expressing cells is a result of a reduction of dCK activity and an increase in efflux, the latter possibly depending on P-gp. A combination of CsA or PSC with AraC may improve the effect of AraC in vivo.
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PMID:Cross-resistance to cytosine arabinoside in a multidrug-resistant human promyelocytic cell line selected for resistance to doxorubicin: implications for combination chemotherapy. 1155 80

The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.
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PMID:Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5'-nucleotidase mRNA measurement in cell lines and in patients with leukemia. 1189 43

To determine whether the human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), cytoplasmic 5'-nucleotidase (5NT), cytidine deaminase (CDD), topoisomerase I (TOPO I) and topoisomerase II alpha (TOPO II) are involved in clinical resistance to cytarabine (ara-C), we analyzed the level of expression of these parameters by reverse transcriptase polymerase chain reaction (rt-PCR), at diagnosis in the blast cells of 77 acute myeloid leukemia (AML) patients treated with ara-C, including 31 for whom samples were collected at first relapse. By univariate and/or multivariate analyses, patients with expression of 5NT or hENT1 deficiency at diagnosis had significantly shorter disease-free survival (DFS) and overall survival (OS). These results suggest that expression of 5NT and reduced hENT1 in leukemic blasts at diagnosis are correlated with clinical outcome and may play a role in resistance mechanisms to ara-C in patients with AML.
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PMID:Potential mechanisms of resistance to cytarabine in AML patients. 1200 78

Factors that reduce the intracellular concentration of triphosphorylated cytarabine (ara-CTP), the active form of cytarabine (ara-C), may induce chemoresistance in acute myeloid leukaemia (AML) patients. These factors include reduced influx of ara-C by the hENT1 transporter, reduced phosphorylation by deoxycytidine kinase (dCK), and increased degradation by high Km cytoplasmic 5'-nucleotidase (5NT) and/or cytidine deaminase (CDD). Increased levels of DNA polymerase alpha (DNA POL) and reduced levels of topoisomerase I (TOPO I) and topoisomerase II (TOPO II) have also been detected in ara-C-resistant cell lines. To determine whether these factors are implicated in clinical ara-C resistance, we analysed the expression of these parameters at diagnosis, using reverse transcription polymerase chain reaction, in the blast cells of 123 AML patients treated with ara-C. At diagnosis, hENT1, dCK, CDD, 5NT, TOPO I, TOPO II, DNA POL and MDR1 were expressed in 83%, 22%, 7%, 37%, 59%, 37%, 39% and 16% of patients respectively. In univariate analysis, patients with expression of 5NT or DNA POL at diagnosis had significantly shorter disease-free survival (DFS). In multivariate analysis, DNA POL positivity and hENT1 deficiency were related to a shorter DFS. In univariate analysis, patients with 5NT-positive blasts had significantly shorter overall survival (OS). In multivariate analysis, shorter OS was related to DNA POL positivity. These results suggest that expression of DNA POL, 5NT and hENT1 at diagnosis may be resistance mechanisms to ara-C in AML patients.
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PMID:In vivo mechanisms of resistance to cytarabine in acute myeloid leukaemia. 1206 Jan 21

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.
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PMID:Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance. 1250 99

The cytotoxic activity of cytarabine (ara-C) in leukaemic blasts depends on activating enzymes such as deoxycytidine kinase (dCK) and inactivating enzymes such as the 5'-nucleotidases. We have analysed dCK and 'high-Km' 5'-nucleotidase (cN-II) mRNA expression by the quantitative real-time polymerase chain reaction at diagnosis in leukaemic blasts from 115 acute myeloid leukaemia (AML) patients treated with ara-C. The prognostic value of these parameters as well as that of the cN-II/dCK ratio was determined. In univariate analyses: (1) low levels of dCK, high levels of cN-II and a high cN-II/dCK ratio predicted shorter disease-free survival (DFS); (2) low levels of dCK and cN-II/dCK ratio also predicted shorter overall survival (OS). In a multivariate analysis taking into account other clinical and laboratory variables: (1) high cN-II expression, a high cN-II/dCK ratio, age >/= 60 years and an unfavourable karyotype were independent prognostic factors for DFS; and (2) a high cN-II/dCK ratio, age >/= 60 years and an unfavourable karyotype predicted shorter OS. Age, karyotype and cN-II/dCK ratio were used to define a prognostic score that permitted the identification of high- and low-risk groups. Our results suggest that dCK and cN-II mRNA expression in leukaemic blasts at diagnosis is correlated with clinical outcome and may play a functional role in the resistance to ara-C in patients with AML.
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PMID:Deoxycytidine kinase and cN-II nucleotidase expression in blast cells predict survival in acute myeloid leukaemia patients treated with cytarabine. 1282 45

Administration of the adenosine analogue fludarabine (FLU) in vivo induces a profound and prolonged T lymphopenia which mainly affects CD4(+) cells. To better understand the mechanistic basis underlying this preferential depletion, we analyzed the in vitro susceptibility of T cell subsets to FLU-induced apoptosis. Contrasting with observations in vivo, our results showed that treatment of peripheral blood mononuclear cells with FLU induced a higher level of apoptosis in CD8(+) than in CD4(+) T lymphocytes. This increased sensitivity of CD8(+) T cells to FLU was observed in samples from both, healthy donors and B cell chronic lymphocytic leukemia patients, and resulted in higher CD4:CD8 ratios in FLU-treated than in untreated cultures (P<0.01). Expression of factors involved in FLU transport and metabolism was then evaluated by quantitative real time-PCR in normal T cell subsets. It was found that mRNA levels of human equilibrative nucleoside transporter-1 nucleoside transporter were higher whereas deoxycytidine kinase and IMP/GMP selective 5'-nucleotidase mRNA levels were lower in CD4(+) cells. However the dCK/cN-II ratio was 2-fold greater in CD8(+) than in CD4(+) T lymphocytes, which could account for the higher apoptosis levels observed in the CD8(+) subset. These results favor the view that decreased CD4:CD8 ratios in FLU-treated patients should be attributed to differences in cell recovery and/or homing between T cell subsets.
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PMID:In vitro susceptibility of CD4+ and CD8+ T cell subsets to fludarabine. 1460 43

Pre-treatment with bryostatin 1 (bryo) has been shown to potentiate the efficacy of (2-chloro-2-deoxyadenosine, cladribine, 2-CdA) in B-cell chronic lymphocytic leukemia (B-CLL) by increasing the ratio of deoxycytidine kinase (dCK) to 5'-nucleotidase (5'-NT) activity. The bryo-induced increase in dCK/5'-NT activity alone has not been a conclusive indication of final clinical outcome. Therefore, we used an ex vivo assay to investigate factors which may affect the bryo-induced enhancement of 2-CdA efficacy in B-CLL patient-derived samples. Bryo-induced increase in dCK/5'-NT was inversely associated with Rai stage CLL (r=-0.86). Increased dCK/5'-NT activity was not correlated with increased efficacy (cell death) or percentage of cellular [8-3H]-2-CdA converted to [8-3H]-2-CdATP ex vivo. Bryo pre-treatment increased the cellular uptake of [8-3H]-2-CdA and incorporation of [8-3H]-2-CdA metabolites into the DNA fraction. Cell death from 2-CdA was inversely correlated with bryo-induced activity of the DNA repair enzyme, DNA-PKcs, (r=-0.77). Thus, the ability of B-CLL to repair damaged DNA may be a more important predictor of the response to bryo/2-CdA and eventual clinical outcome than dCK/5'-NT activity. Additional CLL patients under bryo-2-CdA therapy are needed to verify these important observations.
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PMID:Factors affecting bryostatin 1-enhanced 2-CdA cytotoxicity in resistant B-cell chronic lymphocytic leukemia. 1520 25

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.
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PMID:Mechanisms of cross-resistance between nucleoside analogues and vincristine or daunorubicin in leukemic cells. 1524 Jan 22


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