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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study was undertaken to assess the predictive value of pretherapeutic determinants of ara-C metabolism and proliferative activity of leukemic blasts for early response to antileukemic therapy in the setting of granulocyte-macrophage colony-stimulating factor (GM-CSF)-based priming before and during TAD-9 induction in 36 consecutive patients with de novo acute myeloid leukemia (AML). Ara-C metabolism was assessed by the activities of deoxycytidine kinase (DCK), deoxycytidine deaminase (DCD), DNA polymerase alpha (Poly alpha), and overall polymerase (overall Poly). The fraction of cells in S phase (%S phase) and thymidine kinase (TK) activity were determined as a measure of proliferative activity. Early response to therapy was defined by the percentage of leukemic blasts in the bone marrow 5 to 7 days after completion of TAD-9 with less than 5% signaling an adequate response and greater than 5% indicating an inadequate early reduction, respectively. While neither %S phase, DCK, nor overall Poly activity were predictive for early response, TK and Poly alpha activities were significantly higher for cases with adequate blast cell clearance. The respective median values were for TK 3.8 versus 1.85 pmol/min/mg protein (P = .012), and for Poly alpha 1.9 versus 0.69 pmol/min/mg protein (P = .014). An inverse relation was detected for DCD activity which was significantly lower in responding patients with a median of 0.33 nmol/min/mg protein (range, 0.0 to 29.5) as compared to a median of 5.1 nmol/min/mg protein (range, 0.11 to 8.45) in early nonresponders, (P = .009). Taking the respective median values as arbitrary cut-points for high or low enzyme activities, responders and nonresponders could be discriminated prospectively. Hence, 14 of 16 cases (88%) with DCD activities below the median of 1.56 nmol/min/mg protein responded as compared to only 3 of 14 (22%) patients with higher DCD activities (P = .0004). From the 15 patients with TK activity above the overall median of 3.2 pmol/min/mg protein, 11 cases (73%) achieved an adequate blast cell clearance while only 6 of 17 cases (35%) with lower values responded (P = .035). Similarly, 12 of 15 patients (80%) with high Poly alpha levels (>1.22 pmol/min/mg protein) responded to induction therapy as compared to only 5 of 14 patients (36%) with lower enzyme activities (P = .02). By logistic regression analysis of enzyme activities, DCD activity was found to be the most sensitive parameter to predict an adequate blast cell clearance (P = .032). Activities of DCD and TK were not only associated with initial response but were also found predictive for remission duration. Hence, from 11 patients with low TK levels 8 (73%) relapsed within 1 year, whereas only 2 of 11 (18%) patients with high TK activity experienced a recurrence of their disease (P = .015). Six of 9 (66%) patients with higher than median DCD levels relapsed within 1 year, whereas 10 of 14 patients (71%) with lower DCD levels had a longer remission duration (P = .085). Analysis of DCD gene expression at the mRNA level by a semi-quantitative reverse transcriptase-polymerase chain reaction method showed that a high transcription rate of the DCD gene was associated with high enzyme activities and vice versa. Hence, the observed intraindividual differences in DCD activity are a reflection of differences in gene activity and transcription rate rather than of variants in translation. Although further analyses are needed to elucidate the molecular mechanisms that determine the variation of enzyme activities in individual patients, the present study strongly suggests that pretherapeutic determination of TK and Poly alpha as well as of DCD allows to predict response to TAD-9 + GM-CSF induction therapy and may provide the means for the development of a risk adapted treatment strategy.
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PMID:Activity of thymidine kinase and of polymerase alpha as well as activity and gene expression of deoxycytidine deaminase in leukemic blasts are correlated with clinical response in the setting of granulocyte-macrophage colony-stimulating factor-based priming before and during TAD-9 induction therapy in acute myeloid leukemia. 929 31

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (MDL101,731) is a new deoxycytidine analog which shows potent antitumor activity against several human tumor models. We previously showed that MDL101,731 inhibited human ribonucleotide reductase (RNR) in HeLa S3 human cervical carcinoma cells. Recently, it has been reported that another deoxycytidine analog, 2'-deoxy-2'-methylidenecytidine (DMDC) which also inhibits RNR from Escherichia coli, does not inhibit RNR in intact L1210 murine leukemia cells. MDL101,731 was designed as an inhibitor of RNR, so it is important to know the contribution of the RNR inhibitory activity of the drug on its antitumor efficacy in vivo. Therefore, we examined the relationship between the antitumor activity and RNR inhibitory activity of MDL101,731 using LX-1 human lung carcinoma which was highly sensitive to this drug. MDL101,731 showed strong inhibition of RNR activity in LX-1 lung carcinoma by both i.v. and p.o. administration. Administration of 15 mg/kg i.v. and 30 mg/kg p.o. of MDL101,731, doses which showed almost the same degree of antitumor activity against LX-1 lung carcinoma on a daily 5 day schedule, caused a similar degree and similar kinetics of inhibition of RNR in LX-1 lung carcinoma at least for 12 h after administration. On the other hand, DMDC as well as 1-beta-D-arabinofuranosyl-cytosine (ara-C), which is a well-known deoxycytidine analog and inhibits DNA polymerase alpha, did not inhibit RNR in LX-1 lung carcinoma at doses demonstrating antitumor activity. These results indicate that MDL101,731 exhibited antitumor activity through inhibition of RNR activity in tumor cells in vivo and the mechanism of antitumor action of MDL 101,731 might be different from those of DMDC and ara-C, at least in part.
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PMID:The relationship between the antitumor activity and the ribonucleotide reductase inhibitory activity of (E)-2'-deoxy-2'-(fluoromethylene) cytidine, MDL 101,731. 977 10

Fludarabine and 1-beta-D-arabinofuranosylcytosine (ara-C) are effective nucleoside analogues for the treatment of leukemias when used as single agents or together. Recent trials of the fludarabine and ara-C therapy with or without growth factors suggested an improved clinical response by combining fludarabine and ara-C. The activity of these antimetabolites depends on their phosphorylation to the respective triphosphates, F-ara-ATP and ara-CTP. The principal mechanism through which these triphosphates cause cytotoxicity is incorporation into DNA and inhibition of further DNA synthesis. A model system of DNA primer extension on a defined template sequence was used to quantitate the consequences of incorporation of one or two analogues by human DNA polymerase alpha (pol alpha). The template (31-mer) was designed so that DNA pol alpha incorporated six deoxynucleotides (alternately G and T) on the 17-mer primer, followed by insertion of an A and then a C. The primer was then elongated with G and T to the full-length product. The apparent Kms of DNA pol alpha to incorporate these analogues (0. 053 and 0.077 microM, respectively) were similar to the Km for dCTP (0.037 microM) and dATP (0.044 microM), suggesting that the enzyme recognized these analogues and incorporated them efficiently on the growing DNA primer. The velocity of extension (Vmax) of these primers ranged between 0.53 and 0.77%/min when normal nucleotides were present. Once inserted at the 3'-terminus, F-ara-AMP or ara-CMP were poor substrates for extension. However, in reactions lacking dCTP and dATP and with high concentrations of ara-CTP, ara-CMP was inserted by pol alpha after incorporation of the F-ara-AMP residue. This tandem incorporation of the two analogues resulted in almost complete inhibition (99.3%) of further extension of the primer. In the presence of competing deoxynucleotides, each analogue resulted in a dose-dependent inhibition of DNA synthesis. When present together, inhibition of the primer elongation was more than additive at low concentrations of analogue triphosphates. Based on these results and the intracellular pharmacokinetics of ara-CTP and F-ara-ATP in leukemia blasts, we propose a pharmacodynamic model to explain interactions between these analogues during combination chemotherapy.
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PMID:Incorporation of fludarabine and 1-beta-D-arabinofuranosylcytosine 5'-triphosphates by DNA polymerase alpha: affinity, interaction, and consequences. 981 18

Events accompanying sequential exposure of U937 leukemic cells to the deoxycytidine (dCyd) analogs 1-[beta-D-arabinofuranosyl]cytosine (ara-C) or 2',2'-difluorodeoxycytidine (gemcitabine; dFdC) followed by two protein kinase C (PKC) activators [bryostatin 1 (BRY) or phorbol 12'-myristate 13'-acetate (PMA)] exhibiting disparate differentiation-inducing abilities were characterized. A 24-hr exposure to 10 nM BRY or PMA after a 6-hr incubation with 1 microM ara-C or 100 nM dFdC resulted in equivalent increases in apoptosis, caspase-3 activation, and polyADP-ribose polymerase degradation, as well as identical DNA cleavage patterns. BRY and PMA did not modify retention of the lethal ara-C metabolite ara-CTP or alter ara-CTP/dCTP ratios. Unexpectedly, pretreatment of cells with ara-C or dFdC opposed BRY- and PMA-related induction of the cyclin-dependent kinase inhibitors (CDKIs) p21CIP1 and/or p27KIP1. These effects were not mimicked by the DNA polymerase inhibitor aphidicolin or by VP-16, a potent inducer of apoptosis. Inhibition of PKC activator-induced CDKI expression by ara-C and dFdC did not lead to redistribution of proliferating cell nuclear antigen but was accompanied by sub-additive or antagonistic effects on leukemic cell differentiation. Sequential exposure of cells to ara-C followed by BRY or PMA led to substantial reductions in clonogenicity that could not be attributed solely to apoptosis. Finally, pretreatment of cells with ara-C attenuated PMA- and BRY-mediated activation of mitogen-activated protein kinase, an enzyme implicated in CDKI induction. Collectively, these findings suggest that pretreatment of leukemic cells with certain dCyd analogs interferes with CDKI induction by the PKC activators PMA and BRY, and that this action may contribute to modulation of apoptosis and differentiation in cells exposed sequentially to these agents.
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PMID:Inhibition of protein kinase C activator-mediated induction of p21CIP1 and p27KIP1 by deoxycytidine analogs in human leukemia cells: relationship to apoptosis and differentiation. 1040 25

Transformed cells are characterized by imbalances in metabolic routes. In particular, different key enzymes of nucleotide metabolism and DNA biosynthesis, such as CTP synthetase, thymidylate synthase, dihydrofolate reductase, IMP dehydrogenase, ribonucleotide reductase, DNA polymerase, and DNA methyltransferase, are markedly up-regulated in certain tumor cells. Together with the concomitant down-modulation of the purine and pyrimidine degradation enzymes, the increased anabolic propensity supports the excessive proliferation of transformed cells. However, many types of cancer cells have maintained the ability to differentiate terminally into mature, non-proliferating cells not only in response to physiological receptor ligands, such as retinoic acid, vitamin D metabolites, and cytokines, but also following exposure to a wide variety of non-physiological agents such as antimetabolites. Interestingly, induction of tumor cell differentiation is often associated with reversal of the transformation-related enzyme deregulations. An important class of differentiating compounds comprises the antimetabolites of purine and pyrimidine nucleotide metabolism and nucleic acid synthesis, the majority being structural analogs of natural nucleosides. The CTP synthetase inhibitors cyclopentenylcytosine and 3-deazauridine, the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine, the dihydrofolate reductase inhibitor methotrexate, the IMP dehydrogenase inhibitors tiazofurin, ribavirin, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and mycophenolic acid, the ribonucleotide reductase inhibitors hydroxyurea and deferoxamine, and the DNA polymerase inhibitors ara-C, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and aphidicolin, as well as several nucleoside analogs perturbing the DNA methylation pattern, have been found to induce tumor cell differentiation through impairment of DNA synthesis and/or function. Thus, by selectively targeting those anabolic enzymes that contribute to the neoplastic behavior of cancer cells, the normal cellular differentiation program may be reactivated and the malignant phenotype suppressed.
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PMID:Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. 1041 91

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.
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PMID:Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP). 1085 36

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.
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PMID:Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines. 1133 Oct 76

Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F-ara-A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F-ara-A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F-ara-A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time-dependently and reached a plateau at 4 h. A 2-h pre-incubation with F-ara-A inhibited the repair in a concentration-dependent manner, with the maximal inhibition by 5 mM. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F-ara-A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.
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PMID:Inhibition of nucleotide excision repair by fludarabine in normal lymphocytes in vitro, measured by the alkaline single cell gel electrophoresis (Comet) assay. 1203 53

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

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.
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PMID:Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells. 1237 42


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