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)

Aphidicolin specifically inhibits eukaryotic DNA polymerase alpha, while 2',3'-dideoxythymidine 5'-triphosphate (d2TTP) inhibits DNA polymerase beta and gamma but not alpha. 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase alpha and beta although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-beta-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d2Thd, a precursor of d2TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d2Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d2TTP, was microinjection directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d2TTP effectively inhibited repair synthesis. The microinjection of d2TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis.
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PMID:Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells. 681 74

The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of 3H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine Kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase alpha and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h. 10(-6) M ara-C inhibited the incorporation of 3H-thymidine by 90-95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of 3H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 degrees C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of 3H-thymidine. Ara-C (10(-6) M) abolished also the PHA induced elevation of DNA polymerase alpha and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.
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PMID:Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes. 706 8

Cytosine arabinoside (ara-C) has been used in the treatment of leukemia, but its exact mechanism of cytotoxicity is not yet known. One of the proposed mechanisms for the effectiveness of this drug in treating leukemias suggests that a metabolite of ara-C, i.e., 2'-deoxycytidine 5'-triphosphate (araCTP), competes with cytosine arabinoside 5'-triphosphate (dCTP) for binding to DNA polymerase. The ratio of the drug metabolite to the endogenous nucleotide (araCTP/dCTP) may, therefore, be important in determining the effectiveness of ara-C therapy. This ratio may also play a role in drug resistance. Previously published methods have focused on either araCTP or dCTP, along with metabolites and analogues of one of these compounds. The methods presented here provide two simple, sensitive ways to measure dCTP and araCTP in the same biological sample.
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PMID:Two simple high-performance liquid chromatographic methods for simultaneous determination of 2'-deoxycytidine 5'-triphosphate and cytosine arabinoside 5'-triphosphate concentrations in biological samples. 716 28

Cytosine arabinoside (Ara-C) is a very potent inhibitor of DNA synthesis in mammalian cells. This inhibition does not appear to be due to the direct inhibition of DNA polymerase by Ara-CTP (triphosphate of Ara-C) because it is a very weak competitive inhibitor of this enzyme. The inhibition of DNA synthesis appears to be due to the incorporation of Ara-C into DNA producing chain termination of polydeoxynucleotide elongation.
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PMID:Biochemical pharmacology of cytosine arabinoside. 716 66

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blast cell proliferative activity and sensitivity to GM-CSF in vitro are associated with early response to TAD-9 induction therapy in acute myeloid leukemia. 747 75

Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.
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PMID:Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase. 751 42

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aphS) and resistant (aphR) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [32P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aphR mutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the alpha-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aphR mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N2-(p-n-Butylphenyl)-2' deoxyguanisine-5'-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells. 777 54

Proliferating cell nuclear antigen (PCNA) is a 36-kD nuclear protein that functions as a cofactor of delta DNA polymerase which is regulated in a cell cycle-dependent fashion. PCNA expression also increases when cells are actively engaged in DNA repair. We used Western blotting (WB) to measure the level of expression of PCNA in peripheral blasts of 36 adult acute myelogenous leukemia (AML) patients treated with Ara-C based induction regimens. PCNA levels correlated positively with the percentage of cells in S+G2M of the cell cycle. Logistic regression analysis revealed PCNA (beta = 4.5162; p = 0.0260) together with age (beta = 0.1777; p = 0.0364) as independent variables for remission induction: high PCNA levels were associated with poor response to induction therapy. PCNA expression was not, however, a predictor of survival in this subset of patients. We conclude that PCNA levels in this disease may be important for predicting response to Ara-C based remission induction chemotherapy.
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PMID:Quantitative expression of proliferating cell nuclear antigen by western blot (PCNAWB) in peripheral blasts correlates with remission induction in patients with acute myelogenous leukemia. 853 14

Cytosine arabinoside (Ara-C) is used to treat leukemias, with complete remission induced by combination chemotherapy in approximately 70% of cases of acute myelogenous leukemia (AML). Ara-CTP acts as a competitive inhibitor of DNA polymerase and may also be incorporated into DNA. Accumulation of deoxyribonucleoside triphosphates (dNTPs) induced by Ara-C may indicate disruption of DNA synthesis in susceptible leukemia cells. A procedure has been developed for the quantification of Ara-CTP and dNTPs from small samples of leukaemia cells from patients (4 x 10(7) cells) activated with concanavalin A (10 micrograms/ml, 48 hr) and grown in the presence of [32P]orthophosphate (1.1 microM, 9 x 10(6) Ci/mol, 16 hr). The susceptibilities to Ara-C of the human leukemia cell lines CCRF-CEM (IC50 = 6.30 nM), CCRF-HSB-2 (IC50 = 10.4 nM) and MOLT-4 (IC50 = 10.0 nM) may be correlated with their abilities to accumulate high concentrations of Ara-CTP (> 1000 amol/cell) with increases of between 1.3- and 3.4-fold in dATP, dGTP and dTTP for the four cell lines, while dCTP decreased between 0.23- and 0.78-fold. By contrast, an Ara-C-resistant derivative of HL-60 cells (IC50 = 400 nM) accumulated only low concentrations of Ara-CTP (71 amol/cell) without significant changes in dNTPs. High concentrations of Ara-CTP in leukemia cells induce accumulations of dATP, dGTP and dTTP due to inhibition of DNA synthesis, and depletion of dCTP. This imbalance in the pools of the four dNTPs could lead to genetic miscoding and cell death.
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PMID:Effects of cytosine arabinoside on human leukemia cells. 893 Jan 29

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the intracellular metabolism of cytosine arabinoside (Ara-C) was comparatively analyzed in normal bone marrow mononuclear cells (NBMMC) from eight healthy volunteers and in leukemic blasts from 50 patients with acute myeloid leukemia (AML). Pretreatment with GM-CSF (100 U/ml) for 48 h resulted in a significant enhancement of DNA synthesis in both cell types: 21 of 35 AML specimens were found to be responsive to GM-CSF as defined by an increase of 3H-TdR incorporation into the DNA > 1.5-fold while NBMMC from normal donors were responsive in all cases. In GM-CSF responsive AML blasts, overall DNA polymerase and DNA polymerase alpha activity increased from a median of 84.4 to 96.1 and from 3.45 to 5.2 pmol/min x mg as compared to a median of 96.7 to 189.9 and 1.2 to 2.2 pmol/min x mg in NBMMC (P < 0.05). Median Ara-C-mediated inhibition of DNA synthesis was significantly more effective in AML blasts as compared to NBMMC (76.5 vs 55.0% at 0.05 microM and 99.0 vs 96.0% at 5.0 microM Ara-C, P < 0.01) but was not influenced by GM-CSF pretreatment. Similarly, intracellular Ara-CTP levels were higher in AML blasts as compared to NBMMC (median of 46.9 vs 18.7 at 1 microM, 167.8 vs 48.0 at 10 microM and 337.5 vs 59.5 ng/10(7) cells at 100 microM extracellular Ara-C, P < 0.01) but showed no enhancement in the presence of GM-CSF. Median deoxycytidine (DCK) and thymidine kinase (TK) activity were only slightly increased in AML blasts after GM-CSF priming. In contrast, NBMMC revealed a significant increase in TK activity after GM-CSF pretreatment (from a median of 1.9 to 3.6 pmol/min x mg, P = 0.039). At low; intermediate and high extracellular Ara-C concentrations GM-CSF pretreatment resulted in a significant enhancement of the 3H-Ara-C incorporation into the DNA in both GM-CSF responsive AML blasts and NBMMC (median of 1.3 to 2.1- and 1.4 to 1.6-fold, P < 0.05). GM-CSF non-responsive AML blasts showed no change in 3H-Ara-C incorporation into the DNA in response to GM-CSF at low Ara-C concentrations but significant increases at intermediate and high extracellular Ara-C concentrations (median increases of 1.63-fold at 1.06 microM with P = 0.01 and 1.37-fold at 10 microM extracellular Ara-C with P = 0.0+005). NBMMC revealed significantly lower GM-CSF-induced increases of the 3H-Ara-C incorporation into the DNA as compared to the effect of GM-CSF priming on DNA synthesis (median increases of 1.4 to 1.7-fold vs 2.6-fold, P < 0.05). These data reveal a different effect of GM-CSF priming on the metabolism of Ara-C in normal vs leukemic cells which may cause a preferential increase in the antileukemic cytotoxicity of Ara-C in the presence of GM-CSF.
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PMID:Differential effect of GM-CSF pretreatment on intracellular Ara-C metabolism in normal bone marrow mononuclear cells vs acute myeloid leukemia (AML) blasts. 909 97


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