Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of methotrexate (MTX) in the presence or absence of exogenous thymidine (dThd, 10(-5) M) or hypoxanthine (Hx, 10(-4) M) on cell cycle kinetics and deoxyribonucleoside triphosphate pools (dNTP) were studied in cultured human leukemic T-cells (CCRF-CEM). MTX cytotoxicity was found to increase linearly with drug dose for MTX concentrations between 10(-9) M and 10(-7) M. No further increase in cytoxicity was observed with much higher MTX concentrations (10(-7) M-10(-4) M). A similar dose-response relationship was found for both MTX-induced inhibition of DNA synthesis and changes in dTTP and dGTP pools but not for either MTX-induced inhibition of purine synthesis or changes in dATP and dCTP pools. Exogenous dThd reduced MTX cytotoxicity, at all MTX concentrations examined, but Hx reduced cytotoxicity only at MTX concentrations less than 6 X 10(-8) M and potentiated toxicity with higher MTX concentrations. This potentiation of cytotoxicity was accompanied by substantial elevation of dATP pools. In all instances where dThd or Hx reduced MTX cytotoxicity, a concomitant increase in both dTTP and dGTP levels and in the rate of DNA synthesis was observed. These results suggest a close correlation between MTX-induced alterations of dNTP and inhibition of DNA synthesis and subsequent MTX cytotoxicity. The possible modulation of MTX cytotoxicity by purines is discussed.
Mol Pharmacol 1982 Jan
PMID:Biochemical and cell cycle perturbations in methotrexate-treated cells. 698 94

We investigated the mechanism of action of 2-aminopurine (Apur) in eucaryotic cells. By analogy with studies in procaryotic systems, the base analog is presumed to incorporate into DNA predominantly opposite T where, upon subsequent DNA replication, it can mispair with C, inducing an A:T leads to G:C transition. This model predicts that Apur-induced mutagenesis will be enhanced by factors that favor formation of Apur-C mispairs, e.g., high levels of dCTP or low levels of TTP. We describe the use of a mutant T-lymphosarcoma cell line, AraC-6-1, which has an abnormally high dCTP pool and a low TTP pool, to test this prediction. AraC-6-1 cells were three- to fivefold more mutable by Apur than their parental cell line, NSU-1. This enhanced mutability by Apur could not be explained by altered incorporation of 3H-labeled Apur, by generally impaired ability to repair DNA damage, or by a direct effect of Apur on the endogenous deoxynucleotide pools. The addition of 10 microM thymidine to the growth medium of AraC-6-1 cells lowered their high dCTP pool (two- to threefold), raised the TTP pool (two- to threefold), and abolished their enhanced mutability by Apur. Further manipulation to produce an abnormally high TTP/dCTP ratio suppressed Apur-induced mutagenesis (8- to 10-fold) in both AraC-6-1 and NSU-1 cells. These observations support the hypothesis that Apur induces A:T leads to G:C transitions in mammalian cells by a mispairing mechanism.
Mol Cell Biol 1982 Sep
PMID:Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells. 698 47

Human thymidine kinase TK1 isoenzyme has been purified 1800-fold from placenta to a specific activity of 2.9 nmoles/min/mg of protein. The rapid purification procedure includes affinity chromatography on a thymidine-Sepharose column. At all stages of purification, the enzyme showed irreversible lability. The native molecular weight was determined to be 45000. Human placental TK1 exhibited specificity for ATP and thymidine as substrates, and significant inhibition was found only with thymidine nucleotides. TTP was the most effective inhibitor.
Mol Cell Biochem 1982 Jun 11
PMID:Human thymidine kinase: purification and some properties of the TK1 isoenzyme from placenta. 698 69

The interaction of dTTP and dATP gamma-4-(N-2-chloroethyl-N-methylamino) benzylamidates with E. coli DNA-polymerase I were studied. dTTP and dATP gamma-4-(N-2-hydroxyethyl-N-methylamino) benzylamidates act as competitive inhibitors of DNA-polymerase I. Ki values for gamma-analogues of dTTP and dATP have been determined. Reactive dTTP and dATP derivatives are shown to be affinity reagent for this enzyme.
Mol Biol (Mosk)
PMID:[Affinity modification of Escherichia coli Dna-polymerase I by 4-(N-2-chloroethyl-N-methylamino)-benzyl-gamma-amides of dTTP and dATP]. 699 29

5'-Amino-5'-deoxythymidine (5'-AdThd) was found to antagonize the feedback inhibition exerted by dTTP on dThd kinase (EC 2.7.1.21). This effect was demonstrable in intact cells, cellular extracts, and purified enzyme preparations. Thus, 5'-AdThd markedly stimulated the uptake of dThd in Hela and Vero cells and reduced the inhibitory effects of dTTP on the dThd kinase activity measured in extracts from both cell types. dThd kinase was purified by affinity column chromatography from Hela and Vero cells, and 5'-AdThd was again shown to reduce the inhibition caused by dTTP. The ability of 5'-AdThd to disrupt the homeostatic mechanisms normally regulating the uptake of dThd was sufficient to convert a noncytotoxic concentration of dThd (30 microM) to one that inhibited Hela cell growth by 40%. Since the activity of regulatory enzymes critically influences the pharmacological response produced by many agents, we propose the design of compounds specifically targeted at enzyme regulatory sites as an approach to drug development.
Mol Pharmacol 1982 Sep
PMID:Enzyme regulatory site-directed drugs. MOdulation of thymidine triphosphate inhibition of thymidine kinase by 5'-amino-5'-deoxythymidine. 714 27

5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D-lyxofuranosyl) thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl) thymine and 2',3'-lyxoanhydrothymidine have been shown to be terminator substrates of human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases as well as DNA polymerase I from E. coli. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. The KM values of ltTTP, rtTTP and laTTP incorporation into DNA chain agree closely with each other, being 1.5-2.5 times higher than KM for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times less than Vmax for dTTP. The evidence favours the hypothesis of a great affinity of modified nucleosides with flattened ribose ring of glycone for DNA polymerases active sites.
Mol Biol (Mosk)
PMID:[Modified nucleoside-5'-triphosphates with an additional conjugated through the 2'-3'-fused ring as DNA polymerase substrates]. 751 83

dCMP-deaminase-deficient V79/dC hamster cells have highly imbalanced deoxyribonucleoside triphosphate (dNTP) pools, i.e. a 17-fold larger dCTP pool, a slightly reduced dTTP and a very low dGTP pool, compared to dCMP-deaminase-proficient V79/p cells. Nevertheless, the two lines showed the same rates of spontaneous mutation at the hprt and ouabain-resistance loci. Analysis of spontaneous hprt mutations indicated an increase in misincorporation of C in V79/dC cells, although it was not statistically significant. When the dCTP pool was further increased fivefold by incubating V79/dC cells with cytidine, C misincorporation increased to 88%, but the mutation frequency remained unchanged. The dNTP pools of V79/dC cells were also altered by treatment with thymidine, or with thymidine plus deoxycytidine. After incubation with thymidine alone, the dCTP pool all but disappeared, whereas it maintained a normal level in the presence of deoxycytidine. In both cases dTTP rose to nmol amounts, and dGTP accumulated. Incubation with 10 mM thymidine was the only treatment that increased the mutation frequency; T misincorporation then accounted for 94% of the base substitutions. In the presence of deoxycytidine the cells had a dTTP/dCTP ratio of 0.04, but 86% of the base substitutions involved C misincorporation and most probably originated from G mis-incorporation caused by excess dGTP. Alterations of RNA splicing and hot spots for base substitutions varied with the imbalance, the latter showed "next-nucleotide effects". Our results suggest that the fidelity of DNA replication in V79 cells is only affected by large changes in the pool and is more sensitive to changes in dGTP than in dCTP or dTTP.
J Mol Biol 1995 Oct 06
PMID:Molecular analysis of mutations in the hprt gene of V79 hamster fibroblasts: effects of imbalances in the dCTP, dGTP and dTTP pools. 756 70

To investigate whether DNA replication in malignant cells deviates from that of normal cells we compared DNA polymerases alpha, delta, and epsilon from normal rat liver to the enzymes from fast-growing (malignant) Novikoff hepatoma cells. DNA polymerases were purified 300-fold by three chromatographic steps. Characterization included measurement of physicochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), quantitation of catalytic activities using specific DNA primer templates (Km values) and inhibitors (Ki values), and identification of polypeptides which are strongly associated with DNA polymerases. Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. In contrast, the DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with the specific primer templates gapped calf thymus DNA and poly(dA.dT), were higher. Furthermore, sedimentation and diffusion coefficients, Stokes' radii, and frictional coefficient ratios of DNA polymerases alpha and epsilon from malignant cells significantly deviated. In addition, when the dNTP-binding sites were probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP), significantly lower Ki values were obtained for the polymerases from Novikoff cells indicating lower affinity of the dNTP binding site to deoxyribonucleoside 5'-triphosphates. Altered catalytic and molecular properties are possibly a consequence of malignant transformation. It is to be expected that similar changes occur in DNA polymerases of other tumors. In particular, diminished affinity to primer templates and weakened nucleotide binding leads to lowered specificity of nucleotide selection in the base-pairing process and is therefore likely to cause an enhanced mutation rate during malignant progression.
J Mol Med (Berl) 1995 May
PMID:DNA polymerases alpha, delta, and epsilon of Novikoff hepatoma cells differ from those of normal rat liver in physicochemical and catalytic properties. 767 Sep 30

Kinetic studies on the two major isoforms of Serratia marcescens nuclease, Sm2 and Sm1, have revealed them to be functionally equivalent. Both isoforms display marked substrate inhibition by DNA and RNA. They both require magnesium for optimal activity, but retain low catalytic activity in its absence. Both are moderately inhibited by mononucleotides including 5'-ATP, 5'-AMP, 5'-TTP and 3'5'-pTp. The two strongest mononucleotide inhibitors studied, 5'-ATP and 5'-AMP, display inhibition constants, KI, on the order of 10(-5) M. In assessing the strength of mononucleotide inhibition the type of nucleotide base appears to be more important than the number of phosphate moieties.
Biochem Mol Biol Int 1994 Aug
PMID:Kinetic studies of the Serratia marcescens extracellular nuclease isoforms. 780 50

We have generated transgenic mice that express the simian virus 40 (SV40) large T antigen under the control of a 1109 bp 5'-flanking sequence of the human thyrotropin beta-subunit (TSH beta) gene. The hybrid gene, termed TTP-1, was microinjected into fertilized mouse eggs and 11 transgenic mice were obtained. One of the transgenic mice, a female, a phenotypical dwarf, developed a pituitary tumor and wasted away from 7 to 9 weeks after birth. To establish the transgenic mouse line, her ovaries were transferred to a normal female, whose ovaries were removed beforehand. To examine the tissue specificity of transgene expression, mRNA of SV40 large T antigen was monitored in various tissues from the transgenic mice by the reverse transcriptase-polymerase chain reaction analysis, and was detected only in the pituitary. Histological and immunohistochemical analyses showed that the pituitary tumors of the transgenic mice were composed of poorly differentiated pituitary cells expressing SV40 large T antigen. These results indicated that the 1109 bp sequence of the human TSH beta 5'-flanking region is essential for pituitary-specific expression of SV40 large T antigen in transgenic mice, which exhibited a dwarf phenotype and developed pituitary tumors. The tumors were composed of undifferentiated cells and did not produce thyrotropin. These transgenic mice should provide a valuable animal model for studying the pathogenesis of anterior pituitary tumors.
Mol Cell Endocrinol 1994 Nov
PMID:Targeted pituitary tumorigenesis using the human thyrotropin beta-subunit chain promoter in transgenic mice. 785 21


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