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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In our view, two major areas of the investigation of the aging process have been most fruitful over the past few years: namely, the genetic and hormonal strategies aimed at the understanding of in vitro cellular senescence. The genetic studies have primarily utilized cell fusion techniques and viral probes. Along with cell cycle studies involving the induction of thymidine kinase activity and TTP synthesis, the cell fusion studies are most consistent with a late G1 block in senescent cells. This effect would appear to be distinct from the G0 arrest of density-inhibited or mitogen-restricted cell populations. The hormonal studies which have centered on the regulation of cell proliferation have recently focused on peptide hormones. EGF has been of particular interest since it is so well characterized. This receptor system remains largely unchanged throughout the lifespan with the notable exception of the purified receptor-associated, autocatalytic, tyrosine-specific kinase activity, which decreases with age. The functional significance of this decrease in enzyme activity is unknown, although its growth regulatory importance is implicated in several systems, and may well represent a critical early G0/G1 event which is absent in senescent cells.
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PMID:Cellular senescence: factors modulating cell proliferation in vitro. 299 27

Seventy five human breast cancers were examined in order to search for the presence of thymidine kinase of the fetal-type (TK-F). The presence of TK-F was evidenced in all tumors. Its activity varied from one to another tumor, but it was evident that the increased TK activity observed in mammary cancers could exclusively be related to high TK-F activity. Some relations between TK-F activity and the presence of estradiol and progesterone receptors (ER, PR) were obvious. The highest activities were observed in cancers with high level of ER and PR. Thymidylate kinase activity (d-TTP synthesis) varied in parallel with TK-F activity. In a general way, it was higher in ER+ PR+ than in ER+ PR- cancers.
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PMID:[Demonstration of fetal-type thymidine kinase in cancer of the breast]. 302 50

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.
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PMID:Human thymidine kinase: purification and some properties of the TK1 isoenzyme from placenta. 698 69

The rapidly growing mouse Gaelstein hepatomas 22 and 22a and rat Zajdela hepatoma are characterized both by a high thymidine kinase activity, increased TTP pool and intense 14C-thymidine incorporation into DNA. This is indicative of an intensive thymidylate biosynthesis via a short, "salvage" pathway. The predominance of this pathway for thymidylate is also characteristic for the spleens of normal animals. On the contrast, in rat and mouse thymus, where the TTP pool was the highest of all normal tissues studied, the thymidine kinase activity and thymidine incorporation into DNA were relatively low. The growth of the three hepatomas under study induces involution of tumour carrier thymus, manifested in a decrease of the TTP pool and the rate of labelled thymidine incorporation into DNA, as well as in a 4-fold decrease of the thymidine kinase activity of rat thymus. In the spleen of mice carrying ascite 22a and solid 22 hepatomas an entirely opposite response to the tumour growth was observed, i. e. in the former case the organ weight and all indices of DNA synthesis were sharply reduced, while in the latter case they were substantially enhanced. In the spleen of Zajdela hepatoma carriers the DNA synthesis is suppressed as can be evidenced from the decrease of labelled thymidine incorporation into DNA and of TTP pool; the weight of organ and the thymidine kinase activity, however, exceed the normal level more than 2-fold.
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PMID:[Thymidine kinase activity, intracellular TTP content and DNA synthesis in transplantable hepatomas and lymphoid tissue of the host]. 721 50

Thymidine kinase (ATP : thymidine 5'-phosphotransferase, EC 2.7.1.21), purified to apparent homogeneity from human liver, was found to have Michaelis constants for thymidine and ATP of 5 and 90 microM, respectively. Based on studies of initial velocity and product inhibition, the enzyme kinetic mechanism is compatible with an ordered sequential reaction with thymidine binding first and thymidine monophosphate released last. The activity of various triphosphate nucleosides as phosphate donors for human liver thymidine kinase showed little specificity with ATP greater than CTP greater than UTP greater than GTP and the respective Michaelis constants ranged from 0.10 to 0.30 mM. Among various purine and pyrimidine compounds, only TTp and dCTP were effective inhibitors of the enzyme. Inhibition with TTP was competitive with respect to both thymidine and ATP with Ki values of 13.5 and 8.5 microM, respectively, while the inhibition produced by dCTP was complex. Deoxycytidine was found to be an effective nucleoside substrate for human liver thymidine kinase with a Michaelis constant of 6 microM. This finding suggests that human mitochondrial deoxycytidine and thymidine kinase activity is a single protein.
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PMID:Kinetic mechanism and inhibition of human liver thymidine kinase. 728 1

The effect of mutational loss of thymidine kinase (TK) on the sensitivity to alkylating agents was investigated in promyelocytic, HL-60, and T-lymphoblastoid, Molt-3, human leukemia cell lines. Although both cell lines exhibited approx. 1% residual TK activity, only HL-60 TK deficient cells had a decreased intracellular TTP pool, i.e., 20% of that of the wild-type. When treated with N-methyl-N'-nitronitrosoguanidine or ethyl methanesulfonate, HL-60 TK deficient cells showed significantly increased killing and mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus relative than did wild-type. Pretreatment of cells with O6-benzylguanine, an inhibitor of O6-alkylguanine-DNA alkyltransferase, partially abolished those differences. Molt-3 wild-type and TK deficient cells had similar cell survivals and HGPRT mutation frequencies following treatment with alkylating agents. These results indicate that TK deficiency, only when a concomitant decrease of TTP pool is detected, plays a pivotal role in the sensitivity to the cytotoxic and mutagenic effects of alkylating agents.
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PMID:Thymidine kinase deficient cells with decreased TTP pools are hypersensitive to DNA alkylating agents. 853 43

3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. The aim of the design program was to identify TS inhibitors with different pharmacological characteristics from classical folate analogs and, most notably, to develop non-glutamate-containing molecules which would not require facilitated transport for uptake and would not undergo intracellular polyglutamylation. One molecule which resulted from this program, AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention. In vivo, AG337 was highly active against the thymidine kinase-deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress.
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PMID:AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies. 861 3

Zidovudine (AZT; 3'-azido-3'-deoxythymidine), a thymidine analog, has been a staple of highly active antiretroviral therapy. It is phosphorylated in the host to the triphosphate and functions by inhibiting the viral reverse transcriptase. However, long-term use of AZT is linked to various tissue toxicities, including cardiomyopathy. These toxicities are associated with mitochondrial DNA depletion, which is hypothesized to be caused by AZT triphosphate inhibition of mitochondrial DNA polymerase gamma. In previous work with isolated heart mitochondria, we demonstrated that AZT phosphorylation beyond the monophosphate was not detected and that AZT itself was a potent inhibitor of thymidine phosphorylation. This suggests an alternative hypothesis in which depletion of the TTP pool may limit mitochondrial DNA replication. The present work extends these studies to the whole cell by investigating the metabolism of thymidine and AZT in the intact isolated perfused rat heart. [3H]thymidine is converted to [3H]TTP in a time- and concentration-dependent manner. The level of [3H]TMP is low, suggesting that the reaction catalyzed by thymidine kinase is the rate-limiting step in phosphorylation. [3H]AZT is converted in a time- and concentration-dependent manner to AZT monophosphate, the only phosphorylated product detected after 3 h of perfusion. Both compounds display negative cooperativity, similar to the observations with cloned and purified mitochondrial thymidine kinase 2. The presence of AZT in the perfusate inhibits the phosphorylation of [3H]thymidine with a 50% inhibitory concentration of 24+/-4 microM. These data support the hypothesis that AZT-induced mitochondrial cardiotoxicity may be caused by a limiting pool of TTP that lowers mitochondrial DNA replication.
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PMID:Zidovudine inhibits thymidine phosphorylation in the isolated perfused rat heart. 1722 Apr 3

3'-azido-3'-deoxythymidine (AZT) has been shown to be a potent inhibitor of thymidine kinase 2 in work from this laboratory. Inhibition results in decreased salvage of thymidine to TTP, which may lead to depletion of the TTP pool and result in the mitochondrial dysfunction and mt-DNA depletion observed with AZT toxicity. The effect of AZT on thymidine phosphorylation in growing cells expressing thymidine kinase 1 has not been shown. Three cell lines were used in these experiments: H9c2, derived from rat cardiomyoblasts; U-937, derived from human monocytes; and Raji, derived from human lymphoblasts. AZT inhibited growth in a concentration-dependent manner in U-937 cells, but not the other cell lines. The phosphorylation of [3H]-thymidine or [3H]-AZT was determined during log growth. All cell lines salvaged and phosphorylated thymidine to TTP, with TTP the major product. The U-937 cells had a much more active salvage pathway than the other cells. All cell lines phosphorylated AZT to the triphosphate, but the major product was AZTMP. The AZT inhibition of growth of the U-937 cells did not correlate with levels of the phosphorylated AZT. In contrast, pro-drug AZT was shown to inhibit thymidine phosphorylation in all lines with 50% inhibition concentrations (IC50) ranging from 4.4 to 21.9muM. Since the U-937 cells expressed higher activity of the salvage pathway than the other cell lines, the U-937 cells may rely more heavily on the salvage pathway for TTP synthesis, accounting for AZT inhibition of growth.
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PMID:Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines. 1829 88

A novel nucleoside analogue, 1-[(2S,4S-2-(hydroxymethyl)-1,3-dioxolan-4-yl]5-vinylpyrimidine-2,4(1H,3H)-dione, or HDVD, was evaluated against a wide variety of herpesviruses and was found to be a highly selective inhibitor of replication of the gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). HDVD had also a pronounced inhibitory activity against murine herpesvirus 68 (MHV-68) and herpes simplex virus 1 (HSV-1). In contrast, replication of herpesvirus saimiri (HVS), HSV-2, and varicella-zoster virus (VZV) was weakly inhibited by the compound, and no antiviral activity was determined against human cytomegalovirus (HCMV) and rhesus rhadinovirus (RRV). The HDVD-resistant virus phenotype contained point mutations in the viral thymidine kinase (TK) of HSV-1, MHV-68, and HVS isolates. These mutations conferred cross-resistance to other TK-dependent drugs, with the exception of an MHV-68 mutant (E358D) that exhibited resistance only to HDVD. HSV-1 and HVS TK-mutants isolated under selective pressure with bromovinyldeoxyuridine (BVDU) also showed reduced sensitivity to HDVD. Oral treatment with HDVD and BVDU was assessed in an intranasal model of MHV-68 infection in BALB/c mice. In contrast to BVDU treatment, HDVD-treated animals showed a reduction in viral DNA loads and diminished viral gene expression during acute viral replication in the lungs in comparison to levels in untreated controls. The valyl ester prodrug of HDVD (USS-02-71-44) suppressed the latent infection in the spleen to a greater extent than HDVD. In the present study, HDVD emerged as a highly potent antiviral with a unique spectrum of activity against herpesviruses, in particular, gammaherpesviruses, and may be of interest in the treatment of virus-associated diseases.
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PMID:Activity and mechanism of action of HDVD, a novel pyrimidine nucleoside derivative with high levels of selectivity and potency against gammaherpesviruses. 2334 17


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