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)

We have undertaken to characterize the role of cytoplasmic 5'-nucleotidase (EC 3.1.3.5) in the phosphorylation of the anti-herpes simplex virus and anti-human cytomegalovirus agent ganciclovir (GCV) in MOLT-4 cells, a human T cell line adapted to grow in suspension culture. The rate of formation of GCV triphosphate was found to be approximately doubled by preincubation of nontransfected MOLT-4 cells with agents that cause the accumulation of IMP, such as ribavirin (20 microM) and mycophenolic acid (1 microM), and the reaction rate was found to be unaffected by high levels of thymidine (100 microM). With herpes simplex virus-1 thymidine kinase (HStk) gene-transduced MOLT-4 cells, the rate of GCV phosphorylation was approximately 40-fold faster than that in uninfected cells and, in marked contrast to uninfected cells, the reaction was significantly inhibited both by IMP dehydrogenase inhibitors and by thymidine. These latter effects appear to be the result of 1) the accumulation of high levels of dTTP in IMP dehydrogenase inhibitor-treated cells, with consequent feedback inhibition of HStk, and 2) direct competitive substrate inhibition by thymidine of the HStk-catalyzed phosphorylation of GCV. Thus, agents that enhance 5'-nucleotidase-catalyzed phosphorylation of GCV in uninfected cells do not play a similar role in HStk-transfected cells, a consequence of the quantitative predominance of the viral thymidine kinase-catalyzed reaction over that attributable to endogenous cytoplasmic 5'-nucleotidase.
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PMID:Effects of IMP dehydrogenase inhibitors on the phosphorylation of ganciclovir in MOLT-4 cells before and after herpes simplex virus thymidine kinase gene transduction. 791 Mar 73

The synthesis of 1,5-anhydrohexitol nucleosides is described. These nucleoside analogues were obtained by alkylation of the heterocyclic bases with the tosylate 10 or by alkylation of the bases with the alcohol 12 under Mitsunobu conditions. The compounds were evaluated for antiviral and cytostatic activity. Highly selective activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) was noted for 1,5-anhydro-2,3-dideoxy-2-(5-iodouracil-1-yl)-D-arabino-hexitol 4b at a concentration of 0.07 microgram/mL. This activity must be dependent on a specific phosphorylation by the virus-encoded thymidine kinase (TK), since compound 4b was inactive against TK-deficient mutants of HSV-1. The corresponding cytosine 4c and guanine 4e analogues showed activity against HSV-1, HSV-2, and other herpes viruses (i.e. cytomegalovirus, varicella-zoster virus) at concentrations well below the cytotoxicity threshold (2 and 20 micrograms/mL, respectively). At these concentrations, compounds 4c and 4e proved also inhibitory to the growth of human T-cells (i.e. MT-4, CEM, MOLT-4).
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PMID:Synthesis and antiherpes virus activity of 1,5-anhydrohexitol nucleosides. 839 14

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.
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PMID:Biochemical and cellular pharmacology of 1843U89, a novel benzoquinazoline inhibitor of thymidylate synthase. 842 62

The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein.
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PMID:The role of p16 in the E2F-dependent thymidine kinase regulation. 862 83

Although zinc is known to be involved in cell proliferation and DNA synthesis, the mechanism by which zinc may regulate these processes is not understood. We have studied the role of zinc on cell proliferation and gene expression of a DNA synthesizing enzyme, deoxythymidine kinase (TK), in a T helper human malignant lymphoblastoid cell line (HUT-78). In zinc-deficient and zinc-sufficient media, the cell doubling time (mean +/- SD) of HUT-78 was 59 +/- 8 hours and 32.6 +/- 6 hours, respectively. The effect of zinc was T cell specific, inasmuch as the cell growth of another T malignant lymphoblastoid cell line, MOLT-3 (immature T cells), was not affected by zinc deficiency. Iron, copper, or manganese did not completely correct the cell growth of zinc-deficient HUT-78 cells. TK activity and the relative accumulation of TK-mRNA were significantly decreased in zinc-deficient cells during the G1 phase of cell cycle in comparison with zinc-sufficient cells. Nuclear run-on experiments and actinomycin-D studies showed that the transcription of TK-mRNA was affected adversely by zinc deficiency. Cell cycle studies showed that more zinc-deficient cells remained in S phase and did not undergo mitosis in comparison with zinc-sufficient cells. In conclusion, our data show that zinc is a T cell-specific growth factor and that a decreased gene expression of DNA-synthesizing enzyme TK in zinc-deficient HUT-78 cells in G1 phase affected adversely the DNA synthesis in S phase and delayed cell cycle.
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PMID:Zinc deficiency affects cell cycle and deoxythymidine kinase gene expression in HUT-78 cells. 875 31

This paper reports the results of 31P and 1H nuclear magnetic resonance (NMR) studies on the uptake and phosphorylation of 3'-azido-3'-deoxythymidine (AZT) in the human CD4+ T-lymphoblastoid cell line CCRF-CEM (CEM-1.3) and in its AZT-resistant cell variant MT-500, isolated by prolonged culturing of CEM cells in the presence of increasing AZT concentrations. After 3 hr of incubation in the presence of 0.5 mM AZT, both AZT and its monophosphorylated form (AZT-MP) could be detected in the sensitive cell line in concentrations above the NMR detection levels. In another cell line, MOLT-4, which is less sensitive to AZT effects, the intracellular level of AZT-MP was much lower and was only slightly raised by increasing the concentration of AZT in the extracellular and intracellular compartments. In the AZT-resistant clone MT-500, characterized by a very low thymidine kinase (TK, EC 2.7.1.21) activity with respect to the parental clone, the intracellular AZT-MP concentration was below detection (<0.02 nmol/10(6) cells). Since, however, not only AZT-MP but also AZT signals failed to be detected in MT-500 extracts following cell incubation with AZT, it was concluded that a TK deficiency cannot be the exclusive mechanism of AZT resistance in these cells. The possible effects of additional mechanisms of drug resistance, such as specific AZT cell extrusion and limited permeation, are discussed, together with the new prospects offered by NMR spectroscopy to further evaluate the limiting steps for the utilization of antiretroviral nucleoside analogues.
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PMID:Intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT): a nuclear magnetic resonance study on T-lymphoblastoid cell lines with different resistance to AZT. 937 18

Zidovudine (ZDV) is a thymidine analogue activated to its triphosphate (ZDVTP) by the host's intracellular enzymes. The initial phosphorylation step is conversion to ZDV monophosphate (ZDVMP). The poor affinity of ZDVMP for thymidylate kinase results in intracellular accumulation of ZDVMP. Clinical use of ZDV is associated with cytotoxicity, thought to be mediated through mitochondrial damage. It has been suggested that ZDV cytotoxicity correlates with intracellular ZDVMP. Here we have further studied the role of ZDVMP in cytotoxicity and some of the mechanisms involved. Intracellular metabolism of ZDV in five lymphocyte/monocyte cell lines, U937, BSM, MOLT 4, JJAHN, and RAJI (4 x 10(6) cells), was investigated following 24 h incubation with [(3)H]ZDV (1.2 microCi; 0.1 microM) and cytotoxicity was determined by the MTT assay. Cytotoxicity was closely related to intracellular concentrations of the major metabolite (ZDVMP) but not with the active metabolite ZDVTP. ZDVMP was the only metabolite detected following incubation of viable mitochondria isolated from U937 cells with ZDV (1.2 microCi; 0.1 microM; 24 h) with mitochondrial levels of 0.27 +/- 0.11 pmol/microg protein (mean +/- SD; n = 3). No MTT toxicity was seen in isolated mitochondria. Following phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells there was an increase in ZDV cytotoxicity compared to unstimulated cells. The results suggest that the mitochondrial isozyme of thymidine kinase (TK2) plays only a minor part in ZDVMP formation. Following PHA stimulation, activation of the cytosolic thymidine kinase isozyme (TK1) is associated with increased toxicity of ZDV. We conclude that ZDVMP responsible for mitochondrial toxicity is formed in the cytosol.
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PMID:Zidovudine phosphorylation and mitochondrial toxicity in vitro. 1170

The biological evaluation of mononucleotide prodrugs (pronucleotides) of various nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), zalcitabine (ddC) and lamivudine (3TC) was reported in human T-lymphoid MOLT-4/8 cells which were grown continuously for more than 1 year in a medium containing cytarabine (Ara-C). In this cell line, expression of deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) was decreased in comparison to parental cells (3.8 and 2.9-fold, respectively). The lower mRNA level of TK1 correlated significantly with lower enzyme activity, whereas no dCK activity was detectable. In Ara-C-resistant cells, anti-HIV-1 effects of ddC, 3TC and AZT were more than 100-fold lower compared with parental cells. In contrast, the corresponding mononucleoside phosphotriesters bearing S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protection retained anti-HIV-1 activity due to their ability to bypass the first monophosphorylation step catalyzed by dCK or TK1. The results demonstrate that in vitro selection of T-lymphoid cells in the presence of Ara-C results in cross-resistance to deoxycytidine (ddC, 3TC) and thymidine (AZT) analogs and that these cellular resistance mechanisms can be bypassed by the use of bis(SATE) pronucleotides.
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PMID:S-acyl-2-thioethyl (SATE) pronucleotides are potent inhibitors of HIV-1 replication in T-lymphoid cells cross-resistant to deoxycytidine and thymidine analogs. 1175 Sep 40

The nucleoside analogs 9-beta-D-arabinofuranosylguanine (araG) and 1-beta-d-arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 rho(0) cells (lacking mtDNA). The mRNA levels and activities of deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p<0.01) in the rho(0) cells, whereas the TK1 mRNA level was lower (p<0.05). The enzyme activity of the S-phase restricted TK1 was also lower (p<0.05) in the MOLT-4 rho(0) cells, whereas the activities of dGK, dCK and TK2 were similar in MOLT-4 wt and rho(0) cell lines. The sensitivities to different cytotoxic nucleoside analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells.
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PMID:Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA. 1948 40

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