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)

Local recurrence of sarcoma is due to residual tumor cells remaining after surgical resection and is associated with decreased survival. We implemented adenoviral-mediated transfer of the herpes simplex thymidine kinase (HSTK) gene with subsequent ganciclovir (GCV) administration to treat a model of residual sarcoma, [3H]Thymidine uptake in MCA sarcoma cells was determined after infection with replication incompetent adenovirus of the AdMLP.HSTK construct in the presence of GCV. In vivo efficacy was evaluated in a model of residual sarcoma when 9 mg of MCA tumor was implanted into the latissimus muscle of Fischer 344 rats. Three days after implantation, animals were randomized to receive AdMLP.HSTK, AdCMV. Null, or viral suspension buffer intratumorally. From Day 4, animals were administered b.i.d. GCV (50 mg/kg) or saline ip. Tumors were excised on Day 14 and weighed. Statistical analysis was by Mann-Whitney U test. In vitro: [3H]thymidine incorporation was significantly decreased in MCA sarcoma cells infected with AdMLP.HSTK in the presence of GCV (P < 0.05). In vivo: Growth of MCA sarcoma treated with AdMLP.HSTK and GCV was significantly inhibited. Final tumor weights in the AdMLP.HSTK/GCV group were lower than all control groups (P < 0.05). A significant antitumor growth effect on MCA sarcoma was seen with adenoviral-mediated transfer of the HSTK gene and GCV administration, both in vitro and in an in vivo model of residual disease. This prodrug gene therapy strategy warrants investigation as an adjuvant modality in the management of sarcoma.
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PMID:Adenoviral thymidine kinase prodrug gene therapy inhibits sarcoma growth in vivo. 922 20

Adenovirus-mediated herpes simplex thymidine kinase/ganciclovir (HSV-tk/GCV) system has been demonstrated to be efficient for the treatment of experimental brain tumors. However, no study has been directed to the elimination of proliferating cellular populations in other pathological conditions. In this study we used this suicide gene approach in a primary culture of astrocytes, as a model of reactive gliosis, in order to evaluate its efficiency as a therapeutic strategy for post-traumatic astrogliosis in vivo. First, we evaluated the peak of astrocytic proliferation to characterize our model. Second, the efficiency of adenovirus-mediated lacZ gene transfer is shown to be dependent on vector multiplicity of infection (MOI). As expected, the cells transfected with the HSV-tk gene showed an increase in sensibility to GCV compared with cells transfected with lacZ gene. Finally, an unexpected interaction between the adenoviral vector and bromodeoxyuridine (BrdU) or [3H]-Thymidine ([3H]-Thy) was evidenced in transfected cultures, whose interpretation is discussed. The present study demonstrates that a recombinant adenoviral vector carrying the tk gene confers to in vitro cultured astrocytes a cytotoxic sensibility to GCV, and that this system constitutes a potentially efficient tool to eliminate the hyperplasia of astrocytes following injury to the central nervous system in vivo.
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PMID:Adenovirus-mediated suicide gene therapy in an in vitro model of reactive gliosis. 993 75

The cytotoxicity of LY231514 was only partially alleviated by thymidine addition (5 microM) in GC3 human colon carcinoma cells, and complete protection required the addition of both hypoxanthine (100 microM) and thymidine. In contrast, the cytotoxic activity of tomudex (raltitrexed, ZD1694) was completely reversed by thymidine alone. MCF-7 human breast and H630 human colon carcinoma cells selected for resistance to tomudex and 5-fluorouracil, respectively via thymidylate synthase (TS) amplification demonstrated only modest resistance to LY231514 compared to tomudex. LY231514-induced cytotoxicity in these resistant cell lines was completely prevented by the addition of hypoxanthine (100 microM), indicating inhibition of purine de novo biosynthesis as a secondary target for LY231514 action. Thymidine at physiologic levels in mouse plasma (approximately 1 microM) produced only a 2.6-fold shift in the IC50 for LY231514-mediated cytotoxicity in GC3/cl1 cells compared to a 128-fold shift for tomudex. LY231514 treatment (i.p., qd x 10) significantly delayed tumor growth in the GC3 carcinoma xenograft model. However, a thymidine kinase-deficient mutant of this same tumor line demonstrated heightened sensitivity to the in vivo antitumor activity of LY231514 with complete regression of established tumors and a large number of tumor-free survivors after one course of treatment. The data demonstrate that inhibition of thymidylate synthase is a prominent mechanism for antitumor activity by LY231514, but important secondary sites of action exist for this multitargeted molecule.
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PMID:Role of thymidylate synthase in the antitumor activity of the multitargeted antifolate, LY231514. 1022 79

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [(3)H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S-transferase (GST) fusion protein showed that the enzyme has a comparatively high K(m) for thymidine (dThd) of approximately 33.2 microM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3'-azido-3'-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK(-) cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [(3)H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.
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PMID:Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir. 1062 30

This report documents the presence of an active thymidine kinase (TK) system within Mesocestoides vogae tetrathyridia as quantified by tritiated thymidine ([3H]-TdR) incorporation using liquid scintillation counting. A 100-fold increase in [3H]-TdR incorporation was observed at 37 degrees C when compared with its incorporation at 0 degrees C. Thymidine's competitive analogue, BrdU, competed for sites within newly replicated DNA. Immunohistochemical trials performed here using antibodies against BrdU identified cells that have entered and passed through S-phase. Positively stained nuclei were most numerous at the anterior tip of tetrathyridia especially within the ganglia, lesser numbers of these cells occurred along the growing commissure and amongst surface tegumental cytons suggesting that stem cells do not exist in one region but are found throughout the entire body. As M. vogae has no internal organ systems the major sites for cell proliferation are those exhibiting maximal cell recruitment and undergoing tissue repair. These results show that it is possible to monitor changes in the cell recruitment pattern within this cestode. Thus use of BrdU and immunohistochemistry demonstrates how spatial arrangement and cellular reorganization can be successfully traced within M. vogae.
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PMID:Tritiated thymidine ([3H]-TdR) and immunocytochemical tracing of cellular fate within the asexually dividing cestode Mesocestoides vogae (syn. M. corti). 1108 29

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.
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PMID:Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers. 1224 48

TAS-102 is a new oral anti-cancer drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-trifluorothymidine (FTD) and 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). TAS-102 currently undergoing clinical trials, has been demonstrated to have at least two mechanisms, inhibition of TS and incorporation into DNA. We hypothesized that the thymidine metabolism enzyme may be a crucial factor that affects the antitumor activity of TAS-102. In the present study, we measured the enzyme activity of thymidine kinase (TK), thymidine phosphorylase (TP) and thymidilate synthase (TS) in human cancer xenografts to investigate the contribution of these enzymes to the sensitivity of TAS-102. Antitumor activity of TAS-102 appears to be associated with TK, tumor growth and TS. However, the most related factors in this study were the TK and TP ratio. There was a significant correlation (p=0.04) between tumor growth inhibition and this ratio. These results suggested that the activation and degradation pattern of FTD plays an important role in the efficacy of TAS-102 and that it is possible to use the TK/TP ratio to predict response to TAS-102 therapy. We also studied the influence of TPI on the capacity of exogenous dThd to reverse FTD-dependent growth inhibition. Thymidine (dThd) levels rescued the effect of FTD in vitro and significantly increased in serum after administration of TAS-102 or TPI alone but not FTD alone. This may suggest the possibility of a decrease in antitumor effect. However, our study indicated that the therapeutic index was clearly increased by FTD combined with TPI, compared with FTD alone, suggesting FTD-induced toxicity to sensitive host tissue can be selectively reversed with dThd. In conclusion, TK and TPI effects on TP play important roles in the cytotoxic action of TAS-102, and it is possible to use the TK/TP ratio to predict more precisely individual resistance or sensitivity.
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PMID:Thymidine kinase and thymidine phosphorylase level as the main predictive parameter for sensitivity to TAS-102 in a mouse model. 1471 72

Thymidine analogs containing o-carboranylalkyl groups at the 3-position were screened as potential substrates for human thymidine kinase 1 (TK1), an enzyme that is selectively expressed in a variety of rapidly proliferating cells, including tumor cells. On the basis of previous studies, 12 of these were identified as potential delivery agents for boron neutron capture therapy, a therapeutic method used for the treatment of high-grade brain tumors. Compound 4 with a pentylene spacer between the o-carborane cage and the thymidine scaffold and compound 10, which has an additional dihydroxypropyl substituent at the o-carborane cage, were the best substrates for TK1 with kcat/Km values of 27% and 36% relative to that of thymidine, respectively. These compounds showed partial competitive inhibition for thymidine phosphorylation by TK1. Neither compound was a substrate of recombinant human thymidine phosphorylase nor were their respective 5'-monophosphates substrates of 5'-deoxynucleotidase 1, thereby indicating potential in vivo stability. The octanol/water partition coefficient for compound 10 was 2.09, suggesting that it has excellent physiochemical properties for crossing the blood brain barrier and penetrating brain tissue. The in vitro cytotoxic effect of the 12 analogs was moderate to low in mammalian cell cultures with IC50 values between 10 and 160 micromol/L. Compounds 4 and 10 were taken up selectively and retained by the murine fibroblast L929 cell line, in contrast to its TK1-deficient variant. These findings suggest that compound 10 is a promising candidate for selective delivery of boron-10 to malignant cells, and additional in vivo studies are planned to evaluate it for boron neutron capture therapy of brain tumors.
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PMID:Evaluation of human thymidine kinase 1 substrates as new candidates for boron neutron capture therapy. 1534 16

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.
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PMID:Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme. 1633 73

Several radiolabeled thymidine analogs as metabolic probes of cell proliferation were developed specifically addressing DNA synthesis. Thymidine analogs containing carboranylalkyl groups for neutron capture therapy at the N-3 position were found to be good substrates for cytosolic thymidine kinase 1 (TK1). According to this approach, a DO3A macrocycle in N-3 position was attached to thymidine. The 3-DO3A thymidine analog was labeled with 68Ga and 111In. Different lipophilicities of the corresponding radiometal-thymidines were detected via RP-HPLC. [111In]DO3A-thymidine ([111In]D3T) was evaluated for cellular uptake in different cell lines (HL60 and DoHH2). Cellular uptake was low in both cell lines. Phosphorylation of the radioconjugates by TK1 was negligible. Although stable complexation of radiometals to thymidine was obtained, introduction of the macrocycle DO3A reduced the affinity to cytosolic TK1 drastically. Low cellular uptake can be ascribed to missing substrate specificity of [111In]DO3A-thymidine for TK1. The absence of substrate specificity may be due to the bulky macrocyclic chelator and partial charges remaining on the coordination sphere due to a more complex solution structure.
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PMID:Synthesis and evaluation of a radiometal-labeled macrocyclic chelator-derivatised thymidine analog. 1663 Oct 84


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