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

---

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

We have previously reported that 5'-aminothymidine (5'-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro. In this work we have analyzed the factors that could potentially account for the differential effect of 5'-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 and HU cells, 5'-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5'-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5'-AdThd. Furthermore, [3H]-5'-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5'-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5'-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15-26 microM) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2-4 microM). This suggests that thymidine kinase is in a strongly inhibited state in situ. To test the sensitivity of thymidine kinase (in situ) to regulation by dTTP we investigated: (a) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and (b) the effect of pH on the uptake of dThd and its perturbation by 5'-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T1/2 and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Basis for the differential modulation of the uptake of 5-iododeoxyuridine by 5'-aminothymidine among various cell types. 270 29

Cells commonly resist growth inhibition by purine and pyrimidine bases and nucleosides by restricting intracellular formation of the corresponding 5'-mononucleotides. Nucleotide derivatives that can act as effective membrane-transport precursors of the poorly membrane-permeable nucleotides have not been identified so far. We studied the bis(pivaloyloxymethyl)ester (I) of FdUMP (5-fluoro-dUMP) and a cyclic phosphodiester (II) of FdUMP derived from 1,3-dihydroxyl-1-C-(pivaloyloxy-methyl)propane which are active in vivo against a 5-fluoro-2'-deoxyuridine (FUdR)-resistant mouse leukemia and are attacked by carboxylic esterases under physiological conditions to produce FdUMP by elimination of formaldehyde and acrolein respectively. The assay for intracellular FdUMP was the inhibition of DNA synthesis due to inhibition of TMP synthetase in cultured mouse LM(TK-) fibroblasts genetically devoid of thymidine kinase (TK) and thus unable to convert FUdR directly to FdUMP. At 10(-6)M, I, II, or FUdR inhibited DNA synthesis in 2 hr by 99, 80, and 35% respectively; at 10(-5)M. maximal inhibition was attained after less than 15, 30 and 90 min respectively. Inhibition of DNA synthesis in TK+ cells by 10(-5) M I, II, or FUdR was reversed completely by 10(-5)M thymidine (TdR) but unaffected by 10(-5)M UdR, confirming TMP synthetase as the locus of inhibition. At 10(-5)M, bis(pivaloyloxymethyl) esters of phenyl phosphate or a p-substituted benzylphosphonic acid did not inhibit significantly DNA synthesis in TK+ cells. From this finding, and from effects produced by V (see below), we conclude that pivalic acid and CH2O arising from I contribute little to its above inhibitory effects. In TK- cells in which DNA synthesis is prevented by blockade of TMP synthetase with aminopterin, the bis(pivaloyloxymethyl) ester (V) of TMP, at 0.9 x 10(-4) M, induced a 4-fold faster rate of DNA synthesis than did 10(-3)M TMP, whereas 10(-3) M TdR did not affect the rate. After 3 hr the rate with V was 80% that in the absence of aminopterin. In the above systems the nucleotide diesters I, II and V appear to be acting as effective extracellular sources of active intracellular FdUMP and TMP, in processes that involve loss of the two esterifying groups.
...
PMID:Evidence for acyloxymethyl esters of pyrimidine 5'-deoxyribonucleotides as extracellular sources of active 5'-deoxyribonucleotides in cultured cells. 281 20

The experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK polypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.
...
PMID:Evidence that deletion of coding sequences in the 5' end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products. 282 63

Several 5-methoxymethyldeoxyuridine (MMdU)-resistant mutants of herpes simplex virus type 1 (HSV1) were classified by measuring their sensitivities to the deoxythymidine kinase (dTK)-dependent antiviral drugs 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV), 1-beta-D-arabinofuranosylthymine (araT), and E-(2)-5-bromovinyldeoxyuridine (BVdU) and to the dTK-independent antiviral drug phosphonoacetate (PAA). Compared to wild-type (WT) virus, all five of the dTK- mutants were highly resistant (greater than or equal to 500-fold) to BVdU and MMdU, moderately resistant to ACV (50- to 100-fold) and araT (10- to 20-fold), but not resistant to PAA. The dTK of the mutant MMdUr-20 (dTK+) appeared to phosphorylate dTMP less well than that of the WT virus, while its affinity for deoxythymidine was not altered. Two other drug-resistant HSV mutants-S1 (isolated against ACV) and B3 (isolated against BVdU)--also showed reduced phosphorylation of dTMP. This suggests that alterations in both dTK and thymidylate kinase activities may determine sensitivity to antiviral drugs.
...
PMID:HSV1-specific thymidylate kinase activity in infected cells. 299 73

Cells made permeable by exposure to lysolecithin following infection by HSV-1 synthesize DNA (in greater amounts than non-infected cells) in the presence of the four deoxyribonucleoside-triphosphates (dNTPs) : dATP, dCTP, dGTP, and dTTP. DNA synthesis also occurs if dTTP is replaced by dT or dTMP, indicating activity of enzymes such as thymidine kinase, thymidylate kinase, deoxyribonucleoside-diphosphate kinase and ADN polymerase. Examination of DNA synthesis in permeabilized cells enables detection of antiviral activity of agents incapable of penetrating into intact cells and therefore ineffective in cell cultures. No detectable protein-tyrosine kinase activity was found in HSV-1 infected cells.
...
PMID:[Use of permeabilized cells in the study of inhibitory products of herpes virus replication]. 300 58

Carcinoma of the colon was induced in rats by injection of a carcinogen 1,2-dimethylhydrazine (DMH), and thymidylate synthetase (TS) and thymidine kinase (TK) activities, which catalyze the biosynthesis of dTMP by the de novo pathway and the salvage pathway of pyrimidine synthesis, respectively, were measured in normal control colon, DMH-treated normal colon, and DMH-induced colon carcinoma with or without administration of two doses of an anti-cancer drug UFT (a combination of tegafur and uracil). TS and TK activities were both increased after treatment with DMH, markedly in colon carcinoma tissue, and to a lesser degree in normal-appearing colon tissue. This phenomenon is well explained by the hypothesis that biochemical alterations of DNA-synthesizing enzyme activities occur as a preliminary step prior to the development of overt cancerous transformation. A low dose of UFT inhibited TS activity but enhanced TK activity, therefore, the salvage pathway may compensate for the reduced level of the de novo synthesis. On the other hand, a large dose of UFT reduced both TS and TK activities, perhaps due to cytotoxic effects of UFT incorporation into RNA.
...
PMID:Thymidylate synthetase and thymidine kinase activities in DMH-induced colon carcinomas in rats and effects of UFT. 310 3

Resistance of human CCRF-CEM leukemic cells in tissue culture to 5-fluoro-2'-deoxyuridine (FdUrd) has been examined following a single drug exposure (FS sublines). In two FS sublines generated by soft agar cloning of FdUrd sensitive cells in the presence of 10 nM FdUrd, the level of drug resistance was maintained at 22- to 30-fold following 1 month growth in the absence of FdUrd. Characteristic of the FS sublines was a decreased accumulation and retention of free intracellular 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) averaging 3% of FdUrd sensitive cells, a more rapid rate of disappearance of free FdUMP and FdUMP-bound thymidylate synthase (EC 2.1.1.45, 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase), and enhanced alkaline and acid phosphatase activities. There was no significant difference in the number of nucleoside transport sites per cell among the FS sublines and FdUrd-sensitive cells, indicating that the decreased accumulation of FdUMP in the resistant sublines was not the result of impaired FdUrd transport across the plasma membrane. The more rapid turnover of FdUMP-bound TMP synthase observed in the FS sublines was neither accompanied by a decreased stability of the TMP synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex, nor an enhanced rate of degradation of FdUrd to the less potent agent, 5-fluorouracil. In addition, the growth rates of the two FS sublines were similar to that of FdUrd sensitive cells in medium containing hypoxanthine, methotrexate, and thymidine, indicating that there was no depletion of thymidine kinase (EC 2.7.1.21, ATP : thymidine-5'-phosphotransferase) in the FS sublines. Therefore, we propose that enhanced activities of acid and alkaline phosphatases, which influence the intracellular accumulation and retention of FdUMP, are important determinants of stable FdUrd resistance in CCRF-CEM cells.
...
PMID:Resistance of CCRF-CEM cloned sublines to 5-fluorodeoxyuridine associated with enhanced phosphatase activities. 315 14

Fourteen patients received 5-iodo-2(1)-deoxyuridine (IdUrd) before surgery for placement of a hepatic arterial catheter. Biopsy specimens were obtained at the time of surgery and incorporation of IdUrd into deoxyribonucleic acid (DNA) in tumor and normal hepatic tissue was measured by HPLC and used as an index of drug selectivity. Over a 3-day intravenous infusion of IdUrd at 1000 mg/m2/day, substitution for thymidine in tumor DNA averaged 3.1%. Normal hepatic DNA contained less than 1% substitution by IdUrd. Arterial delivery of IdUrd increased levels in DNA, whereas modulation with fluorodeoxyuridine produced mixed results. In six patients, flow cytometric analysis showed that the tumor contained a median of 32% of tumor cells that had incorporated IdUrd in 3 days, corresponding to a potential doubling time of only 10 days. Thymidylate synthetase activity in tumors was 20-fold greater than in normal liver tissue, whereas thymidine kinase activity was twofold greater in tumors. These pharmacologic studies encourage further clinical trials of IdUrd as a cytotoxic agent or radiosensitizer.
...
PMID:Selective incorporation of iododeoxyuridine into DNA of hepatic metastases versus normal human liver. 316 89

Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Growth studies in defined medium show that as little as 8 micrograms thymidine/ml supports maximal growth of HPS 401, whereas 50 micrograms/ml is required by HPS 400. In contrast, both strains require over 40 micrograms thymidylate/ml to achieve maximal growth. HPS 401 exhibits thymidineless death when grown without thymidine; relative viability decreases to less than 0.01% after 190 h incubation. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the "salvage" pathways of nucleotide metabolism.
...
PMID:Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401. 316 46

The effects of a variety of 5-, 5'-, and 3'-substituted deoxyuridine derivatives on the cytoplasmic thymidine kinase (EC 2.7.1.21) purified from a human colon carcinoma cell line, HCT 116, were determined. Of particular interest was elucidation of the structural features important for antagonism of the feedback inhibition of thymidine kinase exerted by thymidine triphosphate. Substitutions at the 5-position altered the potency of the 5'-modified compounds. The replacement of the 5-hydrogen with a methyl group or an iodine greatly increased the affinity of compounds for the thymidine kinase. This was evident for enzyme substrates with 5'-hydroxyl groups [2'-deoxyuridine (dUrd), 2'-deoxythymidine (dThd) and 5-iodo-2'-deoxyuridine (IdUrd)], feedback inhibitors with 5'-triphosphate substitutions (dUTP, dTTP and IdUTP), and for 5'-amino derivatives [5'-amino-2',5'-dideoxyuridine (5'-AdUrd), 5'-amino-2'-5'-dideoxythymidine (5'-AdThd) and 5-iodo-5'-amino-2',5'-dideoxyuridine (5'-AIdUrd)]. Qualitatively, however, the 5-substitutions did not affect the nature of the interactions with dThd kinase. For example, in the presence of dTTP, 5'-AdUrd stimulated dThd kinase activity as much as 5'-AdThd, but approximately a 100-fold greater concentration of 5'-AdUrd was required. Similar results were obtained using intact cells in which substitutions at the 5-position affected the potency, but not the efficacy, of the 5'-amino derivatives to stimulate dThd phosphorylation. In contrast, substitutions at the 5'-position did alter the nature of the interaction with dThd kinase. Thus, the 5'-hydroxyl compounds, dUrd, dThd and IdUrd, did not reverse the enzyme inhibition produced by dTTP nor did they stimulate dThd uptake in intact cells. 5'-Deoxy-5'-(ethylthio)thymidine, 5'-deoxy-5'-[(2-hydroxyethyl)thio]thymidine, and dTMP, but not dTDP, also antagonized the inhibition of dThd kinase produced by dTTP. In comparison to 5'-AdThd, the 3'-amino derivatives, 3'-AdThd and 3'-5'-diAdThd, were much less potent, but still efficacious, antagonists of feedback inhibition. These results indicate that a wide range of dUrd derivatives can disrupt the regulation of dThd kinase and provide leads for the development of new nucleotide analogues.
...
PMID:Structure-activity analysis of antagonism of the feedback inhibition of thymidine kinase. 335 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---