Intracellular Thymidylate Synthase Inhibition by Trifluorothymidine in FM3A Cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5‐Fluorouracil (5FU). TS activity reduced to 9% (0.1 µM TFT) and 58% (1 µM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK^?) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Mitotic control of dTTP pool: a necessity or coincidence?

The fidelity of DNA replication in eukaryotic cells requires a balanced dNTP supply in the S phase. During the cell cycle progression, the production of dTTP is highly regulated to coordinate with DNA replication. Intracellular thymidine is salvaged to dTTP by cytosolic thymidine kinase (TK1) and thymidylate kinase (TMPK), both of which expression increase in the G1/S transition and diminish in the mitotic phase via proteolytic destruction. Anaphase promoting complex/cyclosome (APC/C)-mediated ubiquitination targets TK1 and TMPK to undergo proteasomal degradation in mitosis, by which dTTP pool is minimized in the early G1 phase of the next cell cycle. In this review, we will focus on regulation of TK1 in the post-S phase and the importance of mitotic proteolysis in controlling dNTP balance, replication stress and genomic stability. Finally, we discuss how thymidine pool and oligomeric forms of TK1 can affect mitotic control of dTTP. This article is for the special issue of IMB 20^th anniversary.     

The association of thymidine kinase activity and thymidine transport in Escherichia coli

We have constructed a series of mutants within the putative nucleoside-binding site of the herpes simplex type-1 virus (HSV-1) thymidine kinase (TK)-encoding gene (tk), contained within an expression vector. While most mutations within this sequence produce an inactive protein, we find no absolute requirement for the wild-type Ile166 and Ala167. The uptake of thymidine (dT) into Escherichia coli tdk-, lacking functional endogenous TK activity, is proportional to the amount of TK activity expressed from the heterologous HSV-1 tk gene. In contrast, there is no enhancement in deoxycytidine uptake into E. coli producing (HSV-1) TK. These results imply a specific role for TK in the active transport of dT into E. coli.     

Altered thymidine kinase or thymidylate synthetase activities in 5-fluoro deoxyuridine resistant variants of mouse neuroblastoma.

Abstract: Abstract-We have previously described a 5-fluorodeox yuridine (FUdR) resistant neuroblastoma variant, possessing normal levels of ATP: thymidine-5-phosphotransferase (EC 2.7.1.21) [trivial name: thymidine kinase (TK)] but an 8-fold elevation in methy1enetetrahydrofolate:dUrd-5′P C-methyltransferase (EC 2.l.l.b) [trivial name: thymidylate synthetase (TS)] relative to the drug-sensitive parental clone. This variant possesses elevated levels of the parental TS species, 30% of which is uninhibitable by in vivo pulses of FUdR, suggesting the subcellular compartmentalization of this enzyme. We contrast this variant with a second FUdR resistant clone isolated from an ethyl-methane-sulfonate mutagenized population of the parental clone. This variant displays a 96% reduction in TK specific activity, despite normal FUdR and thymidine uptake rates, demonstrating the independence of thymidine phosphorylation and uptake. Grown without drug, its resistance declines (half-life of 15 cell divisions) with its TK specific activity rising to a plateau of 16% of the parental level after 56 cell divisions. Thymidine (1.0μM) protects the TK+ but not the TK- variants from FUdR induced growth inhibition but is without effect on TS specific activity. Unlike Tetrahymena (DICKENS et al., 1975), neuroblastoma TS activities appear not to be regulated by adenosine or guanosine cyclic nucleotide levels.     

Molecular Cloning of the Wild-Type and Mutant thyA Gene from Shigella flexneri Y

The thyA gene which codes for thymidylate synthase has been cloned and sequenced from the wild-type Shigella flexneri Y strain SH4 and a thyA mutant TSF21 after amplifying the gene by polymerase chain reaction (PCR). The nucleotide sequence revealed 98% homology to the E. coli K-12 thyA gene. The sequence of the wild-type thyA gene of Shigella flexneri Y was identical with that of the thyA mutant except that the residue T at position 345 was replaced by residue A in the thyA mutant. This change would cause a predicted amino acid substitution of leucine at position 44 in the polypeptide product of the wild type by glutamine in the mutant. Thus, Leu⁴⁴ may be critical in enzymatic activity of the thyA gene product thymidylate synthase.     

Bacteriophage Resistance of a ΔthyA Mutant of Lactococcus lactis Blocked in DNA Replication

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 ΔthyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.     

A mathematical model of human thymidine kinase 2 activity

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ～0.5 (apparent negative cooperativity) for dT and ～1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.     

Generation and Characterization of Thymidine/d-Alanine Auxotrophic Recombinant Lactococcus lactis subsp. lactis IL1403 Expressing BmpB

Genetic engineering of Lactococcus lactis to produce a heterologous protein may cause potential risks to the environment despite the industrial usefulness of engineered strains. To reduce the risks, we generated three auxotrophic recombinant L. lactis subsp. lactis IL1403 strains expressing a heterologous protein, BmpB, using thyA- and alr-targeting integration vectors: ITD (thyA ⁻ alr ⁺ bmpB ⁺), IAD (thyA ⁺ alr ⁻ bmpB ⁺), and ITDAD (thyA ⁻ alr ⁻ bmpB ⁺). After construction of integration vectors, each vector was introduced into IL1403 genome. Integration of BmpB expression cassette, deletion of thyA, and inactivation of alr were verified by using PCR reaction. All heterologous DNA fragments except bmpB were eliminated from those recombinants during double crossover events. By using five selective agar plates, we also showed thymidine auxotrophy of ITD and ITDAD and d-alanine auxotrophy of IAD and ITDAD. In M17G and skim milk (SYG) media, the growth of the three recombinants was limited. In MRS media, the growth of IAD and ITDAD was limited, but ITD showed a normal growth pattern as compared with the wild-type strain (WT). All the recombinants showed maximal BmpB expression at an early stationary phase when they were cultivated in M17G supplemented with thymidine and d-alanine. These results suggest that auxotrophic recombinant L. lactis expressing a heterologous protein could be generated to reduce the ecological risks of a recombinant L. lactis.     

Herpes simplex virus type 1 virion‐derived US11 inhibits type 1 interferon‐induced protein kinase R phosphorylation

The herpes simplex virus type 1 (HSV‐1) VRTK^? strain that was previously isolated in our laboratory as an acyclovir‐resistant thymidine kinase (TK)‐deficient mutant, is more sensitive to type 1 interferon than is the parent strain VR3. The properties of this mutant were investigated to clarify the mechanism for its hyper‐sensitivity to interferon (IFN). It was found that: (i) IFN‐pretreated cells, but not those treated with IFN after adsorption, are hyper‐sensitive to IFN; (ii) the mutant cannot inhibit protein kinase R phosphorylation efficiently during the early stage of replication (2 hrs post‐infection); (iii) expression of US11 in infected cells and its incorporation into the virion is reduced in the mutant compared to the wild type, despite the fact that a similar degree of DNA synthesis occurs during replication of both strains and; (iv) over‐expression of wild‐type viral TK has no effect on the phenotype of the VRTK^? strain, indicating that the phenotype is induced by a mutation(s) that does not involve the TK gene. These results suggested that the presence of US11 in the virion, but not that expressed after infection, plays an important role in the escape function of HSV‐1 from the antiviral activity of type 1 IFN.

     

Intracellular thymidylate synthase inhibition by trifluorothymidine in FM3A cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5-Fluorouracil (5FU). TS activity reduced to 9% (0.1 microM TFT) and 58% (1 microM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK-) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Isolation and characterization of thymidylate synthetase mutants of Xanthomonas maltophilia

Thymidylate synthetase mutants of Xanthomonas maltophilia ATCC 13270 were isolated on a solid minimal medium containing 50 mg/l thymidine and a high concentration of trimethoprim (500 mg/l). It was found that a high concentration of trimethoprim was required to prevent background growth of the wild-type strain. The isolated mutants could grow on thymidine or dTMP at a concentration of 50 mg/l while they were unable to grow on 1000 mg/l thymine or 50 mg/l deoxyridine. Thymidylate synthetase activity was assayed in the wild-type cells and in the mutant cells but only the wild-type cells contained measurable enzyme activity.     

An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.     

Altered thymidylate kinase substrate specificity in mammalian cells selected for resistance to iododeoxyuridine

A clone of Syrian hamster melanoma cells was selected for resistance to high levels of the thymidine (dT) analog 5-iododeoxyuridine (IdU). Unlike cell lines previously isolated as IdU resistant (IdU^r), these IdU^r lines had normal levels of thymidine kinase (EC 2.7.1.21) activity, grew in HAT medium, and readily incorporated exogenous dT and 5-bromodeoxyuridine (BrdU) into DNA. However, these IdU^r cells were found to preferentially exclude IdU from their DNA. Analyses of the soluble nucleotide pools of the IdU^r cells showed that they were able to take up and phosphorylate exogenous dT as well as the wild-type cells, and both mutant and wild-type cells accumulated dTTP as the major phosphorylated component. In contrast, while the wild-type cells in the presence of exogenous IdU accumulated significant levels of IdUTP (as well as IdUMP), the IdU^r cells accumulated only IdUMP. Thus, the mutant cells appear to have a markedly decreased ability to phosphorylate IdU beyond the monophosphate level. Assays of thymidylate kinase (EC 2.7.4.9) activity in extracts of the IdU^r cells indicated a marked preference for dTMP as substrate over IdUMP (in comparison to the wild-type enzyme activity). The cell lines described in this study represent a new phenotype arising from selection for resistance to a halogenated dT analog. The resistance appears to involve a change in the substrate specificity of thymidylate kinase, such that the enzyme in the IdU^r cells has an enhanced ability to discriminate between very closely related compounds.     

Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate

BACKGROUND: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yield ADP and TDP. In addition to its vital role in supplying precursors for DNA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. RESULTS: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF(3), TDP and ADP, and the bisubstrate analog TP(5)A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group to a catalytically unfavorable position, which rotates back in the AlF(3) complex to a position suitable for in-line attack. In the fully closed state observed in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly with the 3'-hydroxyl group of TMP. CONCLUSIONS: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.     

Modulation of K+ translocation by AKT1 and AtHAK5 in Arabidopsis plants

Root cells take up K⁺ from the soil solution, and a fraction of the absorbed K⁺ is translocated to the shoot after being loaded into xylem vessels. K⁺ uptake and translocation are spatially separated processes. K⁺ uptake occurs in the cortex and epidermis whereas K⁺ translocation starts at the stele. Both uptake and translocation processes are expected to be linked, but the connection between them is not well characterized. Here, we studied K⁺ uptake and translocation using Rb⁺ as a tracer in wild‐type Arabidopsis thaliana and in T‐DNA insertion mutants in the K⁺ uptake or translocation systems. The relative amount of translocated Rb⁺ to the shoot was positively correlated with net Rb⁺ uptake rates, and the akt1 athak5 T‐DNA mutant plants were more efficient in their allocation of Rb⁺ to shoots. Moreover, a mutation of SKOR and a reduced plant transpiration prevented the full upregulation of AtHAK5 gene expression and Rb⁺ uptake in K⁺‐starved plants. Lastly, Rb⁺ was found to be retrieved from root xylem vessels, with AKT1 playing a significant role in K⁺‐sufficient plants. Overall, our results suggest that K⁺ uptake and translocation are tightly coordinated via signals that regulate the expression of K⁺ transport systems.     

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells II. Test validation and interpretation

The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFT^R mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample log_et test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.