Thymidylate Synthase Ternary Complex Research Articles

Thymidine Kinase 1 Loss Confers Trifluridine Resistance without Affecting 5-Fluorouracil Metabolism and Cytotoxicity.

Acquired resistance to therapeutic drugs is a serious problem for patients with cancer receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analogue-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1-TK1 -/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The current data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. Mol Cancer Res; 16(10); 1483-90. ©2018 AACR.

Pretreatment with insulin enhances anticancer functions of 5-fluorou-racil in human esophageal and colonic cancer cells

To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anticancer functions and its mechanisms in the human esophageal cancer cell line (Eca 109) and human colonic cancer cell line (Ls-174-t). The effect of insulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After insulin treatment or insulin/5-FU treatment, cell cycle distribution of both cell lines was analyzed by flow cytometry. Western blot assay was used to assess the expression of caspase-3 and thymidylate synthase (TS). Apoptosis was detected by flow cytometry, DNA fragmentation assay, and terminal transferase dUTP nick end labeling assay (TUNEL). Moreover, the changes of 5-FU uptake after insulin pretreatment were detected by HPLC assay and Western blot analysis. We found that insulin enhanced the inhibitory effect of 5- FU on cell proliferation when Eca 109 cells and Ls-174-t cells were pretreated with insulin for the appropriate time. Insulin increased the cell number of the S phase and the uptake of 5-FU. Insulin/5-FU treatment enhanced apoptosis of tumor cells and upregulated the expression of cleaved caspase-3 compared with 5-FU treatment. Moreover, insulin/5-FU treatment induced the changes of free TS and the TS ternary complex level compared with 5-FU treatment in Eca 109 and Ls-174-t cells. These data suggest that insulin enhances anticancer functions of 5- FU when it is treated before 5-FU for the appropriate time in human esophageal and colonic cancer cell lines.

Stacked beta-bulges in thymidylate synthase account for a novel right-handed rotation between opposing beta-sheets

The beta-sandwich in thymidylate synthase comprises two six-stranded mixed beta-sheets, each contributed by one subunit of the dimeric molecule. In contrast to other proteins of known structure in which beta-sheets stack face to face, the central beta-sheets in the thymidylate synthase dimer are related by a right-handed rather than a left-handed twist. Using a highly refined model of an Escherichia coli thymidylate synthase ternary complex, we show that the individual beta-sheets in each subunit are severely distorted by an unusual series of stacked beta-bulges, which partitions each larger sheet into two smaller beta-sheets approximately orthogonal to one another. These stacked beta-bulges are locally stabilized by hydrogen bonding involving eight conserved residues. This extended structure anchors the phosphate of bound dUMP and controls the precise orientation of the catalytically essential active site cysteine. Stereochemical factors associated with the pronounced crease caused by these stacked bulges account for the right-handed twist of opposing beta-sheets.

A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A slow-binding interaction.

The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.