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Results for “Thymidylate Synthase”

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At least 19 recordsLinked to original sources
PubMed2000

Inhibition of the mammalian transcription factor LSF induces S-phase-dependent apoptosis by downregulating thymidylate synthase expression.

The thymidylate synthase (TS) gene, which is induced at the G(1)-S transition in growth-stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP-1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G(1)-S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant-negative LSF (LSFdn) prevents the increase in TS enzyme levels during G(1)-S, and induces apoptosis in growth- stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5-fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S-phase by targeting a single gene that is critical for cell survival.

Amino Acid Sequence↗
PubMed1977

Presence and properties of thymidylate synthase in trypanosomatids.

High speed centrifugal supernatant fractions of homogenates of a number of trypanosomatids were assayed for thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) activity using the method of Lomax and Greenberg (1967) J. Biol. Chem. 242, 109-113). Similar activities were detected in Crithidia fasciculata, Crithidia oncopelti, the blood forms of Trypanosoma brucei, Trypansoma congolense and Trypanosoma lewisi and the blood, intracellular and culture forms of Trypanosoma cruzi, suggesting that all species synthesize at least some thymidylate de novo. The properties of the activities in C. fasciculata and the three forms of T. cruzi were compared with those of the isofunctional bacterial and mammalian enzymes. The trypanosotamid enzyme was inhibited by Mg2+, was much more sensitive to mercaptoethanol, had higher apparent Km values for substrate (dUMP) and cofactor (tetrahydrofolate), had a higher apparent molecular weight and was markedly more sensitive to inhibition by suramin. It is, therefore a possible target for chemotherapeutic attack, either on its own or in combination with a dihydrofolate reductase inhibitor. No evidence was obtained for the regulation of the trypanosomatid enzyme, either by its product, dTMP, or by dTDP or dTTp. This result agrees with previous studies which suggested that in trypanosomatids, the level of dTMP was regulated, at least in part, by a catabolic pathway consisting of a thymidylate phosphatase and a thymidine phosphorylase which degraded the excess of dTMP to thymine.

Animals↗
PubMed2026

Resveratrol Attenuates Gemcitabine Resistance in Hepatocellular Carcinoma Cells by Inhibiting Thymidylate Synthase.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Gemcitabine (Gem) is a commonly used drug against HCC, but its efficacy is limited by the development of resistance. Resveratrol (Res), a natural polyphenol with antitumor activity, may reverse Gem resistance in HCC, although the mechanism remains unclear. METHODS: The effects of Res on the proliferation, apoptosis, cell cycle, and invasion of Hep3B and HuH-7 cells were assessed via cell counting kit-8 (CCK-8), clonogenic, flow cytometry, and Transwell assays, respectively. Potential Res targets were predicted by network pharmacology, and markers of HCC prognosis were identified from the cancer genome atlas (TCGA) data. The interaction between Res and thymidylate synthase (TYMS) was validated by molecular docking and dynamics simulation. A Gem-resistant HuH-7 cell line (HuH-7/GR) was established, and when these cells were treated with Res combined with Gem, the effect on Gem sensitivity was detected by CCK-8 assay, clonogenic assay, and flow cytometry. Finally, a subcutaneous nude mouse model of HCC was used to evaluate the in vivo effects of Res combined with Gem. RESULTS: Res inhibited HCC cell proliferation, induced apoptosis and G2/M arrest, and suppressed invasion in a concentration-dependent manner. Network pharmacology and TCGA analysis identified TYMS as an important target gene for Res. TYMS was highly expressed in HCC tissues and correlated with poor prognosis. Res treatment reduced TYMS expression, while molecular docking and simulation showed stable binding of Res to TYMS. TYMS levels were elevated in HuH-7/GR resistant cells. Res combined with Gem was found to reverse drug resistance, inhibit proliferation and colony formation, and induce apoptosis. The Res + Gem combination group showed the smallest tumor volume in the in vivo model. CONCLUSION: By attenuating Gem resistance through TYMS inhibition, Res holds promise as a clinically viable adjunct to Gem-based chemotherapy, offering a potential strategy to improve outcomes in HCC patients.

Resveratrol↗
PubMed1979

31P NMR studies on the interaction of deoxyuridylate with thymidylate synthase.

The 31P nuclear magnetic resonance signal of deoxyuridylate was studied in the presence and absence of thymidlate synthase. In the absence of enzyme the chemical shift of deoxyuridylate is pH dependent with a pKa of 6.25. In the presence of enzyme, a peak corresponding to the dianioinc form of deoxyuridylate is observed which is independent of pH between pH 5.7 and pH 7.4. The pKa of the phosphate in the deoxyuridylate-thymidylate synthase complex is therefore less than 5. The release of inorganic phosphate from deoxyuridylate catalyzed by contaminating phosphatase was also observed.

Deoxyuracil Nucleotides↗
PubMed1978

Cytotoxicity of 5-fluoro-2'-deoxyuridine: requirement for reduced folate cofactors and antagonism by methotrexate.

Protein in vitro inhibition of thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by 5-fluoro-2'-deoxyuridylate requires 5,10-methylenetetrahydrofolate. The cytoxicity of 5-fluoro-2'-deoxyuridine towards cultured L1210 mouse leukemia cells is reduced when intracellular reduced folates are depleted, either by limiting the source in media or by inhibition of dihydrofolate reductase with methotrexate. Likewise, the intracellular amount of 5-fluoro-2'-deoxyuridylate covalently bound to thymidylate synthase in L1210 cells treated with 5-fluoro-2'-deoxyuridine is greatly diminished when cells are depleted of folate cofactors. The folate requirement for optimal growth of L1210 cells is lower than that required for maximal cytotoxicity of 5-fluoro-2'-deoxyuridine. These findings provide a biochemical rationale that may be useful in designing clinical protocols that use 5-fluorinated uracil analogs.

Animals↗
PubMed2026

Fluoropyrimidine Cardiotoxicity: Role of Uridine Triacetate and Pharmacogenomic Insights from a Case of 5-FU-Induced Cardiogenic Shock.

Fluoropyrimidines, including 5-fluorouracil and capecitabine, are widely used antimetabolite agents and remain central to the treatment of several solid tumors, particularly gastrointestinal malignancies. However, they are a well-established cause of chemotherapy-related cardiotoxicity. Although coronary vasospasm is the best recognized manifestation, fluoropyrimidine cardiotoxicity encompasses a broad clinical spectrum, ranging from chest pain and arrhythmias to acute heart failure, and, rarely, fulminant cardiogenic shock. This review discusses severe fluoropyrimidine-associated cardiotoxicity through the illustrative presentation of a young woman without previous cardiovascular disease who developed acute biventricular dysfunction and cardiogenic shock shortly after first exposure to FOLFIRINOX, requiring temporary mechanical circulatory support. Administration of uridine triacetate within the recommended therapeutic window was associated with rapid recovery of ventricular function. Cardiac magnetic resonance imaging demonstrated diffuse myocardial edema without late gadolinium enhancement, consistent with reversible toxic-inflammatory myocardial injury. Expanded genomic analysis identified dihydropyrimidine dehydrogenase and thymidylate synthase variants not detected by standard pretreatment pharmacogenetic screening. In this study we examine the pathophysiological mechanisms of fluoropyrimidine cardiotoxicity, the rationale for uridine triacetate in severe presentations, and the potential role of expanded pharmacogenomic profiling within a precision cardio-oncology framework.

Humans↗
PubMed1979

Role of thymidylate synthetase activity in development of methotrexate cytotoxicity.

Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.

Animals↗
PubMed1975

Participation of exogenous thymine and thymidine in deoxyribonucleic acid synthesis in Lactobacillus acidophilus.

The degree of participation (DP) of exogenous thymidine and thymine in overall DNA synthesis was studied in Lactobacillus acidophilus R-26. The DP of thymidine remains constant under a variety of conditions (except at low thymidine concentrations, when the DP is influenced by deoxyribonucleosides and folic acid). A 5-bromodeoxyuridine-resistant mutant was isolated, which displayed cross-resistance to 5-bromouracil and a significantly lower DP of thymidine than the parental strain. Thymine was poorly incorporated in the parental strain even in the presence of deoxyribosides. The results of this investigation would be compatible with the possibility of an alternative pathway for thymidylate synthesis other than the known thymidylate synthase pathway.

Bromodeoxyuridine↗
PubMed1977

Thymidine labeling index of human breast carcinoma. Enhancement of in vitro labeling by 5-fluorouracil and 5-fluoro-2'-deoxyuridine.

Inhibitors of thymidylate synthetase, 5-fluoro-2'-deoxyuridine (FUDR) and 5-fluorouracil (FU), enhanced in vitro thymidine labeling of human breast carcinoma cells. Their use resulted in an increase in the measured thymidine labeling index (TLI) of breast carcinomas by increasing detectability of labeled nuclei in autoradiographs. The TLI was measured with FU or FUDR enhancement in primary breast carcinomas from nine women younger than age 50, and from 30 women 50 years or older. The mean and geometric mean TLI were 8.0 and 6.3 respectively for the younger group, and 4.0 and 2.8 respectively for the older group. Similar significant age-associated differences were noted in a series of 133 TLI measurements without FU or FUDR. The TLI was not significantly correlated with primary breast carcinoma size or number of axillary nodal metastases. The capacity to form axillary metastases must be related to factors other than the rate of cell replication in breast carcinomas.

Breast Neoplasms↗
PubMed1978

Thymidylate synthetase and dihydrofolic acid reductase in the stimulated human lymphocyte.

The phytohemagglutinin (PHA)-stimulated human lymphocytes demonstrated trace or no activity (dihydro) folate acid reductase using three methods including a radioassay, but demonstrated ample activity of thymidylate synthetase. This was true regardless of the day of harvest, (first through seventh) of the stimulated lymphocyte. The lymphocyte extracts revealed no inhibitor to the reductase enzyme when these extracts were added before the liver extracts to the assay system. When methotrexate (MTX) was added to the culture media of the lymphocytes, there was, as expected, an increase in the synthetase activity, but the expected rise in the reductase activity did not occur, it remained nil. On the other hand, MTX did influence the incorporation of nucleosides by the stimulated lymphocytes in a fashion similar to its action on the incorporation of the same nucleosides by other cells.

Cells, Cultured↗
PubMed1977

Fluorescent inhibitors of thymidylate synthetase.

Two fluorescent derivatives of 2'-deoxy-5-fluorouridine 5'-p-aminophenyl phosphate were prepared by treatment of this compound with fluorescein isothiocyanate in dimethyl sulfoxide or 5-(dimethylamino)naphthalene sulfonyl chloride in pyridine. The products of the reactions were isolated by diethylaminoethylcellulose chromatography and were shown to be homogeneous by polyacrylamide electrophoresis and TLC. Confirmation of the structure was provided by elemental analysis, absorption and fluorescence spectra, PMR measurements, and liberation of nucleotide upon hydrolysis with snake venom phosphodiesterase. The fluorescent derivatives are good competitive inhibitors (Ki approximately10(-6) M) of thymidylate synthetase from a methotrexate-resistant strain of Lactobacillus casei.

Fluoresceins↗
PubMed1978

Antitumor agents XXX: Evaluation of alpha-methylene-gamma-lactone-containing agents for inhibition of tumor growth, respiration, and nucleic acid synthesis.

Evidence is presented that a number of sesquiterpene lactones isolated from plants and synthesized pyrimidines containing the alpha-methylene-gamma-lactone moiety are potent inhibitors of Walker 256 carcinosarcoma and Ehrlich ascites tumor growth and marginal inhibitors of P-388 lymphocytic leukemia and Lewis lung tumor growth. In vitro aerobic basal respiration and oxidative phosphorylation processes of Ehrlich ascites cells were inhibited by these agents as well as deoxyribonucleic acid polymerase and thymidylate synthetase enzymatic activities. These studies indicate that the alpha-methylene-gamma-lactone moiety, the beta-unsubstituted cyclopentenone ring, and the alpha-epoxycyclopentanone system are the essential moieties for inhibition of these biochemical parameters.

Animals↗
PubMed1979

Characterization of an amber suppressor in Pneumococcus.

Partial revertant has been isolated, with resistance to aminopterin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su+) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su+ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidylate synthetase gene are suppressible. Thymidylate synthetase activity is partially restored by su+. Optochin mutants can also be suppressed. Thus su+ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycin is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor. The t-RNA extracted from the suppressor strain su+ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su+ gene is amber specific. Thus su+ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su+; this is additional evidence that both categories contain point mutations.

Alleles↗