Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Cytotoxicity to 5-fluoro-2'-deoxyuridine (FdUrd) derives from its conversion to 5-fluorodeoxyuridine-5'-monophosphate, which binds to and inhibits thymidylate synthase (TS) in the presence of the cofactor, 5,10-methylenetetrahydrofolate. We have selected FdUrd-resistant variants of the human cell line HEp-2 following adaptation to stepwise increases in drug concentration. In the initial selection, maximal drug resistance was associated with a 26-fold increase in the cellular level of TS. Greater TS overproduction (80-fold) was obtained by selection for FdUrd resistance in the presence of 10 microM folinic acid and 100 microM deoxyinosine. The latter agents were included to expand the folate pool to ensure adequate levels of cofactor during the selection process. Using cDNA plasmid pMTS-4, which is complementary to mouse TS mRNA, we show that TS overproduction in the HEp-2 variants is accompanied by a 100-fold increase in TS mRNA and a 100-fold amplification of the TS structural gene. Thus, TS overproduction and gene amplification is a mechanism of resistance to FdUrd in human cells.
Mol Pharmacol 1985 Nov
PMID:Thymidylate synthase overproduction and gene amplification in fluorodeoxyuridine-resistant human cells. 293 32

A panel of seven human colorectal cell lines of differing phenotype has been examined to elucidate the role of thymidylate synthase (TS) in the response to 5-fluoro-2'-deoxyuridine (FdUrd). Although TS is a major target of FdUrd, no consistent relationship was observed between the intracellular levels of TS and the response to FdUrd among the cell lines. Levels of thymidine kinase and dihydrofolate reductase, enzymes that are involved in generation of ligands that form the inhibitory ternary complex with TS, do not correlate with FdUrd response. Two cell lines that exhibit innate resistance to FdUrd, relative to the other cell lines, have variations in TS enzyme structure or gene structure. Cell line HCT 116 contains two forms of TS, as defined by isoelectric focusing. One form, which is unique to HCT 116, is more basic than the common form, which is present in all the cell lines. Cell line RCA contains a variation in the TS structural gene, as defined by restriction fragment-length analysis. These structural variations, which are associated with reduced response to FdUrd, may serve as markers for reduced clinical response to TS-directed chemotherapy.
Mol Pharmacol 1988 Oct
PMID:Thymidylate synthase as a determinant of 5-fluoro-2'-deoxyuridine response in human colonic tumor cell lines. 297 71

Inhibition of thymidylate synthase (TS) is an important mechanism of action of fluoropyrimidine antimetabolites. Thus, TS structure and expression are expected to be determinants of response to these agents. The role of TS in fluoropyrimidine response has been analyzed in a panel of human colonic tumor cell lines. Previous work has demonstrated that there is little correlation between TS concentration and sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd) among these cell lines, suggesting that parameters other than the TS levels are responsible for the variations in drug response. One such parameter has been identified in cell line HCT 116. This line, which is relatively resistant to FdUrd, produces two structural forms of TS, as determined by mobility of the enzyme in isoelectric focusing polyacrylamide gels. One form is common to all the cell lines, whereas a variant form, which is more basic and is encoded by a separate structural gene, is unique to HCT 116. Cells expressing one or the other TS form have been isolated and used to demonstrate that the variant form is associated with FdUrd resistance. Kinetic experiments indicate that the variant TS has reduced affinities for 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, which are ligands involved in formation of a stable inhibitory complex with the enzyme. Thus, the innate resistance of cell line HCT 116 to FdUrd is derived, at least in part, from production of an altered structural form of TS having reduced affinity for ligands.
Mol Pharmacol 1988 Oct
PMID:A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2'-deoxyuridine in a human colon tumor cell line. 297 72

We have previously isolated fluorodeoxyuridine-resistant mouse fibroblast (LU3-7) and neuroblastoma (FUdR-R) cell lines that overproduce thymidylate synthase and the mRNA for this enzyme up to 50-fold as compared to the parental cell lines. We have also cloned cDNA corresponding to mouse thymidylate synthase mRNA into pBR322. In the present study, we used this cloned cDNA as a hybridization probe in Southern blot analysis of DNA from the parental and overproducing cell lines. These analyses showed that the thymidylate synthase gene is amplified 50-100 fold in LU3-7 cells and about 30-fold in FUdR-R cells when compared to the respective parental cells. The sizes of the restriction fragments were the same in the parental and overproducing cells of each type, suggesting that extensive rearrangements have not occurred in the vicinity of the thymidylate synthase gene during the amplification process. However, not all of the fragments in the parental cells were amplified in the overproducing cells, suggesting that there may be multiple genes or pseudogenes for the enzyme. Restriction fragment length polymorphisms were detected when analyzing DNA from several different mouse cell lines. When LU3-7 cells were grown in the absence of selective pressure, the level of thymidylate synthase overproduction and the number of copies of the thymidylate synthase gene decreased in parallel.
Mol Pharmacol 1985 Jul
PMID:Thymidylate synthase gene amplification in fluorodeoxyuridine-resistant mouse cell lines. 299 33

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.
Mol Pharmacol 1985 Dec
PMID:Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene. 300 99

We have examined the pattern of transcription exhibited by four genes in the dTTP biosynthetic pathway of Saccharomyces cerevisiae. Consistent with the results reported previously by Storms et al. (1984), the TMP1 (or CDC21) gene encoding thymidylate synthase was found to be transcribed in a periodic manner during the cell cycle with maximal mRNA levels occurring just prior to the onset of DNA replication. Three other genes in this pathway DCD1, DUT1 and DFR1 encoding dCMP deaminase, dUTP pyrophosphatase and dihydrofolate reductase, respectively, exhibited relatively constant levels of transcription throughout the cell cycle. These results, particularly for DFR1, are in marked contrast with those obtained in other eukaryotic systems which have suggested that, in general, genes encoding enzymes involved in DNA precursor synthesis are subject to cell cycle regulation. Thus, periodic transcription is not a property common to all genes involved in DNA replication in this eukaryote.
Mol Gen Genet 1986 Sep
PMID:Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae. 302 Mar 75

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).
Mol Cell Biol 1986 May
PMID:Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae. 302 2

5-Fluorouracil, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorocytidine (FCyd), 5-fluoro-2'-deoxycytidine (FdCyd), 5-trifluoro-2'-deoxythymidine (F3dThd), and the 5'-monophosphates and 3',5'-cyclic monophosphates thereof were found to inhibit thymidine kinase-deficient (TK-) mutant strains of herpes simplex virus (HSV) at a much lower concentration than the wild-type (TK+) HSV strains. Other 5-substituted 2'-deoxyuridines that have previously been recognized as potent thymidylate synthase inhibitors behaved in a similar fashion. The activity of FdUrd, FdCyd, F3dThd, and their 3',5'-cyclic monophosphates against TK-HSV was readily reversed by 2'-deoxythymidine (dThd) but not by 2'-deoxyuridine (dUrd). These compounds also inhibited the incorporation of [6-3H]dUrd into DNA at a concentration which was up to 5 orders of magnitude lower than the concentration at which the incorporation of [methyl-3H] dThd was inhibited. Thus, while not being a target for the well established anti-HSV compounds in TK+HSV-infected cells, thymidylate synthase appears to be an important target in TK-HSV-infected cells. In addition to dTMP synthase, TK-HSV-infected cells appear to reveal other therapeutically exploitable targets such as OMP decarboxylase (towards pyrazofurin), CTP synthase (towards carbodine and its cyclopentenyl analogue), dihydrofolate reductase (towards methotrexate), and S-adenosylhomocysteine hydrolase (towards neplanocins).
Mol Pharmacol 1987 Aug
PMID:Potent activity of 5-fluoro-2'-deoxyuridine and related compounds against thymidine kinase-deficient (TK-) herpes simplex virus: targeted at thymidylate synthase. 303 43

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.
J Mol Biol 1986 Sep 05
PMID:Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei. 309 84

We have demonstrated the effect of different media on meiotic recombination in Drosophila melanogaster. Recombination is more frequent when the medium is deprived of bases, nucleosides and nucleotides. We have shown that two inhibitors of thymidylate (dTMP) synthesis - aminopterin inhibiting dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR) inhibiting thymidylate synthetase - result in a significant increase in meiotic recombination in the yellow/white region on the X chromosome of Drosophila melanogaster. Moreover the addition of thymidine to the richest medium significantly lowers normal recombination. Such studies represent a powerful tool for future studies on the mechanism of meiotic recombination.
Mol Gen Genet 1985
PMID:Inhibitors of thymidylate synthesis increase whereas thymidine decreases meiotic recombination in Drosophila melanogaster. 315 97


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