Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.

Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.     

Induction of mutations by 5-fluorodeoxyuridine: A mechanism of self-potentiated drug resistance?

In medium containing concentrations of deoxycytidine that occur $\text{in vivo}$, 5-fluorodeoxyuridine induced mutation frequencies 6–90 fold greater than spontaneous mutant frequencies at two genetic loci in Chinese hamster cells. In medium lacking deoxycytidine, 5-fluorodeoxyuridine was more cytotoxic but induced no mutants. Hence, the effectiveness of cancer therapy with 5-fluorodeoxyuridine may be limited by self potentiated development of 5-fluorodeoxyuridine-resistant mutants and enhanced and prolonged by manipulating deoxycytidine metabolism.     

Crystallization of human thymidylate synthase

Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3.0 A. The protein was cloned and expressed in Escherichia coli and then crystallized from ammonium sulfate in the presence of beta-mercaptoethanol at a variety of pH values. The crystals are trigonal in the space-group P3(1)21; the unit cell dimensions are a = b = 96.7 A, c = 84.1 A.     

Mechanism of resistance of Pediococcus cerevisiae strains to 5-fluorodeoxyuridine.

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.     

Expression of human thymidylate synthase in Escherichia coli

A cDNA clone encoding thymidylate synthase (TS) has been isolated from a human T-cell library and modified in the 5'-untranslated region to incorporate several unique cloning sites. The gene has been cloned as a cassette into several Escherichia coli expression vectors which did not provide detectable amounts of the enzyme. A successful approach used a constitutive E. coli expression vector developed for the enzyme from Lactobacillus casei. A 115-base pair 5'-untranslated region from the L. casei TS which contains a ribosomal binding site and other regulatory sequences has been fused to the coding region of the human TS gene to provide a construct that is expressed in E. coli. The level of expression was further enhanced by altering the nucleotide sequence of the first 90 base pairs to accommodate common codon use in E. coli. In our best expression system, catalytically active human TS is expressed to a level that represents about 1.6% of the total soluble protein. The recombinant human TS has been purified and characterized; except for the presence of an amino-terminal blocking group, the enzyme has physical and kinetic properties similar to the enzyme isolated from human cells.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Analysis of mRNA recognition by human thymidylate synthase

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2′-deoxyuridine-5′-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix–loop–helix domain on the protein surface was identified as the putative RNA-binding site.     

Isolation of functional cDNA clones for human thymidylate synthase

Thymidine auxotrophic mutants of mouse FM3A cells due to thymidylate synthase deficiency can be transformed into prototrophs by DNA-mediated gene transfer using total human DNA (Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., and Seno, T. (1983) J. Biol. Chem. 258, 48-53). From one such transformed cell clone, cloned recombinant lambda phages containing DNA fragments were obtained recently that were concluded by circumstantial genetic evidence to have been derived from the human thymidylate synthase gene (Takeishi, K., Ayusawa, D., Kaneda, S., Shimizu, K., and Seno, T. (1984) J. Biochem. (Tokyo) 95, 1477-1483). Using a DNA segment derived from the cloned genomic DNA fragment and free of repetitive sequences as a probe, functional cDNA corresponding to thymidylate synthase mRNA could be cloned from a cDNA library of SV40 transformed human fibroblasts constructed by Okayama and Berg (Okayama, H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289). The cloned cDNA plasmid containing an insert of approximately 1.7-kilobase transformed mouse thymidine auxotrophic mutant cells to thymidine prototrophic cells at a frequency of 2-3 transformants/micrograms of DNA/10(5) cells, a value almost comparable to the highest so far reported. The resultant transformants retained the introduced cDNA and expressed human thymidylate synthase protein sufficient for supporting normal growth of otherwise auxotrophic mouse cells.     

A comparison of thymidylate synthetase activities from 5-fluorodeoxyuridine sensitive and resistant variants of mouse neuroblastoma.

Abstract— We have previously described a 5-fluorodeoxyuridine (FUdR) resistant variant of mouse neuroblastoma possessing an 8-fold elevation in methylenetetrahydrofolate: dUrd-5′-P C-methyltransferase (EC2.1.1.b) [trivial name: thymidylate synthetase] specific activity relative to that of the sensitive parental clone. This increased specific activity is not associated with a change in cytoplasmic inhibitors or activators, a decreased degradation rate of the enzyme, or the synthesis of a new species with an increased V_max, but appears to represent an increased synthesis of the enzyme species found in the sensitive parental clone. More resistant cell populations demonstrate even higher specific activities of this enzyme. The enzymatic activities from both the FUdR sensitive and resistant cells have identical stabilities to sonication, freezing, heat, urea, and sodium dodecyl sulfate, are equally and strongly inhibited by 5-fluorodeoxyuridine-5′-phosphate, and have the same affinity for the substrate 2′-deoxyuridine-5′-phosphate (K_m= 1·4 = 10⁻⁶m ). Both are stimulated by the addition of mercaptans and partially protected from heat denaturation in the presence of substrate. Unlike Don Chinese hamster cells (Conrad & Ruddle 1972) an actinomycin d pulse of neuroblastoma cells in monolayer culture did not increase the thymidylate synthetase specific activity. Mixed growth of FUdR sensitive and resistant cells produced only additive activities.     

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.     

Role of genomic factors beyond thymidylate synthase in the prediction of response to 5-fluorouracil

ABSTRACT

5-Fluorouracil (5FU) is still a major drug in combinations regimens for the treatment of colorectal cancer (CRC) both in the adjuvant and palliative setting. 5FU or its oral prodrug capecitabine is usually combined with irinotecan/oxaliplatin and the novel agents bevacizumab/cetuximab. Although this improved the outcome, the overall prognosis in patients with metastasized disease is still relatively poor. Although the target for 5FU, thymidylate synthase was shown to have a predictive value, this could only predict response in a subset of patients. Given the heterogeneous and complex nature of CRC, it is likely that other aberrations can affect therapeutic response. As an alternative, we investigated Copy number alterations using oligonucleotide-based high-throughput array-comparative-genomic-hybridization (aCGH) to obtain an unbiased screening of the whole genetic spectrum. Chromosomal aberrations have been identified in 85% of CRC patients and include genomic regions harboring copy number alterations in the DNA. These alterations may change the expression of many genes and might explain the differential response to therapy as shown in recent studies with several 5FU combinations. In order to clarify new predictive parameters for 5FU, we used aCGH in a historical cohort of patients, which received treatment with single agent 5FU, and an unsupervised clustering analysis showed a statistical (p < 0.05) difference between responding and nonresponding patients. We also find that several regions showed differences between responders/non-responders, such as losses in 12p12.3–12q15 and in 18p (where TS is located) in responding patients. Genome-wide analysis may provide an additional tool to discriminate between responders and nonresponders.     

A specific increase in chloroplast gene mutations following growth of Chlamydomonas in 5-fluorodeoxyuridine.

Summary Growth of Chlamydomonas in the thymidine analog 5-fluorodeoxyuridine (FdUrd) leads to a reduction in the amount of chloroplast DNA and also alters the pattern of chloroplast gene transmission in crosses (Wurtz et al., 1977). We have now found that growth of Chlamydomonas in FdUrd also increases at least 10 to 20 fold the frequency of cells expressing antibiotic resistant or non-photosynthetic mutations in the chloroplast genome with no concomitant increase in nuclear gene mutations with similar phenotypes. Clearly this effect is not locus specific since the non-photosynthetic chloroplast mutations thus far isolated comprise 9 recombinationally separate loci in the chloroplast genome (Shepherd et al., 1977, 1979). Only with the use of FdUrd has isolation of this important class of non-photosynthetic mutations been possible. The efficiency of recovery of chloroplast gene mutations rises as FdUrd concentration increases from 0.1 to 1.0 mM. At higher concentrations of FdUrd, growth rates and mutant yields are reduced. We propose the analog increases the yield of chloroplast mutations by a two-step process in which mutations are first induced as a result of thymidine starvation and then become expressed because the chloroplast DNA has been greatly reduced in ploidy and possibly damaged. FdUrd has its maximal effect on both recovery of chloroplast gene mutations and chloroplast gene transmission in crosses after cells grown in the presence of the analog for several generations remain at stationary phase for about 24 h. These observations suggest that chloroplast DNA metabolism is very active in non-dividing stationary phase cells of Chlamydomonas.     

Structural determinants for the intracellular degradation of human thymidylate synthase

Thymidylate synthase (EC 2.1.1.45) (TS) catalyzes the conversion of dUMP to dTMP and is therefore indispensable for DNA replication in actively dividing cells. The enzyme is a critical target at which chemotherapeutic agents such as fluoropyrimidines (e.g., 5-fluorouracil and 5-fluoro-2'-deoxyuridine) and folic acid analogues (e.g., raltitrexed, LY231514, ZD9331, and BW1843U89) are directed. These agents exert their effects through the generation of metabolites that bind the active site of TS and inhibit catalytic activity. The binding of ligands to the TS molecule leads to dramatic changes in the conformation of the enzyme, particularly within the C-terminal domain. Stabilization of the enzyme and an increase in its intracellular level are associated with ligand binding and may be important in cellular response to TS-directed drugs. In the present study, we have examined molecular features of the TS molecule that control its degradation. We find that the C-terminal conformational shift is not required for ligand-mediated stabilization of the enzyme. In addition, we demonstrate that the N-terminus of the TS polypeptide, which is extended in the mammalian enzyme and is disordered in crystal structures, is a primary determinant of the enzyme's half-life. Finally, we show that TS turnover is carried out by the 26S proteasome in a ubiquitin-independent manner. These findings provide the basis for a mechanistic understanding of TS degradation and its regulation by antimetabolites.     

Characteristics of thymidylate synthase purified from a human colon adenocarcinoma

Thymidylate synthase has been purified greater than 4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH2-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH2-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu1-7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlu1 and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folypolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 microM) or CH2-H4PteGlu (4.3 microM) were detected when polyglutamates of [6R]CH2-H4PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGlu5. In addition, CH2-H4PteGlu4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH2-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH2-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.     

Polymorphisms of folate pathway enzymes (methylenetetrahydrofolate reductase and thymidylate synthase) and their relationship with thymidylate synthase expression in human astrocytic tumors

Two important polymorphisms of folate cycle enzymes, methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase (TS) enhancer region (TSER) 28-bp tandem repeat, are related to risk of various types of cancer, including brain tumors, although there are few studies on this subject. A case-control study of these two polymorphisms in astrocytomas of different grades was carried out using polymerase chain reaction-restriction fragment length polymorphism, also determining the immunohistochemical expression of TS. The MTHFR 677 TT genotype was less associated with astrocytic tumors (odds ratio [OR]=0.00; p=0.0238), but the TSER polymorphism did not show any significant association. Combined genotype TT-double repeats/triple repeats (2R/3R) had a protective effect against astrocytomas (OR=0.00; p=0.0388). Expression of TS protein was observed in the majority of cases, with grade IV tumors being the exception. Moreover, the median H-score for the pilocytic astrocytomas was significantly higher when compared with that for diffuse tumors. There was an inverse correlation between the 2R/2R genotype and the highest TS-expressing tumors, and 3R/3R was relatively more frequent among the tumors grouped in the third and fourth quartiles. Our results provide support for the role of MTHFR and TS polymorphism in gliomagenesis, possibly because of the alteration of DNA methylation and repair status. Moreover, high levels of TS expression were detected in these tumors.