Developmental arrest in Caenorhabditis elegans dauer larvae causes high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae.

Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.

A non-classical type of alveolar macrophage response to Trichinella spiralis infection.

Studies of arginase expression and activity in guinea pig alveolar macrophages during Trichinella spiralis infection, prompted by earlier observation of innate lung response to the parasite, showed the macrophages to express both activity and protein of arginase type I. In cultured macrophages part of the enzyme was found to be always released to the extracellular medium. Whereas BCG in vivo treatment, alone or preceded by T. spiralis infection, stimulated arginase activity, T. spiralis infection alone affected the enzyme distribution between intracellular and extracellular fractions, and properties (K(m) and V(max)), rather than total (intracellular + extracellular) activity, with TGF-beta apparently responsible for a part of the effect. Anti-TGF-beta antibody treatment of the animals influenced both arginase activation by Mn(2+) and dependence of the enzyme-catalysed reaction on pH. Whereas T. spiralis infection activated guinea pig alveolar macrophages by the type II macrophage activation, as indicated by constant arginase expression, associated with previously demonstrated lack of stimulation of nitric oxide production, BCG treatment invoked an alternative type of activation mechanism, reflected by stimulation of macrophage arginase, but not iNOS, activity.

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.

5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.

Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1-24 months after infection) and Trichinella pseudospiralis (isolated 5.5-13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.

An isotopic assay of dUTPase activity based on coupling with thymidylate synthase.

A new rapid, sensitive and convenient procedure is presented allowing determination of dUTPase activity. With [5-(3)H]dUTP used as the substrate, dUTPase, converts it to the corresponding monophosphate and is coupled with thymidylate synthase-catalyzed reaction, resulting in tritium release from [5-(3)H]dUMP. Following charcoal absorption of the labeled nuleotides, radioactivity of tritiated water is determined. The new assay was tested to show comparable results with a previously described assay, based on measuring dUTPase-catalyzed [5-(3)H]dUMP production.

Acyclic analogues of 5-fluoro-dUMP and 5-fluoro-2'-deoxyuridine: synthesis and inhibition of thymidylate synthase and tumour cell growth.

1-[(2-Hydroxyethoxy)methyl]-5-fluorouracil (HEMFU) and 1-[(1,3-dihydroxy-2-propoxy)methyl]-5-fluorouracil (DHPFU) were prepared by alkylation of the di-O-TMS derivative of 5-fluorouracil and phosphorylated with the use of the wheat shoot phosphotransferase system to their monophosphates, HEMFUMP and DHPFUMP. 1-(2-Phosphonylmethoxyethyl)-5-fluorouracil (PMEFU) was obtained by condensation of diethyl-2-chloroethoxymethanephosphonate with 5-fluorouracil and cleavage of the alkylphosphoester with trimethylbromosilane. Inhibition of highly purified thymidylate synthase from mouse tumour Ehrlich carcinoma and leukemia L1210 cells by each of the nucleotide analogues, DHPFUMP, PMEFU and HEMFUMP, and of L5178Y mouse leukemia cell growth by the nucleoside (HEMFU) analogue, were studied. DHPFUMP proved to be the strongest inhibitor, non-competitive vs dUMP, with K(i)app 2.8 microM for time-independent interaction with the enzyme and N5,N10-methylenetetrahydrofolate (CH2H4PteGlu). In the presence of CH2H4PteGlu, DHPFUMP exhibited time-dependent inactivation of the enzyme, the inactivation rate plots being biphasic and pointing to Ki values in the microM range (10(3)-fold higher than for 5-fluoro-dUMP). HEMFUMP and PMEFU were much weaker inhibitors of the enzyme, with K(i)app values of 0.26 mM (non-competitive vs dUMP) and 30 mM (non-competitive vs dUMP), respectively. HEMFU, despite the weak interaction of its nucleotide analogue with the enzyme, proved to be a strong cell (L5178Y) growth inhibitor, with IC50 in the range 10(-5) M.

Synthesis and interaction with thymidylate synthase of 5'-dithiophosphate and 5'-fluorothiophosphate of thymidine.

Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.

Synthesis and interactions with thymidylate synthase of 2,4-dithio analogues of dUMP and 5-fluoro-dUMP.

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cells, with K(i) values of 32 microM and 55 microM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K(i) value was 10(3)-10(4)-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximately 10(-5)M) than FdUrd (IC50 approximately 10(-9)M), 2-thio-FdUrd(IC50 approximately 10(-7)M) or 4-thio-FdUrd (IC50 approximately 5x10(-8)M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine.

Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues.

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

Comparison of the effect of hydroxyurea and methotrexate on DNA fragmentation at various reaction conditions.

Using the changes in DNA breakage as a marker of DNA damage, the direct action of hydroxyurea (HU) and methotrexate (MTX) on DNA was examined. The experimental design was to expose isolated DNA to HU and MTX alone or HU and MTX with accelerators of free radical reaction (H2O2, Fe..) and to determine DNA fragmentation assessed by electrophoresis. The results indicated that HU can damage DNA, but to demonstrate this ability it needs H2O2, Fe.. or prolonged incubation in solution. Unlike HU, MTX with H2O2 was ineffective; MTX with Fe.. at certain degree protected DNA against lesions induced by Fe.. alone. It is concluded that despite several common features of HU- and MTX-induced toxic side-effects in the cells suggesting interference of these drugs with free radical reactions, their direct effect on DNA under oxidizing conditions is quite different at least at the concentrations used by us.

Enhancement of methotrexate-induced growth inhibition, cell killing and DNA lesions in cultured L5178Y cells by the reduction of DNA repair efficiency.

Acute treatment of L5178Y cells by methotrexate (MTX) caused concentration-dependent post-treatment growth inhibition and cell killing. The effects were potentiated in the presence of caffeine (CAF). At the same experimental conditions the CAF-dependent increase in mature and newly formed DNA lesions was found. The results suggest that even short MTX treatment can cause DNA lesions which are normally, at least partially, repaired. By the reduction of DNA repair efficiency with CAF, these lesions can be expressed what finds its reflection in the enhancement of MTX cytotoxicity.

Modification by caffeine of acute cytotoxic response of cultured L5178Y cells to hydroxyurea treatment.

The effect of caffeine (CAF) on acute cytotoxic response of L5178Y lymphoblasts to hydroxyurea (HU) treatment was studied. The following events were examined: abnormal cell enlargement (giant cell formation), the rate of recovery of cell reproduction and DNA synthesis after releasing the cells from the HU blockage, parental DNA breakage and cell death. The presence of CAF at nontoxic concentration prevented giant cell formation, enhanced cell growth inhibition and cell killing. The effect of CAF was variable, dependent on the duration of exposure to HU and the time of exposure to CAF. To obtain maximal effect, the continuous presence of CAF during HU treatment and posttreatment time was necessary. Hydroxyapatite chromatography assay of single strand (ss) and double strand (ds) fractions in parental DNA and the measurement of the rate of post-treatment recovery of DNA synthesis indicated that CAF enhanced HU-induced DNA lesions. It is concluded that the results give further evidence that even short HU treatment can damage not only newly formed but also parental DNA. The lesions are normally, at least partly repaired and can be expressed under the conditions of DNA repair inhibition.

Mechanism of unbalanced growth-induced cell damage. II. A probable relationship between unbalanced growth, DNA breakage and cell death.

This study examines the relationship between unbalanced growth, DNase II activity, DNA breakage and cell survival during the exposure of L5178Y cells to hydroxyurea (HU), excess thymidine (dThR) or HU with excess of four deoxyribonucleosides (dNR). It has been found that in the cells arrested by HU or dThR, but still appearing viable with the trypan blue exclusion test, Protein/DNA imbalance and abnormal cell volume are correlated with enhancement of DNase II activity in the cells and in the medium and with moderate increase in parental DNA breakage. The incidence of DNA breaks was markedly potentiated in the presence of non-toxic concentration of caffeine (CAF), used to inhibit DNA repair. In HU+dNR arrested cells, in which unbalanced growth was abolished, enhancement of DNase II activity and of DNA breakage in the presence or absence of CAF was substantially prevented. Comparison of posttreatment cell survival in the presence or absence of CAF confirmed the differential effect of CAF: while in HU or dThR arrested cells the presence of CAF induced marked cell killing, in HU+dNR arrested cells the influence of CAF was negligible. Only a slight effect of CAF was observed in cells in which dThR-induced arrest and unbalanced growth were reversed by deoxycytidine (dCR) addition. It is suggested that the involvement of DNA nucleases in the unbalanced growth-induced overproduction of numerous hydrolytic enzymes, with their progressive leakage through the cell membranes, can lead to progressive DNA digestion. DNA breaks produced in this way are normally, at least partly, repaired. Concomitant exposure of such cells to DNA repair inhibitor can markedly enhance the level of breaks, leading to potentiation of unbalanced growth-induced cell killing.

Giant cell formation in cultured L5178Y lymphoblasts induced by hydroxyurea treatment.

The conditions leading to hydroxyurea-induced abnormal cell enlargement (giant cell formation) were studied in L5178Y lymphoblasts in the culture. Exposure for only 1 hour to a concentration of 1 mM hydroxyurea (HU) was sufficient to produce an abnormal enlargement of about 25 percent of the cells. The maximal proportion of giant cells reached the value of about 50 percent after treatment with 1 mM HU for 5 hours and was not changed by a further prolongation of exposure time and/or by increasing the concentration of HU to 10 mM. Comparison of cell populations with various proportions of giant cells as regards their ability to reproduce, DNA synthesis and persistence in the culture suggested that at least some part of giant cells lost the ability to divide, their DNA synthesis was markedly suppressed or retarded and more than 50 percent of them disappeared earlier than 48 hours after the termination of HU exposure. It is concluded that at least at certain HU concentrations and in suitable stage of target cells the response of cells to short HU treatment can resemble that produced by X-rays or other DNA damaging agents.