Thymidylate synthetase-positive and -negative murine mammary FM3A carcinoma cells as a useful system for detecting thymidylate synthetase inhibitors

The murine mammary FM3A/O and the thymidylate (dTMP) synthetase-deficient FM3A/TS^? carcinoma cell lines can be considered as a novel and useful test system for the detection of nucleoside analogues which are directly aimed at the thymidylate synthetase. These compounds should be inhibitory for FM3A/O but not for FM3A/TS^? cells, and their inhibitory effects on FM3A/O cell growth should be readily reversed by exogenous dThd within the concentration range of 5–20 μM.     

Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Thymidylate Synthetase-Positive and -Negative Mouse Mammary Carcinoma Cell Lines: Useful Model for Monitoring the Incorporation of Pyrimidine Nucleoside Analogues into Host Cell DNA and for Detecting Thymidylate Synthetase Inhibitors

Abstract

The dTMP synthetase-positive and -negative murine mammary FM3A carcinoma cell lines can be proposed as a useful system for identifying specific dTMP synthetase inhibitors, and for measuring the incorporation of pyrimidine nucleoside analogues into host cell DNA.     

Arabinosyl nucleosides. XII. Influence of arabinofuranosylthymine on growth of L5178y cells

The antibiotic 1β-D-arabinofuranosylthymine (araThd) is a potent inhibitor of the growth of mouse lymphoma cells (L5178y). The ED₅₀ concentration was found to be 9.8 μM. The cells die as a consequence of an unbalanced growth. The cytostatic activity of araThd can be abolished by coincubation with dThd and dUrd but not with Urd.At cytostatic concentrations araThd selectively blocks DNA synthesis; RNA- and protein synthesis are unaffected. Intracellularly araThd is rapidly phosphorylated to araTTP. This enzymic phosphorylation does not influence the synthesis of the naturally occuring, related triphosphate dTTP.AraTMP is incorporated into DNA during DNA synthesis; 1 mol of araTMP is incorporated/19, 500 molecules of dTMP.     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides

The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from sodium dodecyl sulfate-gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogues was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FdUrd), 3'-azido-2',3'-dideoxythymidine (AZT), and 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 microM as compared to 0.5 microM for dThd and 9 microM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; negative cooperativity with dThd. Negative cooperativity was also observed with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxy-thymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 microM for TK1, and 2, 10, and 78 microM for TK2, respectively. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogues intended for use in cells with different expression of TK1 and TK2.     

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.     

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.     

Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.     

Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis.

The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.     

Differential affinities of pyrimidine nucleoside analogues for deoxythymidine and deoxycytidine kinase determine their incorporation into murine leukemia L1210 cells

A number of 5-substituted pyrimidine deoxyribonucleoside (dThd or dCyd) derivatives have been evaluated for their effects on the incorporation of dThd and dCyd into the nucleotide pool and nucleic acids of murine leukemia L1210 cells. Several observations indicate that the dThd kinase and dCyd kinase activity of the cells and the differential affinities of these enzymes for the pyrimidine deoxyribonucleosides determine the incorporation of dThd and dCyd into the cells: (i) dThd and dCyd were not incorporated into mutant L1210 cells deficient in either dThd kinase or dCyd kinase activity; (ii) for a series of 5-substituted dThd and dCyd analogues a strong correlation was found between their inhibitory effects on the incorporation of dThd or dCyd into cell material and their Ki/Km for dThd kinase and dCyd kinase (r = 0.92 and 0.97, respectively); (iii) inhibitors of DNA synthesis (i.e. araC) and RNA synthesis (i.e. actinomycin D) suppressed the incorporation of dThd, most likely due to an inhibitory activity at the dThd kinase level (through the allosteric action of dTTP or slow regeneration of dThd kinase).     

Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells

Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell line. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient (TK-) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK- or TK-proficient (TK+) cells was examined. As a result, the growth of TK- cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK+ cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the supernatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in confluent culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant growth in the presence of certain growth inhibitors produced by density-inhibited V79 cells.     

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Uptake of [3H] thymidine and cell DNA synthesis during the early multiplication phase of herpesvirus hominis in BHK cells.

Experiments about the interaction of herpes viruses with BHK-cells during the first 6 h after infection concerning uptake and incorporation of dThd have been reported. During adsorption and penetration, the inhibition of uptake and of incorporation of [3H] dThd is sensitive to heat, but not to ultraviolet irradiation or cycloheximide. The eclipse is characterized by a strongly increased uptake of [3H] dThd and by inhibition of cell DNA synthesis. Both are sensitive to ultraviolet irradiation of the particles and cycloheximid treatment of the cells. It is concluded that the events during adsorption and penetration are dependent on the particles themselves, whereas the events during the eclipse depend on the activity of the viral genome. The implications of the findings are discussed.     

A study of the influence of newly synthesized acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline derivatives on deoxythymidine and deoxycytidine kinase activities in human neurofibrosarcoma and ovarian cancer

The influence of nine newly synthesized uracil acyclonucleosides, and 36 derivatives of 1,2,3,4-tetrahydroisoquinoline on the activity of enzymes catalysing dTMP and dGMP synthesis, on the content of dTTP and dGTP in acid soluble fraction and on the incorporation of [14C]dThd and [14C ]dGuo into DNA in tumour homogenates was studied. The influence of the compounds was studied in the cytosol from intraoperatively excised human tumours - neurofibrosarcoma and ovarian cancer. It was shown that dTMP and dGMP synthesis is inhibited competitively by 34.1+/-4.0% in both types of tumours by 0.2 mM 1-N-(3'-hydroxypropyl)-6-methyluracil (1) and 0.2 mM 1-N-(3'-hydroxypropyl)- 5,6- tetramethyleneuracil (2). The mentioned acyclonucleosides reduced the content of dTTP and dGTP in the acid soluble fraction of tumours (59.7+/-3.1% of control). 1-(4-chlorophenyl)-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (3), 1-(2,3-dichlorophenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (4) and 1-(3-methoxyphenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (5) at 0.2 mM concentration caused a mixed type inhibition of the synthesis of dTMP and dGMP by, on average, 33.2+/-4.4%, and reduced the content of dTTP and dGTP in the acid soluble fraction (52.6+/-3.7% of control) but were active only in the cytosol of neurofibrosarcoma. While acyclonucleosides undergo phosphorylation in the cytosol by cellular kinases, with their triphosphates being active acyclonucleoside metabolites, active 1,3,4,5-tetrahydroisoquinoline derivatives (compounds not containing a deoxyribose moiety), cannot be phosphorylated. ACN and THI derivatives which inhibit dThd and dCyd kinase activities, inhibit also the incorporation of [14C]dThd and [14C]dGuo (ACN - 50.2+/-2.7%, THI - 53.4+/-3.9% of incorporation inhibition) into tumour DNA. The obtained results point to the mechanism of uracil acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline biological activity consisting in inhibiting the synthesis of DNA components.