Biology of 5-fluoro-2'-deoxyuridine sensitivity of Drosophila melanogaster larvae

Using axenically cultured Drosophila melanogaster, grown on defined medium, the lethal effect of 5-fluoro-2′-deoxyuridine (FUdR) at a concentration of 10^?6 M has been ascribed to inhibition of the enzyme thymidylate synthetase; in the presence of dietary thymidine the lethal dose of FUdR is increased 100-fold. Mortality under these higher concentrations is probably due to the effect of FUdR upon RNA synthesis. Larval transfer between media with and without FUdR indicates a prolonged period during larval life when the base analogue is effective, both under conditions where suppression of DNA and RNA synthesis seem to cause death.The response of fruit-fly larvae to FUdR probably reflects the similarity of deoxyribopyrimidine metabolism in Drosophila to that in other organisms. However, finding that deoxycytidine is almost as efficient as thymidine in relieving the killing effect of low concentrations of FUdR suggests that some aspects of nucleotide metabolism in Drosophila remain to be elucidated.     

Thymidylic Acid Synthesis in Eimeria tenella (Coccidia) Cultured In Vitro*

The source of thymidylic acid for DNA synthesis in 1st generation stages of Eimeria tenella cultured in vitro was investigated using 5-bromo-2′-deoxyuridine, a thymidine analog, and 5-fluoro-2′-deoxyuridine, an inhibitor of de novo thymidylic acid synthesis. Results show that the parasite is unable to utilize either exogenous thymidine or 5-bromo-2′-deoxyuridine but is dependent upon the methylation of deoxyuridylic acid via the thymidylate synthetase reaction to supply thymidine for DNA synthesis.     

A method for the determination of total,free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts

A radiochemical assay for thymidylate synthase (EC 2.1.1.45, dTMP synthase), which permits the accurate determination of total, free, and 5-fluoro-2′-deoxyuridylate (FdUMP)-bound enzyme in cells exposed to the 5-fluoropyrimidine anticancer agents, is described. The total intracellular concentrations of dTMP synthase (free plus FdUMP-bound enzyme) in extracts from CCRF-CEM leukemic cells incubated with 5-fluoro-2′-deoxyuridine were determined following dissociation of the covalent dTMP synthase-5,10-methylenetetrahydrofolate-FdUMP ternary complex in the presence of the substrate, 2′-deoxyuridine-5′-monophosphate. The addition of substrate prevented reformation of the ternary complex during the dissociation procedure, and allowed complete recovery of FdUMP binding sites in cells exposed to a high concentration of 5-fluoro-2′-deoxyuridine. After removal of the substrate by charcoal adsorption, the concentration of total FdUMP binding sites was determined by titration of the enzyme with a saturating concentration of [6-³H]FdUMP and 5,10-methylenetetrahydrofolate. The concentration of FdUMP-bound dTMP synthase was then calculated as the difference between the total and free (without prior ternary complex disruption) enzyme values. The high sensitivity of this assay coupled with its ability to accurately quantitate both free and FdUMP-bound dTMP synthase in cells exposed to a wide range of fluoropyrimidine concentrations should make it useful for a variety of experimental and clinical studies.     

Viability of dopaminergic neurones is influenced by serum and astroglial cells in vitro

Transplantation of embryonic nigral grafts into the striatum of Parkinson's disease patients is not optimal, mainly due to low survival of grafted neurones. Current strategies focus on enhancing neuronal survival by transplanting enriched neuronal cell populations. There is growing evidence for the importance of astroglia in neuronal survival.To characterise the effects of glial cells on dopaminergic neurones, 5-fluoro-2′-deoxyuridine was added to embryonic rat ventral mesencephalic cultures in the presence or absence of serum. The survival and morphology of glial fibrillary acidic protein immunopositive astroglia and tyrosine hydroxylase immunopositive dopaminergic neurones was examined. In serum-containing medium, astroglial cells predominated and 5-fluoro-2′-deoxyuridine had no significant effect on either astroglia or dopaminergic neurone survival. In serum-free medium, astroglial growth was attenuated and numbers were significantly lower in 5-fluoro-2′-deoxyuridine treated compared with untreated cultures. There was no significant difference in the numbers of dopaminergic neurones between 5-fluoro-2′-deoxyuridine treated and untreated cultures. However, by the eighth day in vitro, there were differences in the morphology of these neurones between treated and untreated cultures. This study shows that the use of 5-fluoro-2′-deoxyuridine and serum-free medium can produce a neurone-enriched culture. However, the dopaminergic neurone population present in these cultures appeared to be morphologically dissimilar to those found in control cultures as neurites were retracted and the cell somas of these cells appeared enlarged. These results provide information on the effects of astrocytes on dopaminergic neurones in ventral mesencephalic cultures and thus have implications for transplantation in Parkinson's disease.     

The effect of 2′-fluoro-2′-deoxycytidine on herpes virus growth

The effect of 2′-fluoro-2′-deoxycytidine (dCfl) on the growth of certain viruses of the herpes type was investigated. It is shown that the compound has considerable anti-viral activity against HSV-I, HSV-II, pseudorabies virus and equine abortion virus. It has an effect comparable to that of araC and is more efficient than br⁵dC, but less so than acyclovir. Experiments with thymidine kinase-negative strains of HSV-I indicated that dCfl was phosphorylated by the viral kinase, and its K_m appears to be low and close to that of thymidine. Density gradient centrifugation enabled us to show that dCfl was incorporated into cellular and viral DNA and RNA. The cytotoxic activity of dCfl appears to be about 10-times smaller than that of araC. Removal of the nucleoside analog, washing and replacement with deoxycytidine reversed this effect, indicating rather a cytostatic than cytotoxic effect.     

Synthesis of 5-Halogeno-6-amino-2′-deoxyurldines and their Analogs as Potential Inhibitors of Thymidine Phosphorylase

ABSTRACT

5-Halogeno-6-amino-2′-deoxyuridines were synthesized from 2′-deoxyuridine as potential thymidine phosphorylase (ThdPase) inhibitors. Among the compounds synthesized, 5-bromo-6-amino-2′-deoxyuridine (6) and 5-iodo-6-amino-2′-deoxyuridine (9) were found to inhibit ThdPase activity with IC50 values of 1.3 μM and 6.5 μM, respectively. In vitro cell culture studies showed that compound (6) can significantly enhance the cytotoxic effects of 5-fluoro-2′-deoxyuridine against a human colon cancer HCT-8 cell line.     

5-Bromovinyl 2′-Deoxyuridine Phosphorylation by Mitochondrial and Cytosolic Thymidine Kinase (TK2 and TK1) and Its Use in Selective Measurement of TK2 Activity in Crude Extracts

Mitochondrial thymidine kinase (TK2) is responsible for phosphorylation of thymidine and deoxycytidine and plays a crucial role in mitochondrial DNA precursor synthesis. TK2 is expressed in all tissues at low levels complicating accurate determinations, especially in tissues with high cytosolic thymidine kinase (TK1) activity. Recently, 5-bromovinyl 2 ′-deoxyuridine (BvdU) at 0.2 μ M was used to measure TK2 activity selectively. BvdU phosphorylation by pure human TK2 and TK1 was tested here, and the ratio of BvdU phosphorylation by TK2/TK1 was 91 at 0.2 μ M but was 500 at 2.5 μ M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.     

Substrate Specificities of Mitochondrial Thymidine Kinase and Cytosolic Deoxycytidine Kinase Against 5-Aryl Substituted Pyrimidine-2′-deoxyribose Analogues

Abstract

Some 5-aryl-2′-deoxyuridine and -deoxycytidine analogues, many with known antiviral activity, were evaluated as substrates for pure deoxycytidine kinase (dCK) and pure mitochondrial thymidine kinase (TK2). Some of the deoxyuridine compounds were also tested with pure cytosolic thymidine kinase (TK1). TK2 showed the highest activity with this type of analogues.     

Different Mechanisms of Inhibition of DNA Synthesis by (E)-5-(2-Bromovinyl)-2′-deoxyuridine in Cells Transfected with Gene for Thymidine Kinase of Herpes Simplex Virus Type 1 and in Cells Infected with the Virus

Abstract

The effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) on deoxyribonucleoside 5′-triphosphate pools was studied in cells transfected with gene for thymidine kinase of herpes simplex virus type 1 and cells infected with the virus. When infected cells were treated with BVDU, the triphosphate form of the nucleoside analog was detected. When transfected cells were treated with BVDU, the triphosphate form was not detected and the pattern of changes in the pools was the same as after 5-fluoro-2′-deoxyuridine treatment. BVDU seems to inhibit DNA synthesis differently in the two cell lines and nucleotide metabolism in the transfected cells was not the same as in the infected cells.     

Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2′-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 μM, were gradually increased to 100 μM. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2′-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/0). Relative to thymidine uptake by HeLa/0 cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzlthionosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLa/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.     

Thymidine kinase-deficient mutants of Physarum polycephalum : Biochemical characterization

Thymidine kinase (TK) and deoxycytidine kinase (dCK) activity levels, [³H]thymidine (TdR) and 5-bromo-2′-deoxyuridine (BUdR) incorporation and 5-fluoro-2′-deoxyuridine (FUdR) sensitivity have been compared in TK-deficient (TU63 and TU84) and normal (TU291 and M₃b) strains of the myxomycete, Physarum polycephalum. The mutants had about 2% of the TK and 100% of the dCK activity of wild-type (wt) strains. They incorporated some TdR into both nuclear (nDNA) and mitochondrial DNA (mtDNA) but incorporated too little BUdR to give a buoyant density shift in nuclear DNA. They grew in the presence of levels of FUdR which completely blocked DNA synthesis in TU291. The FUdR sensitivity of strain M₃b could be increased by supplementing growth medium with folic acid.     

Synthesis of (E)-5-(2-cIOdovinyl)-3′-0-(1-Methyl-1,4-Dihydropyridyl-3 -Carbonyl)-2′-Fluoro-2′-Deoxyuridine (Ivfru-Cds) for Brain Targetted Delivery of Ivfru,an Antiviral Nucleoside

Abstract

(E)-5-(2-lodovinyl)-2′-fluoro-3′-0-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11) was synthesized for future evaluation as a lipophilic, brain-selective, pyrimidine phosphorylase-resistant, antiviral agent for the treatment of Herpes simplex encephalitis (HSE). Treatment of (E)-5-(2-iodovinyl)-2′-fluoro-2′-deoxyuridine (6) with TBDMSCI in the presence of imidazole in DMF yielded the protected 5′-O-t-butyldimethylsilyl derivative (7). Subsequent reaction with nicotinoyl chloride hydrochloride in pyridine afforded (E)-5-(-2-iodovinyl)-2′-fluoro-3′-O-(3-pyridylcarbonyl)-5′-O-t-butyldimethylsily-2′-deoxyuridine (8). Deprotection of the silyl ether moiety of 8 with n-Bu₄N⁺F^? and quaternization of the resulting 3′-O-(3-pyridylcarbonyl) derivative 9 using iodomethane afforded the corresponding 1-methylpyridinium salt 10. The latter was reduced with sodium dithionite to yield (E)-5-(2-iodovinyl)-2′-fluoro-3′-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11).     

The biosynthesis of natural and unnatural polyoxins by Streptomyces cacaoi

The biosynthesis of the pyrimidine moiety and the uronic acid moiety of the polyoxins and the formation of unnatural polyoxins has been studied in Streptomyces cacaoi. Experimental evidence is provided for the biosynthesis of thymine via a pathway that is independent of thymidylate synthetase. This new thymine pathway is based on two experimental approaches. First, two known inhibitors of DNA synthesis (1-formylisoquinoline thiosemicarbazide and 5-fluoro-2′-deoxyuridine), when added to polyoxin-producing cultures of S. cacaoi, inhibit the synthesis of TMP from exogenously supplied uracil but do not inhibit the synthesis of the thymine or hydroxymethyluracil in the polyoxin complex. Second, exogenously supplied thymine and hydroxymethyluracil are taken up by S. cacaoi but are not incorporated into the thymine or hydroxymethyluracil of the polyoxin complex. The thymine is incorporated into the DNA. The uracil in polyoxin L could be the parent pyrimidine chromophore with C-1 additions occurring at carbon-5 to form thymine and hydroxymethyluracil. Carbon-3 of serine but not the methyl group of methionine is a one-carbon source for the formation of the thymine and hydroxymethyluracil in the polyoxin complex.S. cacaoi can synthesize unnatural polyoxins, as evidenced by the incorporation of 5-fluoro, 5-bromo, and 6-azauracil into the polyoxins; 5-iodo-, 2-thio-, or 4-thiouracil is not a substrate. Two new polyoxin analogs synthesized and characterized when 5-fluorouracil is added to the cultures are 5-fluoropolyoxin L and 5-fluoropolyoxin M. There is a marked change in the molar ratio of the uracil:thymine:hydroxymethyluracil chromophores in the polyoxin complex following the incorporation of 5-fluoro-, 5-bromo-, or 6-azauracil. Apparently, the unnatural polyoxins inhibit the addition of the C-1 unit to carbon-5 of uracil in the polyoxin complex. Polyoxin L and polyoxin C do not inhibit Escherichia coli and Streptococcus faecalis, but 5-fluoropolyoxin L and 5-fluoropolyoxin C inhibit both these organisms. There is little or no difference in the inhibition of the fluorinated and natural polyoxins against leukemia L-1210 cells. The fluoro group on carbon-5 of the uracil ring does not affect the enzyme-inhibition complex with chitin synthetase since the inhibition constant of fluoropolyoxins L is the same as has been reported for polyoxins A, D, and L.The ¹⁴C-labeling pattern in the 5′-amino-5′-deoxy-d-allofuranosyluronic acid moiety of the polyoxins from ¹⁴C-labeled glucose, allose, and glycerol suggests that the formation of this unique C-6 uronic acid in the polyoxins does not proceed via the direct oxidation of either d-glucose or d-allose to the -onic or -uronic acids. Glucose is converted to two three-carbon trioses, followed by either (i) the oxidation of one of the trioses to a threecarbon acid and subsequent condensation with another three-carbon sugar to form the C-6 uronic or (ii) an 80:20 equilibrium of the two trioses followed by condensation to a hexose which is then oxidized to the C-6 uronic acid.     

SYNTHESIS OF 5-ETHYNYL-2′-DEOXYURIDINE-5′-BORANOMONO PHOSPHATE AS A POTENTIAL THYMIDYLATE SYNTHASE INHIBITOR

The 5-ethynyl-2′-deoxyuridine nucleoside and the 5′-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2′-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2′-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.     

3′-O- and 5′-O-Propargyl Derivatives of 5-Fluoro-2′-Deoxyuridine: Synthesis,Cytotoxic Evaluation and Conformational Analysis

A series of new 3′-O- and 5′-O-propargyl derivatives of 5-fluoro-2′-deoxyuridine (1–4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2′-deoxyuridine analogues (1–4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3′-O-propargyl-5-fluoro-2′-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.     

The effect of 5-Iodouracil on the growth and biosynthetic processes of bacteriophage T4td8 in the absence of light

5-Iodouracil (IUra)-substituted progeny bacteriophage T4td8 were grown under conditions such that, upon CsCl equilibrium isopycnic gradient centrifugation, progeny with density distributions about the median similar to that of unsubstituted phage are obtained. In the absence of light a monotonie relationship exists between decreasing progeny viability and increasing percent IUra substitution. IUra is equivalent to thymine as a growth factor on a molar basis, and at concentrations of IUra plus thymine above that required for maximum particle production, the percent IUra substitution in phage DNA is determined by the mole fraction of IUra in the medium. The lethal effects of 5-iodo-2'-deoxyuridine (IdUrd) and IUra are equivalent, and are not produced by a direct effect on the phage particles. At equivalent percent substitution in phage DNA the order of lethality is IUra > 5-bromouracil (BrUra) > 5-chlorouracil (ClUra). There is no interference with the transfer of thymine from host cell to progeny phage by the presence of IUra in the medium, and IUra affects neither the time of lysis nor the content of phage DNA in the infected cells.     

Preferential uptake of thymidine by thymineless enterobacteria: its significance in DNA labelling.

Minimum satisfactory concentrations of thymine and thymidine were determined for the growth of a high thymine-requirng (thy) mutant to Escherichia coli strain J5-3. Cultures were then grown in the presence of these concentrations of non-radioactive ('cold') pyrimidine together with 5 microCi/ml [methyl-3H)thymine, or [methyl-3H)thymidine (specific activities 5 Ci/m mole), and the uptake of radioactivity into ice cold trichloroacetic acid insoluble material determined. By far the most efficient labelling system was obtained if the label was supplied as radioactive thymidine and growth requirements satisfied by thymine alone. The addition of deoxyadenosine to the labelled thymidine/unlabelled thymine system dramatically reduced uptake of label. The addition of radioactive thymine with either thymine or thymidine to ensure satisfactory growth gave poor labelling. Using the [methyl-3H] thymidine/thymine system it was possible to increase the concentration of thymine from 8 to 64 microgram/ml with only a 25% reduction in label uptake after a 2 h period. The same system was also shown to be most efficient for labelling a thy derivative of another K12 strain, a thymine low-requiring (tir) K12 strain, a thy mutant of Klebsiella aerogenes 418 and a tir derivative of Salmonella typhimurium LT2.     

New Synthesis of 5-Carboxy-2′-deoxyuridine and Its Incorporation into Synthetic Oligonucleotides

Abstract

5-Carboxy-2′-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2′-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2′-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

Nucleoside and Oxypurine Homeostasis in Adult Rabbit Cerebrospinal Fluid and Plasma

Abstract: In adult New Zealand white rabbits, the effects of food deprivation and of massive elevations of plasma uridine or thymidine concentrations on CSF and plasma nucleoside and oxypurine concentrations were studied. Nucleoside and oxypurine levels were determined by high performance liquid chromatography using unequivocal methods of compound identification. After 48 and 96 h of food deprivation, the concentrations of uridine, cytidine, inosine, thymidine, deoxycytidine, deoxyuridine, hypoxanthine, xanthine, and uric acid in CSF and plasma were not different than in controls, except at 96 h, when the plasma uridine concentration was 35% lower (p < 0.05). After elevation of the plasma and CSF thymidine concentrations to ∼200 and 100 μM, respectively, with intravenous thymidine for 5 h, there was a large increase in CSF and plasma thymine to ∼100 μM and a smaller increase in plasma and CSF deoxyuridine concentrations. After elevation of the plasma and CSF uridine concentrations to 0.6 and 0.2 mM, respectively, there was a large increase in CSF and plasma uracil and a smaller increase in plasma and CSF deoxyuridine concentrations. Elevated plasma concentration of thymidine and uridine significantly decreased the CSF to plasma ratios of deoxyuridine and thymidine; however, only elevated plasma uridine concentrations decreased the CSF to plasma ratio of uridine. These results document the powerful homeostatic mechanisms that regulate the concentrations of the principal nucleosides and oxypurine bases in CSF.     

Temperature Sensitive Mutants of BHK 21 Cells

Temperature sensitive mutant cells were isolated from animal cells by multiple culturing in the presence of 5-fluoro-2′-deoxyuridine. Properties of the mutants are described.