Immunocytochemistry of 5-bromo-2′-deoxyuridine labelled oligonucleotide probes

Summary A synthetic oligonucleotide probe, complementary to oxytocin m-RNA was labelled enzymatically with 5-bromo-2-deoxyuridine (5-BrdU) and, with [-³²P]-ATP. The labelled probes were used for in situ, hybridization of histological sections of the mouse hypothalamus. A monoclonal antibody to 5-BrdU and the streptavidine-peroxidase technique were used in order to visualize hybridization with the 5-BrdU labelled probe. In situ hybridization with [³²P] labelling was detected autoradiographically. With both methods hybridized neurons were visible in the magnocellular hypothalamic nuclei. While immunostaining and radio-labelling provided similar localization of oxytocin m-RNA, only the immunocytochemical technique showed clear cellular resolution of the reaction product. In situ hybridization with 5-BrdU labelled probes followed by 5-BrdU immunocytochemistry seems to be a powerful alternative to common autoradiographic techniques.     

Induction of alkaline phosphatase and the glycoprotein hormone α subunit in HeLa cells by inhibitors of DNA polymerase

Levels of the glycoprotein hormone α subunit and alkaline phosphatase activity were increased in cultures of HeLa S₃ cells exposed to aphidicolin (0.2–10 μg/ml) or phosphonoformic acid (0.1–3 mm), inhibitors of DNA polymerase α. Induction was dependent on both the concentration and duration of exposure to the inhibitors and was prevented by cycloheximide and actinomycin D. Limited characterization of the induced α subunit and alkaline phosphatase activity suggest that they are similar to the uninduced proteins expressed by this cell line. Induction of both proteins by aphidicolin and phosphonoformic acid was enhanced by the simultaneous addition of 3 mm sodium butyrate but was depressed by 1 mm hydroxy urea. In contrast, both butyrate and hydroxy urea cause induction of these proteins when added alone to HeLa cultures. It is unlikely that a direct relationship exists between protein induction and the inhibition of DNA synthesis produced by aphidicolin and phosphonoformic acid since the concentrations required to produce half-maximal induction are 5 to 10 times greater than those needed to inhibit replication by 50%.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2′-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Parathyroid cell polarity as revealed by cytochemical localization of ATPases,alkaline phosphatase and 5′-nucleotidase

Summary Activities of Ca²⁺-dependent ATPase, Mg²⁺-dependent ATPase, Na⁺-K⁺-dependent ATP-ase, alkaline phosphatase, and 5-nucleotidase were demonstrated after incubation of 40-m vibratome sections of bovine parathyroids and subsequent visualization by electron microscopy. Prior to sectioning, parathyroid tissue was fixed with 1% glutaraldehyde for localization of alkaline phosphatase, and with 2% formaldehyde and 1% glutaraldehyde for demonstration activities of ATPases and 5-nucleotidase. The activities of the five enzymes were found at the apicolateral domain of the plasma membrane in parathyroid cells, i.e. at the site parathyroid cells face neighbouring parenchymal cells. Ca²⁺-ATPase activity was also seen on mitochondria, Golgi complex and RER. The presence of these plasma membrane associated enzymes at the apicolateral domain only indicate polarity in parathyroid cells. It further suggests that many processes including transmembrane transport take place at the apicolateral domain, the site of parathyroid cells opposing blood capillaries.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone α-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Differential tolerance ofArabidopsis thaliana crown gall tumors and calli to 5-bromo-2′-deoxyuridine

The degree of tolerance of two crown gall tumors and leaf calli ofArabidopsis thaliana to BUdR was compared. The nopaline producing teratoma tumor tolerated BUdR in concentration as high as 2.10^?4 M. The tolerance of octopine producing unorganized crown gall tumor to BUdR was lower, but both exceeded significantly the degree of tolerance to BUdR of untransformedA. thaliana calli, where 10^?5 M BUdR already show some inhibitory effect on the growth rate.     

Effects of 5-bromo-2′-deoxyuridine on arachidonic acid metabolism of neuroblastoma and leukemia cells in culture: A possible role of endogenous prostaglandins in tumor cell proliferation and differentiation

Effects of 5-bromo-2′-deoxyuridine (BrdU) were studied on two neuroblastoma and two leukemia cell lines, in terms of the relationship between prostaglandin (PG) synthesis and cell growth/differentiation. After treatment with BrdU (5 μg/ml), cell growth of the 4 cell lines was inhibited and one neuroblastoma cell line (GOTO) showed flattened morphology with positive S-100 protein, one of the differentiation markers for Schwann or glial cells. In the 4 cell lines, BrdU treatment reduced [1-¹⁴C]-arachidonic acid incorporation into phosphatidylinositol and phosphatidylethanolamine and was associated with an increase into phosphatidylcholine and triglyceride. BrdU treatment also increased fractions of 6-keto-PGF_1α and PGF_2α , with a decreased TXB₂ fraction. The decreased ratio of TXB₂ /6-keto-PGF_1α or increased 6-keto-PGF_1α fraction correlated significantly with cell growth inhibition, suggesting that the changes in the balance of endogenous PGs might be associated with BrdU-induced cell growth inhibition with or without differentiation of neuroblastoma and leukemia cells in culture.     

Insulin-dependent release of 5′-nucleotidase and alkaline phosphatase from liver plasma membranes

Insulin treatment of isolated liver plasma membranes induced the release of 5′-nucleotidase and alkaline phosphatase. This effect was maximal at physiological hormone concentrations, being 36% and 17% for 5′-nucleotidase and alkaline phosphatase respectively, and was fully mimicked by the phosphatidylinositol specific phospholipase C (PI-PLC), thus confirming the presence of a glycosyl-phosphatidylinositol anchoring-system for these exofacial enzymatic proteins. The complete inhibition of insulin dependent enzyme release by neomycin is strongly supportive of an involvement of membrane-located PI-PLC activity. In addition, the insulin-like effect on enzyme release induced by the GTP non-hydrolysable analog, GTP-γ-S, and its sensitivity to the pertussis toxin are in favour of a mediatory role exerted by the G proteins system, in the transduction of some actions of insulin.     

Model system studies of the use of 5-bromo-2′-deoxyuridine for selection of deficient mutants in plant cell suspension and protoplast cultures

Experiments using separate growing and arrested cell suspensions showed that treatment with the appropriate level of BUdR selectively killed the growing cultures. This effect was not light-dependent. A model system comprising leaf protoplasts of 2 different mutants of Nicotiana tabacum, only one of which was sensitive to valine, was developed to test the ability of this method to select non-dividing from dividing cells in mixed cultures. Following treatment of cultures with BUdR in the presence of valine, colonies recovered were tested on media which clearly differentiated the cell types on the basis of valine resistance and the ability to green in the presence of streptomycin (the marker for the valine-sensitive line). BUdR treatment of valine-inhibited cultures increased the percentage of valine-sensitive colonies recovered from mixtures containing up to 99.9% valine-resistant cells, although recovery of sensitive colonies was poor where these cells only represented a small proportion of the culture.     

Modulation of diphthamide synthesis by 5′-deoxy-5′-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of an aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5′-deoxy-5′-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Dose-, time-, and tissue-dependent effects of 5-bromo-2′ -deoxyuridine on the in-vitro organogenesis of Arabidopsis thaliana

Vigorous organogenesis can be induced from hypocotyl and root explants of Arabidopsis thaliana using a two-step culture procedure consisting of preculture on callus-inducing medium (CIM) and subsequent culture on shoot-inducing medium (SIM) or root-inducing medium (RIM). With this culture system, we examined the influence of 5-bromo-2′-deoxyuridine (BrdU), a thymidine (dT) analogue, on plant organogenesis in vitro. Treatment with BrdU during SIM or RIM culture had negative effects on shoot and root redifferentiation over a broad range of concentrations. When explants were exposed to low concentrations of BrdU during preculture and then transferred onto BrdU-free SIM, shoot redifferentiation was accelerated significantly. At higher doses, BrdU treatment during the pre-culture inhibited shoot redifferentiation strongly in hypocotyl explants, but not in root explants. This suggests that a target of the BrdU action lies within the process of acquisition of cell proliferation competence specifically involved in hypocotyl dedifferentiation. These effects of BrdU were counteracted by the simultaneous addition of excess dT. BrdU-pretreated and untreated explants did not differ significantly in the phytohormone dependency of shoot redifferentiation. Our results provide a basis for future studies on plant organogenesis combining pharmacological analysis with BrdU as a probe and molecular genetics with Arabidopsis mutants.     

Measurement of human placental 5′-AMP deaminase activity by radiometric assay

The level of 5′-AMP deaminase in homogenates of human term placenta has been measured by means of a simple radiometric assay. The assay uses ¹⁴C-labeled AMP as substrate and incorporates conditions of pH and K⁺ concentration, which optimize the 5′-AMP deaminase activity, and inhibitors of 5′-nucleotidase and adenosine deaminase to reduce interference from these enzymes. Assay products are separated by descending paper chromatography and quantitated by liquid scintillation counting. The activity of 5′-AMP deaminase in human term placenta determined by this assay was 474 ± 37 nmol min^?1 g^?1 at 30°C and was less than the 5′-AMP phosphatase activity evident under the same assay conditions. The assay is suitable for measurement of 5′-AMP deaminase in extracts of other tissues in which high levels of phosphatases and adenosine deaminase preclude assay of 5′-AMP deaminase by such techniques as ultraviolet absorption changes or ammonia estimation.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Structure and Conformation of the Antiviral Agent 5-Methoxymethyl-2′-deoxyuridine

Abstract

The three dimensional structure of the activiral agent, 5-methoxymethyl-2′-deoxyuridine (MmdUrd) was determined by x-ray diffraction methods. MMdUrd crystallized in space group P2₁2₁2₁ of the orthorhombic system with a = 9.166(1)A, b, = 25.348(1)Amm c = 5.270(1)A and Z = 4. The conformation of the glycosyl bond is anti (χ = 233.30), the deoxyribose ring has the C(2′)-endo envelope conformation (²E), the CH₂OH side chain has the g⁺ conformation and the methoxy group at the C(5) position is on the same side of pyrimidine plane as the 0(4′) oxygen. NMR spectroscopy was used to determine the conformation in solution. The spectra indicate that the sugar ring exists in a 60:40 equilibrium of the S- and N-states. The population of the three rotamers about the exocyclic c(4′)–C(5′) bond were estimated to be g⁺:t:g::61%:31%:8%. The correlaiton of molecular conforation with antiviral activity is discussed.     

Synthesis and anticancer activity of some 5-fluoro-2′-deoxyuridine phosphoramidates

Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3′-O-(t-butoxycarbonyl)-5-fluoro-2′-deoxyuridine (3′-BOC-FdU) (9a–9j) and 5-fluoro-2′-deoxyuridine (FdU) (10a–10j) were synthesized by means of phosphorylation of 3′-BOC-FdU (4) with 4-chlorophenyl phosphoroditriazolide (7), followed by a reaction with the appropriate amine. Phosphoramidates 9a–9j were converted to the corresponding 10a–10j by removal of the 3′-t-butoxycarbonyl protecting group (BOC) under acidic conditions. The synthesized phosphoramidates 9a–9j and 10a–10j were evaluated for their cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Two phosphoramidates 9b and 9j with the N-ethyl and N-(methoxy-(S)-alaninyl) substituents, respectively, displayed remarkable activity in all the investigated cancer cells, and the activity was considerably higher than that of the parent nucleoside 4 and FdU. Among phosphoramidates 10a–10j compound 10c with the N-(2,2,2-trifluoroethyl) substituent showed the highest activity. Phosphoramidate 10c was more active than the FdU in all the cancer cell lines tested.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Structures of Oligonucleotides Containing 5-(methoxymethyl)-2′-deoxyuridine Determined by NMR Spectroscopy

Abstract

Base pairing of 5-(methoxymethyl)-2′-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU. Abase pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH₂ and CH₂?O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU. G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.