In vivo determination of 5-bromo-2′-deoxyuridine incorporation into DNA tumor tissue by a new P-postlabelling thin-layer chromatographic method

The halopyrimidine 5-bromo-2′-deoxyuridine (BUDR) can serve as one of many indicators of tumor malignity, complementary to histologic grade. We have developed a thin-layer chromatographic (TLC) technique that can assess tumor DNA base composition and analogue (BUDR) incorporation which vies with immunochemistry for BUDR. This requires post-labeling DNA by nick-translation and radioactive 5′-phosphorylation of representative ³²P-α-dNMPs (deoxynucleotide monophosphates). Subsequent 3′-monophosphate digest exchanges a radioactive ³²PO₄ for the neighboring cold nucleotide. Separation in two dimensional PEI-cellulose TLC is carried out in acetic acid, (NH₄)₂SO₄, and (NH₄)HS0₄. TLC of dNMPs was applied to control HeLa DNA, and HeLa cells receiving BUDR. BUDR is detected in 10⁶ HeLa cells after 12–72 h incubations. Findings in HeLa DNA demonstrate normal TLC retention factors for all ³²P-dNMPs. Two dimensional R_F (x,y axes in cm) demonstrate: dAMP=1.4, 9.4; dCMP=10.0, 13.5; dGMP=4.6, 4.4; dTMP=9.0, 7.4; and BUDRMP 6.4, 6.6. This technique quantifies BUDR-which parallels tumor S phase, and serves as an indicator of labelling index (LI).     

Iododeoxyuridine administered to mice is de-iodinated and incorporated into DNA primarily as thymidylate.

Iododeoxyuridylic acid, a structural analog of thymidylic acid, is extensively de-iodinated in vivo by the enzyme thymidylate synthetase. Substantial amounts of the deoxyuridylic acid formed by this process are subsequently methylated and incorporated into DNA as thymidine. As a result, when mice are given tritiated iododeoxyuridine, most of the tritium incorporated into their DNA is present in thymidine rather than in iododeoxyuridine. Some, but not nearly as much, tritium from tritiated bromodeoxyuridine is also incorporated into DNA thymidine.     

Acute response of the mouse bladder to derivatives of 4-ethylsulphonylnaphthalene-1-sulphonamide and bladder carcinogens measured by the uptake of (125 I)5-iodo-2 -deoxyuridine

The structural features of 4-ethylsulphonylnaphthalene-1-sulphonamide (ENS) responsible for the induction of DNA synthesis in the mouse bladder have been investigated using a method in which DNA synthesis is measured by the uptake of a thymidine analogue, [¹²⁵I]5-iodo-2′-deoxyuridine (IUdR). The ability to stimulate DNA synthesis was unaffected by the nature of the alkyl group in 4-alkylsulphonylnaphthalene-1-sulphonamide. The sulphonamide group appeared to be essential to the activity of the molecule because naphthalene-1,4-disulphonamide was active whereas 1,4-diethylsulphonylnaphthalene was not. Maximum activity was found when the sulphonamide group was attached to an aromatic system (benzene or naphthalene) containing an alkylsulphonyl or a sulphonamide group. Bladder carcinogens other than ENS failed to stimulate the uptake of [¹²⁵I]IUdR sufficiently to produce statistically significant results. The reasons for the large variation in response between individual mice are discussed, as are the implications of the structure activity relationships to the mode of action of ENS.     

5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells

The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [³H]thymidine or 5-bromo-2′-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via “click” chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2′-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2′-deoxyuridine

Summary Growth of choriocarcinoma cells in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effect of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing: BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Synthesis and Properties of DNA Containing Cyclonucleosides

Here, we present efficient syntheses of the R and S diastereomers of 8,5′-cyclo-2′-deoxyadenosine and 6,5′-cyclo-2′-deoxyuridine. We incorporated these interesting nucleosides into DNA to study how the cyclo linkage affects the stability of duplex formation.     

Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography: Application to a pharmacokinetic study

This paper describes a relatively simple and sensitive high-performance liquid chromatographic assay (HPLC) with ultraviolet absorbance detection for 5-fluorouracil (5-FUra) and its two main metabolites, 5-fluorouridine (5-FUrd) and 5-fluoro-2′-deoxyuridine (5-FdUrd), in plasma. In this study, two plasma clean-up procedures involving addition of internal standard, solid-phase and liquid-liquid extractions have been developed. A reversed-phase Kromasil C₁₈ column was used. The detection was performed at 268 nm for 5-FUra and at 275 nm for the two metabolites. Linear detection responses were obtained for concentrations ranging from 25 to 1000 ng/ml. The average recovery from plasma was 35, 42 and 48% for 5-FUra, 5-FUrd and 5-FdUrd, respectively. Precision, expressed as C.V., ranged from 2.7 to 13% and the mean recovery from 94 to 105%. The limits of quantitation and detection of the three analytes were 20 and 10 ng/ml, respectively. The method was used to monitor the pharmacokinetic profile of 5-FUra and its two metabolites in patients with metastatic colorectal cancer.     

Biodistribution of [77Br]-5-Bromo-2-Deoxyuridine in Rats

Abstract

The feasibility of 5-bromo-2′-deoxyuridine as tumour imaging agent for PET studies in comparison with thymidine is discussed.     

Cellular Response to Efficient dUTPase RNAi Silencing in Stable HeLa Cell Lines Perturbs Expression Levels of Genes Involved in Thymidylate Metabolism

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2′-deoxyuridine and 5-fluoro-uracil. The catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated.     

Characterization of the Pre-meiotic S Phase through Incorporation of BrdU during Spermatogenesis in the Rat

Seminiferous tubules in mammals have histological arrangements defined by the associations between somatic cells and germ cells. The processes of DNA synthesis in meiotic and mitotic cells have different features that are not easily distinguishable through morphological means. In order to characterize the pre-meiotic S phase, 5-bromo-2’-deoxyuridine (BrdU) was injected intraperitoneally into Wistar rats, which were sacrificed 30 min, 2 hr, and 24 hr after injection. We found three different labeling patterns. One of these patterns was characterized by a distribution of the label in the form of speckles, most of which were associated with the nuclear envelope (labeling type I). We suggest that this pattern is due to mitotic DNA synthesis of type B spermatogonia. Labeling type II consisted of labeled foci scattered throughout the nuclear volume, which can be correlated with preleptotenic cells in pre-meiotic DNA synthesis. After 24 hr of incorporation, a third type of labeling, characterized by large speckles, was found to be related to cells in the “bouquet” stage; that is, cells in transition between the leptotene and zygotene phases. Our results indicate that BrdU incorporation induces different labeling patterns in the mitotic and pre-meiotic S phases and thus makes it possible to identify somatic and germinal cells.     

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.     

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.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

DNA repair in lymphoblastoid cell lines established from human genetic disorders

Lymphoblastoid cell lines (LCLs) established from chromosomal breakage syndromes or related genetic disorders have been used to study the effects of mutagens on human lymphoid cells. The disorders studied include xeroderma pigmentosum, ataxia telangiectasia, Fanconi's anemia, Bloom's syndrome and Cockayne's syndrome. Three approaches were used to assess the cells' ability to cope with a particular mutagen: (1) assaying recovery of DNA snythetic capabilities as measured by [³H]thymidine (dT) incorporation; (2) measurements of classical excision DNA repair by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (3) determining cell survival by colony formation in microtiter plates. LCLs established from xeroderma pigmentosum showed increased sensitivities to ultraviolet (354 nm) light and N-acetoxy-2-acetylaminofluorene (AAAF) as determined by DNA synthesis or colony formation and had diminished levels of excision-repair. Cockayne's syndrome LCLs, on the other hand, had increased sensitivities to ultraviolet (UV) light, AAAF and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) while showing near normal levels of DNA-repair after treatment with each agent. An LCL established from ataxia telangiectasia had decreased DNA repair synthesis and defective colony-forming ability following treatment with MNNG. LCLs, in addition to ease of establishment, appear likely to provide useful material for the study of DNA repair replication and its relationship to carcinogenesis.     

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.     

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.     

Novel Enamine Derivatives of 5,6-Dihydro-2′-Deoxyuridine Formed in Reductive Amination of 5-Formyl-2′-Deoxyuridine

Reductive amination of 5-formyl-3′,5′-di-O-acetyl-2′-deoxyuridine with primary amines and sodium triacetoxyborohydride (NaBH(OAc)₃) afforded novel enamine derivatives of 5,6-dihydro-2′-deoxyuridine as a result of unexpected 1,4-conjugate reduction of intermediate Schiff bases in addition to the secondary amine derivatives of 2′-deoxyuridine, typical 1,2-reduction products.     

Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-³H]-2′-deoxyuridine or [5-³H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-³H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.     

Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro

Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach to monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to visualize newly synthesized mtDNA in individual cells to study mtDNA replication within subcellular compartments of neurons. The technique combines the incorporation of 5-bromo-2-deoxyuridine (BrdU) and/or 5-ethynyl-2′-deoxyuridine (EdU) into mtDNA, together with a tyramide signal amplification protocol. Employing this technique, we visualized and measured mtDNA biogenesis in individual cells. The labeling procedure for EdU allows for more comprehensive results by allowing the comparison of its incorporation with other intracellular markers, because it does not require the harsh acid or enzyme digests necessary to recover the BrdU epitope. In addition, the utilization of both BrdU and EdU permits sequential pulse–chase experiments to follow the intracellular localization of mtDNA replication. The ability to quantify mitochondrial biogenesis provides an essential tool for investigating the alterations in mitochondrial dynamics involved in the pathogenesis of multiple cellular disorders, including neuropathies and neurodegenerative diseases. (J Histochem Cytochem 58:207–218, 2010)     

Synthesis and in Vitro Antiviral Activities of [(Dihydrofuran‐2‐yl)oxy]methyl‐phosphonate Nucleosides with 2‐Substituted Adenine as Base

The synthesis of [(2′,5′‐dihydrofuran‐2‐yl)oxy]methyl‐phosphonate nucleosides with a 2‐substituted adenine base moiety starting from 2‐deoxy‐3,5‐bis‐O‐(4‐methylbenzoyl)‐α‐L ‐ribofuranosyl chloride and 2,6‐dichloropurine is described. The key step is the regiospecific and stereoselective introduction of a phosphonate synthon at C(2) of the furan ring. None of the synthesized compounds showed significant in vitro activity against HIV, BVDV, and HBV.