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Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy.

It is well recognized that many metabolic enzymes play essential roles in cancer cells in producing building blocks such as nucleotides, which are required in greater amounts due to their increased proliferation. On the other hand, the significance of enzymes in preventing the accumulation of their substrates is less recognized. Here, we outline the evidence and underlying mechanisms for how many metabolites normally produced in cells are highly toxic, such as metabolites containing reactive groups (e.g., methylglyoxal, 4-hydroxynonenal, and glutaconyl-CoA), or metabolites that act as competitive analogs against other metabolites (e.g., deoxyuridine triphosphate and l-2-hydroxyglutarate). Thus, if a metabolic pathway contains a toxic intermediate, then we may be able to induce accumulation and poison a cancer cell by targeting the downstream enzyme. Furthermore, this poisoning may be cancer cell selective if this pathway is overactive in a cancer cell relative to a nontransformed cell. We describe this concept as illustrated in selenocysteine metabolism and other pathways and discuss future directions in exploiting toxic metabolites to kill cancer cells.

Kinetoplastid and apicomplexan parasites comprise a group of protozoans responsible for human diseases, with a serious impact on human health and the socioeconomic growth of developing countries. Chemotherapy is the main option to control these pathogenic organisms and nucleotide metabolism is considered a promising area for the provision of antimicrobial therapeutic targets. Impairment of thymidylate (dTMP) biosynthesis severely diminishes the viability of parasitic protozoa and the absence of enzymatic activities specifically involved in the formation of dTMP (e.g. dUTPase, thymidylate synthase, dihydrofolate reductase or thymidine kinase) results in decreased deoxythymidine triphosphate (dTTP) levels and the so-called thymineless death. In this process, the ratio of deoxyuridine triphosphate (dUTP) versus dTTP in the cellular nucleotide pool has a crucial role. A high dUTP/dTTP ratio leads to uracil misincorporation into DNA, the activation of DNA repair pathways, DNA fragmentation and eventually cell death. The essential character of dTMP synthesis has stimulated interest in the identification and development of drugs that specifically block the biochemical steps involved in thymine nucleotide formation. Here, we review the available literature in relation to drug discovery studies targeting thymidylate biosynthesis in kinetoplastid (genera Trypanosoma and Leishmania) and apicomplexan (Plasmodium spp and Toxoplasma gondii) protozoans. The most relevant findings concerning novel inhibitory molecules with antiparasitic activity against these human pathogens are presented herein.

The kinetics of PCR amplification has been studied using fluorescently labeled zwitterionic deoxyuridine triphosphates that contain different fluorophores linkers of different lengths between the fluorophore and nitrogenous base. The concentration dependence of the modified substrate on the yield of the PCR product has been studied. Taq and Vent (exo–) DNA polymerases without 3'-5' exonuclease activity have been used for amplification, and the subsequent hybridization analysis of amplification products has been performed. Hydrophobicity and spatial size of the fluorophore have been found to affect inhibition of polymerases in PCR, while the length of the linker between the fluorophore and heterocyclic base has a significant impact on the substrate properties of the labeled deoxyuridine derivatives, i.e., on their incorporation in the PCR product. Electroneutral derivatives of deoxyuridine triphosphate with an average linker length have appeared to be most effective in PCR and subsequent microarray hybridization analysis. The studied fluor-dUTP are suitable for the incorporation of labels in PCR products using Taq and Vent (exo–) DNA polymerases in the subsequent hybridization analysis.

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

OBJECTIVE Radioiodine-131 is a radionuclide that is used for therapeutic purposes in hyperthyroidism and thyroid cancer. The aim of this study was to evaluate apoptotosis and proliferative changes in radioiodine-related kidney damage. MATERIALS AND METHODS Three groups (n=10/group) of rats were used as follows: the rats were in group 1 untreated, and the rats in groups 2 and 3 were treated once with oral radioiodine (111 MBq). The animals in group 2 were killed at the end of the seventh day and the rats in group 3 were killed at the end of the 10th week. The kidneys were removed and evaluated immunohistochemically. The presence of radioiodine in the kidneys was shown by the Na+/I-symporter antibody and proliferating cell nuclear antigen, Ki-67, caspase-3, caspase-8, caspase-9, and terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay were used to detect cell proliferation and apoptosis. RESULTS Na+/I-symporter protein accumulation in the kidneys was observed to be significantly greater in group 2 than in group 3 (P

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

Deoxyuridine triphosphate derivatives (dUTPs) modified at the C5 position of the pyrimidine ring with various aromatic hydrocarbon substituents of different hydrophilicities have been synthesized. The aromatic hydrocarbon substituents were attached to dUTPs via a CHCHCH2NHCOCH2 linker. The efficiency of the PCR incorporation of modified dUMPs using Taq, Tth, Vent (exo-) and Deep Vent (exo-) polymerases and a model DNA template containing one, two and three adjacent adenine nucleotides at three different sites within the sequence was investigated. For all the polymerases used, the yield of the modified PCR product was significantly increased with increasing hydrophilicity of the aromatic hydrocarbon substituent. In particular, for the above polymerases, the efficiency of the incorporation of dUMPs modified with the most hydrophilic of the studied aromatic hydrocarbon substituents, a 4-hydroxyphenyl residue, was 60-85% of the efficiency of dTMP incorporation. At the same time, the relative efficiencies of the incorporation of dUMPs modified with 2-, 4-methoxyphenyl, phenyl and 4-nitrophenyl substituents ranged from 20 to 50% and were 2-18% for the 1-naphthalene and 4-biphenyl groups, which were the most hydrophobic of the studied aromatic hydrocarbon substituents.

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1-methylcyclopropene (1MCP) group at the 7-position of 7-deaza-2'-deoxyadenosine and applied for primer extension experiments. The real-time kinetic data reveals that this 1MCP-modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP-modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site-specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.

In order to establish the Diels-Alder reaction with inverse electron demand for postsynthetic DNA modification a 1,2,4-triazine-modified 2'-deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4-triazine group was attached at the 5-position of 2'-deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions using a mixture of dATP, dGTP, dCTP and the modified 2'-deoxyuridine triphosphate with the KOD XL and Vent polymerases. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN-modified rhodamine at room temperature in yields of up to 82%. Dual (and triple) modifications were successfully achieved and obtained in quantitative yields if the modification sites were separated by three base pairs. These results establish the 1,2,4-triazene as bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5-tetrazine for postsynthetic labeling by the Diels-Alder reaction with inverse electron demand.

Etimicin (ETM) can accumulate in kidneys and cause tubular epithelial cell cytotoxicity. This article aims to study ETM elimination in kidneys and its nephrotoxicity, apoptosis, and histopathological insults of renal tubular epithelial cells, after repeated administration. A total of 36 rats were randomly divided into ETM-treated group and vehicle control group. Rats in ETM-treated group were treated intraperitoneally (i.p.) with 100 mg/kg/day ETM and rats in control group received physiological saline (i.p.) for 5 consecutive days. Determination of ETM concentrations accumulated in rat kidneys was carried out by high-performance liquid chromatography on the basis of derivatization with o-phthalaldehyde and by ultraviolet detector. Apoptotic renal tubular epithelial cells were identified by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Histopathological insults in kidneys were evaluated by hematoxylin and eosin staining. On day 1 after cessation of ETM administration, the accumulation concentration was 347.50 ± 193.30 μg/g tissue; on day 15, ETM concentration became 16.71 ± 9.99 μg/g tissue. Elimination half-life of ETM in rat kidney was about 3.05 days. Apoptotic renal tubular epithelial cells induced by etimicin was recovered gradually from 1544 ± 138 n/mm2 on day 1 to 716 ± 208 n/mm2 on day 15. Histopathological damage was also gradually recovered from vacuolation of tubular epithelial cells as well as renal tubular edema on days 1, 3, and 7 to nearly normal on day 15. From these results, we concluded that renal tubular epithelial cell cytotoxicity induced by ETM can gradually restore with its decreasing concentration in rat kidneys.

Fluorescent nucleic acids were prepared utilizing the polymerase extension (PEX) reaction to incorporate fluorescent molecules. 2’-Deoxyuridine triphosphate (dUTP) derivatives possessing pyrene molecules as fluorophores were synthesized using the aqueous-phase Sonogashira coupling between 5-Iodo-dUTP and acetylene-linked pyrene molecules. The incorporation of the pyrene (Py)-labeled deoxyuridine triphosphates (PyU) into DNA. into DNA by polymerase was evaluated by polyacrylamide gel electrophoresis, demonstrating that the PyU can work as a good substrate for the PEX reaction. The fluorescent properties of the functionalized DNA prepared by the PEX reaction were characterized by steady-state fluorescence measurements. The Py-conjugated DNA showed typical emission spectra of the pyrene, and the DNA with two pyrene molecules connected to each other by a diethylene glycol linker exhibited a presence of the functional group at this position. broadened emission attributed to the electronic interaction between the Py molecules. Nucleic acids modified with functional groups/molecules are of significant interest as potential applications in chemical biology, diagnostic tools 1or nanotechnology and material science.

Somatic hypermutation (SHM) of Ig genes is the result of a two-phase process initiated by activation-induced cytidine deaminase, relying on two different strategies for the introduction of mutations at CG pairs (phase I) and at AT pairs (phase II). To explain the selectivity of phase II, two mechanisms were proposed: AT-selective error-prone DNA-polymerases, deoxyuridine triphosphate (dUTP) incorporation, or both. However, there has been no experimental evidence so far of the possible involvement of the latter. We have developed a ligation-anchored PCR method based on the formation of single-strand breaks at uracils. In this study, we show the presence of uracil in hypermutating VkOx genes in wild type (AID+/+) mice, demonstrating that dUTP incorporation via DNA polymerases could be a major mechanism in SHM. Thus, error-prone DNA polymerases would participate in SHM via low-fidelity replication and incorporation of dUTP.

To help understand the pathogenesis of megaloblastic anaemia, we have studied folate-deprived HL60 cells. Cells grown with no added folic acid developed macrocytosis, a prolonged doubling time, a grossly increased deoxyuridine-suppressed value, and markedly reduced thymidylate synthetase activity. Cells in medium containing 50 nmol/l added folic acid became macrocytic with similar biochemical changes, but their doubling time was only marginally prolonged. In 100 nmol/l added folic acid, there was slight macrocytosis and a normal doubling time, but marked biochemical alterations were still present. The findings demonstrate that folate deficiency causes diminished thymidylate synthetase activity and macrocytosis in human myeloid cells, but that such cells may nevertheless demonstrate no prolongation of their doubling time, indicating either that their supply of thymidine triphosphate (TTP) is sufficient, or that mis-incorporation of deoxyuridine triphosphate (dUTP) is occurring. This suggests that the ineffectiveness of haemopoiesis in folate deficiency may result from damage to bone marrow cells in some way other than arrest in S-phase by a reduced delivery of TTP to the DNA replication fork.

We explore the potential of the Diels–Alder cycloaddition for the functional tagging of DNA strands. A deoxyuridine triphosphate derivative carrying a diene at position 5 of the pyrimidine base was synthesized using a two-step procedure. The derivative was efficiently accepted as substrate in enzymatic polymerization assays. Diene carrying strands underwent successful cycloaddition with maleimide-terminated fluorescence dyes and a polymeric reagent. Furthermore, a nucleotide carrying a peptide via a Diels–Alder cyclohexene linkage was prepared and sequence-specifically incorporated into DNA. The Diels–Alder reaction presents a number of positive attributes such as good chemoselectivity, water compatibility, high-yield under mild conditions and no additional reagents apart from a diene and a dienophile. Furthermore, suitable dienophiles are commercially available in the form of maleimide-derivatives of fluorescent dyes and bioaffinity tags. Based on these advantages, diene- and cyclohexene-based nucleotide triphosphates are expected to find wider use in the area of nucleic acid chemistry.

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the α-phosphate.

The purpose of this study is to provide a detailed protocol and quality control steps for measuring sperm DNA fragmentation (SDF) by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay using a new bench top flow cytometer, determine the reference value of SDF, and assess sensitivity, specificity, and distribution of SDF in infertile men and controls with proven and unproven fertility.Semen specimens from 95 controls and 261 infertile men referred to a male infertility testing laboratory were tested for SDF by TUNEL assay using Apo-Direct kit and a bench top flow cytometer. Percentage of cells positive for TUNEL was calculated. Inter- and intraobserver variability was examined. TUNEL cutoff value, sensitivity, specificity, and distribution of different cutoff values in controls and infertile patients were calculated.The reference value of SDF by TUNEL assay was 16.8 % with a specificity of 91.6 % and sensitivity of 32.6 %. The positive and negative predictive values were 91.4 and 33.1 %, respectively. The upper limit of DNA damage in infertile men was significantly higher (68.9 %) than that in the controls (19.6 %).TUNEL assay using flow cytometry is a reproducible and easy method to determine SDF. At a cutoff point of 16.8 %, the test showed high specificity and positive predictive value. The results of this test could identify infertile men whose sperm DNA fragmentation does not contribute to their infertility and confirm that a man who tests positive is likely to be infertile due to elevated sperm DNA fragmentation.

The purpose of this study was to investigate the long- and short-term inflammatory and apoptotic effects of whey protein on the livers of non-exercising rats. Thirty rats were divided into three groups namely (1) control group, (2) short-term whey (WS) protein diet (252 g/kg for 5 days), and (3) long-term whey (WL) protein diet (252 g/kg for 4 weeks). Interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and cytokeratin 18 (CK-18-M30) were assessed using enzyme-linked immunosorbent assay and immunohistochemical methods. Apoptosis was evaluated using the terminal transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. Hepatotoxicity was evaluated by quanitation of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Based on the biochemical levels and immunohistochemical results, the highest level of IL-1β was identified in the WL group ( p < 0.01). The IL-6 and TNF-α results were slightly lower in the WS group than in the control group and were highest in the WL group ( p < 0.01). The CK-18-M30 and TUNEL results were highest in the WS group and exhibited medium intensity in the WL group ( p < 0.01). AST results were statistically significant for all groups, while our ALT groups were particularly significant between the WL and control groups ( p < 0.01). The results showed that when whey protein is used in an uninformed manner and without exercising, adverse effects on the liver may occur by increasing the apoptotic signal in the short term and increasing inflammatory markers and hepatotoxicity in the long term.

Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance.

Mild whole body hyperthermia (WBH) treatment when given prior to radiation exposure is known to protect mice against radiation-induced hemopoietic death. This radioprotective effect seems to be mediated through WBH-induced cytokines and growth factors. Exposure to total body irradiation (TBI) also induces cell death by apoptosis in animal cells. The present studies were therefore carried out to investigate whether WBH pretreatment could protect mice against radiation-induced apoptotic death. The extent of apoptosis in bone marrow cells derived from mice at different time intervals after WBH, TBI and whole body hyperthermia treatment given 20 h prior to total body irradiation (WBH+TBI) treatments was studied by three independent methods namely DNA ladder assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay and caspase-3 assay. The studies have revealed that the mild WBH treatment (39°C, 1 h) when given 20 h prior to TBI (8 Gy) results in reduction in TBI-induced apoptosis. The results imply that WBH-induced impairment of apoptosis may have some role in WBH-induced radioprotection in Swiss mice.

Thymidylate synthase (TS) is an important enzyme catalysing the reductive methylation of dUMP to dTMP that is further metabolized to dTTP for DNA synthesis. Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation. © 2001 Cancer Research Campaign http://www.bjcancer.com

Objective.—To determine if the DNA strand breaks caused by tissue sectioning result in terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end-labeling (TUNEL) reactivity. Methods.—The incidence and location of TUNEL-positive nuclei were determined in 5- and 15-μm sections of human stomach. Five- and 15-μm sections of tonsil were stained as a positive control. Results.—In 5-μm gastric sections, 69% of nuclei were labeled; in 15-μm sections, only 30% were labeled. In the latter sections, almost all labeled nuclei were located at the cut surface of sections. Labeled nuclei did not have apoptotic morphology. Apototic bodies and tingible body macrophages were labeled throughout 15-μm sections of tonsil. Conclusions.—Tissue sectioning creates TUNEL reactivity. The morphologic findings on routine stains should be considered the gold standard for the detection of apoptosis on tissue sections.

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.

The objective of the present study was to study the effect of graded doses of citrinin (CIT) on apoptosis and oxidative stress in male Wistar rats till F1 generation. The animals were divided into four groups comprising 25 males and 25 females each, that is, group I: 1 ppm CIT; group II: 3 ppm CIT; group III: 5 ppm CIT; and group IV was kept as a control. The male and female animals of all the groups were kept separately and were fed basal rations containing the above-mentioned concentrations of CIT for 10 weeks. After 10 weeks, male and female animals of respective groups were kept for mating (one male/two females). After getting 10 pregnant females, the males were killed. These 10 pregnant females were allowed to give birth to young ones (F1 generation) naturally which were fed CIT in the above-mentioned doses till the age of 6 weeks and then were killed. Apoptosis was analysed in kidneys, liver and testes by DNA ladder pattern, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labelling assay and Bcl-2/Bax ratio. Besides, tissue oxidative stress was also analysed. It was concluded in the present study that CIT induces its toxic effects till F1 generation, and apoptosis and oxidative stress both play a very important role in toxicity. The effect of CIT was observed in a dose-dependent manner. However, in kidneys, both the mechanisms (apoptosis and oxidative stress) play their role in inflicting renal damage, while in liver only reactive oxygen species play a major role. Finally, the CIT toxicity did not lead to apoptosis and oxidative stress in male gonads till F1 generation.

Six deoxynucleoside triphosphate (dNTP) analogues were synthesized, having the α,β-P-O-P bond replaced with an imido (P-N-P) functionality. They were all shown to be reasonably potent inhibitors of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). This has permitted, for the first time, an estimate of the relative binding affinities of the parent triphosphates (dATP, TTP, dGTP, and dCTP) toward the enzyme's active site, in that they can be compared indirectly by correlation with the behavior of their α,β-imido analogues. Other complicating processes such as consecutive incorporation into the growing DNA chain can be excluded because the imido linkage cannot be cleaved by HIV-1 RT. The 5-iodo analog of deoxyuridine triphosphate (IdUMPNPP, 5 ) was the most potent inhibitor, having an IC 50 value of 7 μ M . A general route for the phosphorylation of purine and pyrimidine 5′-imidodiphosphates by pyruvate kinase was developed using phosphoenolpyruvate (PEP) as a phosphoryl group donor. Enzymatic phosphorylation was shown to be a more efficient approach than chemical methods.

Chronic alcoholism is associated with increased cancer risk that may be related to ethanol-induced alterations in methionine and deoxynucleotide metabolism. These metabolic relationships were studied in micropigs fed diets for 12 months that contained 40% ethanol or cornstarch control with adequate folate. Ethanol feeding altered methionine metabolism without changing mean terminal liver folate levels. After initial equilibration to diet, ethanol feeding significantly increased monthly serum homocysteine levels while reducing serum methionine levels over the time course of the experiment. After 12 months, hepatic methionine synthase activity and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) were significantly reduced in ethanol-fed animals, whereas the ratio of liver deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) was increased and correlated inversely with methionine synthase activity. These findings were associated with increased frequency of hepatocytes with apoptotic bodies and positivity for proliferating cell nuclear antigen (PCNA) in livers from ethanol-fed minipigs. These studies suggest that chronic ethanol feeding perturbs methionine metabolism by impairment of methionine synthase activity, resulting in deoxynucleoside triphosphate (dNTP) imbalance, increased apoptosis, and regenerative proliferation. These biochemical alterations may provide a promoting environment for carcinogenesis during long-term ethanol exposure.

The development of high-resolution scanning-probe microscopes has reawakened interest in the possibility of sequencing large nucleic acid molecules by direct imaging. Such an approach would be facilitated by the availability of effective methods for increasing contrast by labelling specific nucleotides, and the utility of introducing mercury atoms into complete DNA molecules through the enzymic polymerisation of mercurated pyrimidine deoxynucleoside triphosphates has been re-investigated. A simplified and improved method for the synthesis of a heat- and thiol-stable, mercurated derivative of deoxyuridine triphosphate in high yield and the incorporation of this precursor into full-length copies of a single-stranded phage M13 template are described. The DNA product has been fully characterised and the quantitative and specific replacement of thymidylic acid residues by the mercurated analogue demonstrated.

Two novel photolabile nucleotide triphosphate (NTP) analogues were synthesized through Sonogashira coupling and their enzymatic incorporation into DNA was evaluated with three different DNA polymerases (Taq, Vent exo- and T4) by polymerase chain reaction. Both nucleotide triphosphate analogues were recognized by these DNA polymerases as substrates for primer extension. Light irradiation of PCR products removed the photolabile group and released the amino and carboxyl moieties. Further site-specific dual-labeling for oligodeoxynucleotides (ODNs) and random labeling for a long DNA construct with fluorophores were successfully achieved with incorporation of the photolabile amine modified deoxyuridine triphosphate (dUnTP).

The aim of this study was to determine if a relationship exists between the levels of sperm DNA fragmentation and necrospermia in infertile men. Semen samples obtained from 70 men consulting for infertility evaluation were analyzed according to World Health Organization (WHO) guidelines. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: normozoospermia (30%; n = 20), moderate necrozoospermia (50-80%; n = 30), and severe necrozoospermia (80%; n = 20). DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The sperm DNA fragmentation index (DFI) was 9.28 ± 2.98% in patients with a normal level of necrotic spermatozoa, 20.25 ± 3.21% in patients with moderate necrozoospermia, and 35.31 ± 5.25% in patients with severe necrozoospermia. There was a statistically significant increase of DNA fragmentation in the necrozoospermic group (P0.01). A strong correlation was found between the degree of necrozoospermia and sperm DNA fragmentation. We concluded that patients with necrozoospermia showed a high level of DNA fragmentation compared to normozoospermic men. Severe necrozoospermia (80%) is a predictive factor for increased sperm DNA damage.

For many years mushrooms have been used empirically in traditional medicine to treat several diseases. Study of the maitake mushroom, with its immunomodulatory and antitumoral properties, has led to the isolation of several bioactive compounds. One of these, D fraction, is known to reduce tumor cell viability. This study examined the effect of isolated D fraction on viability and apoptosis of human breast cancer cells (MCF7). These cells were treated with maitake (D fraction) extract at 18μg/mL, 36μg/mL, 91μg/mL, 183μg/mL, or 367μg/mL or were left untreated (control) for 24 hours. MCF7 incubation with the maitake extract resulted in decreased cell viability [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium assay] in a dose-dependent manner. Apoptosis was statistically significantly increased in a dose-dependent manner at every concentration tested (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay). Upon incubation with D fraction, a microarray assay revealed upregulation of BAK-1 and cytochrome c transcripts, 2 proteins directly involved in the apoptotic pathway. Reverse transcriptase polymerase chain reaction studies confirmed these findings; BAK-1 was one of most overexpressed gene, as observed by microarray assay. These findings confirm the apoptotic effect of maitake D fraction in breast cancer cells and further highlight the involvement of cytochrome c release to the cytoplasm. Cytoplasmic release of cytochrome c, another player in the apoptotic pathway, was also increased after incubation with D fraction in a dose-dependent manner. This finding indicates that the effect of this compound involves mitochondrial dysfunction. The identification of the molecular mechanisms by which D fraction exerts its effects is crucial for the development of preventive and therapeutic strategies for cancer. Fil: Soares, Raquel. Universidad de Porto; Portugal Fil: Meireles, Manuela. Universidad de Porto; Portugal Fil: Rocha, Ana. Universidad de Porto; Portugal Fil: Pirraco, Ana. Universidad de Porto; Portugal Fil: Obiol, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Alonso, Eliana Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Joos, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Balogh, Gabriela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina

Purpose: To explore the anti-tumor effect of the Shenfouweikang in treatment of gastric cancer. Methods: Nude mice grafted with a human gastric adenocarcinoma cell line BGC-823 was used as the experimental model. The mice were divided into three groups, one control and the two drug administration experimental groups. Animals in the two experimental groups received either Shefouweikang or Dele granule over a 60-day period starting at the first day after grafting. Animals received saline as controls. All the mice were sacrificed at 61 days after being grafted. The anti-tumor effect was assessed by two ways: (1) tumor size was periodically measured during the life of the animals and tumor weight was determined by an electron balance immediately after the animals killed. (2) Apoptotic indices (AI) were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method and flow cytometric analysis. Results: Compared with controls and Dele granular powder, tumor growth (size and weight) was significantly inhibited with the Shefouweikang decoction treatment (P

Routine semen analysis is unable to assess alterations in sperm chromatin organization such as DNA damage. Because fertility is based not only on the absolute number of spermatozoa but also on their functional capability, methods for exploring sperm DNA stability and integrity are being used to evaluate fertility disorders. A large number of direct and indirect tests that measure sperm DNA damage have been developed. This chapter focuses on one of those tests – the terminal deoxytransferase mediated deoxyuridine triphosphate (dUTP) nick end-labeling or TUNEL assay – which is increasingly being used in many laboratories.

A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.

Nucleoside phosphates can bind to many functional proteins like G-proteins or other GTP-binding proteins in signal transduction or translation processes. Till now internalization of nucleoside phosphates into live cells remains a challenge. We study the internalization of a fluorescent-labelled deoxyuridine triphosphate into HeLa cells and other adhesion and suspension cells. We use different cell-penetrating peptides and a cocktail suitable for formation of non-covalent complexes with the nucleotide. Internalization is observed by fluorescence microscopy, and the uptake efficiency is quantitatively estimated by fluorescence spectroscopy. The applied concentrations of CPPs and the cocktail were checked on cell viability (MTT test) and membrane integrity (bioluminescence test with peptidyl-luciferin), indicating that the CPPs and the complexes with the nucleotide are cytotoxic above certain concentrations. These concentrations depend on CPP and cell type and are the limiting factors for the cargo uptake.

5-Methoxymethyl-2′-deoxyuridine (MMdUrd) is a selective anti-herpes agent that is dependent upon initial phosphorylation by Herpes simplex virus-induced deoxythymidine kinase. In order to determine its mechanism of action, MMdUrd was converted to the 5′-triphosphate (MMdUTP) and the nature of interaction of MMdUTP and dTTP with DNA polymerase of E. coli, HSV-1, and human α was investigated. The order of utilization of deoxyuridine analogues by bacterial and HSV-1 DNA polymerases for DNA synthesis was: dTTP > MMdUTP. In contrast, 5-methoxymethyl-2′-deoxycytidine-5′-triphosphate (MMdCTP) was a better substrate for HSV DNA polymerase compared to dCTP. MMdUTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dTTP incorporation (Ki= 2.9 × 10−6M). The IC50values of MMdUTP for both HSV and human αDNA polymerases were 4.5 × 10−6M. These data suggest that the selective activity of MMdUrd is due to its preferential phosphorylation by viral thymidine kinase and not at the DNA polymerase level. These results may also account for the difference in anti-HSV activity between MMdUrd and its deoxycytidine analogue.

We have developed a method for purifying DNA fragments containing excision-repair patches which involves incorporation of biotinated deoxyuridine monophosphate into repair patches followed by isolation of biotin-containing DNA fragments using streptavidin and either isopycnic density gradient centrifugation or gel electrophoresis. Normal human fibroblast were damaged with UV radiation, rendered permeable and allowed to perform repair synthesis in the presence of ATP, dATP, dGTP, [ 3 H]dCTP and biotinated deoxyuridine triphosphate. The DNA was purified, sonicated to a number-average molecular weight of 150 bp, then incubated with streptavidin, a protein with a high affinity for biotin and with a density of 1.3 g/ml in cesium trifluoroacetate compared to 1.6 g/ml for DNA. Isopycnic centrifugation in cesium trifluoroacetate resulted in the separation of the streptavidin-DNA complex with little or no dissociation. The streptavidin-DNA complex was also separated from free DNA by electrophoresis in 2% agarose. This method is applicable to any type DNA damage repaired by the excision repair pathways in which thymine is present in the repair patches, including damage from chemical carcinogens and ionizing radiation.

The sera of 39 patients (38 women and 1 man), 16 with limited and 23 with diffuse clinical form of systemic sclerosis (SSc), were tested for anti-centromere (ACA), anti-topoisomerase I (ATA) and anti-RNA polymerase III (ARA) antibodies. The presence of apoptotic cells in cultures of circulating lymphocytes was investigated using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) technique. ACAs were present in 16 (41%), ATA in 15 (38%) and ARA in 8 (21%) cases. The mean frequency of apoptotic lymphocytes was statistically higher in the ARA positive patients with respect to that in the control population (P < 0.001), in ACA (P < 0.001) and in the ATA (P < 0.001) groups. Moreover, apoptosis was distributed homogenously in ACA and ATA positive subjects, but not in the ARA patients. Our results show that there is an increase in apoptosis in the lymphocytes of ARA positive SSc patients.

A non-radioactive dot-blot hybridization assay for the detection of B19 parvovirus infections was developed using a digoxigenin-labelled probe both on nylon and nitrocellulose filters. A 700 bp Bam HI Hin dIII fragment of B19 DNA was used to construct the probe. Probe labelling was carried out by incorporating deoxyuridine triphosphate labelled with digoxigenin. The dot-blot hybridization assay was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. The specificity and sensitivity of digoxigenin-labelled B19 DNA probe was compared with the results obtained with 32 P-labelled B19 DNA probe. Out of the 504 serum samples tested, 3 samples were positive in all the hybridization assays performed and 494 were negative, 7 serum samples gave a weak positive reaction when Dig-B19 probe was used on nitrocellulose filters. The 77 faringeal swabs tested were negative in all the hybridization assays performed. Our hybridization assay showed a high sensitivity and reproducibility and it appears to be a rapid, practical and reliable test for routine screening of B19 parvovirus DNA in large numbers of clinical specimens.

Objective To evaluate the incidence of apoptosis after vitrification warming of mouse ovaries. Design Experimental study. Setting University-based research laboratory. Animal(s) Twelve- to 14-day-old National Medical Research Institute female mice. Intervention(s) Vitrification of mouse ovaries using ethylene glycol. Main outcome measure(s) Follicle viability assessment by trypan blue testing, morphologic examination by hematoxylin–eosin staining and transmission electron microscopy, apoptosis assessment using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method and DNA laddering technique. Result(s) No statistically significant difference in follicle viability was observed between vitrified and nonvitrified ovaries. On transmission electron microscopy, vitrified ovaries showed a well-preserved ultrastructure. No sign of apoptosis was observed morphologically or by transferase-mediated deoxyuridine triphosphate nick end-labeling technique in either fresh or vitrified-warmed mouse ovaries. Despite the presence of a laddering pattern of DNA in control induced thymic tissue, no similar pattern was observed in fresh or vitrified-warmed ovaries. Conclusion(s) The data suggest that the vitrification technique does not induce apoptosis in mouse ovarian tissue investigated just after warming.

In order to enhance a collection of modified deoxynucleoside triphosphates useful for in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) techniques, we designed and synthesized modified analogues of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate bearing a flexible and hydrophilic 7-amino-2,5-dioxaheptyl linker at a C5 position. Both analogues were found to be substrates for thermostable DNA polymerases which belong to an evolutional family B during PCR.

Hair plucking is the most frequently used method of anagen induction within hair follicles. In this study, we found that plucking leads to the entire renewal of the follicular stem cell region of the mouse pelage follicle. Comparative histochemical analysis revealed that S100A4 protein was specifically distributed in the outer layer of the epithelial sac, which has been identified as the stem cell region of the pelage follicle, whereas the slow cycling cells that retained 5-bromo-2'-deoxyuridine label for 8 wk were located in the epithelial sac and also in the hair germ. Combined terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling method and immunohistochemistry revealed that positive cells were detected in the outer layer of the epithelial sac possessing both bromo-2'-deoxyuridine and S100A4 labels 4.5 h after plucking. No terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling signal, however, was observed in the hair germ. Serial inspection of the plucked follicle revealed that almost all regions of the epithelial sac became terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling positive 12 h after plucking. Terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling-positive cells ultimately degenerated without forming apoptotic bodies. Subsequently, the surviving label-retaining cells in the hair germ migrated upward to re-epithelialize the damaged portion. These results indicate that follicular stem cells in the epithelial sac underwent cell death after plucking. It is likely that the hair germ is responsible for the reconstruction of the stem cell region of the hair follicle.

The synthesis of an europium tris-bipyridine cryptate labeled 2'-deoxyuridine-5 '-triphosphate analog (K-11-dUTP) is described. This labeled triphosphate was incorporated into DNA through enzymatic reactions with terminal transferase and DNA polymerases. The enzymatic reactions were monitored by TRACE (Time Resolved Amplification of Cryptate Emission), a homogeneous method using Fluorescence Resonance Energy Transfer (FRET) from an europium cryptate as donor to a modified allophycocyanine as acceptor.

Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.

We wished to verify whether semen processing by discontinuous double-layered density gradient centrifugation could improve sperm apoptotic DNA fragmentation rates using a commercially available deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay in 35 consecutive men presenting for assisted reproductive treatments. Although sperm motility did improve as expected, no effects were observed in sperm apoptotic DNA fragmentation rates, and this should be considered in the routine assisted reproduction setting.

The fluoropyrimidines 5-fluoro-2’-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) are both applied in the treatment of patients with colorectal cancer. These agents exert their action after metabolization to the nucleotide level (Fig. 1). The nucleotides 5-fluorouridine triphosphate (FUTP) and 5-fluoro-2’-deoxyuridine triphosphate (FdUTP) may mediate antiproliferative effects by their incorporation into cellular RNA and DNA, respectively. Anabolism to 5-fluorodeoxyuridine monophosphate (FdUMP) causes an inhibition of the key-enzyme of the pyrimidine de novo synthesis, thymidylate synthase (TS)1, 2. FdUrd’s main mechanism of action is thought to be inhibition of TS3, while the action of FUra is incorporation into RNA and TS inhibition in different magnitudes, depending on the schedule4, 5.