5‐Alkynyl‐2′‐deoxyuridines: Chromatography‐Free Synthesis and Cytotoxicity Evaluation Against Human Breast Cancer Cells.

Meneni, Srinivasarao; Ott, Ingo; Sergeant, Craig D.; Sniady, Adam; Gust, Ronald; Dembiński, Roman

doi:10.1002/chin.200732207

Cited by 2 publications

(2 citation statements)

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“…For this technique, two critical steps are identified: (1) ethynyl deoxyuridine toxicity for some cell lines is higher in comparison to BrdU and (2) the kinetic conditions for alkyneazide cycloaddition are important. EdU has been reported to have antiviral activities, but also affects cell proliferation by inhibition of thymidilate synthetase and deoxynucleoside kinase activity (Cristofoli et al, 2007;Meneni et al, 2007). EdU concentrations used for this assay ideally should be assessed for each cellular model.…”

Section: Critical Parameters and Troubleshootingmentioning

confidence: 99%

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

et al. 2008

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Determination of incorporation of the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle (see UNIT 7.7). However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S‐phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by an alkenyl deoxyuridine (5‐ethynyl‐2′‐deoxyuridine, EdU), which is incorporated into DNA. The nucleotide exposed ethynyl residue was then derivatized by a copper‐catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA‐bound bromouracil moieties were then detected by commercial anti‐BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging. Curr. Protoc. Cytom. 58:7.34.1‐7.34.13. © 2011 by John Wiley & Sons, Inc.

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Section: Critical Parameters and Troubleshootingmentioning

confidence: 99%

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

et al. 2008

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“…On the other hand, the use of EdU and EdC is complicated by their cytotoxicity [16][17][18][19][20][21][22]. Although it was shown that the EdC toxicity is lower than that of EdU [5], the reason was not known.…”

Section: Introductionmentioning

confidence: 99%

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

et al. 2016

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5-Ethynyl-2′-deoxyuridine (EdU) and 5-ethynyl-2′-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.

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5‐Alkynyl‐2′‐deoxyuridines: Chromatography‐Free Synthesis and Cytotoxicity Evaluation Against Human Breast Cancer Cells.

Cited by 2 publications

References 1 publication

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

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