Abstract
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.
Highlights
5-Ethynyl-20-deoxyuridine (EdU) and 5-ethynyl-20-deoxycytidine (EdC), representing analogues of 20-deoxyuridine and 20-deoxycytidine, respectively, were tested as substances with an anti-viral effect during the 1980s [1,2]
As the stimulation effect was suppressed by the cytidine deaminase (CDD) inhibitor tetrahydrouridine and by the CDD and DCTD inhibitor 20-deoxytetrahydrouridine, the authors supposed that EdC is transformed to EdU which is incorporated into DNA
Qu et al [5] interpreted the results of experiments focused on the EdU and EdC toxicity in several cell lines as proof that EdC follows the EdC ! EdC monophosphate (EdCMP) ! EdCDP ! EdCTP pathway as a major metabolic pathway
Summary
5-Ethynyl-20-deoxyuridine (EdU) and 5-ethynyl-20-deoxycytidine (EdC), representing analogues of 20-deoxyuridine and 20-deoxycytidine, respectively, were tested as substances with an anti-viral effect during the 1980s [1,2] Presently, their use is primarily connected with the detection of cellular replicational activity [3,4,5,6,7]. It is supposed that the cellular deaminases are involved in the inactivation of drugs based on the 20-deoxycytidine analogues (e.g. cytarabine and gemcitabine [25]) From this point of view the pair EdU and EdC are an interesting model system with the possibility of quick visualization of the incorporated nucleosides. We have shown here that one of the anti-bromodeoxyuridine monoclonal antibodies that exhibits high affinity to EdU [26] does not effectively react with EdC
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