Dr Jekyll and Mr Hyde: A Strange Case of 5-Ethynyl-2‘- deoxyuridine and 5-ethynyl-2‘- deoxycytidine

Introduction

5-Ethynyl-2‘-deoxyuridine (EdU) and 5-ethynyl-2‘-deoxycytidine (EdC) represent analogues of 2‘-deoxyuridine and 2‘-deoxycytidine, respectively that are used mainly 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. We focused on the efficiency of the conversion of EdC to EdU and particular steps leading to this conversion. Concurrently, we followed the toxicity of both nucleosides and tested the possibility that the toxicity is directly connected with the conversion of EdC to EdU.

Materials/methods

Cells lines used: HeLa cells, 143B PML BK TK, A549, U2OS and HCT116 cells. Methods used: MTT assay, inhibition of CDD and DCTD activity by siRNA or specific inhibitor, run-on replication, immunofluorescence analysis, hypotonic treatment, nucleotide pools analysis by HPLC-MS, western blot, hypotonic treatment, DNA isolation Data analysis The data of particular experiments were analysed using following software: CellProfiller, Microsoft Excel, GraphPad Prism 6 software.

Results and conclusions

We performed the DNA analysis of six randomly chosen human cell lines incubated with EdC. Surprisingly, not a single one of the tested cell lines contained detectable amount of EdC in DNA. Instead, DNA of all lines contained EdU. The amount of incorporated EdU differed in particular cell lines and EdC cytotoxicity was directly proportional to this amount. The results of experiments with the targeted inhibition of the activity of cytidine deaminase and dCMP deaminase in HeLa cells indicated that the dominant role in the conversion pathway of EdC to EdUTP plays cytidine deaminase. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP and to suppress the incorporation of EdC. Although the conversion of EdC to EdCTP occurred with much lesser effectivity than the transformation of EdU to EdUTP the crucial factor leading to the suppression of EdC incorporation in DNA seemed to be rather the mechanisms directly related to replication complex than the suppression of the production ofEdCTP. Overall, the results obtained clearly showed that EdC and its metabolites are a substrate of a whole range of enzymes in the pathway leading to the production of EdCTP as well as in the opposite pathway leading to the degradation of EdCTP. Our results also indicated that the deamination activity plays only marginal role in the effective protection of cells from the EdC incorporation. On the other hand, this activity substantially contributes to EdC toxicity due to the gradual conversion of EdC to EdUTP followed by the incorporation of EdU into DNA. In this respect, deamination activity paradoxically allows the use of EdC as a replicational marker, and concurrently, fundamentally contributes to its toxicity.

This work was supported by the Ministry of Health of the Czech Republic [AZV 15-31604A].