Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2′-deoxycytidine (EdC) and its conversion to 5-ethynyl 2′-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.

脱氧胞苷类似物（dCas）广泛用于治疗恶性疾病。它们通常被胞苷脱氨酶 (CDD) 或脱氧胞苷单磷酸脱氨酶 (dCMP 脱氨酶) 灭活。其他代谢途径，例如磷酸化，可以极大地促进它们的激活（失活）。在这里，描述了一种分析细胞中这些途径的新技术。它基于 5-乙炔基 2'-脱氧胞苷 (EdC) 的使用及其转化为 5-乙炔基 2'-脱氧尿苷 (EdU)。对其使用进行了测试，以评估 CDD 和 dCMP 脱氨酶在五种癌症和四种非癌细胞系中的作用。该技术提供了解决胞苷转运蛋白、CDD、dCMP 脱氨酶和脱氧胞苷激酶对 EdC 代谢的综合影响的可能性。利用这种技术，我们开发了一种快速且廉价的方法来鉴定缺乏 CDD 活性的细胞系。数据显示，与癌细胞相比，研究中使用的所有非癌细胞都表现出较低的 CDD 含量（如果有的话），并且它们的胞苷脱氨酶活性可完全归因于 dCMP 脱氨酶。该技术还证实了脱氧胞苷激酶对于 dCas 代谢的重要性，并表明 dCMP 脱氨酶在 dCas 脱氨和 CDD 中发挥着基础作用。此外，所描述的技术提供了同时测试细胞毒性和 DNA 复制活性的可能性。