CDD plays a pivotal role within the pyrimidine salvage pathway. In this study, a novel, rapid method for the identification of cell lines lacking functional cytidine deaminase was developed. This innovative method utilizes immunocytochemical detection of the product of 5-fluorocytidine deamination, 5-fluorouridine in cellular RNA, enabling the identification of these cells within two hours. The approach employs an anti-bromodeoxyuridine antibody that also specifically binds to 5-fluorouridine and its subsequent detection by a fluorescently labeled antibody. Our results also revealed a strong correlation between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and cytidine deaminase content. On the other hand, no correlation was observed between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and deoxycytidine monophosphate deaminase content. Similarly, no correlation was observed between this ratio and equilibrative nucleoside transporters 1 or 2. Finally, concentrative nucleoside transporters 1, 2, or 3 also do not correlate with the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio.

• Klíčová slova
• 5-fluorocytidine deamination, 5-fluorouridine, cytidine deaminase, equilibrative and concentrative nucleoside transporters,
• MeSH
• buněčné linie MeSH
• cytidin analogy a deriváty   metabolismus   MeSH
• cytidindeaminasa * metabolismus   nedostatek   genetika   MeSH
• lidé MeSH
• uridin analogy a deriváty   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 5-fluorouridine MeSH Prohlížeč
• cytidin MeSH
• cytidindeaminasa * MeSH
• uridin MeSH

Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication. The analysis also showed that very popular synchronisation protocols based on the double thymidine block can result in changes in the UNG2 content and uracil excision rate. In response, we propose a new method for the description of the changes of the content and the activity of different cell components during cell cycle without the necessity to use synchronisation protocols.

• MeSH
• buněčný cyklus MeSH
• kinetika MeSH
• lidé MeSH
• oprava DNA MeSH
• replikace DNA * MeSH
• S fáze MeSH
• uracil-DNA-glykosidasa * metabolismus   MeSH
• uracil metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• uracil-DNA-glykosidasa * MeSH
• uracil MeSH

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.

• MeSH
• cytidin * metabolismus   MeSH
• cytidindeaminasa metabolismus   MeSH
• dCMP-deaminasa * MeSH
• deoxycytidin MeSH
• deoxycytidinkinasa genetika   metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytidin * MeSH
• cytidindeaminasa MeSH
• dCMP-deaminasa * MeSH
• deoxycytidin MeSH
• deoxycytidinkinasa MeSH

Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available. In this review, the basic techniques used for cell quantification, with a special emphasis on techniques based on fluorescent DNA dyes, are described. The main aim of this review is to guide readers through the possibilities of cell quantification with various methods and to show the strengths and weaknesses of these methods, especially with respect to their sensitivity, accuracy, and length. As these methods are frequently accompanied by an analysis of cell proliferation and cell viability, some of these approaches are also described.

• Klíčová slova
• DNA dyes, cell metabolism, cell quantification, enzymatic conversion of substrate,
• MeSH
• biotest MeSH
• DNA analýza   chemie   metabolismus   MeSH
• fluorescenční barviva * MeSH
• počet buněk metody   MeSH
• proliferace buněk MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA MeSH
• fluorescenční barviva * MeSH