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.

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Olomouc Czech Republic

Laboratory of Inherited Metabolic Disorders Department of Clinical Chemistry Palacký University and University Hospital Olomouc Olomouc Czech Republic

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de Sousa Cavalcante L, Monteiro G. Gemcitabine: Metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur. J. Pharmacol. 2014;741:8–16. doi: 10.1016/j.ejphar.2014.07.041. PubMed DOI

Mini E, Nobili S, Caciagli B, Landini I, Mazzei T. Cellular pharmacology of gemcitabine. Ann. Oncol. 2006;17(Suppl 5):v7–12. doi: 10.1093/annonc/mdj941. PubMed DOI

Hamed SS, Straubinger RM, Jusko WJ. Pharmacodynamic modeling of cell cycle and apoptotic effects of gemcitabine on pancreatic adenocarcinoma cells. Cancer Chemother. Pharmacol. 2013;72:553–563. doi: 10.1007/s00280-013-2226-6. PubMed DOI PMC

Grant S. Ara-C: Cellular and molecular pharmacology. Adv. Cancer Res. 1998;72:197–233. doi: 10.1016/S0065-230X(08)60703-4. PubMed DOI

Issa JP, et al. Phase II study of low-dose decitabine in patients with chronic myelogenous leukemia resistant to imatinib mesylate. J. Clin. Oncol. 2005;23:3948–3956. doi: 10.1200/JCO.2005.11.981. PubMed DOI

Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation. Cell. 1980;20:85–93. doi: 10.1016/0092-8674(80)90237-8. PubMed DOI

Heinemann V, et al. Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: A mechanism of self-potentiation. Cancer Res. 1992;52:533–539. PubMed

Xu YZ, Plunkett W. Modulation of deoxycytidylate deaminase in intact human leukemia cells. Action of 2',2'-difluorodeoxycytidine. Biochem. Pharmacol. 1992;44:1819–1827. doi: 10.1016/0006-2952(92)90077-V. PubMed DOI

Gilbert JA, et al. Gemcitabine pharmacogenomics: Cytidine deaminase and deoxycytidylate deaminase gene resequencing and functional genomics. Clin. Cancer Res. 2006;12:1794–1803. doi: 10.1158/1078-0432.CCR-05-1969. PubMed DOI

Jansen RS, Rosing H, Schellens JH, Beijnen JH. Deoxyuridine analog nucleotides in deoxycytidine analog treatment: Secondary active metabolites? Fundam. Clin. Pharmacol. 2011;25:172–185. doi: 10.1111/j.1472-8206.2010.00823.x. PubMed DOI

Hubeek I, et al. The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia. Br. J. Cancer. 2005;93:1388–1394. doi: 10.1038/sj.bjc.6602881. PubMed DOI PMC

Kufe DW, Major PP, Egan EM, Beardsley GP. Correlation of cytotoxicity with incorporation of ara-C into DNA. J. Biol. Chem. 1980;255:8997–8900. doi: 10.1016/S0021-9258(19)70512-2. PubMed DOI

Major PP, Egan EM, Beardsley GP, Minden MD, Kufe DW. Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA. Proc. Natl. Acad. Sci. U.S.A. 1981;78:3235–3239. doi: 10.1073/pnas.78.5.3235. PubMed DOI PMC

Lamba JK. Pharmacogenomics of cytarabine in childhood leukemia. Pharmacogenomics. 2011;12:1629–1632. doi: 10.2217/pgs.11.148. PubMed DOI

Hamada A, Kawaguchi T, Nakano M. Clinical pharmacokinetics of cytarabine formulations. Clin. Pharmacokinet. 2002;41:705–718. doi: 10.2165/00003088-200241100-00002. PubMed DOI

Gandhi V, Xu YZ, Estey E. Accumulation of arabinosyluracil 5'-triphosphate during arabinosylcytosine therapy in circulating blasts of patients with acute myelogenous leukemia. Clin. Cancer Res. 1998;4:1719–1726. PubMed

de Vos D, van Overveld W. Decitabine: A historical review of the development of an epigenetic drug. Ann. Hematol. 2005;84(Suppl 1):3–8. doi: 10.1007/s00277-005-0008-x. PubMed DOI PMC

Bergman AM, Pinedo HM, Peters GJ. Determinants of resistance to 2',2'-difluorodeoxycytidine (gemcitabine) Drug Resist. Updates. 2002;5:19–33. doi: 10.1016/S1368-7646(02)00002-X. PubMed DOI

Funamizu N, et al. Is the resistance of gemcitabine for pancreatic cancer settled only by overexpression of deoxycytidine kinase? Oncol. Rep. 2010;23:471–475. PubMed

Giovannetti E, et al. Cytotoxic activity of gemcitabine and correlation with expression profile of drug-related genes in human lymphoid cells. Pharmacol. Res. 2007;55:343–349. doi: 10.1016/j.phrs.2007.01.003. PubMed DOI

Morita T, Matsuzaki A, Kurokawa S, Tokue A. Forced expression of cytidine deaminase confers sensitivity to capecitabine. Oncology. 2003;65:267–274. doi: 10.1159/000074480. PubMed DOI

Funamizu N, et al. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels. Plos One. 2012;7:e37424. doi: 10.1371/journal.pone.0037424. PubMed DOI PMC

Beumer JH, et al. Pharmacokinetics, metabolism, and oral bioavailability of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine in mice. Clin. Cancer Res. 2006;12:7483–7491. doi: 10.1158/1078-0432.CCR-06-1250. PubMed DOI

Beumer JH, et al. Concentrations of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) and its cytotoxic metabolites in plasma of patients treated with FdCyd and tetrahydrouridine (THU) Cancer Chemother. Pharmacol. 2008;62:363–368. doi: 10.1007/s00280-007-0603-8. PubMed DOI

Kreis W, et al. Effect of tetrahydrouridine on the clinical pharmacology of 1-beta-D-arabinofuranosylcytosine when both drugs are coinfused over three hours. Cancer Res. 1988;48:1337–1342. PubMed

Ciccolini J, et al. Cytidine deaminase residual activity in serum is a predictive marker of early severe toxicities in adults after gemcitabine-based chemotherapies. J. Clin. Oncol. 2010;28:160–165. doi: 10.1200/JCO.2009.24.4491. PubMed DOI

Mameri H, et al. Cytidine deaminase deficiency reveals new therapeutic opportunities against cancer. Clin. Cancer Res. 2017;23:2116–2126. doi: 10.1158/1078-0432.CCR-16-0626. PubMed DOI PMC

Ligasová A, et al. A new sensitive method for the detection of mycoplasmas using fluorescence microscopy. Cells. 2019;8:1510. doi: 10.3390/cells8121510. PubMed DOI PMC

Ligasová A, et al. Dr Jekyll and Mr Hyde: A strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine. Open Biol. 2016;6:150172. doi: 10.1098/rsob.150172. PubMed DOI PMC

Ligasová A, Strunin D, Friedecký D, Adam T, Koberna K. A fatal combination: A thymidylate synthase inhibitor with DNA damaging activity. PLoS One. 2015;10:e0117459. doi: 10.1371/journal.pone.0117459. PubMed DOI PMC

Milanesi G, et al. BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli. Mol. Cell Biol. 1984;4:1551–1560. PubMed PMC

Ligasová A, Rosenberg I, Bocková M, Homola J, Koberna K. Anchored linear oligonucleotides: The effective tool for the real-time measurement of uracil DNA glycosylase activity. Open Biol. 2021;11:210136. doi: 10.1098/rsob.210136. PubMed DOI PMC

Romero-Calvo I, et al. Reversible ponceau staining as a loading control alternative to actin in Western blots. Anal. Biochem. 2010;401:318–320. doi: 10.1016/j.ab.2010.02.036. PubMed DOI

Schindelin J, et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Madrova L, et al. Mass spectrometric analysis of purine de novo biosynthesis intermediates. PLoS One. 2018;13:e0208947. doi: 10.1371/journal.pone.0208947. PubMed DOI PMC

Van Rossum G, Drake FL. Python 3 Reference Manual. Createspace; 2009.

Harris CR, et al. Array programming with NumPy. Nature. 2020;585:357–362. doi: 10.1038/s41586-020-2649-2. PubMed DOI PMC

McKinney, W. In Proceedings of the 9th Python in Science Conference, Vol. 445 51–56 (2010).

Berg S, et al. ilastik: Interactive machine learning for (bio)image analysis. Nat. Methods. 2019;16:1226–1232. doi: 10.1038/s41592-019-0582-9. PubMed DOI

Carpenter AE, et al. Cell profiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100. doi: 10.1186/gb-2006-7-10-r100. PubMed DOI PMC

Kamentsky L, et al. Improved structure, function and compatibility for cell profiler: Modular high-throughput image analysis software. Bioinformatics. 2011;27:1179–1180. doi: 10.1093/bioinformatics/btr095. PubMed DOI PMC

Virtanen P, et al. SciPy 1.0: Fundamental algorithms for scientific computing in Python. Nat. Methods. 2020;17:261–272. doi: 10.1038/s41592-019-0686-2. PubMed DOI PMC

Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 2008;105:2415–2420. doi: 10.1073/pnas.0712168105. PubMed DOI PMC

Ligasova A, Koberna K. Quantification of fixed adherent cells using a strong enhancer of the fluorescence of DNA dyes. Sci. Rep. 2019;9:8701. doi: 10.1038/s41598-019-45217-9. PubMed DOI PMC

Pilger BD, et al. Substrate diversity of herpes simplex virus thymidine kinase. Impact of the kinematics of the enzyme. J. Biol. Chem. 1999;274:31967–31973. doi: 10.1074/jbc.274.45.31967. PubMed DOI

Qu D, et al. 5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: Detection of proliferating cells. Anal. Biochem. 2011;417:112–121. doi: 10.1016/j.ab.2011.05.037. PubMed DOI

Liboska R, Ligasová A, Strunin D, Rosenberg I, Koberna K. Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine—The method for the effective suppression of this cross-reactivity. PLoS One. 2012;7:e51679. doi: 10.1371/journal.pone.0051679. PubMed DOI PMC

Ligasová A, Koberna K. DNA replication: From radioisotopes to click chemistry. Molecules. 2018;23:3007. doi: 10.3390/molecules23113007. PubMed DOI PMC

Ligasová A, Strunin D, Liboska R, Rosenberg I, Koberna K. Atomic scissors: A new method of tracking the 5-bromo-2'-deoxyuridine-labeled DNA in situ. Plos One. 2012;7:e52584. doi: 10.1371/journal.pone.0052584. PubMed DOI PMC

Wiley JS, Jones SP, Sawyer WH, Paterson AR. Cytosine arabinoside influx and nucleoside transport sites in acute leukemia. J. Clin. Invest. 1982;69:479–489. doi: 10.1172/JCI110472. PubMed DOI PMC

Yamauchi T, et al. Intracellular cytarabine triphosphate production correlates to deoxycytidine kinase/cytosolic 5'-nucleotidase II expression ratio in primary acute myeloid leukemia cells. Biochem. Pharmacol. 2009;77:1780–1786. doi: 10.1016/j.bcp.2009.03.011. PubMed DOI

Galmarini CM, Jordheim L, Dumontet C. Role of IMP-selective 5'-nucleotidase (cN-II) in hematological malignancies. Leuk. Lymphoma. 2003;44:1105–1111. doi: 10.1080/1042819031000077142. PubMed DOI

Mahmood T, Yang PC. Western blot: Technique, theory, and trouble shooting. N. Am. J. Med. Sci. 2012;4:429–434. doi: 10.4103/1947-2714.100998. PubMed DOI PMC

A Rapid Approach for Identifying Cell Lines Lacking Functional Cytidine Deaminase

The kinetics of uracil-N-glycosylase distribution inside replication foci