Method of determining the activity of enzymes converting cytosine derivatives to uracil derivatives in cells, tissues and organisms
Nucleoside analogs are widely used drugs in the chemotherapy of malignant and viral diseases. Their application and effectiveness is considerably influenced by a wide range of mechanisms involving transport, phosphorylation, catabolism and as well mutual competition with natural nucleosides. Cytosine specific enzymes participate in the deamination of analogues of deoxycytidine and their monophosphates into uracil derivatives. Important examples of such analogues are some medicaments, e.g. ara-C (1-β-arabinofuranosylcytosine), dFdC (2’,2’-difluoro-2’-deoxyuridine), PSI-6130 (β-D-2’-deoxy-2’-C-methylcytidine), L-dC (β-L-2’-deoxycytidine) or 5-aza-dC (5-aza-2’-deoxycytidine). In this respect, it is expected that their deamination could influence the results of a treatment. Currently, however, there is a lack of quick and reliable procedures for determining the activity of cytosine deaminases. The most common approach for determining the activity of cytosine deaminases in cells is the analysis of the products of deamination after the disintegration of the cells, or tissues. However, when using current techniques, it is a relatively long process, often with the usage of radioactive markers and special equipment, which allows the acquisition of data on the average activity in a relatively large population of cells, but not in small cell populations, or in individual cells.
The developed method allows a quick determination of the activity of enzymes transforming cytosine derivatives into uracil derivatives in a sample of tissues, or cells using analogues of cytosine nucleosides. These substances are transformed into uracil derivatives by deamination, and are subsequently detected in DNA or RNA. The amount of the uracil nucleoside analogue is then determined and the signal can be analyzed by i.e. classical microscopic techniques or by flow cytometers or plate readers.
The method has been developed for the use in solutions to allow detection of deaminase activities e.g. in sera or cell suspensions.
The main benefit of the developed method is quick and reliable identification of the activity of enzymes converting cytosine derivatives to uracil derivatives and can help in research focused on the seeking of new inhibitors of deaminases or testing various cell lines from the perspective of deaminase activities or in testing the effect of DNA or RNA oligonucleotides (antisense oligonucleotides and siRNA) targeted at the inhibition of the expression of deaminases. In the diagnostics, it can help with the application of medicaments based on cytosine analogues of nucleosides.
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PV 2014-579 PCT/CZ2015/000097 EP 15774495.4
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague Institute of Applied Biotechnologies, a.s.