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Cellular deoxynucleoside kinases as markers for cytotoxicity of nucleoside therapeutics

Objective

To determine the role of the four cellular deoxynucleoside kinases in the cytotoxicity of antiviral and anticancer nucleosides.
To study the enzymology and molecular biology of the cellular deoxynucleoside kinases, particularly with respect to new nucleoside analogs.
To establish reliable pre-clinical cell and animal models for the development of new chemotherapy's with higher efficacy and minimal toxic side effects.

Deoxynucleoside kinases are required for the activation of nucleoside analogs used in chemotherapy. There are four deoxynucleoside specific kinases in animal cells i.e. cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). These enzymes have different specificity and are expressed at different levels in cells and tissues. Detailed characterization of the enzymatic properties of the four enzymes with purified recombinant enzymes will be done in case of TK1 and dCK. Crystallisation experiments with TK1 and dCK for structural determinations will be performed. A major goal of the project is to clone the genes for TK2 and dGK and to perform detailed biochemical and molecular biology studies with these enzymes.
The expression of all four enzymes will be determined in tumors, drug treated and untreated normal and infected cells with selective enzyme assays, immunochemical methods and quantitative mRNA assays. There are important species differences in the substrate specificity of human and rodent dCK-the molecular basis for this differences will be investigated. The role of the four kinases in deoxynucleoside metabolism will be tested by selection and construction of cell culture, organelle (e.g. isolated synaptosomes) and animal model systems where one or several of the enzyme functions will he eliminated.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

The Swedish University of Agricultural Sciences
Address
3,Husargatan
751 23 Uppsala
Sweden

Participants (3)

Foundation for Research and Technology-Hellas
Greece
Address
1527,Vasilika Vouton P.o.box
711 10 Heraklion
Semmelweis University of Medicine
Hungary
Address
9,Puskin U
1444 Budapest
Vrije Universiteit Amsterdam
Netherlands
Address
1117,De Boelelaan 1117
1081 HV Amsterdam