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Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine
1Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston 77555-1019, USA.
Abstract:
We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 microCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [3H] deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [3H] deoxyadenosine selection, lymphoma cell clones resistant to 15 microCi/ml [3H] deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [3H] Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.
Insights
Tritiated deoxyadenosine selectively kills wild-type S49 cells. Resistant mutants lacking deoxycytidine kinase can be specifically isolated using this radiolabeled nucleoside as a selective agent.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Tritiated deoxyadenosine selectively kills wild-type S49 murine lymphoma cells.
- Resistant mutants previously identified lacked adenosine kinase but retained deoxyadenosine phosphorylating activity.
Purpose of the Study:
- To further investigate the specificity of tritiated deoxyadenosine selection.
- To derive and characterize lymphoma cell clones resistant to high concentrations of tritiated deoxyadenosine.
Main Methods:
- Derivation of resistant cell lines (S49-dA15) using high-dose tritiated deoxyadenosine.
- Assessment of resistance to non-radioactive nucleosides (deoxyadenosine, deoxyguanosine, thymidine).
- Enzyme assays to determine kinase activities and analysis of nucleoside excretion.
Main Results:
- The resistant line S49-dA15 showed cross-resistance to deoxyadenosine and deoxyguanosine but remained sensitive to thymidine.
- S49-dA15 cells lacked deoxycytidine kinase, an enzyme that also phosphorylates deoxyadenosine.
- Mutant cells over-excreted deoxycytidine, indicating a salvage defect.
Conclusions:
- Tritiated deoxyadenosine is a potent selective agent for isolating deoxycytidine kinase-negative mutants.
- The study elucidates the specific roles of adenosine and deoxycytidine kinases in nucleoside salvage pathways.
- Understanding these pathways is crucial for developing targeted therapies and drug resistance studies.