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Adenosine kinase deficiency in tritiated deoxyadenosine-resistant mouse S49 lymphoma cell lines
K J Sastry1, C Huang, T S Chan
1Department of Microbiology, University of Texas Medical Branch, Galveston 77550.
Abstract:
Mutant sublines were derived of S49 mouse T-lymphoma cells that were resistant to tritiated deoxyadenosine. Twenty-five isolates that were selected in 1 microCi/ml of the nucleoside were cross-resistant to 6-thioguanine, were sensitive to HAT (hypoxanthine, aminopterin, and thymidine), and contained less than 1% of hypoxanthine phosphoribosyltransferase activity in wild-type cells. One of the mutant clones, S49-dA2, was further subjected to selection in a medium containing 2 microCi/ml tritiated deoxyadenosine and 1 microgram/ml deoxycoformycin, an inhibitor of adenosine deaminase. All resistant subclones were cross-resistant to tubercidin, 6-methylmercaptopurine riboside, and arabinosyladenine. One of the subclones, S49-12, was completely devoid of adenosine kinase and was partially deficient in deoxyadenosine kinase. This subclone, however, contained wild-type levels of deoxycytidine kinase. DEAE chromatography of the wild-type cell extracts revealed two deoxyadenosine phosphorylating activities, one of which coeluted with adenosine kinase and was the enzyme missing in S49-12. The other species phosphorylated both deoxyadenosine and deoxycytidine, of which deoxycytidine was the preferred substrate.
Insights
Mutant mouse lymphoma cells resistant to deoxyadenosine were identified. These cells showed cross-resistance to other nucleosides and lacked key enzyme activity, revealing new insights into purine metabolism.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- S49 mouse T-lymphoma cells are a model for studying purine metabolism.
- Resistance to nucleoside analogs can reveal information about cellular salvage pathways.
Purpose of the Study:
- To isolate and characterize mutant S49 cell lines resistant to tritiated deoxyadenosine.
- To investigate the enzymatic basis of this resistance and its implications for purine salvage pathways.
Main Methods:
- Selection of mutant cell lines resistant to tritiated deoxyadenosine.
- Cross-resistance assays with various nucleoside analogs.
- Enzyme activity assays, including hypoxanthine phosphoribosyltransferase and kinase activities.
- DEAE chromatography to separate enzymatic activities.
Main Results:
- Isolated mutant sublines resistant to deoxyadenosine were cross-resistant to 6-thioguanine and sensitive to HAT.
- One subclone, S49-12, was deficient in adenosine kinase and partially deficient in deoxyadenosine kinase but retained deoxycytidine kinase activity.
- DEAE chromatography identified two deoxyadenosine phosphorylating activities, one absent in S49-12, which coeluted with adenosine kinase.
Conclusions:
- Mutant S49 cells resistant to deoxyadenosine provide a tool to study purine salvage pathways.
- Adenosine kinase plays a significant role in deoxyadenosine phosphorylation in S49 cells.
- The identified enzymatic activities highlight the complexity of nucleoside metabolism in T-lymphoma cells.