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Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells
Abstract:
Using the S49 T-cell lymphoma system for the study of immunodeficiency diseases, we characterized several variants in purine salvage and transport pathways and studied their responses to the cytotoxic action of adenosine (5-20 micron) in the presence of adenosine deaminase (ADA) inhibitors. Both an adenosine transport deficient mutant and a mutant lacking adenosine (ado) kinase activity are resistant to the cytotoxic effects of adenosine up to 15 micron. Variants lacking hypoxanthine-guanine phosphoribosyl transferase or adenine phosphoribosyltransferase are sensitive to the killing action of adenosine. We monitored the intracellular concentrations of purine and pyrimidine nucleotides, orotate, and PPriboseP in mutant and wild-type cells following the addition of adenosine and an ADA inhibitor. We conclude that at low concentrations, adenosine must be phosphorylated to deplete the cell of pyrimidine nucleotides and PPriboseP and to promote the accumulation of orotate. These alterations account for one mechanism of adenosine toxicity.
Insights
Adenosine toxicity in immunodeficiency disease research is mediated by its phosphorylation, which depletes pyrimidine nucleotides and promotes orotate accumulation. This mechanism explains how adenosine harms cells, particularly in purine salvage pathway variants.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Investigating immunodeficiency diseases requires understanding purine metabolism and cellular responses to cytotoxic agents.
- Adenosine's role in cellular toxicity is complex and influenced by various metabolic pathways.
Purpose of the Study:
- To characterize S49 T-cell lymphoma variants in purine salvage and transport pathways.
- To determine the response of these variants to adenosine cytotoxicity in the presence of adenosine deaminase (ADA) inhibitors.
- To elucidate the intracellular mechanisms underlying adenosine toxicity.
Main Methods:
- Utilized the S49 T-cell lymphoma system with characterized variants in purine metabolism.
- Assessed cellular resistance and sensitivity to varying concentrations of adenosine with ADA inhibitors.
- Monitored intracellular nucleotide concentrations (purine, pyrimidine), orotate, and PPriboseP in mutant and wild-type cells.
Main Results:
- Mutants deficient in adenosine transport or adenosine kinase activity exhibited resistance to adenosine cytotoxicity up to 15 µM.
- Variants lacking hypoxanthine-guanine phosphoribosyl transferase or adenine phosphoribosyltransferase were sensitive to adenosine's killing effects.
- Adenosine phosphorylation was identified as crucial for depleting pyrimidine nucleotides and PPriboseP, leading to orotate accumulation.
Conclusions:
- Cellular resistance to adenosine is linked to defects in adenosine transport and phosphorylation.
- Adenosine toxicity at low concentrations is primarily mediated by its phosphorylation.
- The depletion of pyrimidine nucleotides and PPriboseP, coupled with orotate accumulation, represents a key mechanism of adenosine-induced cell death.