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Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells
Abstract:
Incubation of mouse T lymphoma (S-49) cells with the inosinate dehydrogenase inhibitor mycophenolic acid produced a depletion of both GTP and dGTP, and resulted in growth inhibition, partial reduction in RNA synthesis, and drastic inhibition of DNA synthesis. Similar results suggested to others that the depletion of dGTP is primarily responsible for toxicity. However, guanosine was as effective as deoxyguanosine at preventing mycophenolic acid toxicity although deoxyguanosine was more effective at elevating dGTP levels. Moreover, in hypoxanthine-guanine phosphoribosyltransferase-deficient mutants of S-49 (6MPR-3-3) deoxyguanosine was unable to prevent mycophenolic acid toxicity or to re-establish normal DNA synthesis, although it returned cellular dGTP but not GTP levels to normal. No other nucleotide levels changed in a way which could account for the toxicity. Incubation of cells with a combination of deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine produced a selective depletion of dGTP to levels similar to that produced by mycophenolic acid, but did not affect cell growth. Studies with cells synchronized by centrifugal elutriation show that the toxicity of mycophenolic acid is specific to the S-phase of the cell cycle. Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acid-treated S-49 cells. These results suggest that the depletion of GTP rather than that of dGTP produces toxic effects in S-49 cells and that GTP is required for DNA synthesis.
Insights
Mycophenolic acid inhibits cell growth by depleting GTP, not dGTP. This GTP depletion specifically impacts DNA synthesis during the S-phase of the cell cycle in mouse T lymphoma cells.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Pharmacology
Background:
- Mycophenolic acid (MPA) is an inosinate dehydrogenase inhibitor.
- MPA causes depletion of GTP and dGTP, leading to growth inhibition and reduced RNA/DNA synthesis.
- Previous studies suggested dGTP depletion is the primary cause of MPA toxicity.
Purpose of the Study:
- To investigate the specific nucleotide depletion responsible for MPA toxicity in S-49 cells.
- To determine the role of GTP and dGTP in DNA synthesis and cell cycle progression.
- To elucidate the mechanism of MPA-induced cytotoxicity.
Main Methods:
- Treatment of S-49 cells and hypoxanthine-guanine phosphoribosyltransferase-deficient mutants with MPA.
- Analysis of GTP and dGTP levels, RNA and DNA synthesis rates.
- Cell cycle synchronization using centrifugal elutriation.
- Assessment of toxicity using deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine combinations.
- Addition of actinomycin D to assess GTP's role.
Main Results:
- MPA treatment depleted both GTP and dGTP, inhibiting growth and DNA synthesis.
- Guancine and deoxyguanosine differentially affected MPA toxicity and nucleotide levels.
- Selective dGTP depletion without GTP depletion did not inhibit cell growth.
- MPA toxicity was specific to the S-phase of the cell cycle.
- Restoration of GTP levels via actinomycin D rescued DNA synthesis.
Conclusions:
- GTP depletion, not dGTP depletion, is responsible for the toxic effects of MPA in S-49 cells.
- GTP is essential for DNA synthesis.
- These findings clarify the mechanism of MPA toxicity and its impact on cellular processes.