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.
Highlights
We present evidence that itis the depletion of GTP rather than dGTP which is toxic to cells, that GTPdepletion is associated with the inhibition of DNA synthesis, and that cells whichare replicating DNA suffer the majority of toxicity
Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acidtreated 5-49 cells.These results suggest that thedepletion of GTP rather than that of dGTP produces toxic phohydrolase; EC 3.1.3.2)type 111, from Escherichia coli, DNase Itype VI, from Crotalus adamanteus were purchased from Sigma, St
Cell Culture-Mouse T lymphoma (S-49)cells were grown in suspension at 37 "C and 10% COa in Dulbecco's modified Eagle's medium supplemented with 10%horse serum
Summary
Toxicity ofMycophenolicAcid-The growth inhibitory and cytotoxic effects of MA on wild type S-49 cells and on HGPRTase-deficient (6MPR-3-3) S-49 cells were examined. Neither Guo nor dGuo were able to prevent MA toxicity in HGPRTase-deficient S-49 cells. HGPRTase-deficient (A-A) S-49 cells were incubated with a toxic concentration of mycophenolic acid and increasing concentrations of guanosine for 24 h.'Wild type (M an) d HGPRTase-deficient (A-A) 5-49 cells were alsa incubated with increasing concentrations of guanosine in the absence of mycophenolicacid. Results are those of a singletypical experiment. Incubation of HGPRTase-deficient cells with 1.2 pM MA for 3 h resulted in nucleotide level changes similar to those in wild type S-49 cells (Table I).
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