Effects of methotrexate on ribonucleotide pools in growing and in growth-arrested tumor cells and antagonism by RNA synthesis inhibitors.

We examined the mechanism of ribonucleotide pool changes in methotrexate-treated Ehrlich ascites tumor cells in culture. In exponentially growing cells, methotrexate inhibited ATP and GTP formation and stimulated UTP and CTP formation by the de novo pathways. Over the following 4 h, ATP and GTP decreased and UTP and CTP increased. During this period RNA synthesis rates decreased by 30 to 40%. Inhibitors of nucleolar RNA synthesis (actinomycin D, doxorubicin, or cycloheximide), but not of DNA synthesis (cytosine arabinoside), prevented ATP and GTP depletion. These results suggest that ATP and GTP were depleted in methotrexate-treated cells because the shift-down of RNA synthesis occurred more slowly than the inhibition of ATP and GTP supply. UTP and CTP pools increased because of increased de novo synthesis and because of decreased consumption in RNA synthesis. Ribonucleoside triphosphates were increased and their syntheses by de novo pathways were inhibited in cells treated with RNA synthesis inhibitors. ATP decreased slightly and GTP remained unchanged in growth-arrested cells treated with methotrexate. In growth-arrested cells, de novo purine nucleotide synthesis was inhibited less and RNA synthesis rates were lower than in growing cells.