DUP 785 (NSC 368390): Schedule-dependency of growth-inhibitory and antipyrimidine effects

Abstract

DUP 785 (NSC 368390; Brequinar sodium) is a new inhibitor of pyrimidine de novo biosynthesis with antitumor activity against several experimental tumors. DUP 785 inhibits the mitochondrial enzyme dihydroorotate dehydrogenase, blocking the conversion of dihydroorotate to orotate. We examined the influence of exposure time to DUP 785 on its growth-inhibitory effects in L1210 murine leukemia and WiDR human adenocarcinoma cells and the effects of pyrimidine (deoxy) nucleosides on reversal of growth-inhibition. The results were correlated with changes in intracellular pyrimidine nucleotide pools and cell cycle distribution. In L1210 cells, a continuous exposure to 25 μM DUP 785 up to 96 hr caused complete growth inhibition. A 2 hr exposure of cells to the drug did not affect growth. In WiDR cells, exposure to the drug for 1–24 hr, followed by cultivation in drug-free medium resulted in recovery of growth. However, cells exposed to the drug for 48 hr or longer were not able to resume growth when recultured in drug-free medium. Reversal studies were performed to know whether selective depletion of one of the pyrimidine (deoxy) nucleotides might be related to the growth-inhibitory effects of DUP 785. Neither thymidine, deoxycytidine alone, deoxycytidine plus tetrahydrouridine; nor cytidine plus tetrahydrouridine added after 24 hr were able to reverse cell growth inhibition induced by 25 μM DUP 785. However, uridine and cytidine alone reversed growth inhibition. UTP and CTP pools in L1210 cells decreased to about 30–40% of control levels after 4 hr of drug exposure, while dTTP and dCTP pools decreased to about 30% of control levels. There were no significant changes in purine nucleotide pools. In WiDR cells, UTP and CTP pools decreased rapidly after drug exposure and were substantially depleted after 24 hr. Reculture of cells in drug-free medium resulted in a significant recovery of UTP and CTP levels only for cells exposed to DUP 785 for 1–24 hr. For cells exposed to the drug for 48 and 72 hr recovery of nucleotide pools was minimal. In L1210 cells, a 12-hr exposure to the drug caused an accumulation of cells in the early S-phase. In WiDR cells, there was a clear accumulation of cells in the S-phase of the cell cycle after 24 hr drug exposure. After culture in drug-free medium cells continued to traverse through the cell cycle. Our results demonstrate that prolonged exposure of cells to DUP 785 was necessary for a long-lasting depletion of pyrimidine nucleotides and a substantial suppression of RNA and DNA synthesis. Selective repletion of one of the (deoxy) ribonucleotides did not restore cell growth. Only repletion of all nucleotides by uridine and/or cytidine restored cell growth. The failure of short-term drug exposure to cause effects may be explained by the rapid recovery of pyrimidine nucleotide pools in drug-free medium, presumably allowing cells to continue RNA and DNA synthesis and to reverse the S-phase block in the cell cycle.