Purine nucleosides as cell-specific modulators of 5-fluorouracil metabolism and cytotoxicity

Purine nucleosides and ribose- 5-phosphate (Rib- 5-P) were used to modulate the metabolism and cytotoxicity of 5-fluorouracil ( 5-FU) in order to get better understanding of the mechanism of action of 5-FU. In extracts from five different cell lines both Rib- 5-P and inosine were relatively good precursors for Rib- 1-P, but deoxyinosine was a moderate to poor precursor for deoxyRib- 1-P. In the human colon carcinoma WiDr and the human epithelial intestinal Intestine 407 inosine enhanced Rib- 1-P concentrations 3–6-fold. Incubation with deoxyinosine resulted in the appearance of deoxyRib- 1-P in both cell lines in levels comparable to those of Rib- 1-P. dIMP had the same effect as deoxyinosine in Intestine 407 cells, but not in WiDr cells. Both inosine and deoxyinosine caused a depletion of phosphoribosyl-pyrophosphate. In WiDr cells deoxyinosine (0.1–1.0 mM) clearly potentiated the growth inhibition by 0.1–0.5 μM 5-FU after 24 h of culture, but growth between 24 and 48 h was normal. In Intestine 407 cells the potentiation of 5-FU cytotoxicity by deoxyinosine was even more pronounced at 48 h than at 24 h. In WiDr cells dIMP did not potentiate 5-FU cytotoxity, but in Intestine 407 cells the effect was comparable to that of deoxyinosine. The lack of potentiation in WiDr was accompanied by a low metabolism of dIMP. Growth inhibition by 5-FU and deoxyinosine could be reversed by thymidine in Intestine 407 cells but not completely in WiDr cells. Since the predominant target of the deoxyinosine- 5-FU combination was thymidylate synthase, we analyzed the inhibition of this enzyme by FdUMP and the retention of the inhibition in cell culture. In both cell lines FdUMP was a potent competitive inhibitor of thymidylate synthase with a K i of between 0.5 and 2 nM. Culture of cells in the presence of 5-FU and deoxyinosine resulted in an almost complete inhibition of thymidylate synthase activity after 24 h but after 48 h the activity was partly recovered. In Intestine 407 cells replenishment of the culture medium at 24 h even enhanced the recovery. Analysis of 5-FU anabolism into nucleic acids demonstrated that deoxyinosine inhibited the incorporation of 5-FU into RNA. It is concluded that in Intestine 407 cells addition of deoxyinosine enhanced the effects of 5-FU on growth inhibition due to increased formation of FdUMP leading to enhanced inhibition of thymidylate synthase. In WiDr cells incorporation of 5-FU into RNA might also contribute significantly to cytotoxicity