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

Godefridus J. Peters, Emile Laurensse, Albert Leyva, Herbert M. Pinedo

Research output: Contribution to journalArticle

Abstract

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 Ki 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.

Original languageEnglish (US)
Pages (from-to)1869-1881
Number of pages13
JournalEuropean Journal of Cancer and Clinical Oncology
Volume23
Issue number12
DOIs
Publication statusPublished - 1987
Externally publishedYes

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Keywords

  • 5,10-CHFH, 5,10-methylene tetrahydrofolate
  • 5-FU, 5-fluorouracil
  • dRib-1-P, deoxy-ribose-1-phosphate
  • FdUMP, 5-fluoro-2′-deoxyuridine-5′-monophosphate
  • FUdR, 5-fluorodeoxyuridine
  • FUMP, 5-fluoro-uridine-5′-monophosphate
  • FUR, 5-fluorouridine
  • IC, concentration that causes 50% growth inhibition
  • OPRT, orotate phosphoribosyl transferase
  • PRPP, 5-phosphoribosyl-1-pyrophosphate
  • Rib-1-P, ribose-1-phosphate
  • Rib-5-P, ribose-5-phosphate
  • TCA, trichloroacetic acid

ASJC Scopus subject areas

  • Oncology

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