Effect of excess folates and deoxyinosine on the activity and site of action of 5-fluorouracil

R. Mark Evans, Jeffrey D. Laskin, Maire T. Hakala

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Abstract

The present study concerns the basis of the effects of high levels of folinic acid (CF) and deoxyinosine (dlno) on the potency and site of action of 5-fluorouracil (FUra) in mouse sarcoma (Sarcoma 180) and human carcinoma cells (Hep-2) in vitro. Sarcoma 180 cells are 50 times more sensitive to FUra than are Hep-2 cells, and thymidylate synthetase (dTMP synthetase) inhibition is growth limiting in Sarcoma 180 but not in Hep-2. Cells were exposed for 3 hr to varied concentrations of FUra alone or in combination with 10 μm CF and/or 1 mM dlno, and the following parameters were determined: (a) the subsequent growth (5 or 6 days) in FUra-free Medium 1640 with or without either 30 μm thymidine or 1 mM 2'-deoxyuridine (dUrd); (b) metabolism of FUra; (c) activity of dTMP synthetase in cell extracts; and (d) incorporation of [2-14C]dUrd at 0, 3, 6, 24, and 48 hr following FUra exposure. In both cell lines, dlno increased the cellular content of unbound 5-fluoro-2'-deoxyuridine-5'-monophosphate up to 100 μm and the inhibition of dTMP synthetase. The potency of FUra, as measured by growth inhibition, was increased only against Sarcoma 180 and not against Hep-2. A very rapid recovery of enzyme activity occurred after dlno, and dTMP synthetase inhibition did not become growth limiting for Hep-2. In both cell lines, 10 μm CF (1000 times more than required for growth) potentiated 3-fold the growth inhibition by FUra. This excess CF did not affect the extent of dTMP synthetase inhibition when measured immediately after FUra. Instead, the potentiating effect of CF is due to the stabilization of the enzyme-5-fluoro-2'-deoxyuridine-5'-monophosphate complex as evidenced by (a) a marked decrease in the ability of 1 mM dUrd to rescue Sarcoma 180 cells after treatment with FUra or Hep-2 cells after treatment with 5-fluoro-2'-deoxyuridine, without effect on the rescue with 2'-deoxythymidine and (b) a marked slow-down in the spontaneous recovery of dTMP synthetase activity after FUra or after FUra and dlno. In all conditions, the recovery of dTMP synthetase activity reached a limit at 6 hr after FUra removal. An impressive correlation was observed in Sarcoma 180 cells between the dTMP synthetase activities (measured by dUrd incorporation), which were.

Original languageEnglish (US)
Pages (from-to)3288-3295
Number of pages8
JournalCancer Research
Volume41
StatePublished - Sep 1 1981
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

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