Quantification of 5-Fluorouracil Incorporation into RNA of Human and Murine Tumors as Measured with a Sensitive Gas Chromatography-Mass Spectrometry Assay

G. J. Peters, P. Noordhuis, A. Komissarov, U. Holwerda, R. M. Kok, J. A M Vanlaar, C. L. Vanderwilt, C. J. Vangroeningen, H. M. Pinedo

Research output: Contribution to journalArticlepeer-review

Abstract

5-Fluorouracil (5FU) can exert its cytotoxic activity by either inhibition of thymidylate synthase or incorporation into RNA. The extent and importance of the latter in tumors of patients are not clear, due to the lack of sensitive and reproducible methods. RNA from 5FU-treated human WiDr colon tumor cells was isolated and [14C]5FU incorporation into RNA was measured by traditional scintillation counting while that of nonradiolabeled 5FU was measured with the present, new method. For the latter purpose, isolated RNA was incubated with RNase, alkaline phosphatase, and uridine phosphorylase, resulting in a complete degradation of RNA, nucleotides, and nucleoside to 5FU. 5FU was then measured with gas chromatography coupled to mass spectrometry. For both methods RNA incorporation was 0.4 pmol/h/μg RNA at 25 μM 5FU while a similar time (up to 4 h) and concentration dependence (25 to 50 μM) were found. Reproducibility of the assay was more than 95%. In a murine colon tumor 5FU incorporation into RNA reached a peak of 10 pmol/μg RNA at 2 h after administration of the maximum tolerated dose of 80 mg 5FU/kg, which was retained until at least 72 h at 2.5 pmol/μg. In tumors from patients treated with 500 mg 5FU/m2 incorporation into RNA after 24 h amounted to 1.0-1.5 pmol/μg; RNA. In conclusion, a novel approach, combining different sensitive and reproducible techniques, was established to measure 5FU incorporation into RNA in clinical tumor specimens enabling determination of its clinical relevance.

Original languageEnglish (US)
Pages (from-to)157-163
Number of pages7
JournalAnalytical Biochemistry
Volume231
Issue number1
DOIs
StatePublished - Oct 1995
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

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