Defective Facilitated Diffusion of Nucleosides, a Primary Mechanism of Resistance to 5-Fluoro-2ˊ-deoxyuridine in the HCT-8 Human Carcinoma Line

Alberto F. Sobrero, Robyn D. Moir, Joseph R. Bertino, Robert E. Handschumacher

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

In vitro resistance of HCT-8 cells to 5-fluoro-2ˊ-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 μM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd Phosphorylase, 5ˊ-fluorouridine kinase, 5-fluorouridine Phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 μM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2ˊ-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25°C and at 4°C over a range of extracellular FdUrd concentrations (0.1 to 10 μM). Rapid entrace of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.

Original languageEnglish (US)
Pages (from-to)3155-3160
Number of pages6
JournalCancer Research
Volume45
Issue number7
Publication statusPublished - Jul 1 1985
Externally publishedYes

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ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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