Synergism and lack of cross-resistance between short-term and continuous exposure to fluorouracil in human colon adenocarcinoma cells

A. F. Sobrero, C. Aschele, A. P. Guglielmi, A. M. Mori, G. G. Melioli, R. Rosso, J. R. Bertino

Research output: Contribution to journalArticle


Background: Our recent findings in vitro in the human colon adenocarcinoma cell line HCT-8 suggest that resistance to fluorouracil (5-FU) in patients with advanced colorectal cancer might be overcome by use of a different treatment schedule. Purpose: We tested the hypothesis that HCT-8 cells resistant to short-term 5-FU exposure retain sensitivity to continuous exposure and studied interactions between the two schedules. Methods: HCT-8 cell lines resistant to short-term (pulse) treatment with 5-FU or to continuous exposure were obtained by six exposures to different concentrations of 5-FU for 4 hours or 7 days. We used a monolayer clonogenic assay to determine 5-FU-induced cell kill in resistant HCT-8 cells and sensitive parent cells. Parent cells were exposed to different concentrations of 5-FU for 1, 4, or 24 hours (short term), for 7 days (continuous exposure), or in a combination of both types of schedules. In a study of the mechanism of interaction between short-term and continuous exposure in parent cells, we performed flow cytometric DNA analysis to determine the percentage of cells in S phase and assays of thymidylate synthase inhibition in intact cells and of incorporation of [6-3H]5-FU nucleotides into nucleic acids. Results: Sensitive HCT-8 cells became fully resistant to 5-FU within five or six treatments, and low-dose continuous exposure almost immediately produced resistant clones. HCT-8 cells resistant to 5-FU given every 4 hours retained full sensitivity to continuous exposure, suggesting lack of cross-resistance between the two schedules, but cells resistant to continuous exposure were cross-resistant to short-term treatment. Parent cells showed a statistically significant (synergistic) enhancement of the cytotoxic activity for 5-FU exposure for 1 hour (100, 300, or 500 μM) followed by continuous exposure (0.5, 1, or 2 μM) or 4 hours (10, 30, or 60 μM) followed by continuous exposure (1 or 2 μM). Short-term plus continuous exposure produced a marked increase in percentage of S-phase cells, compared with the percentage for each schedule alone. The combination of 1-hour exposure and continuous exposure (1000 and 2 μM, respectively) produced a marked accumulation of cells in S phase at 24 hours (59%), which lasted up to 96 hours (53%). The combination of the two schedules produced only additive enhancement of thymidylate synthase inhibition as well as incorporation of [6-3H]5-FU nucleotides into nucleic acids of HCT-8 cells. Conclusions: Our findings provide a rationale for the use of bolus 5-FU and continuous infusion 5-FU in sequence. Implications: We are conducting a clinical trial of bolus methotrexate followed by continuous-infusion 5-FU plus leucovorin.

Original languageEnglish
Pages (from-to)1937-1944
Number of pages8
JournalJournal of the National Cancer Institute
Issue number23
Publication statusPublished - 1993


ASJC Scopus subject areas

  • Cancer Research
  • Oncology
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging

Cite this