SYNERGISM AND LACK OF CROSS-RESISTANCE BETWEEN SHORT-TERM AND CONTINUOUS EXPOSURE TO FLUOROURACIL IN HUMAN COLON ADENOCARCINOMA CELLS

Background: Our recent findings in vitro in the human colon adenocarci noma 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 schedu les. Methods: HCT-8 cell lines resistant to short-term (pulse) treatme nt 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 m onolayer clonogenic assay to determine 5-FU-induced cell kill in resis tant 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 shor t-term and continuous exposure in parent cells, we performed flow cyto metric DNA analysis to determine the percentage of cells in S phase an d assays of thymidylate synthase inhibition in intact cells and of inc orporation of [6-H-3]5-FU nucleotides into nucleic acids. Results: Sen sitive HCT-8 cells became fully resistant to 5-FU within five or six t reatments, and low-dose continuous exposure almost immediately produce d resistant clones. HCT-8 cells resistant to 5-FU given every 4 hours retained full sensitivity to continuous exposure, suggesting lack of c ross-resistance between the two schedules, but cells resistant to cont inuous exposure were cross-resistant to short-term treatment. Parent c ells showed a statistically significant (synergistic) enhancement of t he cytotoxic activity for 5-FU exposure for 1 hour (100, 300, or 500 m uM) followed by continuous exposure (0.5, 1, or 2 muM or 4 hours (10, 30, or 60 muM) followed by continuous exposure (1 or 2 muM). Short-ter m 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 muM, 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 thymidylat e synthase inhibition as well as incorporation of [6-H-3]5-FU nucleoti des into nucleic acids of HCT-8 cells. Conclusions: Our findings provi de 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.