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 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.


OBJECTIVE
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 microM) followed by continuous exposure (0.5, 1, or 2 microM) or 4 hours (10, 30, or 60 microM) followed by continuous exposure (1 or 2 microM). 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 microM, 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.


CONCLUSIONS
We are conducting a clinical trial of bolus methotrexate followed by continuous-infusion 5-FU plus leucovorin.
Sobrero
A F
AF
Department of Medical Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
Aschele
C
C
Guglielmi
A P
AP
Mori
A M
AM
Melioli
G G
GG
Rosso
R
R
Bertino
J R
JR
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000201:enzymology; D002453:Cell Cycle / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D004334:Drug Administration Schedule; D004351:Drug Resistance; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D005434:Flow Cytometry; D005472:Fluorouracil / Q000008:administration & dosage* / Q000494:pharmacology; D006801:Humans; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D013940:Thymidylate Synthase; D005472:Fluorouracil