Novel mechanism(s) of resistance to 5-fluorouracil in human colon cancer (HCT-8) sublines following exposure to two different clinically relevant dose schedules

Abstract

Mechanisms of resistance to 5-fluorouracil (FUra) were compared between a cell line resistant to a short-term exposure (4 h) to this agent (HCT-8/FU4hR) and a cell line resistant to a prolonged exposure (7 days) to the fluoropyrimidine (HCT-8/FU7dR). The two cell lines were obtained by repeatedly exposing 2 x 10(5) cells to a constant concentration of FUra (1000 microM for 4 h or 15 microM for 7 days), able to produce 3-4 logs of cell kill. HCT-8/FU4hR cells were still sensitive to FUra given as a 7-day exposure, suggesting different mechanisms of resistance. In addition, HCT-8/FU7dR cells were cross-resistant to fluorodeoxyuridine and, to a lesser degree, methotrexate; while HCT-8/FU4dR cells were not. Both HCT-8/FU4hR and HCT-8/FU7dR cells were similar to parental HCT-8 cells with regard to uptake of FUra as well as the pattern of FUra-metabolizing and FUra target enzymes. Although neither in situ thymidylate synthase (TS) activity nor the degree of its inhibition by FUra showed any evidence of alteration in HCT-8/FU7dR cells, a rapid recovery of TS activity after drug removal was evident in this cell line. The addition of as much as 100 microM leucovorin did not completely inhibit the recovery of thymidylate synthesis after FUra exposure. No differences were detected in the kinetic properties (Km for 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, concentration producing 50% inhibition for fluorodeoxyuridylate) or TS from HCT-8/FU7dR cells as compared to parental HCT-8 TS. Baseline levels of 5,10 methylenetetrahydrofolate were decreased in HCT-8/FU7dR cells, and analysis of the chain length distribution of the polyglutamylated form of the folate cofactor showed that in this cell line the defect in 5,10-methylenetetrahydrofolate levels is accompanied by, and possibly due to, a defect in the polyglutamylation of this cofactor. In contrast, HCT-8/FU4hR cells were similar to the parental cell line with regard to both the degree of in situ TS inhibition by FUra and duration of inhibition after FUra removal. Labeling studies with [3H-6]FUra (4 h exposure, 100 microM) showed that the incorporation of the fluoropyrimidine into RNA is significantly decreased in HCT-8/FU4hR cells as compared to parental HCT-8 cells.(ABSTRACT TRUNCATED AT 400 WORDS)