NOVEL MECHANISM(S) OF RESISTANCE TO 5-FLUOROURACIL IN HUMAN COLON CANCER (HCT-8) SUBLINES FOLLOWING EXPOSURE TO 2 DIFFERENT CLINICALLY RELEVANT DOSE SCHEDULES

Mechanisms of resistance to 5-fluorouracil (FUra) were compared betwee n 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 obtai ned by repeatedly exposing 2 x 10(5) cells to a constant concentration of FUra (1000-mu-M for 4 h or 15-mu-M for 7 days), able to produce 3- 4 logs of cell kill. HCT-8/FU4hR cells were still sensitive to FUra gi ven as a 7-day exposure, suggesting different mechanisms of resistance . In addition, HCT-8/FU7dR cells were cross-resistant to fluorodeoxyur idine and, to a lesser degree, methotrexate; while HCT-8/FU4hR cells w ere not. Both HCT-8/FU4hR and HCT-8/FU7dR cells were similar to parent al 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 t hymidylate synthase (TS) activity nor the degree of its inhibition by FUra showed any evidence of alteration in HCT-8/FU7dR cells, a rapid r ecovery of TS activity after drug removal was evident in this cell lin e. The addition of as much as 100-mu-M leucovorin did not completely i nhibit the recovery of thymidylate synthesis after FUra exposure. No d ifferences were detected in the kinetic properties (K(m) for 2'-deoxyu ridylate and 5,10-methylenetetrahydrofolate, concentration producing 5 0% inhibition for fluorodeoxyuridylate) or TS from HCT-8/FU7dR cells a s compared to parental HCT-8 TS. Baseline levels of 5,10 methylenetetr ahydrofolate were decreased in HCT-8/FU7dR cells, and analysis of the chain length distribution of the polyglutamylated form of the folate c ofactor showed that in this cell line the defect in 5,10-methylenetetr ahydrofolate levels is accompanied by, and possibly due to, a defect i n the polyglutamylation of this cofactor. In contrast, HCT-8/FU4hR cel ls were similar to the parental cell line with regard to both the degr ee of in situ TS inhibition by FUra and duration of inhibition after F Ura removal. Labeling studies with [H-3-6]FUra (4 h exposure, 100-mu-M ) showed that the incorporation of the fluoropyrimidine into RNA is si gnificantly decreased in HCT-8/FU4hR cells as compared to parental HCT -8 cells. The mechanisms of resistance found in these cell lines indic ate that the mechanism of cell kill by FUra differs depending on the d ose schedule used: short-term exposure to high concentrations of FUra kills cells by an RNA effect, while prolonged exposure to low doses is cytotoxic via inhibition of thymidylate synthesis and, consequently, DNA synthesis.