Different Clinically Relevant Dose Schedules Colon Cancer (HCT-8) Sublines following Exposure to Two Novel Mechanism(s) of Resistance to 5-Fluorouracil in Human

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 I(T cells to a constant concentration of FUra (1000 MMfor 4 h or 15 MMfor 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 addi tion, HCT-8/FU7dR cells were cross-resistant to fluorodeoxyuridine and, to a lesser degree, methotrexate; while HCT-8/FU4hR 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 MMleucovorin did not completely inhibit the recovery of thymidylate synthesis after FUra exposure. No differ ences were detected in the kinetic properties (A,,,for 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, concentration producing 50% inhi bition 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-methylenetetra hydrofolate 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 |'I !-<>|lIra (4 h exposure, 100 MM)showed that the incorporation of the fluoropyrimidine into RNA is significantly de creased in HCT-8/FU4hR cells as compared to parental HCT-8 cells. The mechanisms of resistance found in these cell lines indicate that the mechanism of cell kill by FUra differs depending on the dose 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. INTRODUCTION FUra4 has been used for 3 decades for the treatment of human solid tumors (1). Of particular note is its role in the treatment Received 9/6/91; accepted 1/27/92 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by grant 9000140PF70 from Consiglio Nazionale delle Ricerche (Progetto Biotecnologie), grant 1991 from AIRC, and grant CA 47179 from the USPHS. ! Fellow of the Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. Current address: Department of Medical Oncology, Istituto Nazionale per la Ricerca sul Cancro. V.le Benedetto XV, 1