Aschele C, Sobrero A, Faderan M A, Bertino J R
Department of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Cancer Res. 1992 Apr 1;52(7):1855-64.
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)
比较了对短期暴露（4小时）于5-氟尿嘧啶（FUra）产生抗性的细胞系（HCT-8/FU4hR）和对氟嘧啶长期暴露（7天）产生抗性的细胞系（HCT-8/FU7dR）之间对5-氟尿嘧啶的耐药机制。这两个细胞系是通过将2×10⁵个细胞反复暴露于恒定浓度的FUra（1000微摩尔/升，4小时；或15微摩尔/升，7天）而获得的，该浓度能够导致细胞死亡3至4个对数级。HCT-8/FU4hR细胞对7天暴露的FUra仍敏感，提示存在不同的耐药机制。此外，HCT-8/FU7dR细胞对氟脱氧尿苷交叉耐药，对甲氨蝶呤的交叉耐药程度较低；而HCT-8/FU4dR细胞则不然。HCT-8/FU4hR和HCT-8/FU7dR细胞在FUra摄取以及FUra代谢和FUra靶酶模式方面与亲代HCT-8细胞相似。尽管在HCT-8/FU7dR细胞中，原位胸苷酸合成酶（TS）活性及其被FUra抑制的程度均未显示出任何改变的迹象，但在该细胞系中，药物去除后TS活性迅速恢复。加入高达100微摩尔的亚叶酸钙并不能完全抑制FUra暴露后胸苷酸合成的恢复。与亲代HCT-8的TS相比，未检测到HCT-8/FU7dR细胞的TS在动力学特性（对2'-脱氧尿苷酸和5,10-亚甲基四氢叶酸的Km值，对氟脱氧尿苷酸产生50%抑制的浓度）方面存在差异。HCT-8/FU7dR细胞中5,10-亚甲基四氢叶酸的基线水平降低，对叶酸辅因子多聚谷氨酸化形式的链长分布分析表明，在该细胞系中，5,10-亚甲基四氢叶酸水平的缺陷伴随着该辅因子多聚谷氨酸化的缺陷，并且可能是由于后者所致。相比之下，HCT-8/FU4hR细胞在FUra对原位TS的抑制程度以及FUra去除后的抑制持续时间方面与亲代细胞系相似。用[³H-6]FUra（4小时暴露，100微摩尔/升）进行的标记研究表明，与亲代HCT-8细胞相比，氟嘧啶掺入HCT-8/FU4hR细胞RNA中的量显著减少。（摘要截断于400字）
