van Triest B, Pinedo H M, van Hensbergen Y, Smid K, Telleman F, Schoenmakers P S, van der Wilt C L, van Laar J A, Noordhuis P, Jansen G, Peters G J
Department of Medical Oncology, University Hospital Vrije Universiteit, The Netherlands.
Clin Cancer Res. 1999 Mar;5(3):643-54.
Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected human colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics, we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC50s varied from 0.8 to 43.0 microM. Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was variable; IC50s were in the range of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 cell lines had an IC50 for AG between 1.3 and 5.3 microM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC50s of 27 and 29 microM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10(6) cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10(6) cells. Regression analysis showed a significant relation between TS catalytic activity and IC50s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC50s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with [3H]methotrexate (MTX) as a reference compound. [3H]MTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10(6) cells/h. A total of 24 h of [3H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX. In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS activity and TS enzyme kinetics.
胸苷酸合成酶（TS）是胸苷酸从头合成中的关键酶，是氟嘧啶和基于叶酸的TS抑制剂的重要作用靶点。在一组13种未经筛选的人结肠癌细胞系中，我们评估了TS水平在对5-氟尿嘧啶（5FU）和最近已进入临床的四种基于叶酸的TS抑制剂敏感性中的作用：ZD1694（Tomudex、雷替曲塞、TDX）、GW1843U89（GW）、LY231514（LY）和AG337（Thymitaq、AG）。由于后几种化合物具有不同的转运和多聚谷氨酸化特性，我们还将这些参数与药物敏感性相关联，药物敏感性通过药物暴露72小时后的磺酰罗丹明B测定法来衡量。对于5FU，IC50值在0.8至43.0微摩尔之间。亚叶酸（LV）仅在13种细胞系中的4种中增强了5FU的活性。对基于叶酸的TS抑制剂的敏感性各不相同；IC50值范围为：TDX为5.3 - 59.0纳摩尔；LY为11.0 - 1570纳摩尔；GW为0.5 - 8.9纳摩尔。13种细胞系中的11种对AG的IC50在1.3至5.3微摩尔之间。两种细胞系对AG耐药，即Colo201和SW1116，其IC50分别为27和29微摩尔。TS催化活性（dUMP转化为dTMP）在62至777皮摩尔/小时/10⁶个细胞之间变化。FdUMP结合位点的数量在32至231飞摩尔/10⁶个细胞之间变化。回归分析显示TS催化活性与5FU和5FU/LV的IC50之间存在显著关系。FdUMP的Ki与AG和GW的IC50显示出显著的斯皮尔曼等级相关性。以[³H]甲氨蝶呤（MTX）作为参考化合物，确定了抗叶酸转运、积累和多聚谷氨酸化的作用。通过还原型叶酸载体的[³H]MTX流入量在18.6至150飞摩尔/10⁶个细胞/分钟之间变化。叶酸多聚谷氨酸合成酶（FPGS）活性范围为47至429皮摩尔/10⁶个细胞/小时。[³H]MTX积累24小时显示出20倍的变化，从1.2至21.8皮摩尔/10⁶个细胞。FPGS水平与对TDX的细胞毒性显示出斯皮尔曼等级正相关。总之，在一个异质性的未经筛选的人结肠癌细胞系组中，对5FU和5FU/LV敏感性的最佳预测指标是TS活性。多种敏感性决定因素对于基于叶酸的TS抑制剂很重要，包括FPGS活性和TS酶动力学。
