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Thymidylate Synthase Level as the Main Predictive Parameter for Sensitivity to 5-Fluorouracil, but not for Folate-based Thymidylate Synthase Inhibitors, in 13 Nonselected Colon Cancer Cell Lines¹

Department of Medical Oncology, University Hospital Vrije Universiteit, 1007 MB Amsterdam, the Netherlands

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 µM. 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 µM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC₅₀s of 27 and 29 µM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10⁶ cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10⁶ 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/10⁶ cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10⁶ cells/h. A total of 24 h of [³H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10⁶ 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.

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