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 lines

Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of th ymidylate, 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 th e clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY) , and AG337 (Thymitaq, AG). Because the latter compounds have different tra nsport and polyglutamylation characteristics, we also related these paramet ers with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC(50)s varied from 0.8 to 43.0 mu M. Leuc ovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sen sitivity to folate-based TS inhibitors was variable; IC(50)s were in the ra nge of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 c ell lines had an LC50 for AG between 1.3 and 5.3 mu M. Two cell lines were resistant to AG, Colo201 and SW1116, with IC(50)s of 27 and 29 mu M, respec tively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 t o 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 IC(50)s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC(50)s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation w as determined with [H-3]methotrexate (MTX) as a reference compound, [H-3]MT X influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range fro m 47 to 429 pmol/10(6) cells/h, A total of 24 h of [H-3]MTX accumulation sh owed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels sh owed 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 in hibitors, including FPGS activity and TS enzyme kinetics.