Differences in the induction of DNA damage, cell cycle arrest, and cell death by 5-fluorouracil and antifolates

Thymidylate synthase (TS) is an important target for chemotherapy and can b e inhibited by 5-fluorouracil (5-FU) and the antifolates, AG337 (Nolatrexed ) and multitargeted antifolate (MTA or Pemetrexed). In addition, 5-FU can b e incorporated into RNA and DNA, and MTA can inhibit two other enzymes. It is, however, unclear to what extent these differences in drug action will i nfluence activation of downstream mechanisms mediated via TS inhibition. Th erefore, two human colon cancer cell lines, WiDr and Lovo, with a different clonogenic origin, were treated with equitoxic concentrations of 5-FU, AG3 37, and MTA to determine the induction of DNA damage, cell cycle arrest, do wnstream protein expression, and cell death. At these concentrations, the s pecific TS inhibitor AG337 induced more DNA damage (up to 20%) than MTA and 5-FU. FACS analysis showed that all drugs induced S phase arrest in Lovo a nd WiDr that was most pronounced after 5-FU and AG337 exposure (50-70%). We stern blotting showed that p53 induction was not detectable in mutant (mt) p53 WiDr and increased much earlier in wild-type (wt) Lovo cells after 5-FU and MTA (24 h) than after AG337 exposure (72 h). In contrast to 5-FU-treat ed Lovo cells, the bcl-2/bax ratio decreased after antifolate exposure. Nev ertheless, both 5-FU and antifolates induced similar amounts of cell death (up to 60%). These results demonstrate that in human colon cancer cells dif ferences in downstream events between AG337 and 5-FU or MTA are related to the additional effects of 5-FU and MTA, which are not associated with TS in hibition.