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Trifluorothymidine (TFT) is a thymidine analog which is part of the new oral formulation TAS-102, which also contains the potent thymidine phosphorylase (TP) inhibitor TPI. TFT is activated by thymidine kinase (TK) to TFT-MP which inhibits thymidylate synthase (TS). TS converts dUMP to dTMP and its inhibition leads to dTTP depletion in the cell causing dUTP to be misincorporated into DNA resulting in DNA damage. In addition, the triphosphate form of TFT can also be incorporated into DNA, resulting in DNA damage and possibly cell death. TFT is active against 5-fluorouracil (5FU)-resistant colon cancer cells. We studied the mechanism(s) of TFT resistance induced in the human colon cancer cell line H630. This was achieved by exposing cells continuously to TFT and stepwise increasing the concentration (final 20 μM) or by intermittent exposure to TFT (final 250 μM 4hr/week), resulting in the TFT resistant H630 variants H630cTFT and H6304TFT, respectively. Using the SRB cytotoxicity assay we determined that H6304TFT and H630cTFT were >200-fold resistant compared to H630 which was retained for at least 30 days when cultured in drug-free medium. Both cell lines were cross-resistant to 2’deoxy-5-fluorouridine (>170-fold), which is activated by TK, but not to 5FU or 5’deoxy-5-fluorouridine, which are activated by TP. Western blot results showed that TS protein levels remained unchanged in both TFT resistant cell lines, but TK protein levels were significantly decreased in H6304TFT. The cell cycle distribution in H630 cells changed significantly after exposure to [IC75]TFT or higher concentrations: S- and G2M-phase cell populations increased by 16.5 % and 19.9 % respectively (p<0.01). In the resistant cell lines the cell cycle distribution did not change after exposure to TFT concentrations up to [IC75]. Results of enzyme assays showed that total TK activity was significant lower in H6304TFT (>90%) but, surprisingly, total TK activity was increased in H630cTFT cells. There was no difference in TP activity between the TFT resistant cell lines and the parental cell line. Whole Genome Microarray data showed that TS and TP mRNA levels remained unchanged and that TK mRNA levels were decreased, although not significantly. Surprisingly, in H630cTFT we observed a strong increase in mRNA levels of phospholipase A2 (about 45-fold), which plays a role in phospholipid remodelling and prostaglandin synthesis, but also fas-mediated apoptosis, indicating that a strong disturbance in signal transduction was induced. Altogether we can conclude that a decrease in thymidine kinase expression (mRNA and protein levels and TK activity) can be a main mechanism for resistance to TFT and this was most pronounced for the H6304TFT cell line.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]