Role of thymidylate synthase in the antitumor activity of the multitargeted antifolate, LY231514

The cytotoxicity of LY231514 was only partially alleviated by thymidine add ition (5 mu M) in GC3 human colon carcinoma cells, and complete protection required the addition of both hypoxanthine (100 mu M) and thymidine. In con trast, the cytotoxic activity of tomudex (raltitrexed, ZD1694) was complete ly reversed by thymidine alone. MCF-7 human breast and H630 human colon car cinoma cells selected for resistance to tomudex and 5-fluorouracil, respect ively via thymidylate synthase (TS) amplification demonstrated only modest resistance to LY231514 compared to tomudex. LY231514-induced cytotoxicity i n these resistant cell lines was completely prevented by the addition of hy poxanthine (100 mu M), indicating inhibition of purine de novo biosynthesis as a secondary target for LY231514 action. Thymidine at physiologic levels in mouse plasma (approximately 1 mu M) produced only a 2.6-fold shift in t he IC50 for LY231514-mediated cytotoxicity in GC3/cl1 cells compared to a 1 28-fold shift for tomudex. LY231514 treatment (i.p., qd x 10) significantly delayed tumor growth in the GC3 carcinoma xenograft model. However, a thym idine kinase-deficient mutant of this same tumor line demonstrated heighten ed sensitivity to the in vivo antitumor activity of LY231514 with complete regression of established tumors and a large number of tumor-free survivors after one course of treatment. The data demonstrate that inhibition of thy midylate synthase is a prominent mechanism for antitumor activity by LY2325 14, but important secondary sites of action exist for this multitargeted mo lecule.