Characterization of a thymidylate synthase (TS)-inducible cell line: A model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymi dylate, which is required for DNA synthesis and repair and which is an impo rtant target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antif olates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). T o study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with te tracycline-regulated expression of TS termed MTS-5. We have demonstrated th at inducible expression of TS increased the IC,, dose of the TS-targeted th erapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC50 dose for MTA was unobtainable when TS was overexpressed in these ce lls, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic ag ents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS upregu lation. Cell cycle analyses revealed that IC50 doses of 5-FU, TDX and MTA c aused an S-phase arrest in cells that did not overexpress TS, and this arre st was overcome when TS was up-regulated. Furthermore, the S-phase arrest w as accompanied by 2- to 4-fold increased expression of the cell cycle regul atory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key r ole in determining cellular sensitivity to TS-directed chemotherapeutic dru gs by modulating the degree of S-phase arrest caused by these agents. Moreo ver, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in c ells that overexpress TS.