Whole transcriptome sequencing of HCT116 cells with altered DNA repair capacities treated with RTX or 5FdUR

Thymidylate synthase (TS) is a key enzyme in thymidylate biosynthesis and an established target of chemotherapeutics such as 5-fluoro-2’-deoxyuridine (5FdUR) and raltitrexed (RTX). Inhibition of TS disrupts the dUTP:dTTP balance, leading to uracil misincorporation, futile base excision repair cycles, DNA strand breaks, and ultimately cell death. However, active U-DNA repair is apparently not required for the cytotoxic effect of these drugs, indicating that alternative pathways contribute to the observed S-phase arrest. To dissect these mechanisms, we investigated the transcriptomic effects of TS inhibition in HCT116 colon cancer cell lines with altered DNA repair capacities treated with 5-fluoro-2′-deoxyuridine (5FdUR) or raltitrexed (RTX). Both drugs induced similar DNA damage responses and S-phase arrest, yet displayed distinct transcriptional signatures. Our findings underscore the multifaceted impact of TS inhibition and reveal drug-specific differences in cellular responses.