Thymidylate synthase inhibitory drugs induce p53-dependent pathways differently

Abstract

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, triggering futile base excision repair cycles, DNA strand breaks, and ultimately cell death. Interestingly, when the main uracil-DNA repair pathway is inhibited, treatment with TS-inhibitory drugs still leads to cell death.

Introduction

Thymidylate synthase (TS, abbreviations are listed in S1 Table), a key enzyme in thymidylate biosynthesis, forms the sole de novo source of dTMP, a dTTP precursor, by transferring a methyl group from 5,10-methylenetetrahydrofolate (MTHF) to dUMP. Cancer cells are actively dividing, thus targeting nucleotide biosynthesis is a common anticancer strategy, and inhibition of TS is widely used in chemotherapeutic treatment of solid tumors such as colon, breast, and head and neck cancers [1–3]. 

Materials and method

Cell culturing

HCT116, UGI expressing HCT116 (HCT116-UGI), and its MMR proficient version (HCT116-MMR-UGI) cells [12] were all maintained in McCoy’s 5A medium (Thermo Fisher Scientific (Gibco), 16600082) supplemented with 10% FBS (Sigma, F9665-500ML) and 1% PenStrep (Thermo Fisher Scientific (Gibco), 15140122). Mycoplasma contamination was regularly checked. 

Results

RTX and 5FdUR treatments influence cellular gene expression profiles differently
To characterize the differences in the cellular effects of the two TS inhibitory drugs, RTX and 5FdUR, we performed whole transcriptome sequencing of (i) the HCT116-UGI cell line, in which both UNG-initiated base excision repair and MMR are impaired; (ii) the MMR-proficient but UNG-inhibited HCT116-MMR-UGI cell line; and (iii) the parental MMR-deficient HCT116 cell line (data are available at GEO under accession GSE318306). 

Discussion

Thymidylate synthase inhibitory drugs are widely used in chemotherapeutic treatments; however, their exact mechanism of action is not yet fully understood. Here, we provide deeper insight into the cellular response at the RNA level initiated by treatment with the antifolate RTX or the base-analogue 5FdUR. 

Conclusion

We reported differences in cellular response at the RNA level between TS-inhibitory drugs, RTX, and 5FdUR, which are further influenced by the cellular repair status. Despite massive cell-line-specific effects, the 5FdUR-biased induction of p53-related pathways was demonstrated; however, p53 mRNA was not induced. In contrast, an increased p53 protein level was observed selectively after high-dose 5FdUR treatment, which could explain the stronger activation of p53-target genes and might contribute to the diverged cellular response previously characterized in this condition [13].

Acknowledgments

We acknowledged Gábor Tusnády for providing access to computational capacity. We are also grateful to Gergő Róna and András Füredi for providing primary p53 and p21 antibodies. We acknowledge the ENCODE Consortium (ENCODE Project Consortium, 2012) and the ENCODE production laboratory(s) generating the particular dataset(s), as well as the MiRBase, and the STRING databases supporting our research.

Citation: Holub E, Szajkó MB, Felföldi A, Vértessy BG, Békési A (2026) Thymidylate synthase inhibitory drugs induce p53-dependent pathways differently. PLoS One 21(7): e0332491. https://doi.org/10.1371/journal.pone.0332491

Editor: Divijendra Natha Reddy Sirigiri, BMSCE: BMS College of Engineering, INDIA

Received: August 31, 2025; Accepted: June 2, 2026; Published: July 1, 2026

Copyright: © 2026 Holub et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: RNA-seq and the TS-coupled RIP-seq data have been deposited to the GEO database under accession numbers GSE318306 and GSE307531, respectively.

Funding: Project no. 137867 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the OTKA_FK_21 funding scheme for A.B., who was also supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/726/22/8) and by the ÚNKP-22-5 New National Excellence Program of the Ministry of Culture and Innovation from the source of the National Research, Development and Innovation Fund. For E.H.: The scientific work and results published in this article were reached with the sponsorship of Gedeon Richter Talentum Foundation in the framework of the Gedeon Richter Excellence PhD Scholarship of Gedeon Richter Plc. E.H. was also supported by the EKÖP-24 University Excellence Scholarship Program (EKÖP-24-3-II-ELTE-596) of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund. M.B.Sz was supported by the Doctoral Excellence Fellowship Program (DCEP) (DKÖP-25-1-BME-52) that is funded by the National Research Development and Innovation Fund of the Ministry of Culture and Innovation and the Budapest University of Technology and Economics. B.G.V. was supported by the National Research, Development and Innovation Fund of Hungary grants K146890, K135231, 2018-1.2.1-NKP-2018-00005, 2022-1.2.2-TÉT-IPARI-UZ-2022-00003, and the TKP2021-EGA-02. There was no additional external funding received for this study.

Competing interests: The authors have declared that no competing interests exist.