Abstract
The antimetabolite 5-Fluorouracil (5-FU) is used in the treatment of various forms of cancer and has a complex mode of action. Despite 6 decades in clinical application the contribution of 5-FdUTP and dUTP [(5-F)dUTP] and 5-FUTP misincorporation into DNA and RNA respectively, for 5-FU-induced toxicity is still under debate.This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect. We reveal that 5-FU treatment leads to extensive problems in DNA replication fork progression, causing accumulation of cells in S-phase, DNA damage and ultimately cell death. Interestingly, these effects can be reinforced by either depletion or inhibition of the deoxyuridine triphosphatase (dUTPase, also known as DUT), highlighting the importance of (5-F)dUTP accumulation for cytotoxicity.With this study, we not only extend the current understanding of the mechanism of action of 5-FU, but also contribute to the characterization of dUTPase inhibitors. We demonstrate that pharmacological inhibition of dUTPase is a promising approach that may improve the efficacy of 5-FU treatment in the clinic.
Highlights
Even after six decades, targeting thymidine synthesis is still one of the most successful strategies to treat cancer [1, 2]
This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect
We investigated the DNA replication defects induced by 5-FU treatment and the importance of 5-FdUTP accumulation for this effect
Summary
Even after six decades, targeting thymidine synthesis is still one of the most successful strategies to treat cancer [1, 2]. DTMP is the precursor for dTTP production, making this reaction essential for thymidine synthesis [3]. TS forms a homodimer, which contains both a substrate (dUMP), and a cofactor (5,10-CH2THF) binding pocket [4]. The 5-fluoro-substituted uracil analogs are metabolized to 5-FdUMP, which binds and thereby occupies the TS-substrate pocket [4]. Inhibition of TS leads to depletion of thymidine and accumulation of the substrate dUMP, which is phosphorylated to dUTP. The increased dUTP/dTTP and 5-FdUTP/ dTTP ratios promote uracil misincorporation into DNA by DNA-polymerases [6]. Besides DNAassociated toxicity, incorporation of the 5-FU metabolite 5-FUTP into RNA has been shown to contribute to cell death [11,12,13,14]. The metabolism and working mechanism of fluoropyrimidines are complex and the contribution of each of these components for toxicity is often debated
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