Expression of uracil DNA glycosylase (UDG) does not affect cellular sensitivity to thymidylate synthase (TS) inhibition
Introduction
Thymidylate synthase (TS) catalyses the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) to form 2′deoxythymidine-5′monophosphate (dTMP). TS is an important target for cancer chemotherapy as it provides the sole de novo source of thymidylate (dTMP) which is essential for DNA synthesis and repair [1]. TS is an important target for cancer chemotherapy.
The importance of TS as a chemotherapeutic target is now well established. 5-fluorouracil (5-FU), a fluoropyrimidine drug, is widely used in the treatment of breast, gastrointestinal, and head and neck cancers [1]. Several specific TS inhibitors have recently been clinically evaluated. These include the quinazoline antifolates, raltitrexed (Tomudex, ZD1694 [2] and ZD9331 3, 4).
However, the mechanisms of cell death following inhibition of TS are not clearly defined. Following TS inhibition, deoxythymidine triphosphate (dTTP) pools become depleted and dUMP pools increase which may also result in the accumulation of 2′-deoxyuridine-5′triphosphate (dUTP) 5, 6, 7. Since DNA polymerase recognises dUTP and dTTP with equal efficiency [8], uracil may become misincorporated into DNA during periods of TS inhibition if the pyrophospatase dUTPase is overwhelmed, when the ratio of dUTP:dTTP is high [6]. Uracil is not normally found in DNA due to the activity of dUTPase and the base-excision repair enzyme uracil DNA glycosylase (UDG) [9]. If incorporated into DNA, uracil is excised by UDG, but the resulting abasic site is likely to be refilled by uracil due to the continuing high dUTP:dTTP ratio. This may cause a futile cycle of misincorporation, excision, repair and further misincorporation resulting in DNA strand breaks and cell death (reviewed in Ref. [10]).
Studies in prokaryotic and eukaryotic systems have shown that uracil misincorporation may occur after inhibition of TS (reviewed in Ref. [10]). In addition, the extensive misincorporation of uracil into DNA is lethal in bacteria [11] and yeast [12] and likely all cellular systems. Although the reason for this is not completely known, it is thought that UDG-mediated DNA repair is central to the process 13, 14, 15, 16, 17, 18. Ultimately these events may inhibit daughter strand synthesis or result in the induction of extensive single strand breaks leading to hyper-recombination, DNA fragmentation and cell death 15, 16, 17.
Several studies, using isogenic cell line models, have shown that dUTPase plays an important role in the sensitivity to TS inhibition 19, 20, 21, 22 especially during the first 24 h of treatment 6, 22. However, relatively little attention has been paid to the role of UDG in determining response to TS inhibition. In addition, a recent report showed high variability in tumour UDG, elevated tumour: normal tissue ratio, and an association with proliferation for UDG in human colorectal cancer [23].
This study has used isogenic cell lines differing in their expression of UDG to assess the role of UDG in determining response to the specific TS inhibitors ZD9331 and raltitrexed (ZD1694).
Section snippets
Materials and methods
All standard laboratory chemicals used in this study were commercial products of AnalaR® grade purchased from either British Drug Houses (BDH) (Poole, Dorset, UK) or from Sigma (Poole, Dorset, UK). ZD9331 and raltitrexed (ZD1694) were synthesised at Zeneca Pharmaceuticals (Macclesfield, Cheshire, UK) and the procedures for their synthesis have been previously described in Refs. 24, 25, respectively. Both compounds were dissolved at 10 mM in 0.1 M NaHCO3 (pH 8.3). The dissolved compounds were
Characterisation of UDG-transfected cell lines
Human lung carcinoma HX147 cells possess low levels of UDG activity (approximately 6-fold less activity than human lung carcinoma A549 cells) [7]. Fig. 1 confirms this finding as HX147 cells showed significantly lower UDG activity than A549 cells (approximately 4-fold greater activity in A549 cells). This value is slightly lower than that previously reported, but this may reflect differences in the cell lines obtained from different sources and that a different substrate was used in each of the
Discussion
Uracil-DNA glycosylase (UDG) is the base-excision repair enzyme responsible for removing uracil from DNA [36]. TS inhibition has been shown to cause an elevation in dUTP:dTTP ratios which may lead to the misincorporation of uracil into DNA 15, 16, 18, 34, 37, 38. In addition to studies in yeast and bacteria showing dUTPase mutants are only lethal in the presence of UDG, several authors have suggested that both UDG and dUTPase may influence the sensitivity of mammalian cells to TS inhibition 7,
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Cited by (32)
Medicinal chemistry aspects of uracil containing dUTPase inhibitors targeting colorectal cancer
2024, Drug Discovery TodayInhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways
2020, Journal of Biological ChemistryCitation Excerpt :Moreover, ATR is involved in S- and G2-phase arrest by activating intra-S and G2/M checkpoint (33) and is necessary during the HR repair pathway (37). On the other hand, 5-FU is thought to be an inhibitor of the enzyme TS, which plays a role in nucleotide synthesis (38, 39). 5-FU induces unstable conformations in the DNA structure at the S phase, and where too many SSBs are present at stalled replication forks in 5-FU–treated cells, DSBs are induced (9, 10).
Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance
2015, Drug Resistance UpdatesCitation Excerpt :These results seem to be specific for PMX. In fact, other studies have reported that changes in UDG expression have little impact on cellular sensitivity to other TS-inhibitors (Luo et al., 2008; Welsh et al., 2003). In contrast, one study has reported diminished DNA replication rates and increased apoptosis in UNG-deficient cells even in the absence of drug treatment (Pulukuri et al., 2009).
SMUG1 but not UNG DNA glycosylase contributes to the cellular response to recovery from 5-fluorouracil induced replication stress
2013, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisCitation Excerpt :Among the four known mammalian UDGs capable of removing uracil and 5-FU from DNA, nuclear UNG has received the most attention because it is the dominant UDG activity in cells, is up-regulated in S-phase, and co-localizes with replication foci [3]. Yet, most studies that have directly tested the role of UNG have concluded that it does not contribute to differences in cellular sensitivity to any TS inhibitor [4–9]. Examination of the other three mammalian UDGs reveals a complex picture.
DNA damage and homologous recombination signaling induced by thymidylate deprivation
2008, Biochemical Pharmacology
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Present address: Arizona Cancer Center, 1515 N. Campbell Ave., Tucson, Arizona, 85724, USA.