Elsevier

European Journal of Cancer

Volume 39, Issue 3, February 2003, Pages 378-387
European Journal of Cancer

Expression of uracil DNA glycosylase (UDG) does not affect cellular sensitivity to thymidylate synthase (TS) inhibition

https://doi.org/10.1016/S0959-8049(02)00610-XGet rights and content

Abstract

Uracil DNA glycosylase (UDG) is a base excision repair enzyme responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Inhibition of thymidylate synthase (TS), an important target for cancer chemotherapy, leads to deoxythymidine triphosphate (dTTP) pool depletion and elevation of deoxyuridine monophosphate (dUMP) pools which may also result in the accumulation of deoxyuridine triphosphate (dUTP). Large quantities of dUTP are believed to overwhelm the pyrophosphatase dUTPase, leading to misincorporation of uracil into DNA. Uracil is removed from DNA by uracil DNA glycosylase (UDG) resulting in an abasic site, but since the ratio dUTP:dTTP may remain high during continuing TS inhibition uracil can become re-incorporated into DNA causing a futile cycle eventually leading to DNA damage and cell death. This study has used isogenic cell lines differing in their expression of UDG to investigate the role of this enzyme in sensitivity to the specific TS inhibitors, ZD9331 and raltitrexed. The study showed that although increased expression and activity of UDG may lead to increased cell growth inhibition after TS inhibition over the first 24 h of treatment (measured using 3-(4,5-dimethyl (thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), probably due to increased damage to single-stranded DNA, the level of enzyme expression does not affect cell viability or cell death (measured using clonogenic assay, cell counting of attached/detached cells and cleavage of both poly ADP-ribose polymerase (PARP) and caspase 3). Increased expression and activity of UDG did not affect sensitivity to TS inhibition at later time points (up to 72 h treatment). Therefore UDG does not appear to play a major role in the response to TS inhibition, at least in the model used, and the results suggest that other determinants of response previously investigated, such as TS and dUTPase, may be more important for the response to 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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    1

    Present address: Arizona Cancer Center, 1515 N. Campbell Ave., Tucson, Arizona, 85724, USA.

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