Welsh S J, Hobbs S, Aherne G W
CRC Centre for Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Belmont, Surrey SM2 5NG, UK.
Eur J Cancer. 2003 Feb;39(3):378-87. doi: 10.1016/s0959-8049(02)00610-x.
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.
尿嘧啶DNA糖基化酶（UDG）是一种碱基切除修复酶，负责去除DNA中因胞嘧啶脱氨或复制过程中的错误掺入而出现的尿嘧啶。胸苷酸合成酶（TS）是癌症化疗的一个重要靶点，对其抑制会导致三磷酸脱氧胸苷（dTTP）池耗竭以及一磷酸脱氧尿苷（dUMP）池升高，这也可能导致三磷酸脱氧尿苷（dUTP）的积累。大量的dUTP被认为会使焦磷酸酶dUTPase不堪重负，导致尿嘧啶错误掺入DNA。尿嘧啶DNA糖基化酶（UDG）会将尿嘧啶从DNA中去除，从而产生一个无碱基位点，但由于在持续抑制TS的过程中dUTP与dTTP的比例可能仍然很高，尿嘧啶会重新掺入DNA，导致一个无效循环，最终导致DNA损伤和细胞死亡。本研究使用了UDG表达不同的同基因细胞系，以研究该酶在对特定TS抑制剂ZD9331和雷替曲塞的敏感性中的作用。研究表明，尽管在处理的前24小时内，TS抑制后UDG表达和活性的增加可能导致细胞生长抑制增加（使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）测量），这可能是由于对单链DNA的损伤增加，但酶的表达水平并不影响细胞活力或细胞死亡（使用克隆形成试验、贴壁/悬浮细胞计数以及聚ADP-核糖聚合酶（PARP）和半胱天冬酶3的裂解来测量）。在较晚时间点（长达72小时处理），UDG表达和活性的增加并不影响对TS抑制的敏感性。因此，至少在所使用的模型中，UDG似乎在对TS抑制的反应中不发挥主要作用，结果表明，先前研究的其他反应决定因素，如TS和dUTPase，可能对TS抑制反应更为重要。
