Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition (“responsive”). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC₅₀ for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines.

中文翻译：

---

抑制人尿嘧啶 DNA 糖基化酶使大部分结直肠癌细胞对 5-氟脱氧尿苷和雷替曲塞敏感，但对氟尿嘧啶不敏感

以前的短发夹 RNA 敲低研究已经确定，人尿嘧啶 DNA 糖基化酶 (hUNG) 的消耗会使一些细胞系对 5-氟脱氧尿苷 (FdU) 敏感。在这里，我们通过将尿嘧啶 DNA 糖基化酶抑制剂蛋白慢病毒转导到在强力霉素诱导型启动子控制下的一大组癌细胞系中来选择性地抑制 hUNG 的催化活性。这种诱导抑制策略更好地评估了小分子靶向 hUNG 的治疗潜力。总共，11 条结肠直肠系中的 6 条在 hUNG 抑制（“响应”）后显示 FdU 效力增加 6 至 70 倍。用氟尿嘧啶 (FU) 未观察到这种 hUNG 依赖性反应，表明 FU 在体外不通过与 FdU 相同的 DNA 修复机制起作用。胸苷酸合酶抑制剂雷替曲塞 (RTX) 的效力，提高脱氧尿苷三磷酸水平，仅在 hUNG 抑制后逐渐增强（<40%），这表明反应性与 FdU 在 DNA 中的掺入和持久性有关，而不是与脱氧尿苷有关。FU/A 和 FU/G 病变在 FdU 毒性中的重要性得到以下观察结果的支持：补充 dT 完全挽救了 RTX 引起的 U/A 病变的毒性作用，但 dT 仅增加 IC_{FdU 为50}，形成 FU/A 和 FU/G 不匹配。与先前的报道相反，细胞对 hUNG 抑制的反应与 p53 状态或胸腺嘧啶 DNA 糖基化酶表达无关。提出了一个模型，其中在没有 hUNG 活性的情况下，FU/A 和 FU/G 碱基对的持续存在在响应性和非响应性结肠直肠系中引发凋亡 DNA 损伤反应。