Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland (E.S.C., A.G., J.C., M.E., K.J.S., B.O., J.T.S.); Lieber Institute for Brain Development, Baltimore, Maryland (M.D.); and Vanderbilt University Medical Center/Vanderbilt-Ingram Cancer Center, Nashville, Tennessee (B.H.P.).
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland (E.S.C., A.G., J.C., M.E., K.J.S., B.O., J.T.S.); Lieber Institute for Brain Development, Baltimore, Maryland (M.D.); and Vanderbilt University Medical Center/Vanderbilt-Ingram Cancer Center, Nashville, Tennessee (B.H.P.)
Mol Pharmacol. 2021 Jun;99(6):412-425. doi: 10.1124/molpharm.120.000191. Epub 2021 Apr 1.
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 IC 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. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.
先前的短发夹 RNA 敲低研究已经证实，人尿嘧啶 DNA 糖基化酶 (hUNG) 的耗竭使一些细胞系对 5-氟脱氧尿嘧啶 (FdU) 敏感。在这里，我们通过慢病毒转导将尿嘧啶 DNA 糖基化酶抑制剂蛋白转入一组受强力霉素诱导启动子控制的大panel 癌症细胞系，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。总共有 11 个结肠直肠系中的 6 个在 hUNG 抑制后显示出 6 至 70 倍的 FdU 效力增加（“有反应性”）。在用氟尿嘧啶 (FU) 进行的实验中没有观察到这种 hUNG 依赖性反应，表明 FU 在体外并不通过与 FdU 相同的 DNA 修复机制起作用。胸苷酸合成酶抑制剂雷替曲塞 (RTX) 的效力（其升高脱氧尿苷三磷酸水平）仅在 hUNG 抑制时增加<40%，这表明反应性与 FdU 在 DNA 中的掺入和持续存在有关，而不是与脱氧尿苷有关。由于 RTX，dT 补充完全挽救了由 RTX 引起的 FdU 中 U/A 损伤的毒性作用，但 dT 仅增加了 FdU 的 IC，FdU 形成 FU/A 和 FU/G 错配，这支持了 FU/A 和 FU/G 损伤在 FdU 毒性中的重要性。与先前的报告相反，细胞对 hUNG 抑制的反应性与 p53 状态或胸苷酸 DNA 糖基化酶表达无关。提出了一种模型，其中在 hUNG 活性缺失的情况下，FU/A 和 FU/G 碱基对的持续存在会引发有反应性和无反应性结肠直肠系中的凋亡 DNA 损伤反应。意义：嘧啶碱基 5-氟尿嘧啶是治疗晚期结直肠癌的主要化疗药物。在这里，这项研究表明，其脱氧核苷形式 5-氟脱氧尿嘧啶 (FdU) 通过一种独特的 DNA 掺入机制起作用，该机制在人尿嘧啶 DNA 糖基化酶的 DNA 修复酶的抑制作用下得到强烈增强。在所测试的超过 50%的结肠直肠细胞系中存在 hUNG 依赖性机制，这表明在小分子 hUNG 抑制剂存在的情况下，可能会使大量人类癌症对 FdU 敏感。
