PMID- 33795350 OWN - NLM STAT- MEDLINE DCOM- 20210810 LR - 20240425 IS - 1521-0111 (Electronic) IS - 0026-895X (Print) IS - 0026-895X (Linking) VI - 99 IP - 6 DP - 2021 Jun TI - Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil. PG - 412-425 LID - 10.1124/molpharm.120.000191 [doi] AB - 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(50) 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. CI - Copyright (c) 2021 by The American Society for Pharmacology and Experimental Therapeutics. FAU - Christenson, Eric S AU - Christenson ES AD - 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.). FAU - Gizzi, Anthony AU - Gizzi A AD - 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.). FAU - Cui, Junru AU - Cui J AD - 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.). FAU - Egleston, Matthew AU - Egleston M AD - 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.). FAU - Seamon, Kyle J AU - Seamon KJ AD - 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.). FAU - DePasquale, Michael AU - DePasquale M AD - 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.). FAU - Orris, Benjamin AU - Orris B AD - 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.). FAU - Park, Ben H AU - Park BH AD - 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.). FAU - Stivers, James T AU - Stivers JT AD - 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.) jstivers@jhmi.edu. LA - eng GR - F32 AI150561/AI/NIAID NIH HHS/United States GR - R01 GM056834/GM/NIGMS NIH HHS/United States GR - T32 CA009110/CA/NCI NIH HHS/United States GR - T32 GM007445/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20210401 PL - United States TA - Mol Pharmacol JT - Molecular pharmacology JID - 0035623 RN - 0 (Antineoplastic Agents) RN - 0 (Quinazolines) RN - 0 (Thiophenes) RN - 039LU44I5M (Floxuridine) RN - EC 3.2.2.- (Uracil-DNA Glycosidase) RN - FCB9EGG971 (raltitrexed) RN - U3P01618RT (Fluorouracil) SB - IM MH - Antineoplastic Agents/*pharmacology MH - Cell Line, Tumor MH - Colorectal Neoplasms/*pathology MH - DNA Damage MH - Drug Screening Assays, Antitumor MH - Floxuridine/*pharmacology MH - Fluorouracil/*pharmacology MH - Humans MH - Quinazolines/*pharmacology MH - Thiophenes/*pharmacology MH - Uracil-DNA Glycosidase/*antagonists & inhibitors/metabolism PMC - PMC11033954 COIS- The authors report no conflicts of interest. EDAT- 2021/04/03 06:00 MHDA- 2021/08/11 06:00 PMCR- 2021/06/01 CRDT- 2021/04/02 05:54 PHST- 2020/10/27 00:00 [received] PHST- 2021/03/09 00:00 [accepted] PHST- 2021/04/03 06:00 [pubmed] PHST- 2021/08/11 06:00 [medline] PHST- 2021/04/02 05:54 [entrez] PHST- 2021/06/01 00:00 [pmc-release] AID - molpharm.120.000191 [pii] AID - MOL_AR2020000191 [pii] AID - 10.1124/molpharm.120.000191 [doi] PST - ppublish SO - Mol Pharmacol. 2021 Jun;99(6):412-425. doi: 10.1124/molpharm.120.000191. Epub 2021 Apr 1.