Base Excision by Thymine DNA Glycosylase Mediates DNA-Directed Cytotoxicity of 5-Fluorouracil

Abstract

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU–induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU–based chemotherapy.

Genomically Incorporated 5-Fluorouracil that Escapes UNG-Initiated Base Excision Repair Blocks DNA Replication and Activates Homologous Recombination

“…For example, a recent study demonstrated that UNG, but not the other UDGs, removes the vast majority of the uracil and 5-FU from the genomic DNA of cells (Pettersen et al, 2011), suggesting that this glycosylase might play a key role in the toxicity of 5-FU and FdUrd. Surprisingly, however, disrupting UNG did not affect (Andersen et al, 2005;An et al, 2007;Kunz et al, 2009;Grogan et al, 2011;Fig. 1.…”

“…In experimental systems using purified proteins and synthetic DNA substrates, both lesions are substrates for the known uracil DNA glycosylases (UDG): UNG1 (which is localized in the mitochondria); UNG2 (which is localized in the nucleus); and thymine DNA glycosylase (TDG), SMUG1, and MBD4 (Mauro et al, 1993;Petronzelli et al, 2000;Baker et al, 2002;Turner et al, 2004;An et al, 2007;Kunz et al, 2009;Pettersen et al, 2011), which excise the aberrant base, an event that initiates repair by the base excision repair (BER) pathway (Wyatt and Wilson, 2009;Kim and Wilson, 2012). In contrast, studies in a variety of cell lines and animal models have variably implicated individual UDGs in sensitivity to 5-FU and its metabolites.…”

TAS-102: A Novel Antimetabolite for the 21st Century

“…Cells deficient in the TDG and MBD4 glycosylases have been reported to be resistant to 5FU. Excision of DNA-incorporated 5FU by these glycosylases results in DNA-strand breaks and activation of DNA damage signaling cascades, which ultimately inhibits cell cycle progression [31]. However, siRNA-mediated knockdown of TDG or MBD4 had no effect on TFT-mediated cytotoxicity, suggesting that the inhibitory effects of TFT on DNA replication and repair enzymes differ from those of 5FU and FdUrd [27].…”

Functional analysis of a survivin-like gene in Bombyx mori

“…Bm-svv was then expressed in non-permissive or permissive cells to determine its effect on cell activity using the Cell Counting Kit-8 (CCK-8), which has been frequently employed in mammalian cells (Weldon et al 2009;Ding et al 2010;Kunz et al 2009;Tamura et al 2010), and found that this method is also useful for the determination of insect cell activity.…”