Fluorodeoxyuridine Modulates Cellular Expression of the DNA Base Excision Repair Enzyme Uracil-DNA Glycosylase

Abstract

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G 1 and is degraded during the S to G 2 phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA-mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd.Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy. (Cancer Res 2006; 66(17): 8829-37)

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

“…Note that our studies do not rule out a role for BER initiated by other DNA glycosylases such as SMUG1, TDG, and MBD4 in response to genomic 5-FU incorporation [8]. It has been reported that the nuclear isoform of UNG (UNG2) is down-regulated following FdUrd treatment in certain human cell lines, which corresponded to resistance [28]. One possible explanation of the phenotypic differences seen between cells lacking UNG2 versus hUgi-expressing cells in which UNG2 is inhibited might lie in the reported interactions between UNG2 and replication-associated proteins [5,29].…”

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

“…Because previous studies found widely disparate effects of modulating the functions of various UDGs in different model systems (Cortellino et al, 2003;Sansom et al, 2003;Andersen et al, 2005;Fischer et al, 2006;An et al, 2007;Kunz et al, 2009;Grogan et al, 2011;Pettersen et al, 2011;Kemmerich et al, 2012;Nagaria et al, 2012), we performed a systematic analysis in multiple human cell lines derived from tumors that are treated with and respond to 5-FU or FdUrd. In these studies, by using multiple siRNAs targeted to each UDG, we showed that UNG, and only UNG, played a role in preventing cell death after exposure to FdUrd.…”

“…Results for a representative experiment that has been performed three times are shown. Pettersen et al, 2011;Kemmerich et al, 2012;Nagaria et al, 2012) or slightly increase resistance (Fischer et al, 2006) to 5-FU or FdUrd in human, mouse, and chicken models. Similarly, studies of TDG, MBD4, and SMUG1 have also revealed discrepant results.…”

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

“…The nuclear isoform of human uracil DNA glycosylase (hUNG2) was targeted for siRNA knockdown as previously described (63). The siRNA sense sequence was 5′-AUCGGCCAGAAGACGCUCUdTdT-3′ and was purchased from Dharmacon.…”