Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Parsels, Leslie A.; Parsels, Joshua D.; Wagner, Lois M.; Loney, Tania L.; Radany, Eric H.; Maybaum, Jonathan

doi:10.1007/s002800050829

Cited by 22 publications

(18 citation statements)

References 17 publications

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“…At a dose of 10 μM FdUrd, survival was 19% (±3.5, n=3) for the UNG proficient cells and 36% (±11.2, n=3), a less than 2-fold difference that was insignificant. It is known that elevated dUTPase expression can influence cell death caused by TS inhibitors [18][19][20][21]. The results in Figure 4 show that nuclear dUTPase protein levels are similar in the UNG proficient and UNG-inhibited cells.…”

Section: Resultssupporting

confidence: 55%

“…The formation of DNA double strand breaks has been reported following TS inhibition, although double strand breaks have not uniformly correlated with death in response to folatebased TS inhibitors [20,22]. We previously found that RTX induced sister chromatid exchanges at subtoxic doses, which suggested that homologous recombination was invoked during thymidylate deprivation [13].…”

Section: Resultsmentioning

confidence: 99%

“…The inviability of dUTPase deficient strains of E. coli and S. cerevisiae even in the absence of UDG activity suggest that excessive genomic uracil cannot be tolerated [31,32]. Studies that manipulated dUTPase levels during chemotherapy-induced thymidylate deprivation indicate that increased dUTPase activity can only delay death [18][19][20][21]. The time dependence implied that cytotoxicity did not ultimately depend on the DNA damage resulting from uracil incorporation [20,21].…”

Section: Discussionmentioning

confidence: 99%

“…Studies that manipulated dUTPase levels during chemotherapy-induced thymidylate deprivation indicate that increased dUTPase activity can only delay death [18][19][20][21]. The time dependence implied that cytotoxicity did not ultimately depend on the DNA damage resulting from uracil incorporation [20,21]. Furthermore, in some cell types death was seen in TTP-depleted cells despite little or no dUTP accumulation [33], while changes in dATP levels have also been implicated [34].…”

Section: Discussionmentioning

confidence: 99%

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

Luo¹,

Walla²,

Wyatt³

2008

DNA Repair

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Thymidylate synthase (TS) is an important target of several chemotherapeutic agents, including 5-FU and raltitrexed (Tomudex). During TS inhibition, TTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). Thus, BER initiated by uracil DNA glycosylase (UDG) activity has been hypothesized to influence the toxicity induced by TS inhibitors. In this study we created a human cell line expressing the Ugi protein inhibitor of UNG family of UDGs, which reduces cellular UDG activity by at least 45-fold. Genomic uracil incorporation was directly measured by mass spectrometry following treatment with TS inhibitors. Genomic uracil levels were increased over 4-fold following TS inhibition in the Ugiexpressing cells, but did not detectably increase in UNG proficient cells. Despite the difference in genomic uracil levels, there was no difference in toxicity between the UNG proficient and UNGinhibited cells to folate or nucleotide-based inhibitors of TS. Cell cycle analysis showed that UNG proficient and UNG-inhibited cells arrested in early S-phase and resumed replication progression during recovery from RTX treatment almost identically. The induction of γ-H2AX was measured following TS inhibition as a measure of whether uracil excision promoted DNA double strand break formation during S-phase arrest. Although γ-H2AX was detectable following TS inhibition, there was no difference between UNG proficient and UNG-inhibited cells. We therefore conclude that uracil excision initiated by UNG does not adequately explain the toxicity caused by TS inhibition in this model.

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Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Luo¹,

Walla²,

Wyatt³

2008

DNA Repair

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“…Acting as a competitive inhibitor, FdUTP could effectively tie up the enzyme, allowing dUTP pools to build up. Several studies have shown that dUTPase plays an important role in the sensitivity of a cell to thymidylate synthase inhibition (7)(8)(9)35). Expression of this enzyme has been shown to vary widely in tumor tissue.…”

Section: Discussionmentioning

confidence: 99%

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

Fischer¹,

Muller-Weeks²,

Caradonna³

2006

Cancer Research

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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)

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Antimetabolites

Houghton¹

Cancer Drug Discovery and Development

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Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Cited by 22 publications

References 17 publications

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

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

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

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