Uracil Misincorporation Research Articles

Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.

Regulation of Folate-Mediated One-Carbon Metabolism by Glycine N-Methyltransferase (GNMT) and Methylenetetrahydrofolate Reductase (MTHFR).

Folate-mediated one-carbon metabolism is an important therapeutic target of human diseases. We extensively investigated how gene-nutrient interactions may modulate human cancer risk in 2 major folate metabolic genes, MTHFR and GNMT. The biochemical impacts of MTHFR and GNMT on methyl group supply, global DNA methylation, nucleotide biosynthesis, DNA damage, and partitioning of the folate dependent 1-carbon group were carefully studied. The distinct model systems used included: EB virus-transformed lymphoblasts expressing human MTHFR polymorphic genotypes; liver-derived GNMT-null cell-lines with and without GNMT overexpression; and HepG2 cells with stabilized inhibition of MTHFR using shRNA, GNMT wildtype, heterozygotous (GNMT(het)) and knockout (GNMT(nul)) mice. We discovered that the MTHFR TT genotype significantly reduces folate-dependent remethylation under folate restriction, but it assists purine synthesis when folate is adequate. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in human hematological malignancies. GNMT affects transmethylation kinetics and S-adenosylmethionine (adoMet) synthesis, and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes. Restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. The systematic series of studies gives new insights into the underlying mechanisms by which MTHFR and GNMT may participate in human tumor prevention.

Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

Micronutrient, Genome Stabili ty and Degenerative Diseases: Nutrigenomics Concept of Disease Prevention - An Overview

Diet is a key factor in determining genomic stability is more important than previously imagined because it impacts on all relevant pathways like exposure to dietary carcinogens, DNA repair, DNA synthesis, epigenetic damage and apoptosis. Recent research focuses into how a single micronutrient deficiency is leading to genomic instability and development of degenerative diseases in various stages of life.The study aimed at finding the nutrigenomic mechanism of how a marginal deficiency of any single micronutrient is interrupting in DNA repairing, methylation and synthesis by taking nutrient-nutrient and nutrient-gene interaction into consideration. It also focuses on how recommended dietary allowance is important in achieving DNA integrity and genome stability to prevent degenerative diseases.Exhaustive review of research papers in genome health nutrigenomicsis involved in this study to explore, assimilate and analyze data to understand the importance of micronutrient in maintaining methylation of CpG sequence and preventing DNA oxidation or uracil misincorporation in DNA to stop disease occurrence in individuals.The study finds a direct link between micronutrient deficiency and increased epigenomic damage, resulting into elevated risk for adverse health outcomes during various stages of life like infertility, tumor development and cancer. The overview study concludes with a vision for a paradigm shift in disease prevention strategy based on diagnosis and micro-nutritional intervention of genome or epigenome damage on an individual basis, i.e. personalized prevention of degenerative diseases in genome health clinic.

SHMT1 knockdown induces apoptosis in lung cancer cells by causing uracil misincorporation.

Reprogramming of cellular metabolism towards de novo serine production fuels the growth of cancer cells, providing essential precursors such as amino acids and nucleotides and controlling the antioxidant and methylation capacities of the cell. The enzyme serine hydroxymethyltransferase (SHMT) has a key role in this metabolic shift, and directs serine carbons to one-carbon units metabolism and thymidilate synthesis. While the mitochondrial isoform of SHMT (SHMT2) has recently been identified as an important player in the control of cell proliferation in several cancer types and as a hot target for anticancer therapies, the role of the cytoplasmic isoform (SHMT1) in cancerogenesis is currently less defined. In this paper we show that SHMT1 is overexpressed in tissue samples from lung cancer patients and lung cancer cell lines, suggesting that, in this widespread type of tumor, SHMT1 plays a relevant role. We show that SHMT1 knockdown in lung cancer cells leads to cell cycle arrest and, more importantly, to p53-dependent apoptosis. Our data demonstrate that the induction of apoptosis does not depend on serine or glycine starvation, but is because of the increased uracil accumulation during DNA replication.

Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG+/+ and UNG−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG−/− cells accumulated significantly greater uracil and DSBs compared with UNG+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG+/+ cells. Furthermore, DSBs in UNG+/+ and UNG−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG+/+ and UNG−/− cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG−/− but not UNG+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.

Abstract B89: Expression of DNA damage repair enzymes determine the efficacy of a novel dUTPase inhibitor, TAS-114.

Abstract Background: Deoxyuridine 5'-triphosphate nucleotidehydrolase (dUTPase) is a pyrophosphatase, which selectively catalyzes FdUTP and dUTP hydrolysis, and tightly restricts 5-fluorouracil (5-FU) and uracil misincorporation into DNA. TAS-114 is a novel potent inhibitor of dUTPase, and is under clinical development as a fluoropyrimidine enhancer. TAS-114 potentiates the antitumor activity of fluoropyrimidines through increasing misincorporation of 5-FU and uracil into DNA, however, precise mechanisms of cytotoxicity after misincorporation of the aberrant base is unknown. Here, we report relationships between the DNA damage repair function and the efficacy of TAS-114 on the anticancer activity of fluoropyrimidine. Materials and Methods: The antitumor activities of 5-FU or 5-FU/TAS-114 were evaluated in 9 human tumor xenograft models. Intratumor dUTPase protein was measured by Western blotting, and phosphorylation of histone H2AX was detected by immunohistochemistry. Tissue concentrations of FdUMP and dUMP were measured by HPLC and a thymidylate synthase (TS) binding assay, respectively. Suppression of DNA damage repair proteins were performed by RNA interference technology in HeLa cells, and then, the cells were used to assess an antiproliferative activity of FdUrd/TAS-114. Results: Oral administration of TAS-114 inhibited dUTPase and synergistically increased antitumor activity of 5-FU in various human tumor xenograft models. The magnitude of dUTPase inhibition was estimated by the change of dUTPase enzymatic products (FdUMP and dUMP) and it appeared to correlate with dUTPase expression and synergistic effect. These data suggest that 5-FU and uracil misincorporation is a primary mechanism of TAS-114 efficacy. In addition, TAS-114 combination with 5-FU increased phosphorylation of histone H2AX, suggesting a TAS-114 activated DNA damage response pathway. Suppression of DNA damage repair proteins involved in base excision repair (BER) and homologous recombination repair (HR) revealed that these DNA repair pathways were important in TAS-114’s synergistic increase of 5-FU cytotoxicity. Conclusion: Expression levels of DNA damage repair proteins in BER and HR affect TAS-114 sensitivity. DNA repair response appears to be a determinant of TAS-114 efficacy as well as dUTPase expression. These factors will be measured inTAS-114/fluoropyrimidines combination clinical trial. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B89. Citation Format: Sayaka Tsukioka, Wakako Yano, Tatsushi Yokogawa, Takeshi Wakasa, Akio Fujioka, Keisuke Yamamura, Satoko Itoh, Masayoshi Fukuoka, Kenichi Matsuo, Kazuharu Noguchi, Teruhiro Utsugi. Expression of DNA damage repair enzymes determine the efficacy of a novel dUTPase inhibitor, TAS-114. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B89.

Abstract B88: TAS-114, a dUTPase inhibitor, in combination with pemetrexed is a novel strategy for the treatment of NSCLC.

Abstract Background: Deoxyuridine 5'-triphosphate nucleotidehydrolase (dUTPase) is a gatekeeper protein, which prevents uracil misincorporation into DNA in cancer cells. dUTPase may protect cancer cells from aberrant base mediated cytotoxicity during thymidylate synthase (TS) inhibition, and its overexpression is associated with resistance to TS inhibitors. TAS-114 is a novel orally available dUTPase inhibitor and Phase I studies with TS-targeted drugs (S-1 and Capecitabine) are ongoing. Pemetrexed is an antifolate-based TS inhibitor and has been proven efficacious for the treatment of non-squamous non-small cell lung cancer (NSCLC). Here, we evaluated the therapeutic possibility of TAS-114 in combination with pemetrexed in NSCLC models. Materials and Methods: In vitro cell growth inhibition was determined by Crystal Violet, using 14 human lung cancer cell lines. The antitumor activity of TAS-114/pemetrexed was also evaluated in NCI-H2228 (human NSCLC), MX-1 (human breast cancer) and Bu25 TK- (human cervical cancer, thymidine kinase deficient) xenograft mouse models for preclinical proof of concept. MX-1 is a dUTPase highly expressed tumor, and Bu25 TK- is a pemetrexed-sensitve model, due to the lack of thymidine salvage pathway within the tumor cells. Intratumoral dUTPase inhibition was evaluated by measuring the dUTPase enzymatic product dUMP by HPLC. Results: TAS-114 significantly increased the cytotoxicity of pemetrexed in various human NSCLC cell lines in vitro. In an NCI-H2228 xenograft model, pemetrexed alone did not show antitumor activity, but oral administration of TAS-114 significantly enhanced the antitumor activity of pemetrexed. In addition, TAS-114 decreased intratumor dUMP accumulation mediated by pemetrexed-induced TS inhibition. Furthermore, strong antitumor activity of TAS-114/pemetrexed and pharmacodynamic modulation of pemetrexed after administration of TAS-114 were observed in the highly dUTPase expressed tumor MX-1. These results suggested that TAS-114 increased the antitumor activity of pemetrexed by dUTPase inhibition and subsequent uracil misincorporation into DNA. Finally, TAS-114 also enhanced the antitumor activity of pemetrexed in the pemetrexed-sensitive Bu25 TK- xenograft model. Conclusion: TAS-114/pemetrexed combination may represent a novel therapeutic strategy in NSCLC and other solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B88. Citation Format: Wakako Yano, Sayaka Tsukioka, Tatsushi Yokogawa, Satoko Ito, Keiji Ishida, Keisuke Yamamura, Akio Fujioka, Masayoshi Fukuoka, Kenichi Matsuo, Kazuharu Noguchi, Teruhiro Utsugi. TAS-114, a dUTPase inhibitor, in combination with pemetrexed is a novel strategy for the treatment of NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B88.

Abstract B90: First-in-human, phase I dose-escalation study of the pharmacokinetics and safety of single and multiple doses of a first-in-class enhancer of fluoropyrimidines, dUTPase inhibitor (TAS-114) in healthy male volunteers.

Abstract Background: Deoxyuridine triphosphatase (dUTPase) is a gatekeeper enzyme for uracil misincorporation and is considered to be an important factor for resistance to 5-Fluorouracil. TAS-114 is a first-in class oral dUTPase inhibitor, which acts as a modulator of the pyrimidine nucleotide metabolic pathway. Animal toxicology studies revealed no significant adverse events. This was a first-in-human study that investigated the pharmacokinetics (PK) and safety of single agent TAS-114 when given at single and multiple doses in healthy male volunteers. Materials and Methods: This was a single center, Phase 1, double-blind, placebo-controlled, randomized parallel study. For the single-dose cohort, subjects received a single dose of TAS-114 at 6, 18, 60, 150, and 300 mg in a fasting condition. The magnitude of dihydropyrimidine dehydrogenase (DPD) inhibition and the food effect on TAS-114 PK were also investigated. For the multiple-dose cohort, subjects received TAS-114 twice daily on days 1 to 14 consecutively, and then once on day 22 to investigate the auto-induction potential of CYP3A4 and its recovery during the rest period. Results: The study enrolled 25 subjects in the dose-escalating single-dose cohort and 10 subjects in the multiple-dose cohort. PK parameters in the single-dose cohort are shown in the Table. The disposition of TAS-114 followed linear kinetics. The elimination half-life was approximately 2 hours. There was no significant food effect on the TAS-114 AUC. A significant increase in uracil Cmax was observed at administered doses of 150 mg or higher of TAS-114, suggesting that significant inhibition of DPD occurred at these doses. No significant changes in TAS-114 exposure or in urinary 6β hydroxycortisol/cortisol ratio (parameter for the induction of CYP3A4) were observed after repeated doses. No notable safety concerns at these dose levels were reported. Conclusion: TAS-114 has shown both a favorable safety and pharmacokinetic profile after single and repeated doses. Global phase 1 programs of TAS-114 in combination with capecitabine or S-1 are underway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B90. Citation Format: Kaku Saito, Hirotaka Nagashima, Kazuharu Noguchi, Kunihiro Yoshisue, Tatsushi Yokogawa, Eiji Matsushima, Takeshi Tahara, Shigeru Takagi, Teruhiro Utsugi. First-in-human, phase I dose-escalation study of the pharmacokinetics and safety of single and multiple doses of a first-in-class enhancer of fluoropyrimidines, dUTPase inhibitor (TAS-114) in healthy male volunteers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B90.

A novel role of the tumor suppressor GNMT in cellular defense against DNA damage

Glycine N-methyltransferase (GNMT) is a folate binding protein commonly diminished in human hepatoma yet its role in tumor development remains to be established. GNMT binds to methylfolate but is also inhibited by it; how such interactions affect human carcinogenesis is unclear. We postulated that GNMT plays a role in folate-dependent methyl group homeostasis and helps maintain genome integrity by promoting nucleotide biosynthesis and DNA repair. To test the hypothesis, GNMT was over-expressed in GNMT-null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1-carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, as well as in Gnmt wildtype (Gnmt(+/+)), heterozygote (Gnmt(+/-)) and knockout (Gnmt(-/-)) mice under folate deplete, replete, or supplementation conditions. Our study demonstrated that GMMT 1) supports methylene-folate dependent pyrimidine synthesis; 2) supports formylfolate dependent purine syntheses; 3) minimizes uracil incorporation into DNA when cells and animals were exposed to folate depletion; 4) translocates into nuclei during prolonged folate depletion. In conclusion, loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. To our best knowledge, the role of GNMT in folate dependent 1-carbon transfer in nucleotide biosynthesis has never been investigated. The present study gives new insights into the underlying mechanism by which GNMT can participate in tumor prevention/suppression in humans.

Uracil–DNA Glycosylase Expression Determines Human Lung Cancer Cell Sensitivity to Pemetrexed

Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthase. The base excision repair (BER) enzyme uracil-DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG(-/-) human colon cancer cells. Here, we examined the relationship between UNG expression and pemetrexed sensitivity in human lung cancer. We observed a spectrum of UNG expression in human lung cancer cells. Higher levels of UNG are associated with pemetrexed resistance and are present in cell lines derived from pemetrexed-resistant histologic subtypes (small cell and squamous cell carcinoma). Acute pemetrexed exposure induces UNG protein and mRNA, consistent with upregulation of uracil-DNA repair machinery. Chronic exposure of H1299 adenocarcinoma cells to increasing pemetrexed concentrations established drug-resistant sublines. Significant induction of UNG protein confirmed upregulation of BER as a feature of acquired pemetrexed resistance. Cotreatment with the BER inhibitor methoxyamine overrides pemetrexed resistance in chronically exposed cells, underscoring the use of BER-directed therapeutics to offset acquired drug resistance. Expression of UNG-directed siRNA and shRNA enhanced sensitivity in A549 and H1975 cells, and in drug-resistant sublines, confirming that UNG upregulation is protective. In human lung cancer, UNG deficiency is associated with pemetrexed-induced retention of uracil in DNA that destabilizes DNA replication forks resulting in DNA double-strand breaks and cell death. Thus, in experimental models, UNG is a critical mediator of pemetrexed sensitivity that warrants evaluation to determine clinical value.

Reduced nerve growth factor levels in stress-related brain regions of folate-deficient mice

Folate deficiency has been linked to neurodegenerative and stress-related diseases such as stroke, dementia and depression. The role of the neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) in stress-related disorders and neurodegeneration has garnered increasing attention in recent years. Uracil misincorporation is involved in the neuropsychiatric dysfunction induced by experimental folate deprivation. However, the effects of folate deficiency on the expression of NGF and NT-3 in brain tissue have not yet been investigated. In a 2×2 design, aged mice lacking uracil-DNA N-glycosylase (Ung−/−) versus wild-type (Ung+/+) controls were subjected to a folate-deficient diet versus a regular diet for three months. Independent of genotype, folate deficiency led to decreased NGF protein levels in the frontal cortex and amygdala. In the hippocampus, NGF levels were increased in UNG−/− mice on the normal diet, but not under folate deficiency, while in UNG+/+ mice, folate deprivation did not affect hippocampal NGF content. NT-3 protein concentrations were neither affected by genotype nor by folate deficiency. Altogether, the results of our study show that folate deficiency affects NGF levels in the frontal cortex, amygdala and hippocampus. The decrease in NGF content in the hippocampus in response to folate deficiency in Ung−/− mice may contribute to their phenotype of enhanced anxiety and despair-like behavior as well as to selective hippocampal neurodegeneration.

Abstract 4484: Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed.

Abstract Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthetase (TS). The DNA base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG-/- human colon cancer cells (DLD1) and proposed that cytotoxic uracil accumulation leads to collapsed replication forks, double strand breaks and cell death. Here, we examined the relationship between cellular capability to remove uracil-DNA and pemetrexed sensitivity in human lung cancer cells. Viral siRNA and shRNA knockdown of UNG enhanced pemetrexed sensitivity in A549 and H1975 cells, respectively. We further evaluated induction of UNG as a mechanism of pemetrexed resistance. Exposure of H1299 cells to step-wise increasing concentrations of pemetrexed established drug resistant sublines: H1299PR-1 and H1299PR-2. Significant induction of UNG protein was observed in both sublines. Importantly, co-treatment with the BER inhibitor, methoxyamine (MX), overcame pemetrexed resistance in H1299PR-1/2 underscoring the utility of BER directed therapeutics to offset acquired pemetrexed resistance. Lastly, differential expression of UNG in cell lines and primary lung cancer tissues was associated with pemetrexed sensitivity. Cell lines derived from squamous and small cell carcinoma, known to be clinically resistant to pemetrexed, had the highest levels of UNG expression in a panel of eight lung cancer cell lines. Analysis of cDNA microarrays derived from primary human lung cancer tissues also indicated high UNG expression in pemetrexed-resistant histological subtypes. Thus, we propose that UNG is a major determinant of pemetrexed sensitivity in human lung cancer. Citation Format: Lachelle D. Weeks, Pingfu Fu, Lili Liu, Stanton L. Gerson. Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4484. doi:10.1158/1538-7445.AM2013-4484

Low Colonocyte Folate Is Associated with Uracil Misincorporation and Global DNA Hypomethylation in Human Colorectum

Low folate status is a risk factor for colon carcinogenesis; mechanisms proposed to account for this relationship include uracil misincorporation into DNA and global DNA hypomethylation. We investigated whether such biomarkers are related to folate status in isolated colonocytes from colonoscopy patients. In cases with adenomatous polyps (n = 40) or hyperplastic polyps (n = 16), colonocytes were isolated from biopsies from the polyp, from a site adjacent to the polyp, and from normal mucosa 10–15 cm distal to the polyp. In polyp-free controls (n = 53), biopsies were taken from ascending, transverse, and descending areas of colon. Within adenoma cases, there was a trend (P-trend < 0.001) of decreasing colonocyte folate (pg/105 cells, mean ± CI) from the site distal to the polyp (16.9 ± 2.4), to the site adjacent to the polyp (14.7 ± 2.3), to the polyp (12.8 ± 2.0). Correspondingly, there were increases in uracil misincorporation (P-trend < 0.001) and global DNA hypomethylation (P-trend = 0.012) across the 3 sites. Colonocyte folate concentrations were significantly correlated with RBC folate concentrations, but only in individuals with generally lower (≤484 μg/L) RBC folate status (r = 0.54; P = 0.006; n = 24), and were also significantly lower in normal mucosa of cases with adenomatous polyps than in controls matched for colonic segment. In conclusion, localized folate deficiency in specific areas of colon might create carcinogenic fields and affect the development of colorectal polyps through uracil misincorporation and DNA hypomethylation; alternatively, the polyp itself might deplete folate in the surrounding tissue. Folate supplementation trials aimed at colon cancer prevention should target individuals with suboptimal folate status.

Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation, double strand break formation and VSG switching in Trypanosoma brucei

Deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.

Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration

The pathogenesis of age-related macular degeneration (AMD) is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133) in the hOGG1 gene and the c.972G>C polymorphism (rs3219489) in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395) and c.–32A>G (rs3087404) polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2), XRCC1 and ERCC6 (CSB) have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis.

Inhibition of dUTPase Induces Synthetic Lethality with Thymidylate Synthase–Targeted Therapies in Non–Small Cell Lung Cancer

Chemotherapies that target thymidylate synthase (TS) continue to see considerable clinical expansion in non-small cell lung cancer (NSCLC). One drawback to TS-targeted therapies is drug resistance and subsequent treatment failure. Novel therapeutic and biomarker-driven strategies are urgently needed. The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is reported to protect tumor cells from aberrant misincorporation of uracil during TS inhibition. The goal of this study was to investigate the expression and significance of dUTPase in mediating response to TS-targeted agents in NSCLC. The expression of dUTPase in NSCLC cell lines and clinical specimens was measured by quantitative real-time reverse transcriptase PCR and immunohistochemistry. Using a validated RNA interference approach, dUTPase was effectively silenced in a panel of NSCLC cell lines and response to the fluoropyrimidine fluorodeoxyuridine (FUdR) and the antifolate pemetrexed was analyzed using growth inhibition and clonogenic assays. Apoptosis was analyzed by flow cytometry. Significant variation in the quantity and cellular expression of dUTPase was observed, including clear evidence of overexpression in NSCLC cell line models and tumor specimens at the mRNA and protein level. RNA interference-mediated silencing of dUTPase significantly sensitized NSCLC cells to growth inhibition induced by FUdR and pemetrexed. This sensitization was accompanied by a significant expansion of intracellular dUTP pools and significant decreases in NSCLC cell viability evaluated by clonogenicity and apoptotic analyses. Together, these results strongly suggest that uracil misincorporation is a potent determinant of cytotoxicity to TS inhibition in NSCLC and that inhibition of dUTPase is a mechanism-based therapeutic approach to significantly enhance the efficacy of TS-targeted chemotherapeutic agents.

Highlights of This Issue

Next generation sequencing and array CGH analysis of one patient's neuroendocrine prostate cancer revealed a homozygous deletion of the methylthioadenine phosphorylase (MTAP) gene. Concurrently, a patient-derived xenograft model was established. A novel two-drug therapy markedly decreased xenograft growth rate and increased cancer cell apoptosis, without toxicity to the mouse host. Moreover, decreased MTAP expression was strongly associated with poor outcome in prostate cancer. This study suggests a unique therapy for patients with MTAP- deficient cancers, with significant implications for personalized oncology, since MTAP is absent in 10 to 75 % of the most intractable kinds of human cancers.Non-small cell lung cancer (NSCLC) remains one of the most difficult to treat malignancies, and novel therapeutics and patient selection strategies are urgently needed. The uracil-metabolizing enzyme dUTPase is reported to protect tumor cells from aberrant uracil misincorporation into DNA during treatment with thymidylate synthase (TS)-targeted therapies. Wilson and colleagues report that dUTPase is significantly overexpressed in NSCLC cell lines and clinical specimens and show that the silencing of dUTPase expression dramatically sensitizes NSCLC cells to cytotoxicity induced by the TS inhibitors pemetrexed and fluorodeoxyuridine. Targeting dUTPase represents a novel mechanism-based therapeutic approach to significantly enhance the efficacy of TS-targeted chemotherapies.Antibodies that block ligand binding to the type-1 insulin-like growth factor receptor (IGF-1R) have limited clinical antitumor activity. Bid and colleagues examined the effect of the SCH717454 antibody on angiogenesis in vitro and in vivo. Antibody blockade of IGF-1R prevented VEGF-stimulated proliferation of vascular endothelial cells, providing the first direct evidence of the involvement of IGF-1R signaling in angiogenesis. Both in vitro and in a mouse model, IGF-2 circumvented the inhibitory activity of SCH717454. The data yield insight into the mechanism of action and resistance to receptor-targeted antibodies and for improved strategies for targeting the IGF-axis for cancer treatment.To achieve the full potential of single-agent therapies targeting specific signaling proteins, it is essential to explore why drug resistance occurs in the majority of treated patients over time. Chen and colleagues investigated the role of alternative signaling pathways in mediating resistance to lapatinib inhibition of HER2-amplified gastric cancer cells. The data showed that activation of MET receptor in MET-overexpressing cells can overcome HER2 inhibition by restoring common downstream signaling pathways more effectively than the activation of other receptor tyrosine kinases. Thus, this finding suggests MET activation as a potential mechanism of resistance to lapatinib inhibition.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.

Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1

Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.