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The rising global incidence of uterine cancer is linked to the escalating prevalence of obesity. Obesity results in alterations in adipocytokines and IGFs, driving cancer progression via inflammation, increased cell proliferation, and apoptosis inhibition, although the precise mechanisms are still unclear. This study examined a set of six markers, namely, adiponectin, leptin, IL6, TNFα, IGF1, and IGF2 and compared them between fifty age-matched endometrial cancer patients (study group) and non-cancer patients with benign gynaecological conditions (control group). We also assessed the relationship of these markers with obesity and explored the correlation between these markers and various tumour characteristics. In the cancer population, these markers were also assessed 24 h and 6 months post-surgery. Remarkably, low adiponectin levels were associated with a 35.8% increase in endometrial cancer risk. Interestingly, compared to control subjects where IGF levels decreased after menopause, post-menopausal women in the study group showed elevated IGF1 and IGF2 levels, suggesting a potential influence of endometrial cancer on the IGF system, particularly after menopause. Lastly, it is noteworthy that a discernible inverse relationship trend was observed in the levels of adipocytokines and IGFs 6 months post-surgery. This indicates that treatment for endometrial cancer may have a differential impact on adipocytokines and IGFs, potentially holding clinical significance that merits further investigation.

Chronic inflammation has been closely linked to the development and progression of various cancers. The epithelial-mesenchymal transition (EMT) is a process involving the acquisition of mesenchymal features by carcinoma cells and is an important link between inflammation and cancer development. Inflammatory mediators in the tumour micro-environment, such as cytokines and chemokines, can promote EMT changes in cancer cells. The aim of this systematic review is to analyse the effect of cytokines on EMT in gynaecological cancers and discuss their possible therapeutic implications. A search of the databases CINAHL, Cochrane, Embase, Medline, PubMed, TRIP, and Web of Science was performed using the keywords: "cytokines" AND "epithelial mesenchymal transition OR transformation" AND "gynaecological cancer". Seventy-one articles reported that various cytokines, such as TGF-β, TNF-α, IL-6, etc., promoted EMT changes in ovarian, cervical, and endometrial cancers. The EMT changes included from epithelial to mesenchymal morphological change, downregulation of the epithelial markers E-cadherin/β-catenin, upregulation of the mesenchymal markers N-cadherin/vimentin/fibronectin, and upregulation of the EMT-transformation factors (EMT-TF) SNAI1/SNAI2/TWIST / ZEB . Cytokine-induced EMT can lead to gynaecological cancer development and metastasis and hence novel therapies targeting the cytokines or their EMT signalling pathways could possibly prevent cancer progression, reduce cancer recurrence, and prevent drug-resistance.

Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation affects global cellular stress responses is sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver, using mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate how AAG affects alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cells expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knockdown compromised UPR induction and led to a defect in XBP1 activation. To verify the requirements for the DNA repair activity of AAG in this response, AAG knockdown cells were complemented with wild-type Aag or with an Aag variant producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, besides its enzymatic activity, AAG has noncanonical functions in alkylation-induced UPR that contribute to cellular responses to alkylation., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The objective of the study was to document the effect of adipocytokines on endometrial cancer progression. A search of the databases CINAHL, Medline, PubMed, Cochrane, Web of Science, Embase and Google Scholar was performed for English language articles from January 2000 to December 2020 using the keywords: (Endometrial cancer) AND (progression OR metastasis) AND (adipocytokine OR adiponectin OR leptin OR visfatin OR IL-6 OR TNF-α OR adipokine OR cytokine). Forty-nine studies on adipocytokines have been included in this review. Adiponectin has been linked with anti-proliferative and anti-metastatic effects on endometrial cancer cells and is associated with a better prognosis. Leptin, visfatin and resistin are linked to the stimulation of endometrial cancer growth, proliferation, invasion and metastasis and are associated with worse prognosis or with a higher grade/stage of endometrial cancer. IL-6, Il-11, IL-31, IL-33, TNF-α, TGF-β1, SDF-1 and CXCR are involved in endometrial cancer cell growth and metastasis or involved in epithelial mesenchymal transformation (EMT) or associated with advanced disease. Adipocytokines have been found to directly impact endometrial cancer cell proliferation, invasion and migration. These molecules and their signalling pathways may be used to determine prognosis and course of the disease and may also be exploited as potential targets for cancer treatment and prevention of progression., (© 2021. The Author(s).)

Selenium is an essential trace element important for human health. A balanced intake is, however, crucial to maximize the health benefits of selenium. At physiological concentrations, selenium mediates antioxidant, anti-inflammatory, and pro-survival actions. However, supra-nutritional selenium intake was associated with increased diabetes risk leading potentially to endothelial dysfunction, the initiating step in atherosclerosis. High selenium causes apoptosis in cancer cells via endoplasmic reticulum (ER) stress, a mechanism also implicated in endothelial dysfunction. Nonetheless, whether ER stress drives selenium-induced endothelial dysfunction, remains unknown. Here, we investigated the effects of increasing concentrations of selenium on endothelial cells. High selenite reduced nitric oxide bioavailability and impaired angiogenesis. High selenite also induced ER stress, increased reactive oxygen species (ROS) production, and apoptosis. Pretreatment with the chemical chaperone, 4-phenylbutyrate, prevented the toxic effects of selenium. Our findings support a model where high selenite leads to endothelial dysfunction through activation of ER stress and increased ROS production. These results highlight the importance of tailoring selenium supplementation to achieve maximal health benefits and suggest that prophylactic use of selenium supplements as antioxidants may entail risk., (© 2020 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020. Published by Elsevier Ltd.)

DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD + -consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear. To address this question, we temporally profiled repair and metabolism in wild-type and Aag -/- cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although Aag -/- cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation is undetectable in AAG-deficient cells. Accordingly, Aag -/- cells are protected from MMS-induced NAD + depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAG-independent. Furthermore, treatment with FK866, a selective inhibitor of the NAD + salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity and Aag -/- cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an important role in the metabolic response to alkylation that could be exploited in the treatment of conditions associated with NAD + dysregulation.

Objective: The aim of this study was to examine DNA ligase activity and expression of DNA damage response pathway (DDR) genes in patients with stable angina (SA) and non-ST elevation myocardial infarction (NSTEMI) and determine whether they correlate with plaque morphology., Background: Patients with coronary artery disease (CAD) have evidence of deoxyribonucleic acid (DNA) damage in peripheral blood mononuclear cells (PBMCs). It is unclear whether this represents excess damage or defective DNA repair activity., Methods: DNA ligase activity and the expression of 22 DDR genes were measured in PBMCs of patients (both SA (n = 47) and NSTEMI (n = 42)) and in age and gender-matched controls (n = 35). Target lesion anatomical assessment was undertaken with frequency domain optical coherent tomography., Results: DNA ligase activity was different across the three groups of patients (control = 119 ± 53, NSTEMI = 115.6 ± 85.1, SA = 81 ± 55.7 units/g of nuclear protein; ANOVA p = 0.023). Pair wise comparison demonstrated that this significance is due to differences between the control and SA patients (p = 0.046). Genes involved in double strand break repair and nucleotide excision repair pathways were differentially expressed in patients with SA and NSTEMI. In SA patients, fibrocalcific plaques were strongly associated with GTSE1, DDB1, MLH3 and ERCC1 expression. By contrast, in NSTEMI patients the strongest association was observed between fibrous plaques and ATM and XPA expression., Conclusion: PBMCs from patients with CAD exhibit differences in DNA ligase activity and expression of DDR genes. Expression levels of certain DDR genes are strongly associated with plaque morphology and may play a role in plaque development and progression. Trial Registration Number URL: www.Clinicaltrials.gov; NCT02335086., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients' clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG , OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG , but not XRCC1 , sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC., Competing Interests: CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest.

Breast cancer is the commonest form of cancer in women, but successful treatment is confounded by the heterogeneous nature of breast tumours: Effective treatments exist for hormone-sensitive tumours, but triple-negative breast cancer results in poor survival. An area of increasing interest is metabolic reprogramming, whereby drug-induced alterations in the metabolic landscape of a tumour slow tumour growth and/or increase sensitivity to existing therapeutics. Nuclear receptors are transcription factors central to the expression of metabolic and transport proteins, and thus represent potential targets for metabolic reprogramming. We show that activation of the nuclear receptor FXR, either by its endogenous ligand CDCA or the synthetic GW4064, leads to cell death in four breast cancer cell lines with distinct phenotypes: MCF-10A (normal), MCF-7 (receptor positive), MDA-MB-231 and MDA-MB-468 (triple negative). Furthermore, we show that the mechanism of cell death is predominantly through the intrinsic apoptotic pathway. Finally, we demonstrate that FXR agonists do not stimulate migration in breast cancer cell lines, an important potential adverse effect. Together, our data support the continued examination of FXR agonists as a novel class of therapeutics for the treatment of breast cancer., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O(6)-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm(-/-) mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Lysosomes play important roles in multiple aspects of physiology, but the problem of how the transcription of lysosomal genes is coordinated remains incompletely understood. The goal of this study was to illuminate the physiological contexts in which lysosomal genes are coordinately regulated and to identify transcription factors involved in this control., Results: As transcription factors and their target genes are often co-regulated, we performed meta-analyses of array-based expression data to identify regulators whose mRNA profiles are highly correlated with those of a core set of lysosomal genes. Among the ~50 transcription factors that rank highest by this measure, 65% are involved in differentiation or development, and 22% have been implicated in interferon signaling. The most strongly correlated candidate was Stat6, a factor commonly activated by interleukin-4 (IL-4) or IL-13. Publicly available chromatin immunoprecipitation (ChIP) data from alternatively activated mouse macrophages show that lysosomal genes are overrepresented among Stat6-bound targets. Quantification of RNA from wild-type and Stat6-deficient cells indicates that Stat6 promotes the expression of over 100 lysosomal genes, including hydrolases, subunits of the vacuolar H⁺ ATPase and trafficking factors. While IL-4 inhibits and activates different sets of lysosomal genes, Stat6 mediates only the activating effects of IL-4, by promoting increased expression and by neutralizing undefined inhibitory signals induced by IL-4., Conclusions: The current data establish Stat6 as a broadly acting regulator of lysosomal gene expression in mouse macrophages. Other regulators whose expression correlates with lysosomal genes suggest that lysosome function is frequently re-programmed during differentiation, development and interferon signaling.

Many alkylating agents are used as chemotherapeutic drugs and have a long history of clinical application. These agents inflict a wide range of DNA damage resulting in a complex cellular response. After DNA damage, cells trigger a series of signaling cascades promoting cellular survival and cell cycle blockage which enables time for DNA repair to occur. More recently, induction of autophagy has been observed in cancer cells after treatment with different DNA-targeted anticancer drugs, including alkylating agents. Several studies have demonstrated that induction of autophagy after DNA damage delays apoptotic cell death and may therefore lead to chemoresistance, which is the limiting factor for successful chemotherapy. On the other hand, depending on the extent of damage and the cellular context, the induction of autophagy may also contribute to cell death. Given these conflicting results, many studies have been conducted to better define the role of autophagy in cancer cells in response to chemotherapy. In this review, we describe the main alkylating agents used in clinical oncology as well as the cellular response they evoke with emphasis on autophagy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Scope: Resveratrol is a plant-derived polyphenol with chemotherapeutic properties in animal cancer models and many biochemical effects in vitro. Its bioavailability is low and raises the possibility that the metabolites of resveratrol have biological effects. Here we investigate the actions of resveratrol 3-O-D-glucuronide, resveratrol 4-O-D-glucuronide, and resveratrol 3-O-Dsulfate on the growth of colon cancer cells in vitro., Methods and Results: The growth of Caco-2, HCT-116, and CCL-228 cells was measured using the neutral red and MTT assays. Resveratrol and each metabolite inhibited cell growth with IC50 values of 9.8–31 μM. Resveratrol caused S phase arrest in all three cell lines. Resveratrol 3-O-D-glucuronide and resveratrol 4-O-D-glucuronide caused G1 arrest in CCL-228 and Caco-2 cells. Resveratrol 3-O-D-sulfate had no effect on cell cycle. Growth inhibition was reversed by an inhibitor of AMP-activated protein kinase (compound C) or an adenosine A3 receptor antagonist (MRS1191). The A3 receptor agonist 2Cl-IB-MECA inhibited growth and A3 receptors were detected in all cell lines. The resveratrol glucuronides also reduced cyclin D1 levels but at higher concentrations than in growth experiments and generally did not increase phosphorylated AMP-activated protein kinase., Conclusion: Resveratrol glucuronides inhibit cell growth by G1 arrest and cyclin D1 depletion, and our results strongly suggest a role for A3 adenosine receptors in this inhibition., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage., Competing Interests: The authors have declared that no conflict of interest exists.

Background: The cytotoxicity of radiotherapy and chemotherapy can be enhanced by modulating DNA repair. PARP is a family of enzymes required for an efficient base-excision repair of DNA single-strand breaks and inhibition of PARP can prevent the repair of these lesions. The current study investigates the trimodal combination of ABT-888, a potent inhibitor of PARP1-2, ionizing radiation and temozolomide(TMZ)-based chemotherapy in glioblastoma (GBM) cells., Methods: Four human GBM cell lines were treated for 5 h with 5 μM ABT-888 before being exposed to X-rays concurrently with TMZ at doses of 5 or 10 μM for 2 h. ABT-888's PARP inhibition was measured using immunodetection of poly(ADP-ribose) (pADPr). Cell survival and the different cell death pathways were examined via clonogenic assay and morphological characterization of the cell and cell nucleus., Results: Combining ABT-888 with radiation yielded enhanced cell killing in all four cell lines, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER50) ranging between 1.12 and 1.37. Radio- and chemo-sensitization was further enhanced when ABT-888 was combined with both X-rays and TMZ in the O6-methylguanine-DNA-methyltransferase (MGMT)-methylated cell lines with a SER50 up to 1.44. This effect was also measured in one of the MGMT-unmethylated cell lines with a SER50 value of 1.30. Apoptosis induction by ABT-888, TMZ and X-rays was also considered and the effect of ABT-888 on the number of apoptotic cells was noticeable at later time points. In addition, this work showed that ABT-888 mediated sensitization is replication dependent, thus demonstrating that this effect might be more pronounced in tumour cells in which endogenous replication lesions are present in a larger proportion than in normal cells., Conclusions: This study suggests that ABT-888 has the clinical potential to enhance the current standard treatment for GBM, in combination with conventional chemo-radiotherapy. Interestingly, our results suggest that the use of PARP inhibitors might be clinically significant in those patients whose tumour is MGMT-unmethylated and currently derive less benefit from TMZ.

More than 15% of cancer deaths worldwide are associated with underlying infections or inflammatory conditions, therefore understanding how inflammation contributes to cancer etiology is important for both cancer prevention and treatment. Inflamed tissues are known to harbor elevated etheno-base (ε-base) DNA lesions induced by the lipid peroxidation that is stimulated by reactive oxygen and nitrogen species (RONS) released from activated neutrophils and macrophages. Inflammation contributes to carcinogenesis in part via RONS-induced cytotoxic and mutagenic DNA lesions, including ε-base lesions. The mouse alkyl adenine DNA glycosylase (AAG, also known as MPG) recognizes such base lesions, thus protecting against inflammation-associated colon cancer. Two other DNA repair enzymes are known to repair ε-base lesions, namely ALKBH2 and ALKBH3; thus, we sought to determine whether these DNA dioxygenase enzymes could protect against chronic inflammation-mediated colon carcinogenesis. Using established chemically induced colitis and colon cancer models in mice, we show here that ALKBH2 and ALKBH3 provide cancer protection similar to that of the DNA glycosylase AAG. Moreover, Alkbh2 and Alkbh3 each display apparent epistasis with Aag. Surprisingly, deficiency in all 3 DNA repair enzymes confers a massively synergistic phenotype, such that animals lacking all 3 DNA repair enzymes cannot survive even a single bout of chemically induced colitis.

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O⁶-methyldeoxyguanosine lesions, O⁶-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O⁶-mG DNA methyltransferase (MGMT) showed elevated O⁶-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer's disease.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-kappaB, and the major 5'-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 (Apex(+/-)) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-kappaB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex(+/-) mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex(+/-) mice exposed to 2-NP displayed a significant decline in 3'-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER.

O(6)-methylguanine DNA methyltransferase (MGMT) suppresses mutations and cell death that result from alkylation damage. MGMT expression is lost by epigenetic silencing in a variety of human cancers including nearly half of sporadic colorectal cancers, suggesting that this loss maybe causal. Using mice with a targeted disruption of the Mgmt gene, we tested whether Mgmt protects against azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF), against AOM and dextran sulfate sodium (DSS)-induced colorectal adenomas and against spontaneous intestinal adenomas in Apc(Min) mice. We also examined the genetic interaction of the Mgmt null gene with a DNA mismatch repair null gene, namely Msh6. Both Mgmt and Msh6 independently suppress AOM-induced ACF, and combination of the two mutant alleles had a multiplicative effect. This synergism can be explained entirely by the suppression of alkylation-induced apoptosis when Msh6 is absent. In addition, following AOM+DSS treatment Mgmt protected against adenoma formation to the same degree as it protected against AOM-induced ACF formation. Finally, Mgmt deficiency did not affect spontaneous intestinal adenoma development in Apc(Min/+) mice, suggesting that Mgmt suppresses intestinal cancer associated with exogenous alkylating agents, and that endogenous alkylation does not contribute to the rapid tumor development seen in Apc(Min/+) mice.

Vision loss affects >3 million Americans and many more people worldwide. Although predisposing genes have been identified their link to known environmental factors is unclear. In wild-type animals DNA alkylating agents induce photoreceptor apoptosis and severe retinal degeneration. Alkylation-induced retinal degeneration is totally suppressed in the absence of the DNA repair protein alkyladenine DNA glycosylase (Aag) in both differentiating and postmitotic retinas. Moreover, transgenic expression of Aag activity restores the alkylation sensitivity of photoreceptors in Aag null animals. Aag heterozygotes display an intermediate level of retinal degeneration, demonstrating haploinsufficiency and underscoring that Aag expression confers a dominant retinal degeneration phenotype.

Alkylation-induced O(6)-methylguanine (O(6)MeG) DNA lesions can be mutagenic or cytotoxic if unrepaired by the O(6)MeG-DNA methyltransferase (Mgmt) protein. O(6)MeG pairs with T during DNA replication, and if the O(6)MeG:T mismatch persists, a G:C to A:T transition mutation is fixed at the next replication cycle. O(6)MeG:T mismatch detection by MutSalpha and MutLalpha leads to apoptotic cell death, but the mechanism by which this occurs has been elusive. To explore how mismatch repair mediates O(6)MeG-dependent apoptosis, we used an Mgmt-null mouse model combined with either the Msh6-null mutant (defective in mismatch recognition) or the Exo1-null mutant (impaired in the excision step of mismatch repair). Mouse embryonic fibroblasts and bone marrow cells derived from Mgmt-null mice were much more alkylation-sensitive than wild type, as expected. However, ablation of either Msh6 or Exo1 function rendered these Mgmt-null cells just as resistant to alkylation-induced cytotoxicity as wild-type cells. Rapidly proliferating tissues in Mgmt-null mice (bone marrow, thymus, and spleen) are extremely sensitive to apoptosis induced by O(6)MeG-producing agents. Here, we show that ablation of either Msh6 or Exo1 function in the Mgmt-null mouse renders these rapidly proliferating tissues alkylation-resistant. However, whereas the Msh6 defect confers total alkylation resistance, the Exo1 defect leads to a variable tissue-specific alkylation resistance phenotype. Our results indicate that Exo1 plays an important role in the induction of apoptosis by unrepaired O(6)MeGs.

DNA interstrand cross-links (ICLs), widely used in chemotherapy, are cytotoxic lesions because they block replication and transcription. Repair of ICLs involves proteins from different repair pathways however the precise mechanism is still not completely understood. Here, we report that the 3-methyladenine DNA glycosylase (Aag), an enzyme that initiates base excision repair at a variety of alkylated bases, is also involved in the repair of ICLs. Aag(-/-) mouse embryonic stem cells were shown to be more sensitive to the cross-linking agent 4,5',8-trimethylpsoralen than wild-type cells, but no more sensitive than wild-type to the psoralen derivative Angelicin that forms only monoadducts. We show that gamma-H2AX foci formation, a marker for double strand breaks that are formed during ICL repair, is impaired in psoralen treated Aag(-/-) cells in both quantity and kinetics. However, in our in vitro system, purified human AAG can neither bind to the ICL nor cleave it. Taken together, our results suggest that Aag is important for the resistance of mouse ES cells to psoralen-induced ICLs.

Chronic inflammation increases cancer risk. While it is clear that cell signaling elicited by inflammatory cytokines promotes tumor development, the impact of DNA damage production resulting from inflammation-associated reactive oxygen and nitrogen species (RONS) on tumor development has not been directly tested. RONS induce DNA damage that can be recognized by alkyladenine DNA glycosylase (Aag) to initiate base excision repair. Using a mouse model of episodic inflammatory bowel disease by repeated administration of dextran sulfate sodium in the drinking water, we show that Aag-mediated DNA repair prevents colonic epithelial damage and reduces the severity of dextran sulfate sodium-induced colon tumorigenesis. Importantly, DNA base lesions expected to be induced by RONS and recognized by Aag accumulated to higher levels in Aag-deficient animals following stimulation of colonic inflammation. Finally, as a test of the generality of this effect we show that Aag-deficient animals display more severe gastric lesions that are precursors of gastric cancer after chronic infection with Helicobacter pylori. These data demonstrate that the repair of DNA lesions formed by RONS during chronic inflammation is important for protection against colon carcinogenesis.

Endogenous formation of the mutagenic DNA adduct 1,N(6)-ethenoadenine (epsilon A) originates from lipid peroxidation. Elevated levels of epsilon A in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for epsilon A lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair epsilon A lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2(-/-) mice indicates that mABH2 is the principal dioxygenase for epsilon A repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of epsilon A lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of epsilon A, comprise the cellular defense against epsilon A lesions.

DNA glycosylases initiate base excision repair by first binding, then excising aberrant DNA bases. Saccharomyces cerevisiae encodes a 3-methyladenine (3MeA) DNA glycosylase, Mag, that recognizes 3MeA and various other DNA lesions including 1,N6-ethenoadenine (epsilon A), hypoxanthine (Hx) and abasic (AP) sites. In the present study, we explore the relative substrate specificity of Mag for these lesions and in addition, show that Mag also recognizes cisplatin cross-linked adducts, but does not catalyze their excision. Through competition binding and activity studies, we show that in the context of a random DNA sequence Mag binds epsilon A and AP-sites the most tightly, followed by the cross-linked 1,2-d(ApG) cisplatin adduct. While epsilon A binding and excision by Mag was robust in this sequence context, binding and excision of Hx was extremely poor. We further studied the recognition of epsilon A and Hx by Mag, when these lesions are present at different positions within A:T and G:C tracts. Overall, epsilon A was slightly less well excised from each position within the A:T and G:C tracts compared to excision from the random sequence, whereas Hx excision was greatly increased in these sequence contexts (by up to 7-fold) compared to the random sequence. However, given most sequence contexts, Mag had a clear preference for epsilon A relative to Hx, except in the TTXTT (X=epsilon A or Hx) sequence context from which Mag removed both lesions with almost equal efficiency. We discuss how DNA sequence context affects base excision by various 3MeA DNA glycosylases.

DNA bases carrying an exocyclic amino group, namely adenine (A), guanine (G) and cytosine (C), encounter deamination under nitrosative stress. Oxanine (O), derived from deamination of guanine, is a cytotoxic and potentially mutagenic lesion and studies of its enzymatic repair are limited. Previously, we reported that the murine alkyladenine glycosylase (Aag) acts as an oxanine DNA glycosylase (JBC (2004), 279: 38177). Here, we report our recent findings on additional oxanine DNA glycosylase (ODG) activities in Aag knockout mouse tissues and other mammalian tissues. Analysis of the partially purified proteins from the mammalian cell extracts indicated the existence of ODG enzymes in addition to Aag. Data obtained from oxanine DNA cleavage assays using purified human glycosylases demonstrated that two known glycosylases, hNEIL1 and hSMUG1, contained weak but detectable ODG activities. ODG activity was the highest in hAAG and lowest in hSMUG1.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.

We present Version 7 of a database of mouse mutant strains that affect biological responses to DNA damage. This database is also electronically available at http://pathcuricl.swmed.edu/research/research.htm.

We present Version 6 of a database of mouse mutant strains that affect biological responses to DNA damage. This database is also electronically available at http://pathcuric1.swmed.edu/research/research.htm.

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.

Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5'-endonuclease activity and 3'-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex(+/-)). Our data indicate that Apex(+/-) mice are indeed APE-haploinsufficient, as exhibited by a 40-50% reduction (p < 0.05) in APE mRNA, protein, and 5'-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase beta and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase beta-dependent BER response to cellular stress.

The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1,N6-ethenodeoxyadenosine ((epsilon)dA). Previous attempts to determine the in vivo role of Aag in (epsilon)dA repair were complicated by technological difficulties in measuring low levels of (epsilon)dA in genomic DNA. Here we describe the development of a new method for (epsilon)dA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of (epsilon)dA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal (epsilon)dA bases, these lesions can also be eliminated from the genome via an alternative mechanism., (Copyright 2003 Elsevier B.V.)

This article is an extension of previously published papers that periodically update a database of mouse mutant strains that are phenotypically defective in biological responses to DNA damage. Most of the mice listed in the database were generated by conventional targeted gene replacement technologies.

Mice defective in the mismatch repair (MMR) gene Msh2 manifest an enhanced predisposition to skin cancer associated with exposure to UVB radiation. This predisposition is further heightened if the mice are additionally defective for the nucleotide excision repair gene Xpc. To test the hypothesis that the predisposition of Msh2 mutant mice to skin cancer reflects a mutator phenotype associated with increased proliferation of skin cells following exposure to UV radiation, Msh2 mutant mice were exposed to the tumor promoter TPA. Such mice showed a robust proliferative response in the skin, but did not manifest evidence of dysplasia or neoplasia. We conclude that the predisposition of Msh2 mice to UVB radiation-induced skin cancer reflects an interaction between the processes of mismatch repair and some other excision repair mode, the exact nature of which remains to be established.

Apurinic/apyrimidinic endonuclease is a key enzyme in the process of base excision repair, required for the repair of spontaneous base damage that arises as a result of oxidative damage to DNA. In mice, this endonuclease is coded by the Apex gene, disruption of which is incompatible with embryonic life. Here we confirm the embryonic lethality of Apex-null mice and report the phenotypic characterization of mice that are heterozygous mutants for the Apex gene (Apex+/-). We show that Apex heterozygous mutant cells and animals are abnormally sensitive to increased oxidative stress. Additionally, such animals manifest elevated levels of oxidative stress markers in serum, and we show that dietary supplementation with antioxidants restores these to normal levels. Apex+/- embryos and pups manifest reduced survival that can also be partially rescued by dietary supplementation with antioxidants. These results are consistent with a proposed role for this enzyme in protection against the deleterious effects of oxidative stress and raise the possibility that humans with heterozygous mutations in the homologous HAP1 gene may be at increased risk for the phenotypic consequences of oxidative stress in cells.

Mouse models that mimic the human skin cancer-prone disease xeroderma pigmentosum (XP) provide an useful experimental system with which to study the relationship between the DNA repair process of nucleotide excision repair (NER) and ultraviolet- (UV) induced skin carcinogenesis. We have generated Xpc mutant mice and documented their deficiency in the process of NER of UV-induced DNA damage. Xpc mutant mice are highly predisposed to UV-B radiation-induced skin cancer, both in the homozygous and the heterozygous state. The combination of Xpc and Trp53 mutations enhances this predisposition and alters the tumor spectrum observed in single mutant mice. These results suggest a synergism between NER and the function of Trp53 in suppression of cancer. We have examined the mutational spectrum in the Trp53 gene from skin cancers in Trp53+/+ and Trp53+/- mice of all three Xpc genotypes and have found evidence for signature mutations associated with defective NER. In addition, we have demonstrated that Xpc mutant mice are highly predisposed to the induction of lung and liver cancers by treatment with 2-acetylaminofluorene (2-AAF) and N-OH-2-AAF. By combining the Xpc mutation with other mutations in genes involved in repair of DNA damage we have identified additional genetic interactions important in carcinogenesis. The mouse Apex gene is a critical component of the base excision repair (BER) pathway as well as the redox regulation of transcription factors important in growth control and the cellular response to DNA damage. By combining mutations in Xpc, Trp53 and Apex we have obtained genetic evidence for a functional interaction between Apex and Trp53 which probably involves the activation of the Trp53 protein by Apex. Mutations in the mismatch repair (MMR) gene Msh2 also influence the carcinogenesis observed in Xpc Trp53 mutant mice. Our results demonstrate that multiple repair pathways operate in prevention of tumor formation.

Cerebro-oculo-facio-skeletal (COFS) syndrome is a rapidly progressive neurological disorder leading to brain atrophy with calcification, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low-to-normal birth weight; growth failure; brain dysmyelination with calcium deposits; cutaneous photosensitivity; pigmentary retinopathy, cataracts, or both; and sensorineural hearing loss. CS cells are hypersensitive to UV radiation because of impaired nucleotide excision repair of UV radiation-induced damage in actively transcribed DNA. The abnormalities in CS are associated with mutations in the CSA or CSB genes. In this report, we present evidence that two probands related to the Manitoba Aboriginal population group within which COFS syndrome was originally reported have cellular phenotypes indistinguishable from those in CS cells. The identical mutation was detected in the CSB gene from both children with COFS syndrome and in both parents of one of the patients. This mutation was also detected in three other patients with COFS syndrome from the Manitoba Aboriginal population group. These results suggest that CS and COFS syndrome share a common pathogenesis.

Mice that are genetically engineered are becoming increasingly more powerful tools for understanding the molecular pathology of many human hereditary diseases, especially those that confer an increased predisposition to cancer. We have generated mouse strains defective in the Xpc gene, which is required for nucleotide excision repair (NER) of DNA. Homozygous mutant mice are highly prone to skin cancer following exposure to UVB radiation, and to liver and lung cancer following exposure to the chemical carcinogen acetylaminofluorene (AAF). Skin cancer predisposition is significantly augmented when mice are additionally defective in Trp53 (p53) gene function. We also present the results of studies with mice that are heterozygous mutant in the Apex (Hap1, Ref-1) gene required for base excision repair and with mice that are defective in the mismatch repair gene Msh2. Double and triple mutant mice mutated in multiple DNA repair genes have revealed several interesting overlapping roles of DNA repair pathways in the prevention of mutation and cancer.

We have examined the mutational spectrum in the Trp53 gene from UVB radiation-induced skin cancers in Trp53+/+ and Trp53+/- mutant mice of all three possible Xpc genotypes. Mutations were detected in exons 2-10 of the Trp53 coding region in approximately 90% of >80 different skin cancers examined. In contrast to Trp53+/+ mice in which most mutations in the Trp53 gene were located in exons 5-8, the majority of the mutations in Trp53+/- mice were at other exons. We observed a high predilection for C-->T transition mutations at a unique CpG site in codon 122 (exon 4) of the Trp53 gene in Xpc-/- Trp53+/- mice. This site is not part of a pyrimidine dinucleotide. Mutations at this codon, as well as in codons 124 and 210, were observed exclusively in Xpc-/- or Xpc+/- mice. Mutations at the corresponding codons (127 and 213) in the human p53 gene have been reported in skin tumors from human patients with xeroderma pigmentosum. Hence, mutations at codons 122 (125), 124 (127), and 210 (213) may constitute signatures for defective or deficient nucleotide excision repair in mice (humans). In Xpc-/- mice, the majority of mutations were located at C residues in CpG sites, in which the C is presumably methylated. A similar bias can be deduced from studies in human XP individuals.

Mutations in nucleotide excision repair (NER) genes in humans result in the UV-induced skin cancer-prone disease xeroderma pigmentosum (XP). Mouse models that mimic XP have provided an informative experimental system with which to study DNA repair, as well as the molecular pathology of UV radiation-induced skin cancer. We reported previously that mice defective in the Xpc gene (Xpc-/-) are highly predisposed to UVB radiation-induced skin cancer and that the appearance of skin cancer is more rapid in Xpc Trp53 double mutants. Extended studies now demonstrate an increased predisposition to UVB radiation-induced skin cancers in Xpc heterozygous mice compared with normal mice. We also show that Xpc Trp53 double heterozygous mutants are more predisposed to skin cancer than Trp53 single heterozygous mice. No mutations were detected in the cDNA of the remaining Xpc allele, suggesting that haploinsufficiency of the Xpc gene may be operating and is a risk factor for UVB radiation-induced skin cancer in mice. Skin tumors from Xpc-/- mice were exclusively well or moderately well-differentiated squamous cell carcinomas. In Xpc+/+ and Xpc+/- mice, many of the squamous cell carcinomas were less well differentiated. We also documented previously increased predisposition to UV radiation-induced skin cancers in Xpc-/- Apex+/- mice. Here we show the absence of mutations in the cDNA of the remaining Apex allele, a further suggestive indication of haploinsufficiency and its resulting predisposition to skin cancer. The Trp53 and Apex heterozygous conditions altered the skin tumor spectrum to more poorly differentiated forms in all Xpc genotypes.