A DNA repair-independent role for alkyladenine DNA glycosylase in alkylation-induced unfolded protein response.

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.

Adipocytokines and disease progression in endometrial cancer: a systematic review.

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-ß1, 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.

Endoplasmic reticulum stress and oxidative stress drive endothelial dysfunction induced by high selenium.

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.

A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway.

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.

An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG).

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.

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

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 ß-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.

Circulating Adipocytokines and Insulin Like-Growth Factors and Their Modulation in Obesity-Associated Endometrial Cancer.

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.

Reprogramming of lysosomal gene expression by interleukin-4 and Stat6.

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.

DNA alkylation damage and autophagy induction.

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.

The Role of Cytokines in Epithelial-Mesenchymal Transition in Gynaecological Cancers: A Systematic Review.

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-ß, 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/ß-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.

O6-methylguanine-induced cell death involves exonuclease 1 as well as DNA mismatch recognition in vivo.

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.

Aag-initiated base excision repair drives alkylation-induced retinal degeneration in 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.

DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice.

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.

Alkyladenine DNA glycosylase deficiency uncouples alkylation-induced strand break generation from PARP-1 activation and glycolysis inhibition.

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.

3-Methyladenine DNA glycosylase is important for cellular resistance to psoralen interstrand cross-links.

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.

Substrate specificity and sequence-dependent activity of the Saccharomyces cerevisiae 3-methyladenine DNA glycosylase (Mag).

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.

Oxanine DNA glycosylase activities in mammalian systems.

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.

DNA Damage and Repair in Patients With Coronary Artery Disease: Correlation With Plaque Morphology Using Optical Coherence Tomography (DECODE Study).

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 ±â€¯53, NSTEMI = 115.6 ±â€¯85.1, SA = 81 ±â€¯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.

Database of mouse strains carrying targeted mutations in genes affecting biological responses to DNA damage Version 7.

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.