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.

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.

Vitamins D2 and D3 Have Overlapping But Different Effects on the Human Immune System Revealed Through Analysis of the Blood Transcriptome.

Vitamin D is best known for its role in maintaining bone health and calcium homeostasis. However, it also exerts a broad range of extra-skeletal effects on cellular physiology and on the immune system. Vitamins D2 and D3 share a high degree of structural similarity. Functional equivalence in their vitamin D-dependent effects on human physiology is usually assumed but has in fact not been well defined experimentally. In this study we seek to redress the gap in knowledge by undertaking an in-depth examination of changes in the human blood transcriptome following supplementation with physiological doses of vitamin D2 and D3. Our work extends a previously published randomized placebo-controlled trial that recruited healthy white European and South Asian women who were given 15 µg of vitamin D2 or D3 daily over 12 weeks in wintertime in the UK (Nov-Mar) by additionally determining changes in the blood transcriptome over the intervention period using microarrays. An integrated comparison of the results defines both the effect of vitamin D3 or D2 on gene expression, and any influence of ethnic background. An important aspect of this analysis was the focus on the changes in expression from baseline to the 12-week endpoint of treatment within each individual, harnessing the longitudinal design of the study. Whilst overlap in the repertoire of differentially expressed genes was present in the D2 or D3-dependent effects identified, most changes were specific to either one vitamin or the other. The data also pointed to the possibility of ethnic differences in the responses. Notably, following vitamin D3 supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding pathways of the innate and adaptive immune systems, potentially shifting the immune system to a more tolerogenic status. Surprisingly, gene expression associated with type I and type II interferon activity, critical to the innate response to bacterial and viral infections, differed following supplementation with either vitamin D2 or vitamin D3, with only vitamin D3 having a stimulatory effect. This study suggests that further investigation of the respective physiological roles of vitamin D2 and vitamin D3 is warranted.

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.

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.

Transcriptomics and micronutrient research.

This review examines the extent to which transcriptomic methods have lived up to their promise in the context of nutrition research, placing particular emphasis on examples from micronutrient research. A case is made that the high quality platform technologies now available, together with established standards and systems for data storage and exchange and powerful new methods of data analysis, mean that microarrays have reached a level of technical maturity at which they can be exploited to their full potential. In the context of nutrition and micronutrient research, transcriptomic methods have already been widely applied, albeit primarily in studies using cell lines and animal models. Using this type of approach, a multitude of genes regulated at the mRNA level by dietary components has been identified and this, in turn, has provided new insights into the biological processes affected by nutritional parameters. Evidence from the very limited number of published transcriptomics-based nutritional studies performed in human volunteers suggests that, with appropriate study design, it is feasible to apply transcriptomic methods successfully in dietary intervention trials. On the other hand, gene expression-based biomarker development still poses a major challenge. Here the use of expression profile 'signatures', rather than single genes, may provide a solution. Approaches designed to identify such 'signatures' are being developed and tested widely, primarily in the context of medical research. The applicability and power of such approaches should also be evaluated in the context of nutrition.

Variation in gene expression profiles of peripheral blood mononuclear cells from healthy volunteers.

The normal degree of intra- and interindividual variation in gene transcription profiles of healthy human tissues has not been extensively investigated. In the study described here, microarrays were employed to analyze gene transcription in peripheral blood mononuclear cells prepared from serial blood samples that had been obtained, at weekly intervals, from apparently healthy human volunteers. Transcript levels for the majority of genes examined were found to be remarkably consistent within samples from a single donor. Conversely, marked differences were observed in samples obtained from different donors. Genes that exhibited differential expression dependent on sex, age, body mass index, and the presence of varying proportions of different leukocyte subsets were identified. These results emphasize the important contributions of genetic and environmental factors, as well as varying representation of different cell types, in determining the overall gene transcriptional profiles of human tissues. However, the study also provides evidence that, within an individual, the gene transcription profiles of sampled tissues can be comparatively stable over time.

2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting.

BACKGROUND: Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation. METHODS: Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers. RESULTS: Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells. CONCLUSIONS: Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.

Identification of the Eph receptor pathway as a novel target for eicosapentaenoic acid (EPA) modification of gene expression in human colon adenocarcinoma cells (HT-29).

BACKGROUND: The health benefits of polyunsaturated fatty acids (PUFAs), particularly those of the n-3 series are well documented. The mechanisms by which these effects are mediated are not fully clarified. METHODS: We used microarrays to assess the effects on gene expression in HT29 colon adenocarcinoma cells of exposure to the n-3 fatty acid eicosapentaenoic acid (EPA). HT29 cells were cultured with EPA (150 muM) for up to 24 hr prior to harvesting and isolation of RNA. Microarray results were analyzed within the statistical package 'R', and GeneGo MetaCore was used to identify key pathways of altered gene expression. RESULTS: EphB4, Vav2 and EphA1 gene expression were identified as significantly altered by EPA treatment. Statistically significant changes in gene expression after HT29 exposure to EPA were confirmed in a second experiment by real-time RT-PCR (TaqMan), This experiment also compared the effects of exposure to EPA to arachadonic acid (AA, n-6). Corresponding changes in protein expression were also assessed by Western blotting. CONCLUSIONS: Eph receptor mediated signaling is an entirely novel signaling pathway through which EPA may promote a wide range of health benefits, in particular in relation to reduction of colorectal cancer progression.

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics.

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400-1,500 mug protein from platelets, and 100-600 mug from PBMC. 30 muL plasma depleted of albumin and IgG provided 350-650 mug protein. A sample of morning urine provided 0.9-8.6 mg protein/dL, and a sample of saliva provided 70-950 mug protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.

Se-methylselenocysteine alters collagen gene and protein expression in human prostate cells.

The anti-cancer activity of selenium is dose-dependent and species-specific but the mechanism is unclear. Se-methylselenocysteine (MSC), found in selenium-enriched alliums, is one of the most potent forms. We exposed two human prostate cell lines (LNCaP clone FGC and PNT1A) to nutritionally relevant doses of MSC and selenite, ranging from deficient to the equivalent of selenium supplementation in humans. The cells were adapted for one month to attain steady-state selenium status. Two microarray platforms, an in-house printed microarray (14,000 genes) and the Affymetrix U133A array (22,000 genes) were used to probe the molecular effects of selenium dose and form and several selenium-responsive genes were identified, many of which have been ascribed to cancer cell growth and progression. In response to MSC supplementation, the expression of 23 genes changed significantly, including several collagen genes. Quantitative RT-PCR assays were designed and optimized for four of the collagen genes to validate array data. Significant decreases in expression of collagen type I alpha 1 (COL1A1), COL1A2 and COL7A1 genes were observed in cells adapted to MSC supplementation compared to the control and selenite exposed cells. There were significant increases in genes encoding other types of collagen, including significant increases in COL6A1 and COL4A5 in response to MSC dose. Functional changes in collagen type I protein expression in response to MSC were confirmed by ELISA. This study reveals for the first time that MSC can alter the expression of several types of collagen and thus potentially modulate the extracellular matrix and stroma, which may at least partially explain the anti-cancer activity of MSC.

The use of BeWo cells as an in vitro model for placental iron transport.

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 +/- 0.5% of the total dose was transported after 8 h, equivalent to 38.8 +/- 2.1 pmol.cm(-2).h(-1). Transfer of Tf across the cell layer was much more limited; 2.4 +/- 0.2% of the total dose was transported after 8 h, equivalent to 5.0 +/- 0.4 pmol.cm(-2).h(-1). Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.

Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

Increased cellular carotenoid levels reduce the persistence of DNA single-strand breaks after oxidative challenge.

Dietary antioxidants, such as the carotenoids, may protect DNA from oxidative damage. This has been proposed to explain the epidemiological association between higher consumption of fruits and vegetables, which are rich in antioxidants, and lower incidence of cancer. However, this remains to be demonstrated conclusively. The effects of carotenoid supplementation on 1) baseline DNA damage, 2) susceptibility of cellular DNA to oxidative attack, and 3) DNA repair were measured in the human lymphocyte cell line Molt-17. Baseline DNA damage, susceptibility to oxidant attack (100 mumol/l H2O2 for 5 min at 4 degrees C), and disappearance of DNA single-strand breaks (SSB) after oxidative challenge were monitored by single-cell gel electrophoresis. DNA repair patch synthesis activity in cell extracts was determined using assays that measure nucleotide incorporation during repair of oxidative lesions in template DNA. Unlike single-cell gel electrophoresis, the parameters measured with these assays are not dependent on strand break religation. There was no evidence that beta-carotene, lutein, or beta-cryptoxanthin supplementation protected cellular DNA from oxidation under basal conditions or after oxidative challenge. However, only carotenoid-supplemented cells exhibited a significant decrease in numbers of SSB over a 2-h period after treatment with H2O2. Carotenoid supplementation did not provoke any detectable change in repair patch synthesis activity. We conclude that supplementation with carotenoids at 8 mumol/l does not provide significant antioxidant protection for DNA in Molt-17 lymphocytes but may enhance recovery of cells from oxidative challenge, as measured by loss of SSB. We argue that these data are most consistent with carotenoids acting to enhance DNA strand break repair.

Evidence that dietary supplementation with carotenoids and carotenoid-rich foods modulates the DNA damage: repair balance in human lymphocytes.

Epidemiological evidence has shown that the habitual consumption of diets high in fruits and vegetables is associated with reduced risk of cancers. The challenge is to identify causal mechanisms of effect. The aim of the current study was to determine whether an increase in rate of removal of DNA single-strand breaks (SSB) following oxidative challenge could be provoked ex vivo in peripheral blood lymphocytes (PBL). The PBL were isolated from apparently healthy volunteers following dietary intervention with: (1) a mixed carotene capsule; (2) a daily portion of cooked minced carrots; (3) a matched placebo; (4) a portion of mandarin oranges; (5) vitamin C tablets. Single-cell gel electrophoresis was employed to measure baseline levels of SSB and DNA susceptibility to oxidative damage, and to monitor the number of SSB over 4 h, in both unchallenged and H2O2-treated PBL. The enzymatic capacity for repair of different types of DNA oxidative lesions was also measured using two related cell-free assays. There was no evidence that any of the dietary supplementation regimens altered baseline levels of SSB, provided any direct antioxidant protection or altered DNA repair capacity, with two exceptions: the number of SSB following exposure to H2O2 decreased more rapidly in PBL from volunteers given the mixed carotene capsules and repair patch synthesis activity in PBL increased from volunteers given the cooked carrots. These results suggest that carotenoids and carotenoid-rich foods can influence DNA damage:repair by modulation of discrete stages in the DNA repair mechanisms.

DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoids in vitro and in vivo.

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studies in vitro and in vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with beta-carotene, lutein or lycopene at a range of concentrations (0.00-8.00 micromol/l) using a liposome delivery method. Uptake was dose-dependent. beta-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2.00 micromol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 micromol H2O2/l, 5 min, 4 degrees C) following incubation with carotenoids between 0.50 and 1.00 micromol/l; at >1.00 micromol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, beta-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma beta-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damage ex vivo was unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.