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The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation. [ABSTRACT FROM AUTHOR]

Background: Mammalian aging is a highly complex process, a full mechanistic understanding of which is still lacking. One way to help understand the molecular changes underlying aging is through a comprehensive analysis of the transcriptome, the primary determinant of age-related phenotypic diversity. Previous studies have relied on microarray analysis to examine gene expression profiles in different tissues of aging organisms. However, studies have shown microarray-based transcriptional profiling is less accurate and not fully capable of capturing certain intricacies of the global transcriptome. Methods: Here, using directional whole transcriptome RNA-sequencing of aged mouse liver we have identified a comprehensive high-resolution profile of differentially expressed liver transcripts comprised of canonical protein-coding transcripts, transcript isoforms, and non-coding RNA transcripts, including pseudogenes, long non-coding RNAs and small RNA species. Results: Results show extensive age-related changes in every component of the mouse liver transcriptome and a pronounced increase in inter-individual variation. Functional annotation of the protein-coding mRNAs and isoforms indicated broad alterations in immune response, cell activation, metabolic processes, and RNA modification. Interestingly, multiple lncRNAs (Meg3, Rian, Mirg) from the Dlk-Dio3 microRNA locus were found up-regulated in aging liver, classifying this locus as a putative regulatory hotspot locus in aging liver. Moreover, integration of the altered non-coding RNAs and protein-coding transcripts into interaction networks of age-related change revealed inflammation, cellular proliferation, and metabolism as the dominant aging phenotypes in mouse liver. Conclusions: Our analyses provide the first comprehensive dissection of the transcriptional landscape in aging mouse liver. [ABSTRACT FROM AUTHOR]

Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo . The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro . Evidently apoptosis is efficient, even in the absence of wild type nibrin. [ABSTRACT FROM AUTHOR]

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80−/− mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs−/− mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80−/−, dna-pkcs−/− and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver and spleen using a lacZ reporter. Our data confirm that inactivation of Ku80 and DNA-PKCS causes reduced lifespan and bodyweights, which is most severe in ku80−/− mice. All mutant mice exhibited a strong increase in lymphoma incidence as well as other aging-related pathology (skin epidermal and adnexal atrophy, trabacular bone reduction, kidney tubular anisokaryosis, and cortical and medullar atrophy) and severe lymphoid depletion. LacZ mutation frequency analysis did not show strong differences in mutation frequencies between knock out and wild type mice. The ku80−/− mice had the most severe phenotype and the Ku80-mutation was dominant over the DNA-PKCS-mutation. Presumably, the more severe degenerative effect of Ku80 inactivation on lifespan compared to DNA-PKCS inactivation is caused by additional functions of Ku80 or activity of free Ku70 since both Ku80 and DNA-PKCS are essential for NHEJ. [ABSTRACT FROM AUTHOR]

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70-/- cells and ku80-/- cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80-/- mice had a shorter life span than dna-pkcs-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER. [ABSTRACT FROM AUTHOR]

Aging and age-related pathology is a result of a still incompletely understood intricate web of molecular and cellular processes. We present a C57 BL/6 J female mice in vivo aging study of five organs (liver, kidney, spleen, lung, and brain), in which we compare genome-wide gene expression profiles during chronological aging with pathological changes throughout the entire murine life span (13, 26, 52, 78, 104, and 130 weeks). Relating gene expression changes to chronological aging revealed many differentially expressed genes ( DEGs), and altered gene sets ( AGSs) were found in most organs, indicative of intraorgan generic aging processes. However, only ≤ 1% of these DEGs are found in all organs. For each organ, at least one of 18 tested pathological parameters showed a good age-predictive value, albeit with much inter- and intraindividual (organ) variation. Relating gene expression changes to pathology-related aging revealed correlated genes and gene sets, which made it possible to characterize the difference between biological and chronological aging. In liver, kidney, and brain, a limited number of overlapping pathology-related AGSs were found. Immune responses appeared to be common, yet the changes were specific in most organs. Furthermore, changes were observed in energy homeostasis, reactive oxygen species, cell cycle, cell motility, and DNA damage. Comparison of chronological and pathology-related AGSs revealed substantial overlap and interesting differences. For example, the presence of immune processes in liver pathology-related AGSs that were not detected in chronological aging. The many cellular processes that are only found employing aging-related pathology could provide important new insights into the progress of aging. [ABSTRACT FROM AUTHOR]

A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the multiple documented changes with age are likely to involve exceptions to this pattern, possibly associated with specific cellular responses to age-related changes other than genotoxic stress. [ABSTRACT FROM AUTHOR]

Steady-state levels of spontaneous DNA damage, the by-product of normal metabolism and environmental exposure, are controlled by DNA repair pathways. Incomplete repair or an age-related increase in damage production and/or decline in repair could lead to an accumulation of DNA damage, increasing mutation rate, affecting transcription, and/or activating programmed cell death or senescence. These consequences of DNA damage metabolism are highly conserved, and the accumulation of lesions in the DNA of the genome could therefore provide a universal cause of aging. An important corollary of this hypothesis is that defects in DNA repair cause both premature aging and accelerated DNA damage accumulation. While the former has been well-documented, the reliable quantification of the various lesions thought to accumulate in DNA during aging has been a challenge. Here, we quantified inhibition of long-distance PCR as a measure of DNA damage in liver and brain of both normal and prematurely aging, DNA repair defective mice. The results indicate a marginal, but statistically significant, increase in spontaneous DNA damage with age in normal mouse liver but not in brain. Increased levels of DNA damage were not observed in the DNA repair defective mice. We also show that oxidative lesions do not increase with age. These results indicate that neither normal nor premature aging is accompanied by a dramatic increase in DNA damage. This suggests that factors other than DNA damage per se, for example, cellular responses to DNA damage, are responsible for the aging phenotype in mice. [ABSTRACT FROM AUTHOR]

Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species ( ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66 KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lac Z mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lac Z (p66Shc+/+) and lac Zp66 KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lac Zp66 KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66 Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lac Zp66 KO mice treated with H2 O2. These results indicate that p66 Shc plays a role in the accumulation of DNA rearrangements and suggest that p66 Shc functions to clear damaged cells rather than affect DNA metabolism. [ABSTRACT FROM AUTHOR]

RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements. [ABSTRACT FROM AUTHOR]

Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair , interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1-/Δ7, which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1 mice-/Δ7 were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1-/Δ7 mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1-/Δ7 mouse model. [ABSTRACT FROM AUTHOR]

Although several lines of evidence support a role for accumulating somatic mitochondrial DNA (mtDNA) mutations in the etiology of aging, it remains unclear if they are a major cause of age-related deterioration and death. Mouse models that harbor elevated mtDNA mutation frequencies age prematurely; these findings were thought to provide conclusive evidence for a causal role of such mutations in aging. Yet, the presence of several conflicting reports has sparked controversy in the field and this is further aggravated by discrepancies in the estimates of mtDNA mutant fractions, which disagree by orders of magnitude. Here, we briefly review the evidence and some of the unresolved questions surrounding a causative role for accumulating mtDNA mutations in aging. [Copyright &y& Elsevier]

Loss of genome maintenance may causally contribute to ageing, as exemplified by the premature appearance of multiple symptoms of ageing in a growing family of human syndromes and in mice with genetic defects in genome maintenance pathways. Recent evidence revealed a similarity between such prematurely ageing mutants and long-lived mice harbouring mutations in growth signalling pathways. At first sight this seems paradoxical as they represent both extremes of ageing yet show a similar 'survival' response that is capable of delaying age-related pathology and extending lifespan. Understanding the mechanistic basis of this response and its connection with genome maintenance would open exciting possibilities for counteracting cancer or age-related diseases, and for promoting longevity. [ABSTRACT FROM AUTHOR]

Non-homologous end joining (NHEJ) is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs) and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and cH2AX DNA damage foci in Ku80-/- as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements. [ABSTRACT FROM AUTHOR]

Background: Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery. Results: Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd. Conclusion: MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license [1]. [ABSTRACT FROM AUTHOR]

The accumulation of somatic DNA damage has been implicated as a cause of ageing in metazoa. One possible mechanism by which increased DNA damage could lead to cellular degeneration and death is by stochastic deregulation of gene expression. Here we directly test for increased transcriptional noise in aged tissue by dissociating single cardiomyocytes from fresh heart samples of both young and old mice, followed by global mRNA amplification and quantification of mRNA levels in a panel of housekeeping and heart-specific genes. Although gene expression levels already varied among cardiomyocytes from young heart, this heterogeneity was significantly elevated at old age. We had demonstrated previously an increased load of genome rearrangements and other mutations in the heart of aged mice. To confirm that increased stochasticity of gene expression could be a result of increased genome damage, we treated mouse embryonic fibroblasts in culture with hydrogen peroxide. Such treatment resulted in a significant increase in cell-to-cell variation in gene expression, which was found to parallel the induction and persistence of genome rearrangement mutations at a lacZ reporter locus. These results underscore the stochastic nature of the ageing process, and could provide a mechanism for age-related cellular degeneration and death in tissues of multicellular organisms. [ABSTRACT FROM AUTHOR]

Noncanonical DNA structures are postulated to be responsible for some breakpoint hotspots that occur frequently in cancers. We developed a novel mouse model system using the naturally occurring H-DNA structure that deviate from the familiar right-handed helical B form found at the breakage hotspot in the human c-MYC promoter and a Z-DNA-forming CG repeat to test this idea directly. Large-scale chromosomal deletions and/or translocations occurred in 5 (7.7%, 95% confidence interval [l] = 3.7% to 12.8%) of the 65 mice carrying the H-DNA-forming sequences and in 7 (6.6%, 95% Cl = 3.8% to 11.6%) of the 106 mice carrying the Z-DNA-forming sequences, but in 0 of the 63 control mice (P =042 and P = .035, respectively, two-sided test). Thus, the DNA structure itself can introduce instability in a mammalian genome. [ABSTRACT FROM AUTHOR]

Recent reports of premature aging in mutant mice with greatly increased rates of mitochondrial DNA mutagenesis (so-called 'mitochondrial mutator mice') appeared to confirm that accumulation of mtDNA mutations is a key mechanism of normal aging. Now, in a dramatic turnaround, a new study reports that levels of point mutations in tissues of aged normal mice are much lower than in the mutator mice, apparently ruling out a causal role in normal aging. [ABSTRACT FROM AUTHOR]